method development in electrospray ionisation fourier transform

Method Development in Electrospray Ionisation

Fourier Transform Ion Cyclotron Resonance

Mass Spectrometry Study of Plant Oils - Macadamia Oil as

a Model

By

Ahmad Mokhtari-Fard

A thesis submitted in fulfilment of the requirements

for the degree of Doctor of Philosophy

School of Chemistry

The University of New South Wales

Sydney, Australia

July 2008

Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils II

Declaration

I hereby declare that this thesis is my own work and that, to the best of my

knowledge and belief, it contains no material previously published or written by

another nor material which to substantial extent has been accepted for an award of

any other degree or diploma of a university or other institute of higher learning

except where due acknowledgement is made in the text of this thesis.

Ahmad Mokhtari-Fard

Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils III

Abstract

A novel analytical method is developed to examine the chemical composition of

plant oils by electrospray ionisation high-resolution Fourier transform ion cyclotron

resonance mass spectrometry in both positive- and negative-ion modes. To date, this

is the first reported application of this technique for the study of macadamia nut oil.

Samples of macadamia nut oil from the Macadamia Integrifolia- Proteaceae family

(smooth shell) are examined. The fatty acid profile of the oil is obtained by this mass

spectrometric examination of the transesterified and hydrolysed oil samples. The

Fourier transform ion-cyclotron resonance mass spectrometry results are compared to

those obtained from similar samples using gas chromatography-mass spectrometry

techniques. High performance liquid chromatography and Fourier transform ion

cyclotron resonance mass spectrometry are used to separate and assign the isomers

present in the methanol extract of the oils in separate experiments.

Significant results in this study include:

- The first observation and identity of a number of oxidised triacylglycerols in

macadamia oil samples.

- The first observation of oxidised and free fatty acids, measured directly in

hydrolysed oil and in the methanol extract of macadamia oil.

- High resolution Fourier transform ion cyclotron resonance mass spectrometry

in broadband mode which enables isobars to be observed.

Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils IV

- Esterified oil Fourier transform ion cyclotron resonance mass spectrometry

results are consistent with our gas chromatography-mass spectrometry results

and with the results of similar studies on macadamia oil in the literature.

- A number of fatty acids with odd number of carbon atoms are observed in the

oil.

- In electrospray ionisation Fourier transform ion cyclotron resonance mass

spectrometry of oils, the sample preparation is straightforward. The sample is

dissolved in methanol or acetonitrile and the solution is introduced to the

electrospray source directly. Introducing oil samples to the gas

chromatograph-mass spectrometer needs the oils to be esterified prior to the

analysis.

- In this work, state-of-the-art mass spectrometry demonstrates distinct

advantages in comparison to gas chromatography measurements such as

direct identification of free fatty acids in oil samples, whereas this is not

possible in gas chromatography-mass spectrometry due to the required

esterification step prior to the analysis.

- High performance liquid chromatography fraction collection is combined

with Fourier transform ion cyclotron resonance mass spectrometry in off-line

mode and found to improve the sensitivity, selectivity and signal to noise

levels due to the lower number of compounds in each high performance

liquid chromatography fraction compared to the methanol extract of

macadamia oil sample. Also isomers of monoacylglycerols have been

resolved using the high performance liquid chromatography technique.

Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils V

Acknowledgments

Many people have assisted me in this study and I wish to record my appreciation

here for their assistance.

I owe thanks to my supervisor Associate Professor Gary Willett for providing

guidance, encouragement and inspiration during the research project and for help in

writing my thesis. I have been always impressed by his down-to-earth personality

and earnest attitude towards scientific research.

My special thanks go to my co-supervisor Mr. Athol Turner who assisted me to

obtain and inspired me with the research described in this thesis. As a good friend, he

always supported me with many helpful discussions and suggestions. I believe what I

have learnt from him will benefit the rest of my life and my career. His young heart

is a gift that I wish will beat for many more years.

I gratefully appreciate the support and supervision provided by Associate Professor

Stephen Colbran, specially during the last year of my PhD study.

I appreciate the guidance and friendship provided by Professor Bryn Hibbert. I will

always be honored to recognize him as an outstanding colleague.

I would like to pay a special tribute to many people who helped me to complete this

thesis. Dr. Rui Zhang, who is a wonderful teacher and taught me how to use the

FTICR mass spectrometer, Dr. Derek Smith, an expert in theoretical chemistry who

helped me in calibration calculations, Dr. Joe Brophy, who assisted me to obtain the

GC-MS results and discussed them with me and Dr. Keith Fisher who provided me

with helpful ideas and procedures for operating the FTICR mass spectrometer.

Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils VI

Special thanks are also paid to FTICR-MS group members including Khaled Edbey,

John Giffard and Nick Proschogo. Over the years, I have received a great deal of

help from all of them. I also appreciate help from Mansour Ahmad teaching me some

skills in working with fatty acids on the FTICR mass spectrometer.

I also pay a special tribute to Mr. Han Sit Chan for his support in solving many

computer and network problems that I encountered during this research project.

Finally, my special thanks go to my wife (Fariba) and two children (Nazanin and

Reza) for supporting me throughout my PhD studies. They are very tolerant and

patient and have always encouraged me to move forward during long years of my

study. I am a lucky person to have such a supportive family.

Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils VII

Dedication Dedicated to:

My wife and my children, (Fariba, Nazanin and Reza)

my parents,

(my mother and the memory of my father)

and my teachers.

Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils VIII

Table of Contents

Declaration................................................................................................... II Abstract.......................................................................................................III Acknowledgments ........................................................................................ V Dedication ................................................................................................. VII Table of Contents.....................................................................................VIII List of Tables...............................................................................................XI List of Figures ..........................................................................................XIII List of Abbreviations ............................................................................... XVI 1. Introduction .............................................................................................. 1

1.1- Definitions ............................................................................................................... 5 1.1.1- Lipids............................................................................................................................ 5 1.1.2- Fatty Acids .................................................................................................................. 13 1.1.3- Triacylglycerols........................................................................................................... 21

1.1.3.1- Determination of the Positional Distribution of FAs in Fats and Oils ..................... 23 1.2- Macadamia, From Nut to Shelf............................................................................... 24

1.2.1- Introduction................................................................................................................. 24 1.2.2– History and Production of Macadamia Nut .................................................................. 25 1.2.3– Botanical Description.................................................................................................. 27 1.2.4- Soil, Climate and Nutrition .......................................................................................... 28 1.2.5- Harvesting................................................................................................................... 28 1.2.6- Oil Extraction .............................................................................................................. 30 1.2.7– Industrial Macadamia Nut Oil Refining Processes........................................................ 32

1.3- Chemical Reactions Used in Sample Preparation for FTICR-MS Analysis.............. 34 1.3.1– Methanol Extraction of Macadamia Nut Oil to Remove the Triacylglycerols................ 34 1.3.2- Transesterification of Macadamia Nut Oil .................................................................... 35 1.3.3- Alkaline Hydrolysis of Macadamia Nut Oil.................................................................. 36

1.4- Mass Spectrometry of Lipids.................................................................................. 38 1.4.1-Gas Chromatography-Mass Spectrometry ..................................................................... 39 1.4.2- Electrospray Ionisation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry............................................................................................................................................. 42

1.4.2.1- Electrospray Ionisation Source ............................................................................. 43 1.4.2.2- The ICR Cell........................................................................................................ 47

1.4.2.2.1- Ion Trapping................................................................................................. 48 1.4.2.2.2- Ion Cyclotron Motion.................................................................................... 50 1.4.2.2.3- Ion Cyclotron Excitation and Detection ......................................................... 51

1.4.2.3- Fourier Transform ................................................................................................ 55 1.4.2.4- Mass Calibration .................................................................................................. 56 1.4.2.5- Tandem Mass Spectrometry ................................................................................. 57

1.5- High Performance Liquid Chromatography ............................................................ 59 1.6- Kendrick Masses and Mass Defects in the Identification of Homologous Series...... 62 1.7- Normal Probability (Rankit) Plot ............................................................................ 63 1.8- Summary of the Method Development ................................................................... 63

2. Experimental........................................................................................... 68 2.1- Materials................................................................................................................ 69 2.2- Chemical Procedures.............................................................................................. 69

2.2.1- Methanol Extraction of Macadamia Oil........................................................................ 69 2.2.2- Hydrolysis of Macadamia Oil ...................................................................................... 70 2.2.3- Transesterification of Macadamia Oil........................................................................... 70 2.2.4- Sample Preparation for Mass Spectrometry .................................................................. 71

2.3- Instrumentation ...................................................................................................... 71

Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils IX

2.3.1- Electrospray-ionisation Fourier Transform Ion Cyclotron Resonance Mass Spectrometer............................................................................................................................................. 71

2.3.1.1- The Vacuum System ............................................................................................ 74 2.3.1.2- Electrospray Ionisation Source ............................................................................. 75 2.3.1.3- The “Infinity®” ICR cell ....................................................................................... 80 2.3.1.4- Ion Trapping ........................................................................................................ 82 2.3.1.5- Typical Source, Ion-transfer and ICR Cell Parameters Used in Positive- and Negative-ion Modes on the BioApex II 70e FTICR Mass Spectrometer ............................. 83 2.3.1.6- Superconducting Magnet ...................................................................................... 84 2.3.1.7- Collision-Induced Dissociation ............................................................................. 84 2.3.1.8- Pulse Sequence in FTICR-MS Experiments .......................................................... 85 2.3.1.9- Mass Calibration .................................................................................................. 87 2.3.1.10- The BioApex II FTICR Mass Spectrometer Control Software ............................. 88

2.3.2- High Performance Liquid Chromatography.................................................................. 88 2.3.2.1- Gradient Elution................................................................................................... 89

2.3.3- Gas Chromatography- Mass Spectrometry.................................................................... 91 3. Validation of the ESI FTICR-MS Method Developed for the Analysis of Plant Oils..................................................................................................... 92

3.1- Relation between Peak Intensities and Concentration of the Ions ............................ 93 3.2- Reproducibility of the ESI Source and the FTICR Mass Spectrometer .................... 95 3.3- Detection Limits of FTICR-MS in FA Measurement ............................................ 100 3.4- Effect of the Hexapole Ion Trap Delay on the Peak Intensities.............................. 100 3.5- Aging Stability of the Oil Samples ....................................................................... 101 3.6- Fragmentation during Ion Transfer ....................................................................... 102 3.7- Discussion of the Validity of the Mass Spectral Peak Assignments ....................... 105 3.8- Normal Probability (Rankit) Test of the Peak Intensities and Measured Masses .... 106

4. Positive-ion ESI FTICR-MS of Processed Macadamia Oil................. 108 4.1- Introduction ......................................................................................................... 109 4.2- Positive-ion ESI FTICR-MS of Processed Macadamia Oil.................................... 109

4.2.1- Free Fatty Acids and Monoacylglycerols Region, m/z 150-400................................... 112 4.2.2- Diacylglycerol Region, m/z 500-750 .......................................................................... 114 4.2.3- Triacylglycerol Region, m/z 800-1000 ....................................................................... 115

4.3- Positive-ion ESI FTICR-MS of Methanol Extract of Processed Macadamia Oil .... 117 4.4- Positive-ion ESI FTICR-MS of Hydrolysed Processed Macadamia Oil................. 121 4.5- Positive-ion ESI FTICR-MS of Esterified Processed Macadamia Oil.................... 123 4.6- Positive-ion ESI FTICR-MS of Esterified Methanol Extract of Processed Macadamia Oil .............................................................................................................................. 128 4.7- A Comparison of the Fatty Acids Observed in the Positive-ion ESI FTICR Mass Spectra of the Neat, Methanol Extract, Hydrolysed, Esterified and Esterified Methanol Extract of Processed Macadamia oil ............................................................................ 130

5. Negative-ion ESI FTICR-MS of Processed Macadamia Oil ............... 139 5.1- Introduction ......................................................................................................... 140 5.2- Negative-ion ESI FTICR Mass Spectra of Neat Processed Macadamia Oil ........... 141

5.2.1- Introduction............................................................................................................... 141 5.2.2- Free Fatty Acids and Monoacylglycerol Region, m/z 150-400 .................................... 142 5.2.3- Diacylglycerol Region, m/z 500-750 .......................................................................... 145

5.2.3.1- Free Fatty Acid Dimers ...................................................................................... 147 5.2.3.2- Diacylglycerols .................................................................................................. 150

5.2.4- Triacylglycerol Region, m/z 800-1000 ....................................................................... 151 5.3- Negative-ion ESI FTICR Mass Spectra of the Methanol Extract of Processed Macadamia Oil............................................................................................................ 154

5.3.1- Introduction............................................................................................................... 154 5.3.2- Free Fatty Acids and Monoacylglycerol Region, m/z 150-400 .................................... 157

5.3.2.1- Kendrick Mass Defect (KMD) Values ................................................................ 161 5.3.3- Diacylglycerol Region, m/z 500-750 .......................................................................... 162 5.3.4- Triacylglycerol Region, m/z 800-1000 ....................................................................... 168

5.3.4.1- KMD Values of the Assignments in the TAG Region.......................................... 171

Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils X

5.3.5- Stability of the Methanol Extract of Processed Macadamia Oil ................................... 172 5.4- Negative-ion ESI FTICR-MS of the Hydrolysed Processed Macadamia Oil.......... 175

5.4.1- Introduction............................................................................................................... 175 5.4.2- FTICR Mass Spectrum of Hydrolysed Processed Macadamia Oil ............................... 176 5.4.3- Comparison of FTICR Mass Spectra of Macadamia Oils ............................................ 180

5.5- A Summary of the Negative-ion ESI FTICR-MS of the Neat, Methanol Extract and Hydrolysed Processed Macadamia Oil......................................................................... 183

6. Gas Chromatography-Mass Spectrometry of Processed Macadamia Oil................................................................................................................... 189

6.1- Introduction ......................................................................................................... 190 6.2- GC-MS Analysis of Esterified Processed Macadamia Oil ..................................... 192 6.3- GC-MS Analysis of the Esterified Methanol Extract of Macadamia Oil ................ 193 6.4- A Summary of the Positive-ion and Negative-ion ESI FTICR-MS and GC-MS of the Hydrolysed and Esterified Neat Processed Macadamia Oil .......................................... 195 6.5- Conclusions ......................................................................................................... 200

7. Off-line ESI FTICR-MS of HPLC Fractions of the Methanol Extract of Processed Macadamia Oil ........................................................................ 202

7.1- Introduction ......................................................................................................... 203 7.2- HPLC of the Methanol Extract of Processed Macadamia Oil ................................ 205 7.3- ESI FTICR-MS of the HPLC Fractions 19 to 31 of the Methanol Extract of Processed Macadamia Oil............................................................................................................ 207 7.4- ESI FTICR-MS of the HPLC Fractions 32 to 50 of the Methanol Extract of Processed Macadamia Oil............................................................................................................ 212

7.4.1- Fractions 32 to 35 ...................................................................................................... 213 7.4.2- Fractions 36 to 38 ...................................................................................................... 213 7.4.3- Fractions 38 to 40 ...................................................................................................... 215 7.4.4- Fractions 44 to 48 ...................................................................................................... 217

7.5- ESI FTICR-MS of the HPLC Fractions 50 to 60 of the Methanol Extract of Processed Macadamia Oil............................................................................................................ 220 7.6- ESI FTICR-MS of the HPLC Fractions 60 to 63 of the Methanol Extract of Processed Macadamia Oil............................................................................................................ 222 7.7- ESI FTICR-MS of the HPLC Fractions 77 to 83 of the Methanol Extract of Processed Macadamia Oil............................................................................................................ 224 7.8- Positive- and Negative-ion FTICR Mass Spectra of the HPLC Blank Fractions of the Methanol Extract of Processed Macadamia Oil............................................................ 226 7.9- General Discussion: ............................................................................................. 228

8. Conclusions and Future Work ............................................................. 235 8.1- General Conclusion for this Study ........................................................................ 236 8.2- Future Work......................................................................................................... 239

9. References ............................................................................................. 244

Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils XI

List of Tables Table 1.1- Four nomenclature methods of FAs............................................................................... 15 Table 1.2- Some saturated and unsaturated FAs............................................................................ 17 Table 1.3- Composition of 100 g of macadamia nuts and the FA constituent of macadamia nut oil.

............................................................................................................................................. 25 Table 1.4- World production and consumption of macadamia nut-in-shell (NIS) and kernel in year

2000...................................................................................................................................... 26 Table 1.5- Different trapping methods usually used in FTICR mass spectrometry. ......................... 48 Table 2.1- Typical parameters used in positive- and negative-ion FTICR mass spectrometry

experiments.......................................................................................................................... 83 Table 2.2- Solvent programming for HPLC in the gradient elution of macadamia oil methanol

extract. ................................................................................................................................. 90 Table 3.1- Assignment of mass spectral peaks in the FTICR mass spectrum of a solution of fatty

acids mixture solution shown in Figure 3.1. .......................................................................... 96 Table 4.1. Assignment of the mass spectral peaks (>2%) in the positive-ion ESI-FTICR mass

spectrum of neat macadamia oil shown in Fig. 4.1.............................................................. 111 Table 4.2. Assignment of the mass spectral peaks (>2%) in positive-ion ESI-FTICR mass spectrum of

the methanol extract of macadamia oil shown in Figure 4.5............................................... 119 Table 4.3. Assignment of the major mass spectral peaks (>2%) in the positive-ion ESI-FTICR mass

spectrum of hydrolysed macadamia nut oil shown in Figure 4.6......................................... 122 Table 4.4- Assignment of the mass spectral peaks (>2%) in the positive-ion ESI-FTICR mass

spectrum of esterified macadamia nut oil shown in Figure 4.7. .......................................... 124 Table 4.5. Assignment of the mass spectral peaks (>1%) in the positive-ion ESI-FTICR mass

spectrum of the esterified methanol extract of processed macadamia oil shown in Fig. 4.10............................................................................................................................................ 129

Table 4.6- A comparison of the fatty acid components observed in the positive-ion ESI-FT-ICR mass spectrometry experiments.................................................................................................. 134

Table 5.1- Assignment of the mass spectral peaks (>2%) in the expanded fatty acid region (m/z 150-400) of the negative-ion ESI FTICR mass spectrum of processed macadamia nut oil shown in Figure 5.2. ....................................................................................................................... 144

Table 5.2- Assignment of the mass spectral peaks (>0.2% of the base peak in Figure 5.1) in the expanded DAG region (m/z 500-750) of the negative-ion ESI FTICR mass spectrum of processed macadamia oil shown in Figure 5.3. ................................................................... 146

Table 5.3- Assignment of the mass spectral peaks (>2% of the base peak in Figure 5.1) in the expanded TAG region (m/z 800-1000) of the negative-ion ESI FTICR mass spectrum of processed macadamia nut oil shown in Figure 5.5. ............................................................. 152

Table 5.4- Assignment of selected mass spectral peaks (>2%) in the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil shown in Fig. 5.6. ....... 157

Table 5.5- Assignment of the mass spectral peaks (>2%) in the expanded FA region (m/z 150-400) of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil shown in Figure 5.7............................................................................... 158

Table 5.6- Calculated Kendrick mass defects for homologous series in the negative-ion ESI FTICR mass spectrum of methanol extract of macadamia nut oil.................................................. 161

Table 5.7- Assignment of the mass spectral peaks (>2%) in the expanded DAG region (m/z 500-750) of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil shown in Figure 5.8............................................................................... 164

Table 5.8- Assignment of the mass spectral peaks (>2%) and KMD values in the expanded TAG region (m/z 800-1000) of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil shown in Figure 5.9.......................................................... 169

Table 5.9- Assignment of the mass spectral peaks (>2% of the base peak) in the negative-ion ESI FTICR mass spectrum of hydrolysed processed macadamia oil shown in Fig. 5.11. ............. 177

Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils XII

Table 5.10- Relative intensities of the assigned mass spectral peaks (>2% of the base peak) in the FTICR mass spectra of hydrolysed neat macadamia oil in both positive and negative-ion modes and the esterified oil in positive-ion mode. ............................................................. 188

Table 6.1- Assignment of the peaks in the GC-MS TIC of esterified processed macadamia oil...... 192 Table 6.2- Assignment of the peaks in the GC-MS TIC of esterified methanol extract of processed

macadamia oil. ................................................................................................................... 194 Table 6.3- A comparison of the FA ions observed in the positive-ion (Tables 4.3 & 4.4) and

negative-ion (Table 5.9) FTICR-MS as well as the GC-MS analysis (Table 6.1) of the hydrolysed and esterified neat processed macadamia nut oil............................................................... 198

Table 7.1. Assignment of the observed mass spectral peaks in the positive-ion ESI-FTICR mass spectra of the HPLC fractions 19 to 31 in Figure 7.3. ........................................................... 210

Table 7.2. Assignment of the observed mass spectral peaks in the negative-ion ESI-FTICR mass spectra of the HPLC fractions 19 to 31 in Figure 7.4. ........................................................... 211



Table 7.5. Assignment of the observed mass spectral peaks in the negative-ion ESI-FTICR mass spectrum of the HPLC fraction 40 in Figure 7.9. .................................................................. 216

Table 7.6. Assignment of the observed mass spectral peaks in the positive-ion ESI-FTICR mass spectra of the HPLC fractions 44 to 48 in Figure 7.10. ......................................................... 218

Table 7.7. Assignment of the observed mass spectral peaks in the negative-ion ESI-FTICR mass spectra of the HPLC fractions 44 to 48 in Figure 7.11. ......................................................... 219




Table 7.11. Assignment of the major mass spectral peaks in the ESI-FTICR mass spectra of the HPLC fractions of the methanol extract of macadamia nut oil. .................................................... 230

Table 7.12. A comparison of the retention times of the HPLC analysis of the methanol extract of macadamia nut oil with standard FA solutions analysed on same column in a previous study............................................................................................................................................ 234

Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils XIII

List of Figures Figure 1.1- A general classification of lipids...................................................................................... 6 Figure 1.2- Chemical structure of (a) glycerol and (b) a generalized structure of a triacylglycerol. ... 9 Figure 1.3- A naturally occurring TAG in cocoa butter. ..................................................................... 9 Figure 1.4- Structures of some common lipids. .............................................................................. 13 Figure 1.5- Hydrolysis of trioleyl glycerol to yield three oleic acid molecules and the glycerol

molecule............................................................................................................................... 19 Figure 1.6- General TAG molecule.................................................................................................. 21 Figure 1.7- Macadamia nuts on the tree ........................................................................................ 28 Figure 1.8- Cold press equipment used in the industrial production of seed and nut oil. Top: a batch

press, bottom: a hole-cylinder type oil expeller. .................................................................. 31 Figure 1.9- Saponifiable lipids in the hydrolysis reaction................................................................ 37 Figure 1.10- Non saponifiable lipids in the hydrolysis reaction of oils. ........................................... 38 Figure 1.11- Formation of charged drops at the tip of electrospray needle in high intensity electric

field. ..................................................................................................................................... 43 Figure 1.12- The formation of Rayleigh jets at the fissility greater than 1. Fine droplets dispatched

from the main drop are visible in shots c and d. ................................................................... 44 Figure 1.13- Schematic diagram of the electrospray needle, capillary and skimmer (not to scale). 46 Figure 1.14- Schematic diagram of the infinity ICR cell used in Bruker BioApex II. ......................... 47 Figure 1.15- Ions orbit in a plane perpendicular to the direction of a uniform magnetic field, B.

Positive (a) and negative (b) ions rotate in opposite directions in ion cyclotron motion....... 50 Figure 1.16- Incoherent and undetectable ion cyclotron orbital motion (a) is converted to a

coherent and detectable motion by applying an electric field and (b) is detected in the ICR cell at the frequency of the ions of particular m/z value....................................................... 52

Figure 2.1- The passively shielded Bruker BioApex II 70e Fourier transform ICR mass spectrometer used in this thesis. ................................................................................................................ 72

Figure 2.2- Schematic diagram of the Bruker BioApex II 70e ESI Fourier transfer ICR mass spectrometer used in this research....................................................................................... 73

Figure 2.3- The differential pumping on the Bruker BioApex II ESI FTICR mass spectrometer vacuum system. ................................................................................................................................. 74

Figure 2.4- Schematic diagram of the off-axis Analytica ESI source used in this research project... 76 Figure 2.5- The off-axis and on-axis spray needles inside the Analytica ESI cage............................ 77 Figure 2.6- The capillary tip and the end plate. . ............................................................................ 77 Figure 2.7- Schematic diagram of quartz capillary, skimmer and hexapole location in the ion optics

system . ................................................................................................................................ 79 Figure 2.8- Photograph showing the hexapole ion-trap in Analytica ESI source in the BioApex II

FTICR mass spectrometer...................................................................................................... 80 Figure 2.9- The infinity ICR cell in BioApex II FTICR mass spectrometer. ........................................ 81 Figure 2.10- Schematic diagram of the infinity ICR cell used in BioApex II FTICR mass spectrometer.

PV1, EV1 etc are parameters’ name . .................................................................................... 82 Figure 2.11- Pulse sequence used in FTICR-MS analyses for a simple experiment. ......................... 85 Figure 3.1- Negative-ion ESI FTICR mass spectrum of the test solution. ......................................... 96 Figure 3.2- Relative intensities of palmitate anion peaks versus measured masses in 17 consecutive

FTICR mass spectra of the test solution (normalised versus oleate anion peak as 1)............. 98 Figure 3.3- Relative intensities of stearate anion peaks versus measured masses in 17 consecutive

FTICR analyses of the test solution (normalised versus oleate anion peak as 1).................... 98 Figure 3.4- Average deviations from exact masses vs. average measured masses in 17 consecutive

FTICR mass spectra of the test solution. Each point represents the average of 17 mass measurements of a particular FA in the test solution............................................................ 99

Figure 3.5- Positive-ion FTICR mass spectra of methanol solution of neat processed macadamia oil in (a) December 2002, (b) July 2003 and (c) September 2003.............................................. 102

Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils XIV

Figure 3.6- Negative-ion FTICR mass spectra of the FA test solution at three capillary-skimmer voltages, a) 20 V, b) 125 V and c) 300 V............................................................................... 104

Figure 3.7- Rankit plot of the measured masses of palmitate anion (255.2330 Da) in seventeen FTICR-MS analyses of the FA solution in negative-ion mode. .............................................. 107

Figure 3.8- Rankit plot of the peak intensities of palmitate anion (255.2330 Da) in seventeen FTICR-MS analyses of the FA solution in negative-ion mode......................................................... 107

Figure 4.1- Positive-ion ESI-FTICR mass spectrum of neat macadamia nut oil. ............................. 110 Figure 4.2- Expanded FA region of positive-ion ESI-FTICR mass spectrum of neat macadamia oil. 112 Figure 4.3- Expanded DAG region of positive-ion ESI-FTICR mass spectrum of neat macadamia nut

oil. ...................................................................................................................................... 114 Figure 4.4- Expanded TAG region of positive-ion ESI-FTICR mass spectrum of neat macadamia nut

oil. The thin vertical line underneath each peak is produced by the peak-picking routine in Bruker software.................................................................................................................. 116

Figure 4.5- Positive-ion ESI-FTICR mass spectrum of the methanol extract of macadamia nut oil. 118 Figure 4.6- Positive-ion FTICR mass spectrum of hydrolysed macadamia nut oil.......................... 121 Figure 4.7- Positive-ion ESI-FTICR mass spectrum of esterified macadamia oil............................. 123 Figure 4.8- Expanded FA and FAME region of the positive-ion ESI-FTICR mass spectrum of esterified

macadamia nut oil shown in Figure 4.7............................................................................... 125 Figure 4.9- Positive-ion FTICR mass spectrum of esterified macadamia oil in m/z 279.12 to m/z

279.23 region. Peaks 0.03 Da apart are resolved................................................................. 127 Figure 4.10- Positive-ion FTICR mass spectrum of esterified methanol extract of macadamia nut oil.

........................................................................................................................................... 128 Figure 4.11- A comparison of the ESI FTICR mass spectra of the FA region of (a) neat macadamia oil,

(b) methanol extract of macadamia oil, (c) hydrolysed macadamia oil and (d) esterified macadamia oil. ................................................................................................................... 132

Figure 4.12- A graphical comparison of the unsubstituted FA anions observed in the positive-ion FTICR mass spectra of the neat (Table 4.1), the methanol extract (Table 4.2), hydrolysed (Table 4.3) and the esterified (Table 4.4) processed macadamia nut oil.............................. 133

Figure 5.1- Negative-ion ESI FTICR mass spectrum of neat processed macadamia nut oil. ........... 142 Figure 5.2- Fatty acid region of the negative-ion ESI FTICR mass spectrum of neat processed

macadamia nut oil in Figure 5.1, m/z 150-400. ................................................................... 143 Figure 5.3- DAG region of the negative-ion ESI FTICR mass spectrum of neat processed macadamia

nut oil in Figure 5.1, m/z 500-750........................................................................................ 146 Figure 5.4- A comparison of the experimental (a) and simulated (b) isotopic distribution of the

C34H65O4¯ fatty acid dimer anion. ........................................................................................ 148 Figure 5.5- (a) TAG region of the negative-ion ESI FTICR mass spectrum of the neat processed

macadamia nut oil in Figure 5.1, m/z 800-1000, (b) expanded peaks in the TAG region, m/z 870-882............................................................................................................................... 151

Figure 5.6- Negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil. ................................................................................................................................ 156

Figure 5.7- FA region of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil in Figure 5.6, m/z 150-400.................................................... 158

Figure 5.8- DAG region of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil in Figure 5.6, m/z 500-750.................................................... 163

Figure 5.9- TAG region of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil in Figure 5.6, m/z 800-1000, (a) prior to internal calibration and (b) after internal calibration was carried out. ..................................................................... 169

Figure 5.10- A comparison of the negative-ion ESI FTICR mass spectra of the methanol extract of macadamia oil on two dates: (a) 22/01/2003 and (b) 19/08/2004...................................... 173

Figure 5.11- Negative-ion ESI FTICR mass spectrum of hydrolysed processed macadamia oil. ..... 177 Figure 5.12- Comparison of the negative-ion ESI FTICR mass spectrum of (a) hydrolysed processed

macadamia oil, (b) hydrolysed cold pressed oil batch 12 and (c) hydrolysed cold pressed oil batch 13.............................................................................................................................. 181

Figure 6.1- GC-MS TIC of esterified processed macadamia oil. ..................................................... 192 Figure 6.2- GC-MS TIC of esterified methanol extract of macadamia oil....................................... 194 Figure 7.1- HPLC chromatogram of the methanol extract of processed macadamia oil. ............... 206

Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils XV

Figure 7.2- Expansion of HPLC chromatogram of the methanol extract of processed macadamia nut oil (Figure 7.1) at retention times of 19 to 31 minutes. ....................................................... 207

Figure 7.3- Positive-ion ESI FTICR mass spectra of the HPLC fractions 19 to 31 of the methanol extract of processed macadamia nut oil. ............................................................................ 208

Figure 7.4- Negative-ion ESI FTICR mass spectra of the HPLC fractions 19 to 31 of the methanol extract of processed macadamia nut oil. ............................................................................ 209

Figure 7.5- Expansion of the HPLC chromatogram of the methanol extract of processed macadamia nut oil (Figure 7.1) at retention times 32 to 50 minutes...................................................... 212

Figure 7.6- Negative-ion ESI FTICR mass spectra of the HPLC fractions: (a) fraction 34 and (b) fraction 35 of the methanol extract of processed macadamia nut oil. ................................ 213

Figure 7.7- Positive-ion ESI FTICR mass spectra of the HPLC fractions: (a) fraction 36, (b) fraction 37 and (c) fraction 38 of the methanol extract of processed macadamia nut oil...................... 214

Figure 7.8- Positive-ion ESI FTICR mass spectra of the HPLC fractions, (a) fraction 38, (b) fraction 39 and (c) fraction 40 of the methanol extract of processed macadamia nut oil...................... 215

Figure 7.9- Negative-ion ESI FTICR mass spectrum of the HPLC fraction 40 of the methanol extract of processed macadamia oil................................................................................................ 217

Figure 7.10- Positive-ion ESI FTICR mass spectra of the HPLC fractions 44 to 48 of the methanol extract of processed macadamia oil.................................................................................... 217

Figure 7.11- Negative-ion ESI FTICR mass spectra of the HPLC fractions 44 to 48 of the methanol extract of processed macadamia oil.................................................................................... 219

Figure 7.12- Expansion of the HPLC chromatogram of the methanol extract of processed macadamia nut oil at retention times 50 to 60 minutes...................................................... 220



Figure 7.15- Expanded HPLC chromatogram of the methanol extract of processed macadamia nut oil at retention times 77 to 83 minutes............................................................................... 224


Figure 7.17- Positive-ion FTICR mass spectrum of the HPLC blank fraction. ................................. 226 Figure 7.18- Negative-ion FTICR mass spectrum of the HPLC blank fraction. . .............................. 227

Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils XVI

List of Abbreviations

APCI Atmospheric pressure chemical ionisation ATP Adenosine triphosphate CI Chemical ionisation CID Collision induced dissociation DAG Diacylglycerol ECD Electron capture detector ELSD Evaporative light scattering detector EI Electron ionisation ESI Electrospray ionisation FA Fatty acid FAME Fatty acid methyl ester FFA Free fatty acid FTICR Fourier transfer ion cyclotron resonance GC Gas chromatography HPLC High performance liquid chromatography ICR Ion cyclotron resonance IUPAC International Union of Pure and Applied Chemistry KMD Kendrick mass defect LC Liquid chromatography LDL Low density lipoprotein MAG Monoacylglycerol MALDI Matrix assisted laser desorption ionisation MS Mass spectrometry PEG Poly ethylene glycol PUFA Polyunsaturated fatty acid S/N Signal-to-noise TAG Triacylglycerol TOF Time of flight TOC Total ion chromatogram UHV Ultra high vacuum

Mokhtari-Fard – Method Development in ESI FTICR-MS Study of Plant Oils 1

Chapter 1

1. Introduction


Plant oils (edible oils) play an important role in human nutrition due to their every

day consumption and important biologically active compounds present in them. The

chemical composition of the different types of oil is considerably diverse, but in

general they contain significant amounts of triacylglycerols (approximately 97%) and

a large number of important minor chemical compounds. Plant oils are usually

characterized by standard techniques such as gas chromatography-mass spectrometry

(GC-MS) for fatty acid methyl ester (FAME) and reversed phase high performance

liquid chromatography (HPLC) for triacylglycerol (TAG) region. These two

techniques only discriminate between different types of oils such as macadamia,

canola and sunflower but not between different batches of same oil from the same

extracted lipid source, because in the case of FAME analysis the ratio of the fatty

acids present in each oil sample remain virtually constant. The aim of this research is

to develop a novel method for the analysis of oils and fats using electrospray

ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry

(FTICR-MS) which enables the direct analysis of complex mixtures, hence enabling

different batches of the same oil (containing different chemical finger prints) to be

chemically characterized. No other analytical technique has the capability of

analyzing oil samples which contain hundreds of compounds and this is highly

relevant to the worldwide edible oil industry. Traditionally, gas chromatography-

mass spectrometry (GC-MS) has been the favoured method for the analysis of oils

and fats and the majority of studies on the analysis of oils and fats have been

conducted using this technique.[1-5]

ESI FTICR-MS demonstrates several advantages in comparison to GC-MS

technique.


- The high resolution and high mass accuracy of the ESI FTICR-MS technique

allows the direct analysis of macadamia nut oil without prior

chromatographic separation. The GC-MS technique, requiring the oil to be

esterified prior to gas chromatography analysis, significantly alters the

chemical composition of the oil, and is also a time-consuming process.

- The high resolution in broadband mode (average 50,000 full width at half

maximum (FWHM)) is a feature of FTICR-MS which enables isobars in

complex mixtures such as oils to be easily resolved.

- The high mass accuracy of FTICR-MS assists one in assigning possible

chemical formulae to selected mass spectral peaks.[6,7] This facility restricts

the number of possible candidates to a small group depending on the

resolution and the mass accuracy of the technique at the designated molecular

mass. As well, the selection criteria applied by the analyst include acceptable

mass measurement error, nominated participating atoms (elemental

composition including carbon, hydrogen, oxygen, nitrogen, phosphorus and

sulphur) and isotope patterns.[8] However, to postulate more accurate

structural formulae we need to carry out further separation and tandem mass

spectrometric analyses.

- Sample preparation and introduction in ESI FTICR-MS includes dissolution

of the sample in a suitable solvent such as methanol and introduction of the

solution into the ESI source; a comparatively simple process.


- The sample is introduced to the ESI source at room temperature and

atmospheric pressure. No high temperature or low pressure inlet system is

required.

- Collision-Induced Dissociation (CID) can be used to provide information

regarding the molecular structure of components of the oil by selectively

fragmenting molecular ions.[9] GC-MS also provides structural information

by fragmentation of the analyte molecules by electron ionization (EI),

however, the fragmentation is not selective. A further drawback to GC-MS is

that double bonds are prone to migrate on the hydrocarbon chain under EI

conditions. Finally, oil samples need to be transesterified so that the

acylglycerols are converted to fatty acid methyl esters to permit the GC-MS

analysis.

The ESI FTICR-MS technique does not resolve molecular isomers, as separation is

based on m/z only. This problem is partly overcome by applying tandem mass

spectrometry in the form of CID to selectively break up the molecular ions into

smaller fragments.[10]

The alternative is to use HPLC in conjunction with ESI FTICR-MS in off-line or on-

line mode. A combination of HPLC and GC-MS has been used in the analysis of

conjugated linoleic acid isomers by Roach et al.[11]

In the off-line mode, the various compounds in the oil are separated by an HP liquid

chromatograph that is connected to a fraction collector which collects the output at

certain time intervals. The collected fractions are then analysed by ESI FTICR-MS.


In the on-line mode, on the other hand, the output of the HPLC column is connected

directly to the ESI source of the mass spectrometer.

It should be emphasized here that ESI FTICR-MS is not considered to be a

replacement for the older techniques such as GC-MS, but instead as complementary.

The information collected using a combination of chromatography and FTICR-MS

techniques provide unparalleled precise understanding of the chemical constituents

of oils, especially for the characterization of trace compounds, and could eventually

be used to establish a library of the chemical composition of such oils.

1.1- Definitions 1.1.1- Lipids

Lipids are among the most important groups of biological molecules because of their

key roles in nutrition, metabolism and the energy needs of living cells. As a group of

organic compounds of various chemical compositions, lipids are usually divided into

four groups:

- Fatty acids (saturated and unsaturated)

- Glycerols (glycerol-containing lipids)

- Nonglycerol lipids (sphingolipids, steroids, waxes, lipid soluble vitamins)

- Complex lipids (lipoproteins)


The term “Lipid” has not found a widely accepted definition. General organic

chemistry and biochemistry textbooks usually use solubility-based definitions and

have defined lipids as a group of naturally occurring hydrophobic compounds that

are readily soluble in non-polar solvents such as hexane, chloroform, toluene and

ether, and have a very low solubility in water.[12,13]

Figure 1.1 shows the main groups and sub-groups of lipids.

Lipids

Fatty acids Glycerides

Saturated Unsaturated Neutral glycerides

Phospho-glycerides

Nonglyceride lipids

Complex lipids

LipoproteinsSphingolipidsSteroids Waxes

Sphingomyelins Glycolipids

Figure 1.1- A general classification of lipids.

Lipids include a wide range of compounds such as fatty acids (FAs) and their

derivatives, carotenoids, terpenes, steroids, waxes and nonglyceride lipids as

illustrated in Figure 1.1. The structures of the compounds in different classes of

lipids are not necessarily similar.


A more specific definition of lipids than one based on solubility is necessary. Most

lipid chemists tend to use the term “lipid” for FAs and their naturally occurring

derivatives such as esters and amides. This definition can be expanded to include

compounds closely related to fatty acid derivatives through biosynthetic pathways or

by their biochemical or functional properties such as prostanoids, aliphatic alcohols,

aliphatic ethers and cholesterol.

Christie has defined the term lipid to include: “Fatty acids and their derivatives, and

substances related biosynthetically or functionally to these compounds”[14] and this is

the definition adopted for this term in this thesis.

The most common type of lipids in nature consist of FAs and substituted FAs linked

by an ester bond to glycerol or other alcohols such as cholesterol, or by amide bonds

to sphingoid bases, or to other amines. Lipids may have moieties other than FAs,

phosphoric acid, organic bases, carbohydrates and various other compounds that can

be hydrolysed and released using a variety of hydrolytic procedures.

Simple lipids are defined as the lipids that, on hydrolysis, yield two or fewer types of

primary products per mole, and complex lipids are those that on hydrolysis, release

more than two primary molecules per mole of lipid.

Examples of a complex lipid can be either a glycerophospholipid, which contains a

phosphoric acid moiety and a glycerol backbone, or a glycolipid, which contains a

carbohydrate moiety.

In terms of energy storage and release, lipids mainly contain hydrogen, oxygen and

carbon, and represent a highly reduced form of carbon in organic molecules; upon

controlled oxidation in living cells, lipids produce a larger quantity of energy


compared to related molecules such as carbohydrates. Thus, in the living cell

metabolism and energy storage, lipids are the preferred molecules by nature due to

their higher energy release upon oxidation.

The lipids found in living systems are either amphipathic (containing both polar and

nonpolar groups) or hydrophobic (possessing only nonpolar groups). The

hydrophobic nature of lipid molecules enables membranes to behave as barriers to

polar molecules and water.[12]

Fats and oils are mixtures of naturally occurring acylglycerols. At room temperature,

fats are usually solids and oils are liquids. As one of their multiple functions, they

serve in energy storage and production in living systems. Although carbohydrates

(such as glucose) serve as the main energy source in the animal cell metabolism, an

equal weight of fat produces twice as much energy compared to carbohydrates. As

the fats are hydrophobic, the organism that carries fat does not have to carry the

hydration water that accompanies carbohydrates. Thus it is more efficient for an

organism to store fats as an optimal source of energy because it needs less mass for

the same amount of energy compared to carbohydrates and proteins and releases

double the amount of energy in bio-oxidation.[15]

The backbone of any triacylglycerol (TAG) molecule is glycerol. To produce a TAG

molecule, the three hydroxyl functional groups in a glycerol molecule are esterified

by three acyl groups. Figure 1.2 shows glycerol and a generalised structure of a TAG

molecule.


OH

HO

OH

OR

O

O R''

O

R'

O

O

(a) Glycerol (b) Triacylglycerol

Figure 1.2- Chemical structure of (a) glycerol and (b) a generalized structure of a triacylglycerol. R, R’ and R” could be identical or different FA substituents.

Figure 1.3 shows a naturally occurring TAG in cocoa butter, 2-oleyl-1,3-distearyl

glycerol, in which two terminal substituted acyl groups are stearyl, and the third one

is oleyl.

O

O OC17H35

C17H35

C7H14

C8H17C

CC

O

OO

2-oleyl-1,3-distearyl glycerol

Figure 1.3- A naturally occurring TAG in cocoa butter.

Simple lipids include TAGs, diacylglycerols (DAGs), monoacylglycerols (MAGs),

steroids including sterols and sterol esters (cholesterol and cholesterol esters in

animal tissues), waxes, tocopherols (substituted benzopyranols) and free fatty acids

(FFAs).

Glycerophospholipids include a variety of compounds that are different in the

substituted groups on the glycerol phosphate. They include phosphatidic acid (1,2-


diacylglycerol-3-phosphate, trace compound in tissues, precursor of most other

glycerolipids), phosphatidylglycerol (1,2-diacylglycerol-phosphoryl-1’-glycerol,

lung-surfactant and important in plant chloroplasts), diphosphatidylglycerol

(cardiolipin, found in heart muscle mitochondria), phosphatidylcholine (1,2-

diacylglycerol-3-phosphorylcholine or lecithin, most abundant lipid in the

membranes of animal tissues), lysophosphatidylcholine (a powerful surfactant and a

water-soluble lipid), phosphatidylethanolamine (cephalin, the second most abundant

phospholipid in animal and plant tissues and major lipid in micro-organisms),

phosphatidylserine (N-acylphosphatidylserine, a weakly acidic lipid),

phosphatidylinositol (regulating vital processes in the cell), phosphonolipids (a

phosphonic acid esterified to glycerol) and ether lipids.

Glycoglycerolipids are found in photosynthetic plants and have mono- or di-

carbohydrate molecules (usually galactose or glucose) substituted on the glycerol

backbone. The other two hydroxyl groups on the glycerol are substituted by FA

groups.

Sphingomyelins and glycosphingolipids include long-chain bases that are linked by

an amide bond to a FA or to phosphorus-containing moieties such as sphingosines,

phytosphingosines, ceramides, sphingomyelins, ceramide phosphorylethanolamines,

neutral glycosylceramides and gangliosides.[14]

Waxes are esters of long-chain (C14 to C36) saturated and unsaturated fatty acids

with long-chain (C16 to C30) alcohols that are insoluble in water but soluble in

nonpolar organic solvents such as hexane.[15]

Figure 1.4 shows the structure of some common lipids.


OH

OH

C15H31

O

O

HO

C15H31

O

O

C15H31

O

O

(a) Glycerol palmitate (MAG) (b) Glycerol dipalmitate (DAG)

HO

H

H

H

(c) A wax molecule (oleyl stearate) (d) Cholesterol

O

R3

R2

HO

R1

(e) Tocopherol (R1, R2 and R3 are H and/or CH3 for α, β, γ or δ tocopherol)

O

O

PO

OOH

OH

C15H31

O

C15H31

O

(f) Phosphatidic acid (1,2-Dipalmitoylglycerol-3-phosphate)


O

O

PO

O OH

O

C15H31

O

C15H31

O

OH

OH

(g) Phosphatidyl glycerol (1,2-Dipalmitoylglycerol-3-phosphoryl-1'-glycerol)

RCOO CH

CH2

H2C

RCOO

O P O CH2

CHOH

H2C O P O CH2

HC

H2C

OOCR

OOCR

O- H+

OO

O- H+

(h) Cardiolipin (diphosphatidylglycerol)

RCOO CH

H2C

H2C

OOCR

O P O CH2CH2N+(CH3)3

O

O-

(i) Phosphatidylcholine


RCOO CH

H2C

H2C

OOCR

O P O CH2CH

O

O-

NH3+

COO-

(j) Phosphatidylserine (N-Acetylphosphatidylserine)

RCOO CH

H2C

H2C

OOCR

O P O

O

O- H+

OH

OHOH

OH

OH

(k) Phosphatidylinositol

Figure 1.4- Structures of some common lipids. R groups denote any organic substituent.

1.1.2- Fatty Acids

Fatty acids, as they occur in nature, are compounds consisting of a hydrocarbon

chain and a carboxylic acid functional group (-COOH). The general chemical

formula of FAs is RCOOH, in which R denotes a hydrocarbon chain, (RCO known

as an acyl group), usually with an even number of carbon atoms (as it is synthesized

using acetyl groups in the living cells) containing typically 4 to 36 carbons (C4 to

C36). The hydrocarbon chain of fatty acids usually contains an even number of

carbon atoms (commonly 12-24), may be saturated or unsaturated, and can contain

various functional groups such as double bonds, hydroxyl groups, alkyl group


branches on their hydrocarbon chain and a few contain three carbon rings. FAs are

usually found in the fats.[15]

Industrially, FAs are produced by the hydrolysis of the ester linkages in a fat or oil

(both of which generally consist of more than 95% TAGs), with the removal of

glycerol.[16]

The term “fatty acid” is often used to refer to those carboxylic acids that occur

naturally in TAGs; however, many biochemists and chemists refer to all unbranched

carboxylic acids as FAs regardless of their origin and chain length. In this thesis the

term “fatty acid” refers to all carboxylic acids that can occur in natural fats and oils,

regardless of the carbon chain length or number or type of functional groups on the

carbon chain.

Saturated FAs do not contain any double bonds along the acyl chain. Examples of

some saturated FAs that are observed in fats and oils are:

• Butyric (butanoic acid): CH3(CH2)2COOH or C4:0

• Lauric (dodecanoic acid): CH3(CH2)10COOH or C12:0

• Myristic (tetradecanoic acid): CH3(CH2)12COOH or C14:0

• Palmitic (hexadecanoic acid): CH3(CH2)14COOH or C16:0

• Stearic (octadecanoic acid): CH3(CH2)16COOH or C18:0

• Arachidic (eicosanoic acid): CH3(CH2)18COOH C20:0

Unsaturated FAs contain one or more double bonds. In most of these fatty acids,

each double bond has 3n carbon atoms after it, for n an integer in the range of 1 up to


about 8, and in animal fats and plant oils it is usual that all double bonds are in a cis

configuration.

The cis and trans terminology is applicable only to double bonds with two different

substitutions, however, unsaturated FAs usually contain more than two different

substituents on each double bond. Thus the Z and E (replacing cis and trans

respectively) terminology describes the steric configuration of the fatty acid alkyl

chain more precisely, although the cis and trans prefixes are still commonly used by

lipid chemists.

Table 1.1 lists four nomenclature methods of FAs.

Table 1.1- Four nomenclature methods of FAs.

Nomenclature of Fatty Acids Names Abbreviations

Trivial Name IUPAC Name Carboxyl-reference

ω-reference

Palmitic acid Hexadecanoic 16:0 16:0 Stearic acid Octadecanoic 18:0 18:0 Oleic acid 9-Octadecenoic acid 18:1 ∆9 18:1 (ω-9)

Linoleic acid 9,12-Octadecadienoic acid 18:2 ∆9,12 18:2 (ω-6) α-Linolenic acid 9,12,15-Octadecatrienoic acid 18:3 ∆9,12,15 18:3 (ω-3)

Apart from the trivial and IUPAC naming systems, there are two nomenclature

systems to make clear where the double bonds are located in the FA molecules.

These two schemes are illustrated below:

Nomenclature 1:

- Specifies the chain length and number of double bonds, separated by a colon; for

example, the 18-carbon oleic acid with one double bond is abbreviated as C18:1. The

positions of double bonds are specified by superscript numbers following ∆ (delta).


For example, C20:2 ∆9,12 indicates that the double bonds are located at the ninth and

twelfth carbon-carbon bonds counting from the carboxylic group as carbon 1.[15]

Nomenclature 2:

- Omega-3, omega-6 or omega-9 (ω-3, ω-6 or ω-9): The first double bond is the

third, the sixth or the ninth carbon-carbon bond respectively counting from the end of

the chain most distant from the carboxyl group (ω carbon atom).

Examples:

• α-Linolenic acid, C18:3,

CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7COOH, is an ω-3 FA,

Δ9,12,15, while γ-linolenic acid, C18:3, is ω-6 FA, Δ6,9,12.

• Linoleic acid, C18:2, CH3(CH2)4CH=CHCH2CH=CH(CH2)7COOH, Δ9,12,

and arachidonic acid, C20:4 Δ5,8,11,14, are ω-6 FAs.

• Oleic acid, C18:1, CH3(CH2)7CH=CH(CH2)7COOH, Δ9, and erucic acid,

C22:1 Δ13, are ω-9 FAs.

(Note: α-Linolenic acid (n-3) is the trivial name for all-cis-octadeca-9,12,15-trienoic

acid and γ-Linolenic acid (n-6) is the trivial name for all-cis-octadeca-6,9,12-trienoic

acid. [17])

In this thesis the first nomenclature will be used when referring to labeling of FAs.

Table 1.2 briefly lists some saturated and some unsaturated FAs and their IUPAC

and common names.


Table 1.2- Some saturated and unsaturated FAs.

R in RCOOH IUPAC name Common name

a) Saturated fatty acids

CH3- Ethanoic acid Acetic acid CH3CH2- Propanoic acid Propionic acid

CH3CH2CH2- Butanoic acid Butyric acid n-C4H9- Pentanoic acid Valeric acid n-C5H11- Hexanoic acid Caproic acid n-C6H13- Heptanoic acid Enanthic acid n-C7H15- Octanoic acid Caprylic acid n-C8H17- Nonanoic acid Pelargonic acid n-C9H19- Decanoic acid Capric acid n-C11H23- Dodecanoic acid Lauric acid n-C13H27- Tetradecanoic acid Myristic acid

n-C15H31- Hexadecanoic acid Palmitic acid

n-C17H35- Octadecanoic acid Stearic acid

n-C19H39- Eicosanoic acid

C20:0 Arachidic acid

b) Unsaturated fatty acids

CH3(CH2)7CH=CH(CH2)7- (Z)-9-Octadecenoic acid

C18:1 Oleic acid

CH3(CH2)4CH=CHCH2CH=CH(CH2)7- (9Z,12Z)-9,12-Octadecadienoic

acid C18:2 Linoleic acid

CH3(CH2CH=CH)3(CH2)7- (9Z,12Z,15Z)-9,12,15-Octadecatrienoic acid

C18:3 α-Linolenic acid

Hexadecanoic acid or palmitic acid (C16:0) is the most abundant saturated FA in

nature whereas cis-9-octadecenoic acid or oleic acid (C18:1) is the most abundant

monoenoic FA in nature.[12] The C18 polyunsaturated FAs linoleic or Z-9,Z-12-

octadecadienoic acid (C18:2) and α-linolenic acid or Z-9,Z-12,Z-15-octadecatrienoic

acid (C18:3) are essential FAs for animals. They are major components of plant

lipids including vegetable oils. These FAs are biosynthetic precursors of C20 and

C22 polyunsaturated FAs (containing 3-6 double bonds) in animal systems.

Arachidonic acid (C20:4) is derived from linoleic acid via sequential de-saturation

and chain elongation reactions[18] and is an important constituent of the membrane


phospholipids in mammalian tissues, and is a precursor in the synthesis of

prostaglandins and other eicosanoids.[12]

The human body readily makes saturated FAs and unsaturated FAs that have one

double bond (monounsaturated FAs), but does not have the appropriate enzymes to

synthesize polyunsaturated FAs. At least one function of the essential FAs is to serve

as a precursor for the synthesis of eicosanoids, such as prostaglandins, a class of

compounds with hormone-like effects in many physiological processes such as

immune system and blood pressure regulation.[12]

FAs occur in large amounts in biological systems, but only in small amounts in the

free state. They typically are esterified to glycerol (to form acylglycerols) or to

glycerol derivatives such as phosphoglycerols.[12] The majority of FAs in plants and

animals occur in the form of TAGs, either simple (same FAs attached to glycerol) or

mixed (different FAs attached to glycerol).[12]

In vertebrates, free FAs circulate in the blood bound noncovalently to a protein

carrier, serum albumin. However, FA derivatives are present in blood plasma mostly

as esters or amides.[15]

FA derivatives are an important source of energy for many tissues since they can

yield relatively large quantities of adenosine triphosphate (ATP). Typically, many

cell types can use either glucose or FA derivatives to produce energy. Heart and

skeletal muscles prefer FA derivatives as the source of energy. On the other hand, the

brain cannot use FA derivatives as a source of energy, relying instead on glucose, or

on ketones produced by the liver from FA metabolism during starvation.


As fats are composed of more than 95% acylglycerols, hydrolysis of fats

(saponification reaction) produces free FA molecules and glycerol. For example,

trioleyl glycerol yields three oleic acid molecules and glycerol on hydrolysis. Figure

1.5 shows the hydrolysis reaction of this triacylglycerol molecule.

Figure 1.5- Hydrolysis of trioleyl glycerol to yield three oleic acid molecules and the glycerol molecule.

Most naturally-occurring FAs possess an even number of carbon atoms with

unbranched carbon chain. The carbon chains may be saturated or unsaturated with a

varying number of double bonds. Double bonds are almost always in cis (Z)

configuration.[12]

Trans FAs are unsaturated FA molecules containing trans double bonds, which make

the molecule less twisted compared to FAs with cis double bonds. These are

generally a byproduct of industrial processes involving catalytic hydrogenation

(hardening of the oil), or alternatively they are generated by bacterial hydrogenation

of unsaturated FAs in the rumen (first stomach in cud-chewing animals) of

ruminating animals. Industrial hydrogenation of edible oils is used to produce stable

and solid oil products at room temperature, to prolong the oil shelf life and to make

the oil transportation and storage easier. Often associated with this process is the

production of unsaturated trans FAs (~5-10%), of which elaidic acid (trans-C18:1

+ C7H14

C8H17

O

OH

O

OO

O

O

O

C17H35

C17H35

C17H35

Hydrolysis

Acid or base

OH

HO

OH

3


Δ9) is a major component. The bacterial process in the rumen yields predominantly

trans vaccenic acid (trans-C18:1 Δ11), and also conjugated linoleic acids such as Δ9-

cis,11-trans-18:2.[19] Furthermore, some regular kitchen food cooking and processing

techniques convert cis-unsaturated FAs naturally present in food to their trans form.

High dietary intake of trans FAs by humans is known to be combined with a high

probability of myocardial infarction, atherosclerosis, coronary heart disease and

prostate cancer occurrence.[20,21]

Dietary lipids are sources of essential FAs for the human body. FAs are important in

membrane biosynthesis, energy balance, eicosanoid production, acetyl-CoA

production via β-oxidation and production of acylglycerols such as TAG molecules

via esterification, which are a source of energy for the body. The FA substituents

present in the cell membrane phospholipids influence the functional properties of

membranes. Polyunsaturated FAs increase the membrane fluidity, while saturated

FAs decrease it.[22]

Common FAs found in plant tissues are even numbered straight-chain compounds of

C14 to C22 with a saturated alkyl chain or up to four double bonds with cis

configuration. These FAs are also found in animal tissues with a wider range of chain

lengths and up to six double bonds that are separated by methylene groups.

Branched-chain FAs are synthesized by many micro-organisms and occasionally can

be produced in animal tissues. FAs with other substituent groups are found in some

plants and micro-organisms. These substituents include acetylenic and conjugated

double bonds, cyclopropane, cyclopentane and furan rings, hydroxy-, epoxy-, and

keto- groups.


1.1.3- Triacylglycerols

Triacylglycerols are composed of glycerol esterified with three FAs. Figure 1.6

illustrates a general molecular formula of TAG molecules.

C

C

C

H

H

H

H

OOCR"

RCOO

OOCR'

H

Figure 1.6- General TAG molecule

IUPAC rules are used to number the carbons of TAG molecules. The secondary

hydroxyl group is shown to the left of carbon 2. The carbon above it will be carbon 1

and the carbon below it will be carbon 3. The prefix “sn” is used to specify

“stereospecific numbering”. Thus, the name triacyl-sn-glycerol is technically more

correct than triacylglycerol. When the stereochemistry is not specified, the primary

hydroxyl groups are usually named α- and α’-positions and the secondary hydroxyl

group is named β-position.

In living cells, the TAG molecules are synthesized by sequential acylation of free

glycerol via enzyme catalysed reactions, or by the catabolism of glucose and the

enzyme-controlled stepwise esterification of the hydroxyl groups. The product of

each esterification step has defined stereochemistry, and the stereochemistry of the

esterification processes are highly controlled by enzymes.[15]


The position and the type of the FA substituents on the glycerol backbone play a key

role in the digestion, absorption and metabolism of the lipid in the living cell. [23,24]

During the digestion of a TAG molecule in the body, FAs at the sn-1 and sn-3

positions are digested by TAG-hydrolysing enzymes (e.g., Lipases) in the intestine.

The result is two free fatty acid molecules and a MAG molecule with an attachment

at sn-2. Free palmitic acid and stearic acid form insoluble salts with calcium and

magnesium in the intestine and their absorption by the body is reduced. The sn-2

MAG does not form a salt and is readily absorbed.[24,25] MAGs with FA derivatives

on the sn-2 positions are re-esterified and released into blood as TAGs later, thus

they are conserved in this way. In human milk fat more than 60% of the palmitic acid

is esterified at sn-2 position, while in cow milk it is mainly at sn-1 and sn-3

positions.[26-28]

The “pancreatic lipase hydrolysis” was the first method used to study the location of

FAs on the position sn-2 of the TAG molecules from natural oils and fats.[29-33]

Complete positional distribution of FAs is now determined using complex

stereospecific hydrolysis procedures. Due to this historical priority of the analytical

procedure, there has been an inclination to presume that the FA esterified on the

secondary hydroxyl group should be more important than the two primary ones. The

secondary hydroxyl group is important in digestion of TAG molecules in the

intestine of animals. The sn-3 is important in the cellular control mechanism as it is

the last position to be acylated during TAG biosynthesis.[34]


1.1.3.1- Determination of the Positional Distribution of FAs in Fats and Oils

The position of FA substituents on TAG molecules influences their metabolism of

the lipids in the living cell. As discussed above, it has been shown that the FA

substituents on the sn-2 position are absorbed better than on the other two positions.

A higher frequency of occurrence of saturated FAs such as palmitic acid on the sn-2

position in animals increases the atherogenic potential of the lipid without altering

the level of blood lipids.[35]

Brokerhoff devised the first stereospecific analysis procedure for TAGs in 1965.[36]

The basic method includes partial hydrolysis of the TAG sample to prepare the

phospholipid derivatives, then hydrolysis with phospholipase A of snake venom,

followed by a separation and transesterification step of the products to be examined

on GC. The composition of the position sn-2 is independently determined by

pancreatic lipolysis.

NMR has found an increasing application in the locating of the FAs on the TAG

molecules in edible oils during the last fifteen years.[37-40] Both 1H and 13C NMR

have been used as an analytical tool to characterize the structure of lipids and to

assess the quality of the lipid products.[38,41] The problem with the 1H NMR of TAG

molecules is the intensive signal overlap of the aliphatic protons, while 13C NMR

suffers complexity from the small chemical shift differences observed for some

carbon atoms, although 13C NMR spectra are much better resolved than the

corresponding 1H NMR Spectra.[42,43] The fact that 13C chemical shifts are

concentration-dependent makes the problem more complicated.[44] 2D NMR

techniques such as HH- and CH-COSY, HMBC and INADEQUATE have been


implicated to distinguish the position and configuration of double bonds in the acyl

chains.[45,46]

1.2- Macadamia, From Nut to Shelf

1.2.1- Introduction

Macadamia nuts are the only commercially developed indigenous Australian crop.

They were well known to the Australian aboriginal people as a reliable food

source.[47] Countries such as South Africa, United States (mainly in Hawaii and

California), Kenya, Israel, Brazil, Malawi and Guatemala have also ventured into

cultivation and commerce of macadamia products.

Although high in oil, macadamia nut has cholesterol-lowering properties and a

beneficial effect on the lipoprotein profile in humans.[48] A controlled randomized

crossover-designed study of thirty healthy men with normal blood cholesterol levels

showed a macadamia-nut-based diet lowered serum total cholesterol and LDL

cholesterol within 4 weeks.[49] Other researches also revealed the preventive effects

of macadamia oil on risk of coronary artery disease[50] and type-2 diabetes.[51] Table

1.3 shows the composition of 100 g of macadamia nuts and the FA constituent of

macadamia nut oil.[52]


Table 1.3- Composition of 100 g of macadamia nuts and the FA constituent of macadamia nut oil.

Composition of 100 g of macadamia nuts (about 40 nuts)

Fatty acid composition of macadamia nut oil

Nutritional Values Minerals, mg Fatty Acid Percent

Calories kCal 727 K 373 Oleic 67.14

Protein/ g 9.23 P 171 Palmitoleic 19.11

Oil/ g 78.21 Mg 119 Palmitic 6.15

Carbohydrates/ g 7.9 Ca 36 Eicosenoic 1.74

Thiamine/ mg 0.22 Na 6.6 Stearic 1.64

Riboflavin/ mg 0.12 Fe 1.8 Arachidic 1.59

Niacin/ mg 1.16 Zn 1.44 Linoleate 1.34

Cholesterol None Mn 0.38 Myristic 0.75

Cu 0.33 Lauric 0.62

1.2.2– History and Production of Macadamia Nut

Leichardt discovered a non-fruiting variety of the Macadamia ternifolia in 1834.[47]

In 1857 von Mueller and his colleague Hill discovered a specimen of Macadamia

ternifolia species in the southern region of Queensland. The macadamia tree was

called the Kindal Kindal tree by the aborigines and they supplemented their diet with

the nuts from this tree.[47]

In 1882 Macadamia integrifolia was introduced to the Hawaiian Islands, where it

was developed industrially from 1922. Macadamia became fully established as a


commercial crop in Hawaii during 1940s, and then subsequently in California,

Australia and other tropical and sub-tropical countries.[53]

In 1970, the Hawaiian macadamia industry produced approximately 90% of the

world production of macadamia nuts.[54] The Australian macadamia industry

essentially began in 1888 in New South Wales but only began to flourish

commercially from 1970. Production of nut-in-shell (NIS) in 1971/1972 was 93

metric tons and this rose to 2000 metric tons in 1983 and 27500 metric tons NIS in

1997.

Annual production of Australian macadamia nut-in-shell has grown from 19000

metric tonnes in 1994 to 29700 metric tonnes in 2003. In 1997 the Australian

macadamia nut production surpassed USA that had been ranked as the major

producer since the 1940s. Table 1.4 lists the production and consumption of

macadamia nut-in-shell (NIS) in the world.[55]

Table 1.4- World production and consumption of macadamia nut-in-shell (NIS) and kernel in year 2000.

Macadamia NIS Production Macadamia NIS Consumption

Country/ Region NIS Tonnes

Kernel Tonnes Country/ Destination Kernel Tonnes

(estimate)

South Africa 12500 3400 North America 10880

Kenya 8800 1000 Japan 3300

Malawi 4000 1000 Europe 3050

Zimbabwe 900 120 Hong Kong / China 1650

Central America 17000 3100 Australia 1200

Hawaii 22000 5500 Exporter – Own Consumption 1950

Australia 30000 9100 Committed Carry Over 1200

Total 95200 23220

Total 23230


The macadamia industry in Hawaii is based on the smooth shell type Macadamia

integrifolia and its cultivars. The rough shell varieties of Macadamia ternifolia show

unpredictable behaviour, are slow to come into bearing in Hawaii and they produce

lower grade nuts.[56]

1.2.3– Botanical Description

The mature macadamia tree grows to a height of 12-15 m. The leaves are dark green

and shiny and branches bear long sweet smelling racemes of creamy white flowers.

In summer each spray of 40-50 flowers produces 4-15 ‘nutlets’ which, eventually

ripen into large clusters of flowers.[57]

Macadamia is an evergreen tree of the family Proteaceae. There are ten species of the

genus macadamia in this family. Only two of these ten species produce edible nuts:

Macadamia integrifolia Maiden and Betche, known as the smooth-shell type and

Macadamia tetraphylla L.A.S. Johnson, commonly referred to as the rough shell

type, and their hybrids.[53]

The macadamia kernel, laid in a thick hard shell and with a high content of glucose

and sucrose, is a very sweet nut.[58] The most common commercially grown

macadamia nuts are of the Macadamia integrifolia cultivars.[52] The kernels of the

latter eight species are not of commercial value as they are bitter and considered

inedible due to the presence of cyanogenic glycosides which release HCN on

hydrolysis.[59,60] Figure 1.7 illustrates macadamia nuts fully grown on the tree.


Figure 1.7- Macadamia nuts on the tree (Courtesy of Australian Macadamia Society)

1.2.4- Soil, Climate and Nutrition

Macadamia trees need free draining soil rich in organic matter and low in

compactness.[61] The main macadamia growing areas in Australia are on the coastal

regions of northern New South Wales and southeast Queensland. Macadamia trees

grow best in a sub-tropical climate. The shallow root system of the macadamia tree is

sensitive to prevailing winds. It grows best in regions with good drainage levels.[52]

1.2.5- Harvesting

Macadamia nuts are usually harvested from the orchard floor by mechanical

sweepers following natural fruit abscission (fruit falling on the ground from the

trees). The flowering period of many cultivars of macadamia is short in Australian


orchards; however, the abscission can occur over several months period within a

single cultivar and also varies in timing between cultivars.[62,63]

It is essential that the nuts are picked up frequently during the abscission peak period,

and more frequently during wet weather, otherwise the quality of the nut and the oil

deteriorates. As the fallen nuts contain about 30 percent water in the husk and 25

percent in the nut, the damage by mould growth could readily take place. The husks

of the macadamia nuts are removed within 24 hours of harvesting, to facilitate

drying.

The drying is the most critical step in the nut-in-shell processing, as it can maximize

the quality and the shelf life of the oil by preventing undesirable physiological

activities which cause fermentation resulting in the spoilage of the kernel. During the

drying process that can take 2-3 weeks, the moisture content of the nuts is reduced

from about 25% at harvest time down to about 2%. An initial drying temperature of

about 38 °C is applied. If this temperature is exceeded, undesired browning of the

kernel may occur later in the cooking step. After 2-3 weeks, the temperature is raised

to about 50 °C. In the final days of drying this higher temperature does not affect the

quality of the kernels.

After dehydration, the nuts are cracked prior to cooking. The nuts are cracked

between stainless steel drums, and the kernels are separated from the shell by a

combination of sieving and air blasting. It is crucial not to damage the kernel during

the process of steel-cracking the shell. It has been found useful to centrifuge the nuts

before cracking to loosen the kernels inside the shell.


The kernels are stable when kept at -20 °C, however, the quality of the extracted oil

declines with increasing the storage temperature and humidity. Storing kernels at -20

°C is not practical commercially; therefore the nuts need to be processed with a

minimum delay.

The kernels are sorted and graded. Grade 1 and 2 kernels are used in confectionary

and the poorer quality kernels are passed on to oil extraction processes.

In the more recent methods of kernel grading, air floating is used to grade the kernels

eliminating the need for further washing and drying prior to the oil extraction

processes.

1.2.6- Oil Extraction

The oil is extracted from macadamia nuts either by mechanical pressing or by solvent

extraction processes. In mechanical pressing, the kernels are compressed and the oil

is squeezed out of the crushed mass of nuts under a very high pressure. A preheating

step is necessary to break the oil-containing cell walls to release the oil content of the

cells. The oil is collected in an appropriate reservoir. The remaining organic material

(the cake) is usually used in animal feed or is extracted using organic solvents to

retrieve the remaining oil in large-scale systems.

A batch press, processing one batch of nuts at a time, range from small hand-driven

presses that an individual can use, to power-driven commercial presses capable of

processing many tons of nuts a day (Figure 1.8 top)


Figure 1.8- Cold press equipment used in the industrial production of seed and nut oil. Top: a batch press, bottom: a hole-cylinder type oil expeller.[64]

Expellers or continuous screw presses, achieve the pressure needed to press the nuts

by means of a screw rotating inside a cylindrical tube. The kernels, that are

previously heated up to 80 °C, are loaded continuously into the expeller where they

are fed into a horizontal cylinder by means of a rotating screw (worm shaft). Figure

1.8 bottom shows a screw type expeller used in the cold-press extraction of

macadamia nut oil.

None of the expeller machines are able to remove all of the oil from the nuts, and the

remaining cake contains 3-8% oil. In small-scale production situations this is not

important as the resulting cake finds uses for animal feed. To extract the remaining

oil from the cake in large-scale oil production systems, it is necessary to use solvent

extraction.


Solvent extraction is a high-technology process used to extract oil from crushed

kernel or from expeller cake. As the capital costs are high for solvent extraction

processes, solvent extraction is used only in large-scale oil extraction systems. The

process is either a batch or a continuous counter-current extraction of the crushed

kernel or the cake in contact with an organic solvent, usually hexane. In the next

step, the solvent is stripped off under vacuum in a recovery plant and is recycled to

the extraction system, and the extracted oil passes on for further processes such as

refining.[64]

1.2.7– Industrial Macadamia Nut Oil Refining Processes

Refining is the last step in the processing of macadamia nut oil and includes some or

all of the following treatments: filtering, neutralization, winterizing, bleaching,

deodorizing and degumming.

Many crude oils contain short-chain free FAs which can induce unpleasant odours

and flavours. The free FAs are neutralized by treating the oil with a solution of

sodium hydroxide at about 70 °C. The FAs form soap with sodium hydroxide and

dissolve in the aqueous phase of the mixture. In larger refineries, the formed soap is

collected and sold to soap manufacturers.

Winterizing involves applying low temperature to the oil for a certain length of time,

during which higher melting point acylglycerides are solidified and are removed by

filtration.


As oils are extracted from different varieties of macadamia kernels, the colour of the

extracted oil is not always consistent. Some oils are somewhat darker in colour and

are bleached by the addition of a small amount of acid-treated activated clays or

activated carbon prior to filtration. Many commercial plants bleach the crude oil as

routine and then add a controlled amount of colour in order to produce a consistent

and standard final product.

Degumming consists of the processes during which the oil is treated with small

amounts of water and phosphoric acid at about 60 °C. Degumming removes

phospholipids, fibers, protein-like compounds, polysaccharides and mucilage

released as the plant cells rupture during oil extraction. The above treatment, with

heat, causes the gums to coagulate after which they may be removed by

centrifugation or settling.

Deodorizing is steam distillation of the oil under pressure and in absence of

atmospheric oxygen, usually under a nitrogen atmosphere. Deodorizing removes

aromatic acids, aldehydes, free FAs and molecules that are produced in the oil before

the refining processes were started. These compounds include peroxides and some

volatile free FAs that are usually generated as a result of the contact of the oil with

air at the distillation high temperatures and can introduce unpleasant odour in the oil.


1.3- Chemical Reactions Used in Sample Preparation for FTICR-MS Analysis 1.3.1– Methanol Extraction of Macadamia Nut Oil to Remove the Triacylglycerols

In this study the methanol extraction of macadamia oil is carried out to extract the

minor constituents such as free FAs from the oil, while the less soluble TAG

molecules remain in the oil. The extracted solution contains minor quantities of the

more abundant TAG (or more soluble substituted TAG) molecules. The former

comprising more than 98% of the oil,[65] this results in an increase in the signal to

noise ratio of the minor components of the oil in FTICR-MS analysis.

The ideal extraction solvent:

1- Should not dissolve the TAG molecules, but dissolve the remaining

compounds in the oil.

2- Should be immiscible with the oil to produce a separate phase.

3- Should be volatile enough to readily evaporate from the solution at a

temperature of about 40 °C or lower to avoid the loss of volatile oil

components during solvent evaporation.

4- Should not contain any non-volatile impurities such as stabilizers.

5- Must be of the highest purity and absolutely free from FAs.

In practice, there is no organic solvent that acts ideally for this extraction process.

The TAG molecules dissolve to some extent in most common organic solvents such

as methanol, hexane and diethyl ether. To achieve a refined dissolution profile, a

variable temperature is applied.[66] TAG and DAG molecules are precipitated out by


reducing the temperature during the extraction process. Smaller molecules such as

FAs are solubilised into the methanol phase from the nut oil as the solubility of these

compounds in methanol remains reasonably high at the low extraction

temperature.[67]

1.3.2- Transesterification of Macadamia Nut Oil

The transesterification reaction is used in this thesis to esterify the acylglycerols in

macadamia nut oil to yield their FA methyl ester derivatives. This is used to generate

the FA profile of the acylglycerols in the oil. The products from this reaction are also

studied using GC-MS.

In the transesterification of the oil with methanol, the acylglycerol ester bonds cleave

to produce FA methyl esters and glycerol. The acyl groups of the acylglycerols are

released in the reaction as FA methyl esters (FAMEs). Free FAs are also esterified. A

specific example, Reaction 1, shows the transesterification reaction of glycerol

tripalmitate in methanol. The products are methyl palmitate and glycerol.

Glycerol tripalmitate

CH3OH

Methyl palmitate

+HO

OH

HO

Glycerol

C15H31

O

OO

O C15H31

O

O

KOH

C15H31

O

C15H31

O

3

Reaction 1- Transesterification of glycerol tripalmitate (a TAG) in methanol yields methyl palmitate and glycerol.


The transesterification reaction of the macadamia oil effectively alters its chemical

composition by replacing the glycerol moiety in the mono, di and triacylglycerols

with methanol. As a result, most of the FTICR-MS peaks are observed in the lower

mass region of the spectrum of the esterified oil, resulting in a simpler spectrum of

fewer assigned compounds. On the other hand and most significantly, the profile of

the FFA, MAG, DAG and TAG molecules in the original oil are lost. Procedure 2.2.2

details the transesterification procedure used in this study.

1.3.3- Alkaline Hydrolysis of Macadamia Nut Oil

During the hydrolysis reaction of the macadamia oil in boiling methanolic KOH, the

ester bonds in the acylglycerols are broken and FA potassium salts (soaps) are

formed. This reaction is referred to as saponification due to its extensive application

in the soap industry. Reaction 3 shows as an example the hydrolysis (saponification)

reaction of glycerol palmitate in hot methanolic KOH solution. The procedure for the

hydrolysis of macadamia oil is explained in Procedure 2.2.3.

O

O

C15H31

O

O KOH C15H31

O

+K -O

HO

OH

HO

Glycerol tripalmitate

Glycerol

+MeOH

C15H31

O

C15H31

O

3

Potassium palmitate (soap molecule)

Reaction 2- Hydrolysis of glycerol tripalmitate in hot methanolic KOH solution produces potassium palmitate (soap) and glycerol.


In general, saponifiable lipids all contain one or more ester, amide, or glycosidic

bonds. Within the major group of saponifiable lipids, there are several subgroups:

waxes, acylglycerols, phosphoglycerides such as phosphatidates, ether lipids such as

plasmalogens, sphingolipids such as ceramides, sphingomyelins and

glycosphingolipids including cerebrosides and gangliosides. Figure 1.9 illustrates

various types of saponifiable lipids (not necessarily found in macadamia oil).

Figure 1.9- Saponifiable lipids in the hydrolysis reaction.

In general, non saponifiable lipids do not react with hot methanolic KOH solution.

They are separated from the final product mixture by ether extraction. Figure 1.10

shows some of the compounds most likely found in the non saponifiable part of the

hydrolysis reaction of oils. However, the exact composition of the hydrolysis

reaction mixture depends on the lipid nature (animal or plant source).

Saponifiable Lipids

Waxes Glycerolipids

Acylglycerols Phosphoglycerides

Sphingomyelins

Cerebrosides

Plasmalogens

Glycosphingolipids

Gangliosides

Saponifiable Lipids

Waxes Sphingolipids Glycerolipids

Phosphoglycerides

Sphingomyelins

Plasmalogens

Glycosphingolipids

Gangliosides


Nonsaponifiable Lipids

Fat Soluble Vitamins

EicosanoidsCholesterol

Vitamin AVitamin DVitamin EVitamin K

Bile AcidsBile SaltsBile Esters

SteroidsHormones

ProstaglandinsLeukotrienesThromboxanes

Progestins, GlucocorticoidsMineralocorticoidsAndrogens, Estrogens etc.

α, β, γ and δ-Tocopherols

and tocotrienols

Nonsaponifiable Lipids

Fat Soluble Vitamins

EicosanoidsCholesterol

Vitamin AVitamin DVitamin EVitamin K

Bile AcidsBile SaltsBile Esters

SteroidsHormones

ProstaglandinsLeukotrienesThromboxanes

Progestins, GlucocorticoidsMineralocorticoidsAndrogens, Estrogens etc.

α, β, γ and δ-Tocopherols

and tocotrienols

Figure 1.10- Non saponifiable lipids in the hydrolysis reaction of oils.

1.4- Mass Spectrometry of Lipids

Numerous analytical techniques have been implemented in the identification of the

constituents of animal fats/oils and plant oils. Among these are wet chemistry

methods (derivatisation, enzymatic methods, hydrolysis, etc.), as well as instrumental

methods such as chromatography including high performance liquid chromatography

(HPLC),[68,69] capillary electrophoresis (CE),[70] thin layer chromatography (TLC),[71]

gas chromatography (GC)[69,72,73] and mass spectrometry techniques.[1]

Mass spectrometry (MS) is an established method in physics, chemistry,

biochemistry and medicine due to high sensitivity and selectivity. Since early 1990s


a considerable progress was achieved by the invention of soft ionization techniques

such as ESI and matrix assisted laser desorption-ionization (MALDI).[74,75]

A primitive application of MS in organic chemistry was the analysis of simple

organic compounds such as alkanes in late 1920s.[76,77] One of the first applications

of mass spectrometry to the analysis of lipids was on the investigation of the

mechanism of FA oxidation in the living cell (beta-oxidation) in 1944.[78]

Various studies on lipids from sector, ion-trap, single and triple quadrupole, and

time-of-flight instruments incorporating EI, fast atom bombardment (FAB),

atmospheric pressure chemical ionisation (APCI),[79] MALDI[75,80] and ESI ionisation

methods are reported.[79,81-84] Nano-electrospray ionisation combined with FTICR-

MS has recently found applications in lipid analysis.[85]

Various mass analysers are used in the analysis of lipids, using either static or

dynamic magnetic or electric fields, but all operate according to the same law. They

all measure dimensionless quantity of mass-to-charge ratio, m/z, that represents the

ratio of the mass number and the charge number of an ion in the mass analyser.[86]

Quadrupole,[87] ion-trap,[88] time of flight,[89] orbitrap[90] and Fourier transform ion

cyclotron resonance (FTICR)[91] are among the most implemented mass analysers.

Christie has listed 208 review articles published on the analysis of lipids during 2000

to 2007.[92]

1.4.1-Gas Chromatography-Mass Spectrometry

During the early years of mass spectrometry application in chemistry, the types of

samples that could be satisfactorily analyzed on MS were limited due to the


requirement of sample volatility for this gas-phase analytical technique. Gas

chromatography appeared to be an ideal solution to the problem by introducing gas

phase samples to the mass spectrometer and separating the constituents of a possible

composite sample. A major challenge was to develop methods to convert solid lipids

into gaseous derivatives capable of passing into GC and MS instruments.[1]

A non-polar to medium polarity stationary phase GC column separates TAGs

according to increasing acyl carbon number. A 70 eV energy EI is widely used as an

ionization source in GC-MS analysis. As highly unsaturated TAG molecules in some

oils tend to undergo degradation and polymerization reactions at high temperatures

applied in GC columns, GC-MS is not recommended in direct analysis of TAGs.[93]

A combination of GC and triple quadrupole mass spectrometer offers the possibility

of MS/MS (tandem MS) to provide more structural information of the analyzed lipid

molecules.[93,94]

The invention of soft ionization techniques such as ESI and MALDI made it possible

to produce gas-phase molecular ions for most lipids directly regardless of the

molecular weight, polarity and complexity of the molecules.[1] As a matter of fact,

before this invention, there was no direct method of MS analysis of intact lipids such

as oils and fats.

Among mass spectrometry methods, GC-MS has found the most application in the

analysis of lipids. However, a fundamental disadvantage of GC-MS is the

requirement of a volatile and thermally stable analyte. Derivatization can solve the

problems of analyte volatility in GC-MS analysis of simple lipids, however, it may

need multiple step reactions for complex biomolecules.[94]


Another drawback of GC-MS technique is that double bonds on unsaturated FAs

tend to migrate during the fragmentation in the electron ionization source.[95]

Determining the location of double bonds in carbon chains of FAs has been a

challenging problem in mass spectrometry. At the internal energies necessary for

ionization in EI source, migration of the double bonds along unsaturated acyl chains

occurs.[95] To overcome this problem, the double bonds are fixed by attaching

protecting groups to the acyl chain.[96]

Vetter developed the pyrrolidide derivatives to locate double bonds on unsaturated

FAs.[97] Harvey proposed 3-picolinyl esters (an aromatic basic reagent) for

derivatization of unsaturated alkyl chains.[98] Other derivatization methods include

piperidyl and morpholinyl esters,[99] triazolopyridine,[100] nitrobenzyl esters,[101] and

oxazoline derivatives.[102]

The nitrobenzyl ester derivatization technique is reported to improve the signal

intensity in the identification of FA mixtures in negative-ion mode[101] and in the

identification of hydroxy FAs in both positive- and negative-ion modes.[72]

Christie has discussed derivatization of unsaturated lipids for analysis on GC-MS

extensively.[103] He has compared various methods of preparation of lipid derivatives

including methyl esters, pyrrolidides, picolinyl esters, isopropylidenes, TMS ethers

and deuteration. The 3-picolinyl esters appear to yield more easily interpretable mass

spectra than the pyrrolidides. The picolinyl esters contain an aromatic ring that is

easily detectable in UV detectors (used in HPLC instruments).[104] He recommends

the picolinyl esters as the best general purpose derivatives for locating the double

bonds on lipids by GC-MS.


Applications of derivatization include, but are not limited to, encouraging

fragmentation along different pathways, improving the selectivity and sensitivity of

the ionization process, enhancing the abundance of the molecular ion, and reducing

the polarity of FAs to increase the volatility and to promote the stability of

molecules.[105]

The main disadvantage of all FA derivatisations is that the geometry about the

double bond cannot be determined.[95,96]

1.4.2- Electrospray Ionisation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

ESI FTICR-MS is the main technique used in this study. GC-MS is also used as a

supporting technique. The present study has led to the first published application of

ESI FTICR-MS technique in the investigation of plant oils by Fard et al. in 2004.[73]

Wu and co-workers later published an article on the application of FTICR-MS in the

investigation of adulteration in vegetable oils in 2005.[106]

The advantages of the ESI FTICR-MS technique compared to other mass

spectrometry techniques are high resolution, high mass accuracy, high sensitivity and

simplicity of sample preparation and injection. In one report we have resolved more

than 180 different components in a positive-ion ESI FTICR mass spectrum of a

processed macadamia oil sample. Hughey and co-workers have resolved 11,127

compositionally distinct components in positive-ion and 6,118 compounds in

negative-ion ESI FTICR mass spectrum of a crude petroleum oil sample with an


average mass resolving power of 350,000. They claim that FTICR-MS offers a

hundred-fold higher peak capacity than the best single-stage GC or LC.[107]

1.4.2.1- Electrospray Ionisation Source

Lord Rayleigh carried out and published one of the first studies on charged droplets

in late 1800s.[108] In this research, he showed that the spherical shape of a charged

drop remains stable as long as the fissility does not exceed unity. The fissility is a

function of the radius of the drop, the surface tension of the liquid and the charge of

the drop. Figure 1-11 illustrates the formation of charged drops in a high electric

field.

Figure 1.11- Formation of charged drops at the tip of electrospray needle in high intensity electric field.

Once the fissility increases beyond unity, two fine jets tend to form at both ends of

the drop (Rayleigh jets) dispatching fine charged droplets out of the main drop.[108] In

this phenomenon, the drop loses about 0.3% of its mass and about 33% of the charge

Sample solution

Additional gas flow

Capillary needle

±4200 V


to reduce the fissility to less than unity. The generated fine droplets are of the size of

approximately 1.5 µm and the fissility of close to 1, so they are likely to undergo a

Rayleigh instability soon afterwards.[109]

Figure 1.12 illustrates the formation of Rayleigh jets at the increased fissility of a

charged drop. The main drop dispatches fine droplets out in shots c and d. In shot f

the drop obtains some stability until the fissility grows to greater than unity due to

further solvent evaporation.

Figure 1.12- The formation of Rayleigh jets at the fissility greater than 1. Fine droplets dispatched from the main drop are visible in shots c and d. The whole process takes about 200 µS. Scale bar=100 µM[109]

Although this speculation has been examined both theoretically[110,111] and

experimentally,[112] the mechanics of the break-up of the fine charged droplets and

the details of the Rayleigh jet fineness are still unclear.[109] The ion evaporation

model of Iribarne and Thomson[113,114] is also widely accepted. In this model

Columbic explosion of rapidly evaporating liquid droplets of high charge density


results in charged droplets with smaller sizes, in which this sequence continues until

field-assisted ion evaporation occurs.

In electrospray ionisation, the droplets of solution are produced by “pneumatic

nebulization” that is injecting the analyte solution through a needle at whose end a

high voltage is applied. This voltage is high enough to disperse the emerging solution

into a very fine spray of charged drops with the same polarity. The solvent

evaporates, shrinking the drop size and increasing the charge density at the drop

surface.

At the Rayleigh limit, the Columbic repulsion overcomes the drop surface tension

and the drop explodes. This explosion forms a series of smaller droplets with lower

charge densities on their surfaces. By further solvent evaporation, this process of

shrinking and exploding is repeated until individually charged naked analyte ions are

formed.

The charges are statistically distributed on the available charge sites on the analyte,

leading to possible formation of multiple-charge ions in proper conditions. Increasing

the rate of solvent evaporation increases the extent of formation of multiple-charged

ions. Decreasing the diameter of the needle and lowering the analyte flow rate will

create ions with higher m/z ratio, making this technique a gentle ionisation

method.[115]

Figure 1.13 shows a schematic diagram of an ESI source along with the skimmer.

The skimmer provides a differential pressure barrier between the source

compartment and the hexapole region of the ESI source despite a 90% ion loss in this

process.


Figure 1.13- Schematic diagram of the electrospray needle, capillary and skimmer (not to scale).

It was during the early years of 20th century that the importance of electrospray was

first realised.[116] The application of electrospray ionisation to evaporate the charged

droplets to produce gas-phase ions of an analyte in a solution was first investigated

by Dole et al. in late 1960s and early 1970s.[117,118] Based on these results, Yamashita

and Fenn[119-121] and Aleksandrov et al.[122,123] successfully combined electrospray

ionization source with mass spectrometry in the mid 1980s.

Off-axis configuration of electrospray needle increases the efficiency of the

electrospray ionisation process. Larger droplets fall down due to the gravity while the

smaller charged droplets are more likely to be absorbed toward the capillary inlet. A

stream of hot drying nitrogen gas promotes solvent evaporation and blows larger

droplets away from the capillary inlet. Belov and co-workers have developed an

alternative aperture structure, the ion funnel, to improve the ion collection and

increase the signal to noise ratio.[124]

Electrospray needle

Skimmer

Capillary


1.4.2.2- The ICR Cell Figure 1.14 shows a schematic diagram of the Bruker infinity ICR cell used in

BioApex II FTICR mass spectrometer.

Figure 1.14- Schematic diagram of the infinity ICR cell used in Bruker BioApex II.

The ‘infinity’ cell is a modified ICR cell with a uniformed potential applied on the

two trapping plates at each end to solve the common problem in the ordinary ICR

cells, ‘z-ejection’, that occurs due to non linearity in the ion excitation.[125,126] An

example of this non-linear effect is coupling of axial and radial motions and may

cause undesired ion ejection along the z-axis of the ICR cell.[127] To eliminate such

undesired ion ejections, Caravatti and Allemann designed a pair of trapping plates

comprising eleven segments with different potentials applied to each segment to

match the potential gradient of the RF excitation field.[128] Such an ICR cell then

behaves like an infinitely long cell with respect to the electrical RF excitation field.

From the ions’ point of view inside the cell, the two trapping plates act as RF mirrors

imitating an infinitely long RF field. Caravatti and Allemann have shown that the

EV1

PV1 PV2

PV2 quench

EV2a

+ + + + + + +

Excite

Detect

EV2b

B

sidekick:

EV1

PV1 PV2

PV2 quench

EV2a

+ + + + + + +

+ + + + + + + + + + + + + +

EV2b

B

sidekick:


infinity cell eliminates the excitation-ejection efficiently at frequency of ωC+2ωt and

greatly improves the S/N ratio.[128]

1.4.2.2.1- Ion Trapping

Ion trapping in FTICR mass spectrometry can be carried out in numerous ways

including Sidekick™ ion-trapping, gated ion-trapping (static or dynamic) and

collision gas-assisted dynamic ion-trapping. Table 1.5 lists different trapping

methods commonly used in FTICR mass spectrometry.

Table 1.5- Different trapping methods usually used in FTICR mass spectrometry.

Trapping Methods Summary

Method Application

Sidekick Routine, high-speed applications such as auto-sampling, HPLC.

Gated (Static) Mainly used in combination with ECD and IRMPD experiments

Gated (Dynamic)

Ultra-high resolution requirements Good for ECD and IRMPD

Gas Assisted (Dynamic)

Ultra high sensitivity applications Used frequently with larger biopolymers.

The Sidekick™ ion trapping method is mainly used for fast routine mass analysis in

FTICR mass spectrometry. After the ion injection is implemented, a potential

difference DEV2 is applied on the two EV2 half-plates outside the first trapping plate

PV1. This potential difference applies a force on the ions entering the cell. This force


is perpendicular to the z-axis of the cell and pushes the ions to ion cyclotron orbits

larger in diameter. The advantage of Sidekick™ ion trapping method is that a wide

mass range of ions can be trapped in the cell. As most of the ions are pushed away

from the centre of the cell into larger cyclotron orbits, this method is not suitable for

the electron-capture dissociation (ECD) or infrared multiphoton dissociation

(IRMPD) experiments that require the ions to reside close to the z-axis in the cell.[10]

Gated Ion Trapping is based on the time-of-flight of the ions in the gated ICR cell on

a proper time scheduling. This ion-trapping method can be applied in either a static

or dynamic way. In static ion-trapping the trapping potential is set at a constant value

and the detection of the ions critically depends on the time of flight of the ions.

Dynamic ion-trapping applies a pulsed trapping potential on the trapping plates in the

ICR cell. The length of the applied pulse can be tuned manually to improve the

signal intensity. The gated trapping keeps the ions on the z-axis of the cell and is

convenient for ECD and IRMPD experiments. The drawbacks associated with this

method are limited mass measurement range for the ions and relatively low trapping

efficiency.

In the collision gas-assisted dynamic ion-trapping method a pulsed collision gas is

applied in the ICR cell to quench the ions and a pulsed potential on the trapping plate

is applied to trap the ions. This trapping method greatly reduces the ion energy range

that is exhibited by the time of flight of the ions. This method is applicable to a wide

mass range of ions. The pulsed quenching gas has to be pumped out of the ICR cell

prior to detection; therefore, this ion-trapping method is slow. The mass range is

limited by the time of flight effect.


1.4.2.2.2- Ion Cyclotron Motion

An ion of mass, m, carrying charge, q, moving in a uniform magnetic field, B, orbits

about the magnetic field direction as shown in Figure 1.15.

Figure 1.15- Ions orbit in a plane perpendicular to the direction of a uniform magnetic field, B. Positive (a) and negative (b) ions rotate in opposite directions in this ion cyclotron motion.[129]

The "undisturbed" cyclotron (rotational) frequency, ωC, is expressed in SI units as:[91]

ωc = qBm

1-1

υc = Cω2π

= 71.535611×10 B

m/z 1-2

In which υ is the cyclotron frequency of the rotation in Hertz, B is the strength of the

magnetic field in Teslas; m is the mass of the charged particle in micrograms; z is the

charge of the ion in multiples of elementary charge.

a b


A noteworthy characteristic of this latter equation is that all ions of the same mass-

to-charge ratio, m/q, rotate at the same ICR frequency, independent of velocity. This

feature makes ICR especially appropriate to mass spectrometry, since the ion

frequency is reasonably insensitive to kinetic energy, so the translational energy

(focusing) is not crucial for accurate m/z determination.

1.4.2.2.3- Ion Cyclotron Excitation and Detection

As for nuclear magnetic precession,[130] ion cyclotron rotation is incoherent, and does

not produce an observable electrical signal, i.e., there is no net difference in the

charge induced on two parallel detector electrodes by the ion cyclotron rotation of

the ions. At the moment of formation in the ICR cell, an ion may start its cyclotron

rotation randomly at any point around either circle shown in Fig. 1.15. Thus, for a

packet of ions, any charge induced on one detector plate will, on average, be

cancelled out by an equal charge induced by an ion with opposite phase, so that the

net measured charge difference between the two plates is zero. Furthermore, the

cyclotron radius of the ions is too small to produce a measurable signal, even if all

ions possessed the same cyclotron phase.

Thus, prior to any detection phase in the ICR cell, there must be an excitation

produced by applying a spatially uniform electric field of amplitude E0 perpendicular

to the magnetic field direction, and rotating resonant to the cyclotron frequency of

ions of a particular m/z value. During this excitation period, the dimensions of the

initial ion packet remain unchanged[131] and the packet accelerates along a spiral

trajectory, as illustrated in Fig. 1.16 a.


Figure 1.16- Incoherent and undetectable ion cyclotron orbital motion (a) is converted to a coherent and detectable motion by applying an electric field and (b) is detected in the ICR cell at the frequency of the ions of particular m/z value.[91]

The ion packet cyclotron radius subsequent to the resonant irradiation of duration

Texcite increases to (SI units)

r = 0 exciteE T2B

1-3

As described,[91] a coherently orbiting ion packet induces a potential difference on

the two detection plates and may be considered as a current source. The receiver

plates and the wirings have an inherent resistance and capacitance in parallel.[132,133]

The current amplitude is proportional to the number of spatially coherent orbiting

ions. At common ICR frequencies (>10 kHz) the S/N ratio is basically independent

of cyclotron frequency. However, at lower frequencies (<10 kHz), the signal is

expected to be a direct function of the frequency.[132,134] As a result, throughout most

of the broad frequency range of a standard FTICR-MS excitation, the relative current

induced on the detection plates is represented by the S/N ratio. In addition, the

a b


detection limit (i.e., the minimum number of detectable ions from an unchanged

signal in an individual 1 S acquisition period to produce a S/N ratio of 3:1) may be

calculated from [133]

N = d(p-p)

1

CVqA (r) 1-4

in which C is the capacitance of the detection circuit, Vd(p-p) is the peak-to-peak

amplitude of the detected voltage (for a particular calibrated spectrometer), and A1(r)

is a coefficient related to the configuration of the trap, approximately proportional to r

and could be determined graphically.[135] For example, for some typical operating

parameters, namely, a detection circuit capacitance of 50 pF, Vd(p-p) of 3×10-7 V, and

A1(r) = 0.5 (i.e., the ion is excited to approximately half of its maximum cyclotron

radius), an observed S/N ratio of 3:1 corresponds to a detection limit of ~187 ions.

For a molecule of mass ~300 being injected at a flow rate of 120 μl/h with 1 s

delay in the hexapole, 187 ions corresponds to a concentration of ~9×10-15 M in

the solution.

The ICR signal is proportional to the induced current,[132,133,136]

dΔQ/dt = -2q(dy/dt)/d 1-5

Note that ICR signal is independent of magnetic field strength. Further, the induced

current increases linearly with ion cyclotron radius, because the ion y-velocity

component, dy/dt, increases linearly with radius, so the ICR signal increases

linearly with the excited ion cyclotron radius.

The linearity of the ICR signal with ion cyclotron radius is important for several

reasons.


(1) At any frequency, the ICR response is proportional to the excitation spectral

magnitude at that frequency, because the ICR signal varies linearly with the

excited ion cyclotron radius (that is a linear function of the product excitation

voltage amplitude × duration).

(2) A Fourier transform of the time-domain ICR response gives the same

"absorption" spectrum that is otherwise obtained by measuring power absorption

while sweeping slowly across the m/z range.[137]

(3) The "superposition" principle implies that the signals from any number of ions

of arbitrary m/z values simply add at the detector; as a result, ions of a wide m/z

range can be detected simultaneously. The two prior points combine to constitute

the "multi-channel" advantage of pulsed excitation followed by Fourier

transformation to yield a spectrum of N data points in 1/N the time it would

take to scan the spectrum one channel at a time.[137]

(4) The detected signal intensity increases linearly with ion charge, so that ICR is

more sensitive for multiply charged ions. For example, individual DNA ions of

108 Da (each with ~30,000 charges) have been detected by FTICR-MS.[138]

Although image current detection at room temperature is typically less sensitive than

ion counting techniques characteristic of ion beam instruments, FTICR-MS detection

is non-destructive.[137]


1.4.2.3- Fourier Transform

The most unique advantage of FTICR as a mass analyser is that ion mass-to-charge

ratio is experimentally measured as a frequency. Because frequency can be measured

accurately (in low and medium frequencies), FTICR-MS offers inherently higher

resolution than other types of mass spectrometry.

Various other aspects of Fourier transform data reduction related to ICR peak shape

and position have been discussed in the literature and are not discussed in detail in

this thesis: Nyquist sampling and foldover,[139] fast Fourier transformation,[140] zero-

filling,[141] "windowing" or apodisation,[137] deconvolution,[142-144] oversampling,[145]

two-dimensional Hadamard[146] or Fourier[147] MS/MS.

Non-FT methods for obtaining a frequency-domain (and thus mass-domain)

spectrum from a digitised time-domain ICR signal include: the Hartley transform (a

way of performing a Fourier transform on real-only data),[148,149] the Bayesian

maximum entropy method (MEM),[150,151] linear prediction,[152,153] and filter

diagonalisation.[154] However, FT data reduction is still overwhelmingly preferred,

partly because non-FT methods typically need an order of magnitude more data

storage; their computation time for an N-point time-domain data set typically scales

as N2 or N3 (vs. NlogeN for the fast Fourier transform;[140]) and they typically

perform best when the number of spectral peaks is small and the peak shapes are

uniform and known.

Finally, it is worth noting that Fourier transforms also work backwards (from

frequency- to time-domain) as well as forward (from time- to frequency-domain).

Thus, it is possible to specify a desired magnitude vs. frequency excitation spectrum,


and apply an "inverse" Fourier transform to generate the corresponding time-domain

waveform to be applied to the excitation electrodes of an ICR trapped-ion cell. Such

“stored waveform inverse Fourier transform” (SWIFT) excitation[155] is now widely

used in both FTICR[156] and Paul (quadrupole)[157,158] ion traps, for mass-selective

excitation and ejection.

1.4.2.4- Mass Calibration

For precise mass measurement, two parameters need to be taken into account; mass

calibration and mass resolution. The mass resolution is determined by the

instrumental and experimental conditions such as the strength of the applied

magnetic field and the pressure in the ICR cell.

A mass calibration equation for FTICR-MS is defined as:[159]

m = obs

γf

+ 2obs

βf

1-5

Where γ = qB/2π and β = -2qGTVeff/4π2

In equation 1-5 fobs is the observed frequency, GT is the geometry factor for the ICR

cell, Veff is the effective cell trapping potential, q is the charge on the ion and B is the

magnetic flux density in the ICR cell.

To calibrate the cell parameters, a reference compound with a well-defined spectrum

is chosen and introduced into the FTICR mass spectrometer. Peaks over a wide mass

range are measured in high-resolution mode. The mass calibration function in the


FTICR-MS operating software then compares the measured frequencies (fobs) with

the expected frequencies (calculated) and adjusts the two calibration constants γ and

β until the difference between the respective frequencies are minimized. This is

usually referred to as external calibration.

The mass calibration can also be performed using internal references and is referred

to as internal calibration. Here a sample is mixed with reference compounds that

produce ions covering the mass range including those of the sample. By calibrating

the reference ion masses, the sample ions are also calibrated. The latter method is the

most accurate in FTICR-MS with reported accuracy of < 1 ppm.[7,160]

1.4.2.5- Tandem Mass Spectrometry

Tandem mass spectrometry is used to obtain more information regarding the

chemical structure of the ions by fragmenting them. In ESI FTICR-MS,

fragmentation could be induced before ions arrive at the ICR cell either by increasing

the capillary-skimmer potential difference in the ESI source or extending ion

trapping times in the hexapole ion trap. Alternatively, fragmentation could be

performed on the trapped ions in the ICR cell by introducing a collision gas into the

ICR cell through a molecular leak valve.

Fragmentation by increased capillary-skimmer potential and hexapole ion trapping

yields less controlled fragment cations compared to the accurately-controlled

fragmentation obtained by the collision gas method in the ICR cell. It can also lead to


subsequent dissociation of the primary fragmented ions by post fragmentation

acceleration.

In the collision gas CID method, ions are translationally excited by applying a pulsed

electric field in the ICR cell. Depending on the length of the exciting electric field,

two different approaches are available in CID experiments, either on-resonance

irradiation (ORI) or sustained off-resonance irradiation (SORI). In ORI experiments

a short duration (<500 μs) electric field pulse is applied to the ions in the ICR cell.

Subsequent inelastic collisions with a neutral gas molecule target result in

fragmentation. In SORI experiments a long off-resonance pulse is applied to the ions

in ICR cell (≥500 ms).[161] SORI-CID results in sequential activation of ions by

multiple collisions of low kinetic energy ions (<10 eV) with the neutral gas molecule

target.[162] As a consequence, only small increments of internal energy are transferred

to the ion throughout the duration of the electric field pulse.[129] The differences

between ORI and SORI CID involve the maximum translational energy obtained by

the ions, the excitation time and the number of collisions effecting dissociation, that

is the amplitude, time, and frequency of the pulses applied to excite the ions

translationally.[163]

Other applicable fragmenting methods in FTICR-MS are: surface-induced

dissociation (SID), electron-capture dissociation (ECD), infrared multiphoton

dissociation (IRMPD) and blackbody infrared radiative dissociation (BIRD).[161,164]


1.5- High Performance Liquid Chromatography

HPLC methods have been widely used in the analysis of the natural products,[165,166]

lipids[167-169] and particularly plant oils.[170] HPLC combined with single or triple

quadrupole mass spectrometer has been described as the most desirable system for

the lipid ester analysis.[171] HPLC separation in both off-line and on-line mode is

described as a preliminary requirement to avoid the co-infusion of the molecules in

the ESI sources that serves as a cause of signal suppression that can result in failure

to detect the minor constituents of the sample.[168]

Numerous researchers have implemented HPLC separation combined with various

ionisation sources in a range of studies.

Lin and co-workers[172] have used two different gradient elution solvent systems with

UV detector and evaporative light scattering detector (ELSD). They state they have

successfully separated 49 free fatty acids and their methyl esters. On examining

Table 1 in this article, it is quite clear from the retention times of several compounds

that they would not be resolved well enough to be identified. For example, retention

times of 18.36 and 18.36 minutes are reported for free fatty acids linolenic acid and

myristic acid. It is clear that these compounds would not be separated in a mixture

and in order to identify the fatty acids it would be necessary to run individual

standards.

The factors affecting retention times such as chain length, functional groups

(hydroxyl, oxo, keto, aldehydes, double and triple carbon bonds etc.) and geometric

isomerism have been discussed quantitatively. By implementing two C18 reversed-


phase columns in series, they have reported separation of double bond positional

isomers. Batta et al. have studied the separation of underivatized arachidonic acid

isomers using two different solvent systems on a reversed-phase HPLC column with

UV detection at 214 nm.[173] Kempen has described a method to simultaneously

analyse arachidonate metabolites in cultured cancer cells using negative-ion

electrospray tandem mass spectrometry (LC/MS/MS).[174] Byrdwell has studied the

oxidation products of acylglycerols in canola oil using reversed-phase HPLC in

conjunction with a triple-quadrupole mass spectrometer in parallel with an ion-trap

mass spectrometer using ESI and APCI sources with ammonium formate added as

electrolyte.[84] They have reported the formation of diacylglycerol and acylium,

RCO+, fragments as well as ammonium adduct of intact molecular ions of TAG

oxidation products.

Bylund has investigated the metabolites of linoleic and arachidonic acids by human

cytochrome P450 enzymes using reversed-phase HPLC column combined with ion-

trap mass spectrometry.[175] They have reported identifying arachidonic acid

metabolites in complex mixtures formed by enzymes such as cytochrome P450.

Redden has separated and quantified the TAGs of borage oil and evening primrose

oil using reversed-phase HPLC with UV detection. Borage oil is reported to have 34

UV-detectable fractions and evening primrose to have 22 fractions.[176] Steenhorst

has employed normal-phase HPLC combined with mass spectrometry to separate the

non-volatile lipid oxidation products into classes according to the molecular

polarities.[177] The classes include epoxy-TAG, oxo-TAG, hydroperoxy-TAG,

hydroxy-TAG and 2.5 acylglycerols. A 2.5 acylglycerol is defined as a product of the

breakdown of TAG hydroperoxides yielding non-volatile acylglycerol species with


two intact fatty acid chains and one short chain ending in an aldehyde or hydroxy

group. The retention times of TAG oxidation products on the normal-phase HPLC

system and the signal intensity of the MS detector are reported stable enough to

enable quantitative analysis based on external calibration.

Holcapek has implemented reversed-phase HPLC combined with various detection

methods including mass spectrometry, UV detection at 205 nm and ELSD for the

determination of the compounds occurring during the production of biodiesel from

rapeseed oil.[178] Individual mono-, di-, tri-acylglycerols and methyl esters of oleic,

linoleic and linolenic acid and free fatty acids are reportedly separated using a

combined linear gradient with aqueous-organic and non-aqueous mobile phases.

Sajiki has described HPLC-MS as more useful and convenient compared to GC-MS

in the study of free-polyunsaturated fatty acids (f-PUFA) oxidative metabolites in

biological samples due to high specificity and high sensitivity and due to the fact that

it requires no complicated pre-treatments.[68] Jandera and co-workers have studied

TAGs and DAGs in 16 plant oils including hazelnut, pistachio, poppy-seed, almond,

palm, rapeseed, macadamia, soya bean, sunflower, linseed, evening primrose, corn,

Brazil-nut, amaranth, Dracocephalum moldavica, and Silybum arianum using

HPLC-MS equipped with APCI source and UV detection at 205 nm and two C18

columns connected in series.[79] They have reported observation of fatty acids with

odd numbers of carbon atoms such as margaric acid (C17:0) and heptadecenoic acid

(C17:1).

Lee and colleagues have interesterified (exchange of acyl groups among

triacylglycerols) macadamia oil by tributyrin and tricaprylin,[179] and have used


normal-phase and reversed-phase HPLC with ELSD to separate and identify the

newly-formed lipids.

Multidimensional HPLC is also used in the study of complex bioorganic samples

such as humic mixtures.[180-182] However, it would be difficult to use such HPLC

techniques in the analysis of the present samples because solvents used in the

reversed and normal phase HPLC are incompatible.

1.6- Kendrick Masses and Mass Defects in the Identification of Homologous Series

Calculating Kendrick mass defects (KMDs) for organic molecules enables us to

readily recognize homologous series (molecules which are different only in the

number of CH2 groups, and which posses a similar heteroatom composition and the

same number of double bonds, rings, etc.).

In 1963 Edward Kendrick proposed a mass scale based on CH2 = 14.00000 rather

than 14.01565 Da.[183] According to this model, the Kendrick mass of a molecule is

calculated as follows:

Kendrick mass = IUPAC mass × (14.00000/14.01565)

Calculating Kendrick masses enables us to calculate the Kendrick mass defects that

are calculated as follows:

Kendrick mass defect = Kendrick mass – Kendrick nominal mass


Kendrick nominal mass is obtained by rounding the Kendrick mass to the nearest

integer number. KMD values are used in the assignment of homologous series in the

negative-ion mass spectra in Chapter 6.

1.7- Normal Probability (Rankit) Plot

The normal probability (rankit) plot is a graphical technique for assessing whether a

data set is distributed normally.[184] A normal distribution of the data points suggests

that systematic error does not exist in the measurements.

To perform this test, data points are sorted in descending order and each point is

given a rank in the data set. Inverse of the standard normal cumulative distribution of

the probability of the occurrence of the data points (z values) are then sketched

versus data points. The points should reasonably fit onto a straight trend line

resulting in a close correlation coefficient (R) of the trend line to 1. Deviations from

this straight line (R far from 1) indicate deviations from normality. In large samples

from a normally distributed population, such a plot will approximate a straight

line.[185]

1.8- Summary of the Method Development

Plant oils are important as they are used in millions of tones per year in food

industries as edible oil and as a source for the production of biofuels. Section 2.1

describes in more detail the samples studied.


In this thesis a novel method is developed to study plant oils (macadamia nut oil as a

model) by ESI FTICR mass spectrometry. This method would enable the effect of

processing steps and chemical reactions on oils to be directly studied. To develop

this method, a sample of processed macadamia nut oil is studied using FTICR-MS

technique. The industrial refining processes have removed most of the polar

compounds from the processed oil, resulting in a relatively simple oil in terms of the

variety of the chemical constituents. This oil is chosen as a model to develop the

method and to optimize the technique. The ESI FTICR mass spectrometric, GC and

HPLC results of the samples are included in the DVD attached to this thesis.

This technique is capable of providing fingerprint libraries of various unprocessed

oils from different cultivars, climates and sources by detecting the trace compounds

in the plant oils. Three-dimensional diagrams of oil constituent compounds of the

same cultivar and from the same source can be a representative of the variations

caused by environmental parameters such as temperature and air oxidation. These

libraries could be used to optimize the quality of the oil by modifying the parameters

affecting the oil quality during nut growth, harvesting, storage and oil extraction.

This method is not considered to be a replacement for other chromatography and

mass spectrometry methods such as GC-MS, but a complementary tool to analyse

oils which are often very complex mixtures of many hundreds of similar compounds

that do not readily lend themselves to GC.

Sample preparation in oil analysis is usually a tedious and time-consuming process in

other mass spectrometry techniques. In MALDI technique, the matrix needs to be

optimized, and introducing the sample to the instrument usually is a multi-step

procedure.


In quadrupole GC-MS studies of oils, a preliminary esterification reaction is

necessary to increase the volatility of the TAGs, DAGs and MAGs by

transesterification of the acyl groups, and to reduce the polarity of free FA molecules

to be examined on the GC column. This reaction causes a dramatic change in the

composition of the TAG, DAG, MAG and free FA molecules present in the oil by

releasing all of the acyl groups from the glycerol backbones as their esters and also

esterification of all of the free FA molecules present in the oil. However, the GC-MS

provides crucial information regarding the total FA profile of oils.

One of the unique advantages of ESI FTICR-MS compared to GC-MS is its

capability to directly measure the free FA content of oil without any preliminary

chemical reactions; the GC-MS technique is only capable of measuring total FA

profile of oils. However, GC-MS technique is capable of resolving the isomers of

FAs whereas standard FTICR-MS is not. Combining HPLC with ESI FTICR-MS

allows both the free FA content of the oil to be measured and the FA isomers to be

resolved.

The second advantage of ESI FTICR-MS over GC-MS is that in ESI the parameters

are usually tuned to minimize the fragmentation, and most of the peaks are simply

representatives of the corresponding molecular ions, while in ordinary GC-MS

instruments with EI source, the fragmentations make the spectra more complicated,

and the double bonds tend to migrate on the unsaturated FA molecules, making the

interpretation of the mass spectra more complex.

The mass resolving power of the sector GC-MS techniques is usually less than 10,

and the mass accuracy is rarely better than 0.1 mass unit. The FTICR-MS technique

represents a resolving power of several orders of magnitude higher and a mass


accuracy in the range of a few parts per million. The sample preparation, on the other

hand, is as easy as dissolving the sample in a solvent (methanol or acetonitrile) and

injecting the solution to the instrument. A brief comparison of FTICR-MS technique

with the GC-MS reveals the following advantages.

- High mass accuracy (<5 ppm using a combination of external and internal

calibrations)

- High resolution (>50000 and up to ~1000000 or more depending on the

measured mass and experimental parameters).

- No need to perform derivatization reactions or complicated sample

preparations prior to mass analysis.

- Capability of direct detection and quantification of free FAs in the oil.

- Very high sensitivity, detection levels of 10-10 to 10-13 M.

As mentioned previously, FTICR-MS detects molecule ions according to their

masses and is not capable of resolving isomers such as cis and trans isomers. For

example, oleic acid (cis-9-octadecenoic acid) and elaidic acid (trans-9-octadecenoic

acid) cannot be resolved by FTICR mass spectrometry as they have identical

molecular masses. FTICR mass spectrometry is not able to resolve the substituents

on the glycerol in DAGs and TAGs. As an example, whether the sn-1 substituent

acyl on the glycerol is oleate and the sn-2 acyl is palmitate or vice versa is not

distinguished by this latter technique. These last points demonstrate the importance

of implementing chromatography as well as mass spectrometric analysis if isomeric

components in the oils are to be studied.


The FTICR-MS instrument is costly and the complexity of the technique is higher

than the GC-MS technique and operation of the former needs more training and skill.

Standard regular chemistry methods such as titrations are also used to measure some

of the parameters of the oils such as free acid content.

A DVD is attached to this thesis contains the complete set of FTICR-MS, HPLC

fraction collection and GC-MS spectra and the spreadsheets of extracted data of

intensities, mass accuracies, reproducibility and rankit tests.


Chapter 2

2. Experimental


2.1- Materials

One processed macadamia oil from Macadamia Integrifolia (smooth shell) from the

family Proteaceae was used as supplied by Bronson and Jacobs, Homebush Bay,

Sydney, Australia. Two unprocessed macadamia nut oil samples (Batch 12 and Batch

13) produced by a cold press method were used as received from same supplier.

Fatty acids and acylglycerol standards were of reagent grade (Fluka) and used as

supplied. Sodium iodide was of analytical grade (May & Baker Ltd. Dagenham,

England) Polyethylene glycol (PEG-300) was of analytical grade (Fluka). PEG-300

and sodium iodide both were used without further purification.

Methanol HPLC grade (Unichrom) was used for extraction, dissolution of the

samples, washing the instrument before and after FTICR-MS experiments and for

HPLC separations.

2.2- Chemical Procedures

2.2.1- Methanol Extraction of Macadamia Oil

Procedure 2.2.1- The oil sample (0.5 ml) was mixed with equal volume of methanol

(0.5 ml) at room temperature in a 1.5 ml Eppendorf tube. For the solid samples, the

mixture was warmed up in warm water to melt the oil. The mixture was vortex

shaken for one minute, and the sample was stored at 0 °C for 12 h to facilitate the


precipitation of triacylglycerols. The remaining solution was decanted and the

solvent evaporated in a stream of nitrogen gas at ~50 °C. The extract was weighed

and stored in a freezer at –20 °C under nitrogen gas to minimize possible reactions

with atmospheric oxygen and/or effects of ultraviolet light.

An extraction blank was prepared according to the same procedure.

2.2.2- Hydrolysis of Macadamia Oil

Procedure 2.2.2- The oil sample (100 mg) was dissolved in a solution of 0.1 M

potassium hydroxide in 95% (v/v) ethanol (2 ml) and the solution was refluxed for 1

hr or left overnight at room temperature. After cooling, water (5 ml) was added, and

the solution was extracted with diethyl ether (10 ml) to remove any non-saponifiable

material. Centrifugation was used if necessary to break any emulsion that sometimes

formed. The aqueous layer was acidified with dilute HCl and was extracted with

diethyl ether (3×5 ml). The combined ether layers containing the products was

washed with water (5 ml), dried over anhydrous sodium sulphate after which the

ether was removed. The resulting hydrolysis products were stored under nitrogen gas

at -20 °C to protect them from possible effects of atmospheric oxygen and light.

2.2.3- Transesterification of Macadamia Oil

Procedure 2.2.3- Acidic transesterification: The oil sample (100 mg) was dissolved

in toluene (1 ml) and methanol (2 ml) containing 1% (v/v) sulphuric acid was added.


The mixture was left overnight at 50 °C, or alternatively refluxed for 2 hrs after

which, the mixture was cooled and diethyl ether was added (10 ml) followed by

water (5 ml). After thorough shaking, the ether layer was removed, washed with

dilute sodium bicarbonate solution (5 ml), then with water (5 ml) and then dried over

anhydrous sodium sulphate. After the ether was removed by evaporation, the methyl

esters were recovered by dissolving in n-hexane.

2.2.4- Sample Preparation for Mass Spectrometry

The solutions of the neat oil, the methanol extract of the oil (extract), hydrolysed oil,

hydrolysed extract, esterified oil and esterified extract were prepared for mass

analysis by dissolving 1 µl of each of the samples in 1 ml methanol. An aliquot 100

µl of this solution was diluted to 1 ml in methanol. The latter solution was analysed

by ESI FTICR mass spectrometry.

2.3- Instrumentation

2.3.1- Electrospray-ionisation Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

A Bruker BioApex II 70e FTICR mass spectrometer was used in this research. This

instrument is equipped with various external ionization sources including ESI, EI,

APCI, MALDI, nanospray and a supersonic expansion cluster ion source.

In this thesis, only the ESI source was used for the study of the macadamia nut oils.


The BioApex II 70e FTICR mass spectrometer consists of several components

including a 7 T passively shielded superconducting magnet, the vacuum system, the

ion optics voltage supply, the electrospray ionization source and the PC compatible

data processing workstation.

Figure 2.1 shows a photograph of the Bruker BioApex II 70e FTICR mass

spectrometer and Figure 2.2 a schematic diagram of the ESI source, ion optics and

the ICR cell.

Figure 2.1- The passively shielded Bruker BioApex II 70e Fourier transform ICR mass spectrometer used in this thesis.

Magnet

Gas inlet

ESI source

Syringe pump


Figure 2.2- Schematic diagram of the Bruker BioApex II 70e ESI Fourier transfer ICR mass spectrometer used in this research.[186] Instrument control variables are used to control various voltages and pulses (eg XDFL controls the ion beam deflection in horizontal plane).

PL1 PL2 (DPL2) Pulsed

PL4 (DPL4) Pulsed

HVO

(XDFL)

Pulsed

(YDFL)

Gate Valve

FOCL1 FOCL2

PL9 Off axis Electrospray Needle

Quartz Capillary

Skimmer

Gold-plated hexapole

ESI Source Ion Transfer Optics

EV1

PV1 PV2

Excitation

Detection

EV2b

PV2 quench

EV2a (EV2,DEV2)

Infinity Cell

PL1 (DPL2) Pulsed

(DPL4) Pulsed

HVO

Pulsed

Gate Valve

FOCL1 FOCL2

PL9

ESI Source Ion Transfer Optics

EV1

PV1 PV2

Excitation

Detection

EV2b

PV2 quench

EV2a (EV2,DEV2)

EV1

PV1 PV2

Excitation

Detection

EV2b

PV2 quench

EV2a (EV2,DEV2)

PV1 PV2

Excitation

Detection

EV2b

PV2 quench

EV2a (EV2,DEV2)

Infinity Cell


2.3.1.1- The Vacuum System

The sample ions are formed at atmospheric pressure in the ESI source and are then

directed into the ICR cell which operates at ultra-high vacuum (UHV) i.e. ~10-10

mbar and requires substantial differential pumping for proper operation of the

instrument.

Figure 2.3 illustrates the vacuum system on the Bruker BioApex II 70e FTICR mass

spectrometer.

Figure 2.3- The differential pumping on the Bruker BioApex II ESI FTICR mass spectrometer vacuum system.[186]

The gas-phase ions formed at the tip of the electrospray needle travel through a

quartz capillary (coated with a thin film of platinum at both ends) into the capillary-

skimmer chamber. In this first chamber, the pressure is maintained at about 10-2-10-4

mbar by a turbo molecular pump (Edwards EXT250H) backed by a rotary pump. The

hexapole ion trap and an extraction plate are located next to the skimmer in the ion

Hexapole

10-2-10-4 mbar 10-5-10-7 mbar 10-9-10-11 mbar 10-7-10-9 mbar

Skimmer

Endplate

Passive iron shield

Preamplifier


source chamber. The pressure in this second chamber is maintained at 10-5-10-7 mbar

by a cryogenic pump (Edwards CoolStar 800).

The extracted ions are directed into the FTICR cell by a series of ion-steering and

ion-focusing lenses. In the ion-focusing region, the pressure is maintained at ~10-7-

10-9 mbar by a cryogenic pump (Edwards CoolStar 400).

Finally, ions enter the ICR cell that is housed in the UHV chamber. The pressure in

this chamber is maintained at 10-9~10-11 mbar by two cryogenic pumps (Edwards

CoolStar 800 and 400).

Two ion gauges (Granville Philips) are used to continually monitor the pressure in

the capillary-skimmer chamber and the ultra high vacuum chamber in real time.

2.3.1.2- Electrospray Ionisation Source

Figure 2.4 shows the schematic diagram of the Analytica off-axis ESI source used in

this research project.


Figure 2.4- Schematic diagram of the off-axis Analytica ESI source used in this research project.

Figure 2.5 illustrates the off-axis and on-axis needles in the ESI cage and Figure 2.6

shows the inlet tip of the quartz capillary and the end plate. The protecting cap has

been removed to expose the capillary tip.

To the skimmer

Hot drying nitrogen gas flow

Nitrogen sheath gas flow

Larger droplets fall due to the gravity

Small charged droplets are drawn to the capillary tip

Quartz capillary

Off-axis spray needle (~45°)


Figure 2.5- The off-axis and on-axis spray needles inside the Analytica ESI cage.

Figure 2.6- The capillary tip and the end plate. The protective cap is removed to expose the capillary tip.

On-axis needle

Off-axis needle

End plate

Capillary tip

Electrical connections


The solutions containing the oil components were injected into the ESI chamber

using a Cole-Palmer (Vernon Hills, Illinois) syringe pump at a constant flow rate of

90-150 μl/h (typically 120 μl/h). The off-axis spray needle inner diameter was 0.1

mm.

The needle was connected to the ground potential. For negative-ion detection a

potential of about +4 kV was applied to the end of the capillary that faces the needle.

The end plate and the mesh cage surrounding the spray needle were held at about 2.5

kV.

This needle was placed off-axis at a distance of about 3 cm from the end of the

quartz capillary. Originally an on-axis configuration was used for the needle spray,

but the off-axis needle was observed to increase the efficiency and reproducibility of

the ESI process. The larger droplets drop due to the gravity while the smaller charged

droplets were more likely to reach the capillary inlet. Also the capillary was kept

cleaner, as the bulk solution was basically directed away from the tip of the quartz

capillary inlet.

A stream of nitrogen gas at room temperature (known as sheath gas) was diverted out

of the needle sheath and pressure optimization was found to improve the ionization

formation of the spray for lipid ions.

A second countercurrent stream of hot drying nitrogen gas (270 °C) that flows out of

the space between the capillary tip and the end plate (see Figure 2.6) was applied to

promote the evaporation of the solvent and also had the added effect of directing

larger droplets away from the capillary inlet as shown in Figure 2.4.


Figure 2.7 shows a schematic diagram of the quartz capillary, skimmer and hexapole

location in the ion optics system.

Figure 2.7- Schematic diagram of quartz capillary, skimmer and hexapole location in the ion optics system (not to scale).

A conical skimmer is located close to the exit end of the quartz capillary as shown in

Figure 2.7 and varied over 0-20 V relative to the ground. A voltage of – 400 to +400

V was applied to the ion exit end of the capillary versus the skimmer. A 2.0 s time

was used for the delay in the hexapole ion trap.

For negative-ion detection a voltage of +4 kV was applied to the end of the quartz

capillary that faces the spray needle (see Figure 2.4). For positive ion these potentials

are reversed. This creates an electric field gradient between the spray needle and the

capillary and initiates the ion-formation from the droplets that emerge from the spray

needle. The end plate and the mesh cylinder surrounding the spray needle were held

at about +2.5 kV versus the ground (see Figure 2.5). A voltage of –400 to +400 V

Capillary

Hexapole Ion optics

To pump To pump

Ion / gas flow

Skimmer


versus skimmer is applied to the exit end of the capillary to assist in collection and

focussing the generated ions from the spray needle.

In the Analytica ESI source a small hexapole (see Figure 2.8) is used to store ions

from the continuous needle spray. Once a sufficient number of ions have been

collected, typically over 1 to 3 s, they are pulsed toward the ICR cell by lowering a

voltage on a trapping electrode at the ICR cell end of the hexapole.

Figure 2.8- Photograph showing the hexapole ion-trap in Analytica ESI source in the BioApex II FTICR mass spectrometer.

2.3.1.3- The “Infinity®” ICR cell

The infinity® ICR cell implemented in Bruker BioApex II 70e FTICR mass

spectrometer is a special ICR cell developed by Bruker in 1991.[128] This cell is

Trapping plate

Hexapole trap rods


designed to apply a uniform potential on the trapping plates at each end of the cell to

solve the common “z-ejection” problem in simple ICR cells. Figure 2.9 shows a

photograph of the infinity ICR cell used in BioApex II FTICR mass spectrometer.

Figure 2.9- The infinity ICR cell in BioApex II FTICR mass spectrometer. The scale is in mm.

The trapping plates have segmented regions thereby allowing differential potentials

to be applied throughout the exciting pulse.


Figure 2.10 shows a schematic diagram of the infinity ICR cell, where PV1 And PV2

are parameter names for the trap plates and EV2a and EV2b are for the side kick ion

trap elements. Typical values for these variables are listed in Table 2.1.

Figure 2.10- Schematic diagram of the infinity ICR cell used in BioApex II FTICR mass spectrometer. PV1, EV1 etc are parameters’ name for voltages see table 2.1.

All electro-plates are made of titanium and are gold-plated. The trapping plates (PV1

and PV2) are designed and arranged as described by Caravatti et al. in their

paper.[128] The additional plates outside the PV1 are EV1 and two EV2 half-plates

designed to trap ions via a Sidekick® frequency.

2.3.1.4- Ion Trapping

The ICR cell ion trapping in BioApex II FTICR mass spectrometer was carried out in

Sidekick™ ion-trapping mode in this thesis. The instrument is also capable of

EV1

PV1 PV2

PV2quench

EV2a

++++

+ ++

Excitation

Detection

EV2b

B

sidekick:

EV1

PV1 PV2

PV2quench

EV2a

++++

+ ++

++++++++

++ ++++

Excitation

Detection

EV2b

B

sidekick:

Magnetic field

From ESI source


applying the gated ion trapping (static or dynamic) and collision gas-assisted

dynamic ion trapping, but those were not used.

2.3.1.5- Typical Source, Ion-transfer and ICR Cell Parameters Used in Positive- and Negative-ion Modes on the BioApex II 70e FTICR Mass Spectrometer Table 2.1 lists typical parameters used in positive- and negative-ion FTICR mass

spectrometry experiments.

Table 2.1- Typical parameters used in positive- and negative-ion FTICR mass spectrometry experiments.

Parameter Positive ion Value (V)

Negative ion Value (V)

Capillary 35.0 -43.0 Skimmer 2.51 -5.00

Offset 0.27 -1.91

Source

Extract -4.51 -0.87 PL1 43.80 -10.20 PL2 36.5 -75.50

DPL2 22.30 14.30 DPL4 -2.60 -11.50

FOCL1 -9.90 -27.20 FOCL2 -29.60 55.90 XDFL 46.8 -26.2 YDFL 16.1 -26.0 EV1 -1.22 4.12 EV2 -1.158 1.770

Ion-transfer

DEV2 -16.00 9.45 PV1 -1.46 -1.22 PV2 2.01 -1.35 EV1 -1.22 4.12 EV2 -1.158 1.770

DEV2 -16.00 9.45

ICR Cell

PL3 0.5 0.9


2.3.1.6- Superconducting Magnet

The Magnex Magnet 7 T superconducting magnet used in this thesis uses passive

magnetic field shielding. The solenoid comprises a coil wound of NbTi

superconducting wire with single and/or multiple filaments in a copper protective

matrix. The operating temperature for this magnet is kept at 4.2 K by immersing it in

a cryostat of liquid helium (280 L). A liquid nitrogen cryostat surrounds the liquid

helium vessel (250L), and both of these vessels are insulated and separated by

vacuum cases.

The superconducting magnet has a bore diameter of 150 mm, in which the ICR cell

sits at the centre of the homogenous magnetic field. Cooling water circulates around

the ICR cell to keep the magnet cool during the vacuum bake out process of the ion

optics and the ICR cell.

2.3.1.7- Collision-Induced Dissociation

Argon gas was used as the collision agent during the CID experiments performed on

the FTICR mass spectrometer. It was introduced into the ICR cell through a

molecular leak valve at a constant flow rate.

The ion selection in the ICR cell is achieved by an RF excitation pulse containing a

‘mass envelope’ at the selected m/z that ejects all unwanted ions out of the ICR cell.

The selected m/z ions are then excited by an excitation pulse containing a single RF

frequency with an appropriate intensity. A delay is allowed for the collision between


the selected ion and the collision gas. This is followed by the excitation of the

product ions in the cell and the detection.

2.3.1.8- Pulse Sequence in FTICR-MS Experiments The computer program that operates the FTICR mass spectrometer has a sequence of

pulse-delay events. Each pulse-delay sequence executes a specific operation on the

FTICR mass spectrometer. The BioApex II FTICR mass spectrometer came with

several standard pulse sequence programs that can be easily modified to suit different

experimental requirements.

Figure 2.11 shows a standard pulse sequence for a simple FTICR-MS experiment.

Acquire

P1 P2 P4

D1 D2

Figure 2.11- Pulse sequence used in FTICR-MS analyses for a simple experiment. The basic elements in each pulse sequence program include ion generation, ion

trapping, ion excitation and ion detection. The event sequence shown in Figure 2-11

is as follows: prior to the ion generation, a quench pulse-delay P1/D1 is applied to one

of the trapping plates which effectively ejects all the ions from the ICR cell. An ion


generation pulse-delay P2/D2 is then followed to produce ions from the ion source. In

the external ESI ion source, the P2 pulse is the ion accumulation time in the hexapole

ion-trap.

After the ions are trapped in the ICR cell, a P3 pulse applies radio frequency (RF)

“chirp” (50-200 V peak-to-peak and 180o out of phase) to the two excitation plates

and excites ions whose natural cyclotron frequencies are resonant with the applied

RF field. Ions of the same mass that are generated randomly in time and space are

now brought into a phase-coherent motion, moving together in a packet. This phase-

coherent ion packet generates an ion-induced image current on the receiver plates,

and this image current is passed to a differential amplifier circuit, which facilitates

the detection and amplification of the signals.

If a single RF frequency is selected for the ion excitation, the result is defined as

mass detection, which is commonly referred to as “narrow band” mass spectrum.

Alternatively, the excitation and detection of ions in ICR cell can be carried out in a

wide mass range by sweeping the excitation frequency with a fast frequency chirp

over a selected mass range. Ions in this mass range are excited into a phase-coherent

motion in less than a millisecond. This chirp excitation is often referred as “broad

band” mass spectrum. The experiments in this thesis were carried out using the Chirp

Excitation method.


2.3.1.9- Mass Calibration

In external mode calibration, polyethylene glycol, MW=300, (PEG300, a commercial

product used in the production of detergents) and sodium iodide were used in

positive-ion mode, and sodium iodide was also used in negative-ion mode. First

appropriate solutions of PEG300 and NaI (10-4 M to 10-5 M) in methanol were

introduced to the FTICR mass spectrometer, the instrument was calibrated by

identifying several peaks over a mass range of 100- 1100 Da using a “Masscal”

program and setting up appropriate calibration table. A mass accuracy of <10 ppm

was typically achieved using this external calibration method.

For high mass accuracy calibration internal mass calibration was carried out using

internal reference compounds. In this method, the observed peaks for the known

compounds in the samples were used to recalibrate the spectra and to calculate the

m/z of the unknown compounds. By using the internal calibration method, a mass

accuracy of <2 ppm was usually achieved.

It can be shown from the theory of FTICR-MS[91] that the number of collected data

points is the highest in the lower mass region of the spectrum, hence, the mass

accuracy is affected by the actual number of data points obtained over the measured

mass and the peak symmetry. Therefore, it is expected to be able to measure the

FTICR-MS peaks with a higher precision and accuracy for fatty acids compared to

TAGs.


2.3.1.10- The BioApex II FTICR Mass Spectrometer Control Software

The operation of the BioApex II FTICR mass spectrometer and the mass

spectrometry data acquisition were conducted by XMASS software package supplied

by Bruker Daltonics Company (Version 6.0.1). This program provides control of the

voltages on different components of the FTICR mass spectrometer, including the ion

source, ion optics and ICR cell. It also possesses many options for data processing.

Details of the functions and techniques for data processing are described in the

XMASS user manual.[187]

A significant feature of the XMASS program is that it facilitates the use of macro

procedures. These predefined macro functions are exhibited as icons in a menu-

driven user-friendly interface. Various pulse sequences for different mass

spectrometry experiments can be carried out simply by selecting the appropriate

icons. These macro routines are accessible to the user and are readily modified.

The mass spectra obtained by the XMASS program can be saved as ASCII files

(rather than binary files) and can then be imported into other software for subsequent

additional processing.

2.3.2- High Performance Liquid Chromatography

HPLC was used to collect various isomers in the methanol extract of various

macadamia nut oils prior to analysing using high mass accuracy FTICR mass

spectrometer. The system used in this research project included:


- Pump: Waters 600 Multisolvent Delivery System (Waters - division of

Millipore, Milford, MA 01757 USA).

- Injector: Waters U6K Fluid Unit (Waters - division of Millipore, Milford, MA

01757 USA). The injector was equipped with a micro-switch to send a trigger

signal to the control program on injection.

- Interface: The data was transferred to the PC via a Shimadzu CBM-101 2-

channel communication bus module interface (Shimadzu Corporation, Japan).

The data was collected and manipulated by LC10 chromatography software

(Shimadzu Corporation, Japan) running on a PC under Microsoft™ Windows™

98 SE operating system.

- Column: Reverse-phase C18 Altex™ Ultrasphere™ ODS, 4.6 mm× 25 cm×

5μm (internal diameter× length× particle diameter).

- Precolumn (guard-column): Nova-Pak® C18 Guard-pak™ precolumn cartridge

(Waters - division of Millipore, Milford, MA 01757 USA).

- Detector: Sedex 55 Evaporative Light Scattering Detector (ELSD) (by

SEDERE 94140 Alfortville France).

- Fraction collector: Isco Foxy 200 (Isco Inc. Lincol, NE, U.S.A.)

2.3.2.1- Gradient Elution

Gradient elution (solvent programming) as detailed in Table 2.2 was used to optimise

the macadamia oil separation efficiency. Methanol and water mobile phase contained

0.05% v/v in glacial acetic acid to keep the pH low to minimise free FA ionisation.

This method gave reproducible chromatograms; hence this enabled the fraction

collection to be performed without the ELSD being connected. Usually when


collecting fractions using an ELSD a splitter is inserted between the outlet of the

column and the ELSD, but this was not required because of excellent reproducibility

of the separation.

Table 2.2- Solvent programming for HPLC in the gradient elution of macadamia oil methanol extract.

Time min Flow Rate (ml/min)

% Acetone

% Water (with 0.05% acetic acid)

% Methanol (0.05% in acetic acid)

Initial 1 0 15 85

40 1 0 0 100

50 1 0 0 100

52 2 0 0 100

70 2 0 0 100

75 2 100 0 0

90 2 100 0 0

91 1 0 15 85

120 1 0 15 85

A solution of macadamia oil methanol-extract was prepared at a concentration of 2

mg/ml in methanol and 25 µl of this solution was injected into the chromatograph.

Fractions were collected over a period of 90 minutes at 1 fraction per minute using a

dedicated fraction collector. The fractions were then examined by ESI FTICR mass

spectrometry in both positive- and negative-ion modes as discussed in Chapter 7.


2.3.3- Gas Chromatography- Mass Spectrometry

For the GC-MS experiments, the GC was a Hewlett-Packard HP 5890 Series II with

a J&W DB-Wax 60 m × 0.5 mm × 0.25 µm, and the mass spectrometer was a VG

QUATTRO triple quadrupole mass spectrometer operated under single quadrupole

conditions. A standard 70 eV EI source was used. A standard mixture of FA methyl

esters was used to calibrate the time component of the GC experiment prior to

injecting each set of samples. The spectra were analyzed using Waters Masslynx

software version 4.0 (Milford Massachusetts 01757 USA).


Chapter 3

3. Validation of the ESI FTICR-MS Method Developed for the

Analysis of Plant Oils


3.1- Relation between Peak Intensities and Concentration of the Ions

A number of studies have been published on the relation of peak intensities to the ion

abundances in FTICR-MS.[152,188-191] The spectral peak heights are not reflective of

the true ion abundances in the ICR cell,[189,190] due to the selective z-axis ejection

artefacts particularly for ions far apart in mass,[192] and due to space charge

interactions for the close-lying peaks.[190]

Comisarow has established a direct relationship between the instantaneous charge

induced on a detection electrode and the number of excited ions in the ICR cell.[132]

There are reports on the effect of more abundant ions in the ICR cell on the peak

intensity and the mass measurement accuracy of the less abundant ions.[193]

Cech and Enke have achieved a linear relationship between the peak intensity and the

concentration of a particular ion in the FTICR-MS for a certain range of

concentration by calibrating the peak intensities using standard solutions of the same

species prior to the injection of unknown samples.[188]

As mentioned above, matrix effects such as salt content, high concentration of co-

existing species, different solvent affinities of the molecules in solution and general

ionization efficiencies are among the factors influencing the peak intensities in ESI

FTICR-MS. Further discrimination can occur during the accumulation in hexapole

and the transmission of ions into the ICR cell according to the hexapole delay and

ion-transfer optics settings.


However, for the ions within comparable range of concentrations, with similar

structures in a close range of masses, using same set of hexapole delay and ion-

transfer optics parameters, a sensible estimate of the relative concentrations of the

ions is achievable from the intensities of the peaks in an FTICR mass spectrum.

An additional complication that should be taken into account in the negative-ion

mode FTICR-MS of the solutions containing free FAs is the degree of completeness

of the dissociation reactions of FAs in the solution and during the evaporation

processes (pKa for stearic acid is 10.15, for oleic acid is 9.85 and for linolenic acid is

8.28 in aqueous solution). In a solution containing stronger FAs, the dissociation of

the weaker FAs and hydroxyl groups can be suppressed, resulting in a decrease in the

relative peak intensity of the weaker FAs and the hydroxyl groups.

On the other hand, in the positive-ion ESI FTICR-MS of free FAs, the ions are

generated by attachment of a cation to non-bonding electrons on the oxygen atoms,

that is a simple ion-dipole interaction and so relative positive-charged adduct ion

affinities of the FA become very relevant to the ionization process.

To verify the effect of acid-base dissociation reactions on the peak intensities in

negative-ion mode ESI FTICR-MS, solutions of the oil and the oil extract in pure

methanol were compared to the solutions of the oil and the oil extract in methanol

containing 10-3 M triethylamine as a volatile organic base. The existence of a basic

molecule in the solution is expected to promote the deprotonation of the acidic

species, resulting in an immense increase in the peak intensities of the anions.

The observed relative intensities for most of the peaks throughout the spectrum

showed a 10-20 percent increase, but the relative peak intensities found to be similar


in two spectra. This is consistent with the previous observations regarding the effect

of an alkaline solution on the deprotonation reactions in negative-ion ESI MS.[194]

3.2- Reproducibility of the ESI Source and the FTICR Mass Spectrometer

A solution containing suberic (C8:0 dicarboxylic acid), palmitoleic (C16:1), palmitic

(C16:0), linoleic (C18:2), oleic (C18:1), eicosenoic (C20:1) and eicosanoic (C20:0)

acids at approximately 10-4 M was prepared from chemicals present in the School of

Chemistry at The UNSW with unknown purities. As in this method development

study we are not performing quantitative analysis or comparing the intensities as a

measure of concentrations, the purity of the chemicals used was not a concern.

This solution was used to validate the method in various experiments discussed in

this section. Figure 3.1 shows the ESI FTICR mass spectrum of the test solution in

negative-ion mode.

The intensities of the peaks assigned to free fatty acids are different due to the fact

that the purities of the used chemicals were unknown. It is also possible that the

difference in the intensities is partly due to the instrumental parameters and relative

ionisation efficiencies.

Table 3.1 lists the assignment of the observed peaks in the negative-ion FTICR mass

spectrum of the prepared test solution.


173.0822

281.2486

369.1532

200 300 400 500 600 700 800 900m/z

255.2335

563.5091 639.5650 905.8527

Figure 3.1- Negative-ion ESI FTICR mass spectrum of the test solution.

Table 3.1- Assignment of mass spectral peaks in the FTICR mass spectrum of a solution of fatty acids mixture solution shown in Figure 3.1.

Formula Measured Mass (Da)

Exact Mass (Da)

Compound Class

C8H13O4‾ 173.0822 173.0820 Di-FA* C9H15O4‾ 187.0976 187.0976 Di-FA* C14H27O2‾ 227.2022 227.2017 FA C16H29O2‾ 253.2177 253.2173 FA C16H31O2‾ 255.2335 255.2330 FA C17H33O2‾ 269.2478 269.2486 FA C18H29O2‾ 277.2201 277.2173 FA C18H31O2‾ 279.2347 279.2330 FA C18H33O2‾ 281.2486 281.2486 FA C18H35O2‾ 283.2636 283.2643 FA C20H37O2‾ 309.2805 309.2799 FA C20H39O2‾ 311.2956 311.2956 FA C16H27O8‾ 347.1717 347.1711 FA dimer C18H25O8‾ 369.1532 369.1555 FA dimer C36H67O4‾ 563.5091 563.5045 FA dimer C36H69O4‾ 565.5217 565.5201 FA dimer C37H73O6‾ 613.5481 613.5413 DAG C39H75O6‾ 639.565 639.5569 DAG C57H109O7‾ 905.8299 905.8179 TAG

* Di-FA: Dicarboxylic fatty acid (HOOC-R-COOH).


Among the assigned peaks in this spectrum, there are 10 FAs, 2 short-chain di-

carboxylic acids, 4 FA dimers, 2 DAGs and one TAG molecule assigned. This table

of chemical compounds clearly demonstrates that the fatty acids used to prepare the

test solution were not pure. The short-chain fatty dicarboxylic acids are possibly

formed by the oxidation of the double bond of unsaturated FAs such as oleic acid.

To investigate the reproducibility of the ESI source and to verify the mass accuracy

of the FTICR mass spectrometer for the free FAs, the solution (containing suberic,

palmitoleic, palmitic, linoleic, oleic, stearic, eicosenoic and eicosanoic acids) was

examined seventeen times on the ESI FTICR mass spectrometer in negative-ion

mode with oleate anion used as the internal calibrant. The observed peaks in the

negative-ion spectra of the solution range from 173.0822 Da for suberic acid at the

lower mass end to 565.5217 Da for oleic-stearic acid dimer at the high mass end. The

ion transfer parameters used in this experiment were optimized and used in the

analysis of the oil and the oil extract samples.

Figure 3.2 illustrates the relative intensities of palmitate anion (255.2330 Da) peak

versus the measured masses in 17 analyses of the test solution. The relative standard

deviation (RSD) of peak intensities is 7.0% and the RSD of mass measurement is

4×10-5 % or 0.4 ppm. Oleate anion (281.2486 Da) is used as the reference compound

for the relative intensities and the internal standard for the mass measurements.


Relative Intensities of Palmitate Anion (Mass 255.2330 Da) in 17

Experiments

0.1

0.11

0.12

0.13

0.14

0.15

0.16

0.17

0.18

0.19

0.2

255.23365 255.2337 255.23375 255.2338 255.23385 255.2339 255.23395 255.234 255.23405Measured Mass (Da)

R.I.

Figure 3.2- Relative intensities of palmitate anion peaks versus measured masses in 17 consecutive FTICR mass spectra of the test solution (normalised versus oleate anion peak as 1).

Figure 3.3 illustrates the relative intensities of stearate anion (283.2643 Da) peak

versus measured masses in 17 analyses of the test solution. The RSD of the peak

intensities is 4.2% and the RSD of mass measurement is 7×10-5 % or 0.7 ppm. Oleate

anion (281.2486 Da) is used as the reference compound for the relative intensities

and the internal standard for the mass measurements.

Relative Intensities of Stearate Anion (Mass 283.2643) in 17 Experiments

0.20

0.21

0.21

0.22

0.22

0.23

0.23

0.24

283.2649 283.265 283.2651 283.2652 283.2653 283.2654 283.2655 283.2656

Measured Mass (Da)

R.I.

Figure 3.3- Relative intensities of stearate anion peaks versus measured masses in 17 consecutive FTICR analyses of the test solution (normalised versus oleate anion peak as 1).


Figure 3.4 illustrates average mass deviation from exact masses for the assigned

peaks in the test solution in seventeen separate negative-ion FTICR-MS analyses.

Only external calibration is performed in this experiment. Mass deviations are

calculated using the following equation:

│ Exact mass – Measured mass │ Mass deviation (ppm) = ──────────────────── × 1000,000 Exact mass

Generally, higher mass deviations are observed at higher ionic masses (about 8.8

ppm at 563 Da) and lower mass deviations are observed at lower ionic masses (about

1.7 ppm at 173 Da).

Average Deviations from Exact Masses vs. Average Measured Masses for 17 ESI FTICR-MS Analyses of the Standard Solution

0

1

2

3

4

5

6

7

8

9

10

150 200 250 300 350 400 450 500 550 600

Average measured mass (Da)

Aver

age

Devi

atio

n fr

om E

xact

Mas

s (p

pm)

Figure 3.4- Average deviations from exact masses vs. average measured masses in 17 consecutive FTICR mass spectra of the test solution. Each point represents the average of 17 mass measurements of a particular FA in the test solution.


3.3- Detection Limits of FTICR-MS in FA Measurement

To verify the detection limit of the FAs in FTICR mass spectrometer, approximately

10-4 M solutions of palmitic, palmitoleic, stearic, oleic, and linoleic acids were

prepared. The criterion was to include saturated and unsaturated FAs in the test

solutions (C16:0, C16:1, C18:0, C18:1, C18:2). Successive dilutions (1/10, 1/100,

1/1000, 1/10000, and so on) were prepared for each of the FAs in methanol. The

solutions were examined on the FTICR mass spectrometer starting with the most

dilute until a signal was observed for that particular acid. The experiment was carried

out in both positive- and negative-ion modes. The detection limits found to be

approximately 10-12 M in positive-ion mode and approximately 10-13 M in negative-

ion mode FTICR-MS (five times of the background noise).

3.4- Effect of the Hexapole Ion Trap Delay on the Peak Intensities

To examine possible contribution to the ions observed in the present analytical

macadamia oil experiments, the hexapole ion trap delay (typically 0-8 S) was

studied. In this experiment, a solution of the processed macadamia nut oil was

injected to the ESI FTICR mass spectrometer with the hexapole trap delay varied

from 0 s to 8 s in 0.5 s increments. At each hexapole delay setting, the spectrum was

recorded, and then the results were compared using the intensities of the observed

peaks in the spectrum.


At hexapole delays longer than 2.5 s the peak intensities did not change dramatically,

while in hexapole delays shorter than 1 s, the intensities of the peaks diminished

drastically. Consequently, a hexapole delay of 2 s was selected for the FTICR-MS

experiments as the optimum setting.

3.5- Aging Stability of the Oil Samples

To investigate the stability of the oil samples, stability tests were performed on the

processed macadamia nut oil. The processed macadamia oil was left in the laboratory

in the daylight at room temperature for up to ten months. The oil was analysed on

ESI FTICR mass spectrometer during this period. Figure 3.5 shows the positive-ion

ESI FTICR mass spectra of neat macadamia oil on three different dates. Figure 3.5-a

shows the FTICR mass spectrum of neat macadamia oil analysed in December 2002,

Figure 3.5-b illustrates the FTICR mass spectrum of neat macadamia oil analysed in

July 2003 and Figure 3.5-c shows the FTICR mass spectrum of neat macadamia oil

analysed in September 2003. As the free fatty acid content of the oil is considered as

a measure of the stability of the oil, it was necessary to investigate the stability of the

macadamia oil sample. The duration of the stability investigation was the total time

spent on the experimental work in this study.

The observed variations in the three FTMS spectra are minor, suggesting that the oil

was stable during the period of the aging stability test.


825.682845.606

853.711

869.715

879.734

897.746

907.765

923.754937.798

955.749 971.749

825.679837.587

853.708

865.625

879.725

907.756

923.743935.771

953.687 967.709

825.689837.601

851.713

879.746

895.747

907.778

923.763939.768

951.765 969.746

820 840 860 880 900 920 940 960 980 m/z

Figure 3.5- Positive-ion FTICR mass spectra of methanol solution of neat processed macadamia oil in (a) December 2002, (b) July 2003 and (c) September 2003.

3.6- Fragmentation during Ion Transfer

At high capillary-skimmer potential differences, ions are highly accelerated and

experience more collisions. Due to this higher number of impacts of the accelerated

ions in the capillary-skimmer region, unnecessary fragmentations may occur.

To investigate the possibility of fragmentation of FAs and the acylglycerols in

positive- and negative-ion modes in the capillary-skimmer and hexapole ion-trap

regions of the ion transfer optics, the test solution of FAs was injected to the FTICR

mass spectrometer while the voltage on the capillary was scanned from the lowest

signal-producing voltage up to the highest possible voltage (~±400 V).

(a)

(c)

(b)


In the positive-ion mode increasing the capillary-skimmer potential to greater than

200 V resulted in the detachment of the attached sodium ion from the FA and

acylglycerol molecular ion complexes, resulting in a corresponding signal loss.

To decrease the possibility of molecular ion fragmentation occurring in both

positive- and negative-ion modes, the capillary-skimmer potential was set to the least

signal-producing voltage in both positive- and negative-ion modes. Typically this

voltage was set to about ± 40 V (10% of the scale) in positive and negative modes to

minimise the possibility of adventitious collision induced dissociation processes (see

Table 2.1).

Figure 3.6 illustrates the effect of capillary-skimmer voltage on the spectrum of the

FA solution in negative-ion mode. At a capillary-skimmer voltage of 300 V the

intensities of the main peaks diminish (approximately 20 times weaker than

intensities at 20 V) and some higher mass peaks appear in the spectrum. The peaks in

the higher mass region are assigned to compounds with high number of oxygen

atoms. At high capillary-skimmer voltages oxidation-reduction reactions are more

likely to occur, producing molecules with high number of oxygen atoms. In addition,

at high capillary-skimmer voltages, excessive CIDs occur resulting in breaking the

FA dimers and also detachment of Na+ from positive ions such as DAGs and MAGs,

causing weak peak intensities and loss of sensitivity in the obtained spectra (See

Figure 3.6 c). Note that in Figure 3.6 (c) the intensity of the spectrum is two orders of

magnitude lower than spectra (a) and (b) (107 vs. 109). If the three spectra in Figure

3.6 are superimposed using same intensity scale, the peaks in spectrum (c) would not

be observed.


On the other hand, a drawback of applying a very low capillary-skimmer voltage is

that dimers of free FAs form in the gas phase in hexapole region. These molecules do

not exist in the oil and are generated in the instrument. However, the dimers peaks

are easily recognised as few peaks appearing only in the 400-600 Da region, and

readily disappear by applying higher capillary-skimmer voltages. The FA dimer

peaks could also be used as internal calibrants for the DAG region peaks.

140.6254

173.0821

212.0756

281.2485

369.1529

140.6250

173.0820

212.0756

281.2487

281.2482

347.1690 413.1159455.3403

509.4622565.5182

639.5568

851.7574907.8229

0.00

0.25

0.50

0.75

1.00

1.25

9x10

In ten s.

0.0

0.2

0.4

0.6

0.8

1.0

1.29

x10

0

2

4

6

8

7x10

200 300 400 500 600 700 800 900 m/z Figure 3.6- Negative-ion FTICR mass spectra of the FA test solution at three capillary-skimmer voltages, a) 20 V, b) 125 V and c) 300 V.

Since the ESI produces ions continuously and the FTICR experiment is gated, the

hexapole ion trap is used to build up the concentration of ions from the ESI source

prior to injection of a packet of ions into the ion optics (Figure 2.2). However, during

(a) 20 V

(b) 125 V

(c) 300 V


this ion trapping process it is possible for some ions to undergo ion molecule

interactions including association and/or CID processes. The longer the ions stay in

the hexapole, the higher the possibility of the CID fragmentation to occur.

3.7- Discussion of the Validity of the Mass Spectral Peak Assignments

The major parameters in assigning empirical formulae to the FTICR mass spectral

peaks in this study is based three parameters: high mass accuracy, high resolution

and knowledge of the chemical composition of macadamia oil obtained in previous

studies and different methods we used such as GC-MS.

High mass accuracy of <2 ppm enables us to assign possible empirical formulae to

the FTICR mass spectral peaks. This high accuracy minimises the number of

possible combinations of the free FAs in a particular ion.

High resolution of approximately 50,000 or higher enables us to differentiate the

isobars (molecules with same nominal masses but different elemental compositions).

Isotopic ratios could be used but in FTICR-MS the isotopic ratios can have an error

of about 10%.

The major FAs in macadamia oil are oleic, palmitoleic and linoleic acid. The MAGs,

DAGs and TAGs all contain various combinations of the above FAs. Each MAG,

DAG and TAG assignment may contain a different possible combination of FAs

giving the same molecular mass. For example, a TAG molecule assigned to C18:1,

C18:1, C16:0 could be assigned to C18:1, C18:0, C16:1. But we would expect the

intensity of the sodium adduct of the first combination to have the highest relative


intensity in the mass spectrum of neat macadamia oil. This is due to the fact that

C18:1 and C16:1 comprise the highest percentage of the oil constituents, while C16:0

and C18:0 are minor constituents of macadamia oil.

3.8- Normal Probability (Rankit) Test of the Peak Intensities and Measured Masses

To investigate possible existence of systematic error in the measurement of the peak

intensities and masses, normal probability (Rankit) tests were carried out. A normal

distribution of the measured peak intensities and masses contradicts the presence of a

systematic error in the measurements. As is explained elsewhere in this thesis

(section 1.4.1.9), the correlation coefficient (R) of the trend line is a measure of the

normality of the distribution of the measured peak intensities and masses. An R value

close to 1 denotes a normal distribution of the points, disagreeing with the existence

of a systematic error in the measurements.

The FA test solution was analysed on the FTICR mass spectrometer 17 consecutive

times in negative-ion mode. The intensities of the peaks and the measured masses

were examined using a Rankit test method. The Rankit test plot yielded an R value of

0.984 for the measured masses and an R value of 0.991 for the measured intensities,

suggesting a normal distribution of the intensities and the measured masses of the FA

peaks in negative-ion mode, ruling out the existence of systematic errors. Figures 3.7

and 3.8 show the rankit plot for the intensities and mass measurements of palmitate

anion in the FA test solution. Similar plots could be produced for other FAs in the

FA solution.


Rankit plot of measured masses (palmitate anion, 255.2330 Da) for 17 FTICR-MS analyses of the FA standard solution in negative-ion mode

R2 = 0.9695

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

255.2336 255.2337 255.2338 255.2339 255.2340 255.2341

m / z (Da)

z

Figure 3.7- Rankit plot of the measured masses of palmitate anion (255.2330 Da) in seventeen FTICR-MS analyses of the FA solution in negative-ion mode.

Rankit plot of relative intensities (palmitate anion, 255.2330 Da) for 17 FTICR-MS analyses of the FA standard solution in negative-ion mode

R2 = 0.9824

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

0.13 0.135 0.14 0.145 0.15 0.155 0.16 0.165 0.17

Relative Intensity

z

Figure 3.8- Rankit plot of the peak intensities of palmitate anion (255.2330 Da) in seventeen FTICR-MS analyses of the FA solution in negative-ion mode.


Chapter 4

4. Positive-ion ESI FTICR-MS of Processed Macadamia Oil


4.1- Introduction

In this chapter the positive-ion ESI FTICR mass spectra of neat, methanol extract,

hydrolysed and esterified processed macadamia oil are discussed. For each spectrum,

only the peaks larger than 2% in intensity are assigned and tabulated, although some

interesting compounds with lower intensities are sometimes listed in the tables.

Comparisons between spectra are made to investigate the validity of the assignments

and various experiments.

For the purposes of this study we have assigned the peaks in the mass spectra

according to elemental analysis and exact-mass measurements in conjunction with

common compounds assigned by GC-MS in other studies undertaken on similar

macadamia nut oil[195,196] and the common knowledge of plant lipid chemistry. Here

however we are able to identify the parent molecular ion species in the neat and

methanol extract samples whereas in the earlier studies such compounds are reduced

to FA methyl esters by derivatization for GC-MS analysis.

4.2- Positive-ion ESI FTICR-MS of Processed Macadamia Oil

The positive-ion ESI-FTICR mass spectrum of neat processed macadamia oil is

shown in Figure 4.1.


319.2249 393.2609

575.5055

603.5352

631.4933659.5219

825.6786

853.7187

879.7338

907.7650

937.7980

300 400 500 600 700 800 900 1000 m/z Figure 4.1- Positive-ion ESI-FTICR mass spectrum of neat macadamia nut oil.

In all, we can identify at least 187 different ionic species in Figure 4.1 though many

of them occur at trace levels (<1%). Application of the Bruker elemental analysis

software, carbon-13 isotope analysis and high-resolution and high-accuracy mass

measurements allows a number of the peaks in Figure 4.1 to be assigned and these

are listed in Table 4.1. The extrapolation from the molecular formulae to the actual

components in the oil are only tentative because no structural analyses have been

carried out on the assigned ions.

TAG Region

DAG Region

MAG and FA Region


Table 4.1. Assignment of the mass spectral peaks (>2%) in the positive-ion ESI-FTICR mass spectrum of neat macadamia oil shown in Fig. 4.1.

Observed Mass# m/z

Exact Mass m/z

Compound Class Assigned Sodium Adduct Compound Normalized

% 319.2249 319.2244 FA Hydroxylinoleic acid- C18:2 13.0 333.2405 333.2400 FAME Hydroxynonadecdienoic acid- C19:2 5.4 353.2665 353.2662 MAG Glycerol palmitate 5.7 367.2401 367.2455 MAG Glycerol hydroxypalmitoleate 5.8 379.2840 379.2819 MAG Glycerol oleate 2.6 393.2609 393.2611 MAG Glycerol-hydroxylinoleate 11.7 411.2654 411.2717 MAG Glycerol-dihydroxyoleate 5.4 575.5022 575.5010 FA Dimer Dimer of C17:0 and C18:1* 31.2 603.5310 603.5323 FA Dimer Dimer of C19:0 and C18:1** 49.5 615.4870 615.4959 DAG Glycerol oleate palmitoleate 9.1 617.5053 617.5115 DAG Glycerol oleate palmitate 13.1 631.4998 631.4908 DAG Glycerol hydroxyoleate palmitoleate 11.2 643.5212 643.5272 DAG Glycerol dioleate 13.7 659.5195 659.5221 DAG Glycerol hydroxyoleate oleate 19.5 675.5127 675.5170 DAG Glycerol dihydroxyoleate 16.9 691.5120 691.5119 DAG Glycerol trihydroxyoleate 10.3 823.6764 823.6786 TAG Glycerol tripalmitoleate 6.1 825.6786 825.6943 TAG Glycerol palmitate dipalmitoleate 9.1 851.7098 851.7099 TAG Glycerol oleate dipalmitoleate 29.3 853.7187 853.7256 TAG Glycerol stearate dipalmitoleate 34.4 879.7338 879.7412 TAG Glycerol dioleate palmitoleate 67.8 881.7537 881.7569 TAG Glycerol dioleate palmitate 61.5 907.7650 907.7725 TAG Glycerol trioleate 100.0 # Three known compounds, glycerol oleate (379.2819 m/z), glycerol dioleate (643.5272 m/z), and

glycerol trioleate (907.7725 m/z) are used as internal standards to correct for the mass errors. The observed masses in the table are before applying internal calibration.

* Could be dimer of C18:1 and methyl palmitate ** Could be dimer of C18:1 and methyl oleate

Three distinct regions are observed in the FTICR mass spectrum of macadamia nut

neat oil in Figure 4.1. The peaks in the region m/z 200-450 are assigned to sodiated

adducts of free FAs, MAGs and some FA dimers that form in the gas phase. The

peaks in the region m/z 500-700 are assigned to sodiated adducts of DAGs as well as

to sodiated adducts of several FA dimers; and the peaks in the region m/z 800-1000

are assigned to sodiated adducts of TAGs present in the oil.


4.2.1- Free Fatty Acids and Monoacylglycerols Region, m/z 150-400

Figure 4.2 illustrates expanded FA region of macadamia oil FTICR mass spectrum.

181.7591

207.1000

221.1153

247.2408

265.2526

279.2312

319.2249

339.2910

353.2617

367.2402

381.2489

393.2609

411.2706

433.2917

175 200 225 250 275 300 325 350 375 400 425 m/z Figure 4.2- Expanded FA region of positive-ion ESI-FTICR mass spectrum of neat macadamia oil.

As mentioned in Section 1.2.7, the neutralising, deodorising and degumming

processes remove the majority of more polar unsaturated free fatty acids (such as

oleic and palmitoleic acids) and other polar compounds from the oil. One might

expect to observe traces of less polar saturated fatty acids such as palmitic acid in the

oil. The detection of free FAs in the neat oil is significant in the context of hydrolytic

rancidity and shows the value of the high-resolution ESI FTICR-MS technique.

Other studies in the literature typically use GC or GC-MS to analyze the FA

components in oils. In these latter cases the oil is first esterified before analysis; a


step that effectively reduces the free FAs and all of the acylglycerols in the oil to

their corresponding methyl esters.

Furthermore, the very high relative population of acylglycerol molecular ions in the

FTICR cell arising from neat oil, has an ion-suppression (cloud) effect on the peak

intensities of trace level ions in the cell, resulting in the observation of only two free

fatty acids in the neat oil spectrum at m/z 319.2249 (C18:2) and m/z 333.2405

(C19:2). The C18:2 cation is assigned to NaC18H31O3+ and could have been formed

from the hydration of the C18:3 linolenic acid. The C19:2 cation is assigned to

NaC19H34O3+ and could have been produced by the hydration of the C19:3

nonadectrienoic acid.

A number of unusual compounds are observed in the expanded FA region spectrum

in Figure 4.2 giving rise to sodiated adducts that can be assigned to acylglycerols

with acyl substituents with odd number of carbon atoms such as glycerol

pentadecenoate, C18H34O4Na+, m/z 337.2359. Alternatively, this sodiated adduct can

be assigned to dihydroxy oleic acid. When the neat oil was esterified, derivatives of

odd numbered fatty acids were not observed, but the hydroxy oleic acid was

observed instead. So we have assigned them in Table 4.1 to be sodiated hydroxy-

adducts. Additional chemical and tandem mass spectrometry experiments are

required to more precisely identify the molecular structure of these compounds. In

such cases peaks are assigned to hydroxy-species (e.g. hydroxy linoleic acid m/z

319.2249) because an ion of this mass is observed from both the neat oil as well as

from the esterified oil sample indicating that it is a hydroxy but not a peroxyacid

compound.


4.2.2- Diacylglycerol Region, m/z 500-750

Figure 4.3 shows an expanded view of the DAG region of macadamia nut oil

spectrum in Figure 4.1.

575.5022

603.5310

617.5053

631.4998

643.5212

659.5195

675.5127

689.4995

707.5155

560 580 600 620 640 660 680 700 m/z Figure 4.3- Expanded DAG region of positive-ion ESI-FTICR mass spectrum of neat macadamia nut oil.

Fatty acids produce dimers in the gas phase by forming a stable hydrogen-bonded

ring.[197] The assignment of two peaks in the expanded DAG region of macadamia oil

spectrum in Figure 4.3 is consistent with the dimerisation reaction of FAs in the gas-

phase. These are m/z 575.5022 which is assigned to a dimer of oleic acid and

heptadecanoic acid and m/z 603.5310 that is assigned to a dimer of oleic acid and

nonadecanoic acid respectively.

Several observed peaks in Figure 4.3 are assigned to DAGs such as m/z 631.4998,

glycerol oleate-palmitoleate with one additional oxygen atom and m/z 659.5195,


glycerol dioleate with one additional oxygen atom. The additional oxygen atom is

expected to be a hydroxyl group on the hydrocarbon chains of the oleate or

palmitoleate acyl groups. A hydroxyl group on the alkyl chain could form by

addition of a water molecule to a carbon double bond on the FA chain. This

“hydration reaction,” is known to occur in the presence of hydrated acids.[197]

Another possibility is the occurrence of electrochemical reactions in the ESI source

due to the electrical discharges at high voltages.[198,199] These reactions can generate

active intermediates during the electrospray ionisation process.[119,120,200] Further

structural investigation is required to more accurately identify the assigned hydroxy

fatty acid compounds, but this was not performed in this study.

4.2.3- Triacylglycerol Region, m/z 800-1000

As might be expected,[201] Figure 4.1 reveals that TAGs and DAGs are the major

constituents of neat macadamia oil. This observation assumes that the ionisation

efficiencies of each group of chemical compounds such as free FAs, MAGs, DAGs

and TAGs are similar within each group. In positive-ion ESI, the mechanisms of

sodium cation attachment to these molecules is similar in each group. No evidence

has been presented for this assumption in present study.

As FTICR-MS resolves compounds according to their masses only, structural

isomers are not resolved by FTICR-MS. Thus, each mass spectral peak can

potentially represent one compound or a mixture of isomers of that compound. In

Chapter 7 we show that such isomers can be isolated using HPLC and then analysed

using the ESI FTICR mass spectrometer in an offline mode.


Figure 4.4 illustrates an expanded view of the TAG region of macadamia nut oil

spectrum in Figure 4.1. The thin vertical line underneath each peak is produced by

the peak-picking routine in Bruker software.

853.7187

879.7338

907.7650

850 860 870 880 890 900 910 m/z Figure 4.4- Expanded TAG region of positive-ion ESI-FTICR mass spectrum of neat macadamia nut oil. The thin vertical line underneath each peak is produced by the peak-picking routine in Bruker software.

The five major peaks in the TAG region of Figure 4.4 are assigned to glycerol-

trioleate (m/z 907.7650), glycerol dioleate palmitate (m/z 881.7425), glycerol

dioleate palmitoleate (m/z 879.7338), glycerol palmitate palmitoleate oleate (m/z

853.7187) and glycerol dipalmitoleate oleate (m/z 851.7098). This result is consistent

with the previous report by Cavaletto where the major FAs associated with

macadamia nut oil are oleic acid 65%, and palmitoleic acid 18% and palmitic acid

7%,[196] and with Maguire where the same major FA components of macadamia oil

are reported as 65%, 17% and 8%respectively.[202] Our GC-MS study discussed in


Chapter 6 shows the major FA components as oleic 58%, palmitoleic 21% and

palmitic 11% methyl esters. The relative peak areas are rounded off to nearest whole

number for comparison purpose.

CID in tandem with a chromatography stage could provide more structural

information about the FAs present in the acylglycerols of the oil. For example, the

sodiated adduct at m/z 851.7098 in Table 4.1 has been assigned to glycerol oleate

dipalmitoleate; alternatively it could have been assigned to glycerol linoleate

palmitoleate palmitate. CID and chromatography experiments would help to refine

the assignments in such cases, but the CID experiments were not attempted in this

study.

4.3- Positive-ion ESI FTICR-MS of Methanol Extract of Processed Macadamia Oil

The purpose behind extraction of the oil in methanol is to eliminate the spectral

interference of triacylglycerols from the oil and thereby enhance the detection of free

FAs in the oil in mass spectra. The methanol extraction achieves this goal, however,

there are some factors observed which are less desirable and complicate

interpretation of the results.

Figure 4.5 shows the positive-ion FTICR mass spectrum of the methanol extract of

macadamia nut oil. The procedure for methanol extraction of macadamia nut oil is

described in section 2.2.1.


Eight sodiated FAs, three MAG and seven DAG ions assigned to the peaks in this

spectrum are listed in Table 4.2.

277.2114

305.2418

351.2479

379.2776

449.1479

587.4582

615.4831

643.5163

907.7481

300 400 500 600 700 800 900 1000m/z

Figure 4.5- Positive-ion ESI-FTICR mass spectrum of the methanol extract of macadamia nut oil.

A comparison of Table 4.2 (extracted macadamia oil) with Table 4.1 (neat

macadamia oil) shows that methanol extraction process has removed the majority of

the acylglycerols such as TAGs from the extracted sample dramatically and

enhanced the concentration and hence improved the detection of free FAs.


Table 4.2. Assignment of the mass spectral peaks (>2%) in positive-ion ESI-FTICR mass spectrum of the methanol extract of macadamia oil shown in Figure 4.5.

Observed Mass m/z

Exact Mass m/z


Intensity % 277.2114 277.2138 FA Palmitoleic acid- C16:1 28.8 279.2285 279.2294 FA Palmitic acid- C16:0 14.5 293.2449 293.2451 FA Heptadecanoic acid- C17:0 2.6 303.2288 303.2294 FA Linoleic acid- C18:2 7.0 305.2418 305.2451 FA Oleic acid- C18:1 47.1 307.2608 307.2607 FA Stearic acid- C18:0 3.9 319.2596 319.2608 FA Nonadecenoic acid- C19:1 10.4 333.2759 333.2764 FA Eicosenoic acid- C20:1 5.0 351.2479 351.2506 MAG Glycerol palmitoleate-C16:1 13.5 375.2499 375.2506 MAG Glycerol linolenate- C18:3 1.0 379.2776 379.2819 MAG Glycerol oleate- C18:1 53.7 449.1479 - - Unknown 23.1 587.4582 587.4646 DAG Glycerol dipalmitoleate- C16:1 30.0 615.4831 615.4959 DAG Glycerol oleate palmitoleate 87.6 617.5134 617.5115 DAG Glycerol oleate palmitate 37.9 633.5049 633.5065 DAG Glycerol hydroxyoleate palmitate 19.6 643.5163 643.5272 DAG Glycerol dioleate 100.0 659.5104 659.5221 DAG Glycerol hydroxyoleate oleate 12.4 675.5121 675.5170 DAG Glycerol dihydroxyoleate 5.7

Figure 4.5 demonstrates the importance of decreasing the ion suppression (cloud)

effect in the FTICR cell by extraction process of the oil, resulting in a significant

decrease in the number of acylglycerol ions in FTICR cell and improvement in the

free FA peak intensities. Eight peaks in Table 4.2 are assigned to free FAs in the

methanol extract of macadamia nut oil, compared to only one in the neat nut oil.

If this extract was to be analysed by GC-MS, all of the acid components would need

to be converted to esters prior to analysis. Consequently, the free FAs and FAMEs (if

present) would not be distinguishable. The alternative approach is to use HPLC

which can separate the ester and acid components. In a further attempt to resolve

such structural isomers, the macadamia oil methanol extract was analyzed by HPLC.

This is discussed later in Chapter 7.


Arachidonic, lauric and myristic acids observed in small amounts (<3%) by previous

workers[196] were detected at trace levels (<1%) in this experiment.

The composition of the free FAs observed in methanol extract of macadamia oil in

Table 4.2 is representative of the free FA composition of macadamia nut oil.

However, it seems significantly different from the composition of FAMEs observed

in esterified macadamia oil in Table 4.4. This shows that the source of the observed

free FAs in the oil is not simple release of the FAs by hydrolysis of acylglycerols in

the oil, but the FAs could have been released selectively via biological routes such as

microorganism enzyme activities as well as possibly the actual processing of the oil.

For example, we compare the relative concentrations of C18:1 and C16:1 in the

FTICR mass spectrum of the methanol extract of the oil and GC mass spectrum of

the esterified oil. This ratio is found to be 47:29 using FTICR-MS in the methanol

extract of the oil, and 60:31 and 56:27 in two distinct GC-MS experiments on the

esterified oil samples. This ratio is reported as 69:20 in literature.[196] The GC

analysis requires an esterification process that releases majority of the FA

substituents from the acylglycerols present in the oil extract as their FAMEs, while

FTICR-MS directly measures the FFA content of the methanol extract of the oil.

This again demonstrates utility of FTICR-MS as a complementary technique in the

analysis of free FAs in such oils.

The mass spectra reveal that the quantity of acylglycerols (mainly TAGs) in the nut

oil (Fig. 4.1) is high compared to the amount of FFAs and the similar compounds

containing additional oxygen atoms in their acyl chains. Since the presence of the

latter compounds in the oil is evidence of oxidation of the glycerols, we take this


observation as evidence of the relative stability of the oil. The ESI FTICR analysis of

the neat and methanol extract of the macadamia oil highlights this point.

4.4- Positive-ion ESI FTICR-MS of Hydrolysed Processed Macadamia Oil

Figure 4.6 shows the positive-ion FTICR mass spectrum of hydrolysed macadamia

nut oil. Nineteen sodiated FAs, two MAGs, two DAGs, two FA dimers and one TAG

ions are assigned to peaks in this spectrum and are listed in Table 4.3. The procedure

for the hydrolysis experiment is described in Section 2.2.2.

The point behind performing the hydrolysis reaction on macadamia oil is to provide

more evidence to confirm the assigned peaks in the neat macadamia oil spectrum

(Figure 4.1) and esterified oil spectrum (Figure 4.7) but more importantly to

investigate the fatty acid composition of acylglycerols present in macadamia oil.

277.2140

305.2453

351.2521

617.5135643.5297

855.7443881.7609

300 400 500 600 700 800 900 m/z Figure 4.6- Positive-ion FTICR mass spectrum of hydrolysed macadamia nut oil.


Table 4.3. Assignment of the major mass spectral peaks (>2%) in the positive-ion ESI-FTICR mass spectrum of hydrolysed macadamia nut oil shown in Figure 4.6.

Observed Mass m/z

Exact Mass m/z


Intensity % 277.2140 277.2138 FA Palmitoleic acid- C16:1 28.2 279.2297 279.2295 FA Palmitic acid C16:0 8.5 301.2151 301.2138 FA Linolenic acid C18:3 2.7 303.2297 303.2295 FA Linoleic acid C18:2 12.9 305.2453 305.2451 FA Oleic acid C18:1 100 307.2593 307.2608 FA Stearic acid C18:0 2.6 319.2254 319.2244 FA Hydroxylinoleic acid C18:2 7.5 319.2615 319.2608 FA Nonadecenoic acid C19:1 5.2 321.2389 321.2400 FA Hydroxyoleic acid C18:1 3.3 333.2774 333.2764 FA Eicosenoic acid C20:1 3.7 337.2342 337.2349 FA Dihydroxyoleic acid C18:1 3.2 351.2521 351.2506 MAG Glycerol palmitoleate 3.2 353.2679 353.2662 MAG Glycerol palmitate 2.3 379.2828 379.2819 MAG Glycerol oleate 1.8 617.5135 617.5115 DAG Glycerol oleate palmitate 11.4 643.5297 643.5272 DAG Glycerol dioleate 8.3 855.7443 855.7412 TAG Glycerol dipalmitate oleate 41.0 881.7609 881.7569 TAG Glycerol dioleate palmitate 44.8 897.7499 897.7518 TAG Glycerol dioleate hydroxy oleate 34.5

A comparison of Table 4.3 with Table 4.4 shows consistency of the assignments. For

example, methyl palmitoleate in Table 4.4 that shows 33% intensity and is consistent

with palmitoleic acid in Table 4.3 which shows 28% relative intensity. The majority

of the FAMEs in Table 4.4 show consistent intensities with corresponding FAs in

Table 4.3.

Figure 4.6 also shows that ESI FTICR-MS is capable of examining whether the

hydrolysis reaction is complete, which in this case demonstrates the fact that the

reaction is partially incomplete as some DAGs and TAGs are observed in the

hydrolysate.


4.5- Positive-ion ESI FTICR-MS of Esterified Processed Macadamia Oil Figure 4.7 shows the positive-ion FTICR mass spectrum of esterified macadamia nut

oil with nine sodiated FA methyl esters, five MAG and three DAG ions assigned to

the peaks in this spectrum that are listed in Table 4.2. The procedure for

esterification is described in section 2.2.3 and is similar to the literature procedures

used for esterification of animal fats and plant oils.[203]

Table 4.4 lists the assignment of the mass spectral peaks in the positive-ion ESI

FTICR mass spectrum of esterified macadamia oil shown in Figure 4.7.

291.2290

319.2599

351.2507

395.2758

617.5111643.5279

200 300 400 500 600 700 800 900 1000 m/z

Figure 4.7- Positive-ion ESI-FTICR mass spectrum of esterified macadamia oil.


Table 4.4- Assignment of the mass spectral peaks (>2%) in the positive-ion ESI-FTICR mass spectrum of esterified macadamia nut oil shown in Figure 4.7.

Observed Mass m/z

Exact Mass m/z

Compound Class Assigned Sodium Adduct Compound Ratio of the

Intensity % 291.2290 291.2295 FAME Methyl palmitoleate- C16:1 32.7 293.2449 293.2451 FAME Methyl palmitate- C16:0 4.8 305.2443 305.2451 FAME Methyl heptadecenoate- C17:1 5.5 307.2606 307.2608 FAME Methyl heptadecanoate- C17:0 2.6 317.2450 317.2451 FAME Methyl linoleate- C18:2 10.8 319.2599 319.2608 FAME Methyl oleate- C18:1 100 333.2391 333.2400 FAME Methyl hydroxylinoleate- C18:2 7.2 337.2348 337.2349 FAME Methyl dihydroxyheptadecenoate 5.6 347.2922 347.2920 FAME Methyl eicosenoate C20:1 5.8 351.2507 351.2506 MAG Glycerol palmitoleate 9.0 367.2456 367.2455 MAG Glycerol hydroxypalmitoleate 2.3 379.2797 379.2819 MAG Glycerol oleate 19.5 393.2593 393.2611 MAG Glycerol hydroxylinoleate 6.2 395.2758 395.2768 MAG Glycerol hydroxyoleate 20.8 615.4940 615.4959 DAG Glycerol oleate palmitoleate 2.3 617.5111 617.5115 DAG Glycerol oleate palmitate 2.4 643.5279 643.5272 DAG Glycerol dioleate 4.1

As a result of the esterification process, most of the DAG and all of the TAG

molecules in the nut oil are converted to their corresponding methyl esters. The

esterification reaction appears incomplete because small amounts (<2%) of sodiated

MAG and DAG ions can be assigned to peaks in Figure 4.7 (e.g., glycerol oleate m/z

379.2797 and glycerol-dioleate m/z 643.5279).

Figure 4.8 shows an expanded FA region of the FTICR mass spectrum of esterified

neat macadamia oil.

The assignment of the base peak in Figure 4.8 at m/z 319.2599 to the sodiated

methyl-oleate adduct confirms the high percentage of oleic acid component of the

DAGs and the TAGs in macadamia nut oil.[196]


277.1132

291.2290

297.2783 305.2443

319.2599

333.2391351.2507

365.2385

379.2797 395.2758

280 300 320 340 360 380 400 m/z

Figure 4.8- Expanded FA and FAME region of the positive-ion ESI-FTICR mass spectrum of esterified macadamia nut oil shown in Figure 4.7.

Other methyl ester ions assigned in this spectrum are eicosenoic (m/z 347.2922),

heptadecanoic (m/z 307.2606), heptadecenoic (m/z 305.2443), linoleic (m/z 317.2450),

palmitic (m/z 293.2449) and palmitoleic (m/z 291.2290) methyl esters. Myristic (m/z

265.2138) and stearic (m/z 321.2764) acid methyl esters were detected in trace level

(<1%) in the esterified macadamia oil. Arachidonic (m/z 341.2451) and lauric (m/z

237.1825) acid methyl esters observed by previous workers in small amounts (<3%)

are also detected at trace levels (<1%) in this experiment.

The relative concentrations of methyl oleate to methyl palmitoleate in the esterified

macadamia nut oil are measured 62:20 using FTICR mass spectrometry, and in the

GC-MS experiments (Chapter 6) the ratio is found as 58:22, whereas in the literature

this ratio is reported as 65:18 using GC-MS technique.[196] Given that the oils used in


these experiments were not identical, the results show a good consistency to within a

~10% interval.

As is expected from the assignments listed in Table 4.1, several odd-numbered

FAMEs (C17:0, C17:1 and C15:1) and one FAME containing an extra oxygen-

bearing functional group in the alkyl chain (methyl hydroxylinoleate, m/z 333.2391)

are assigned and listed in Table 4.4. The presence of hydroxy and methoxy

derivatives of FAs in oils has been reported in the literature previously[177,204-207] and

since any peroxy-, or epoxy-acid components present in the original nut oil would

have been destroyed by the esterification step, we have assigned this latter ion to the

hydroxylinoleate. No further work is undertaken in this thesis to identify the

chemical structure of the ions listed in Table 4.4 as this work was beyond the scope

of the present study.

The results of Table 4.4 which show the FA components of the esterified neat oil,

best correlate with the literature GC-MS results of Cavaletto[196] because they should

contain all the methyl esters produced from the esterification of the free FAs, MAGs,

DAGs and TAGs in the nut oil. In general the literature and present results correlate

well with only relatively small differences observed for the relative concentrations of

the various FA profiles. The principal components are oleic acid at 62% (cf.

65%[196]) and palmitoleic at 20% (cf. 18%[196]). Arachidonic and myristic acids are

observed by Cavaletto but they were observed at trace levels (<1%) in present work.

Eicosenoic and stearic acids were not detected in our GC-MS experiments but were

observed in the ESI FTICR-MS experiments as in the GC-MS experiments by

Cavaletto.


High resolution of FTICR mass spectrometry allows us to resolve peaks with very

close masses. As an example, Figure 4.9 illustrates four peaks in the m/z 279.12 to

m/z 279.23 region resolved in the FTICR mass spectrum of esterified macadamia oil.

279.1924

279.1205

279.1591

279.2294

C14H24O4Na+

C13H20O5Na+

C15H28O3Na+

C16H32O2Na+

279.08 279.10 279.12 279.14 279.16 279.18 279.20 279.22 279.24 279.26 m/z

Figure 4.9- Positive-ion FTICR mass spectrum of esterified macadamia oil in m/z 279.12 to m/z 279.23 region. Peaks 0.03 Da apart are resolved.

Table 4.4 includes a number of compounds that are observed in ESI FTICR mass

spectrum of esterified macadamia oil but are not observed in the GC-MS analysis of

the esterified macadamia oil sample including MAG molecules such as glycerol

palmitoleate and glycerol oleate and DAG molecules such as glycerol oleate

palmitate and glycerol dioleate.

In addition, two hydroxy FAMEs and three hydroxy MAGs are assigned in Table 4.4

including methyl hydroxylinoleate, methyl dihydroxyheptadecenoate, glycerol

hydroxypalmitoleate, glycerol hydroxylinoleate and glycerol hydroxyoleate. Neither


of these compounds are observed in our GC-MS analysis of macadamia oil samples

or are reported in the published GC-MS studies on plant oils.

4.6- Positive-ion ESI FTICR-MS of Esterified Methanol Extract of Processed Macadamia Oil

Figure 4.10 shows the positive-ion FTICR mass spectrum of the esterified methanol

extract of the macadamia nut oil with twelve sodiated FA methyl esters and two

mono-acylglycerol ions assigned to peaks in this spectrum listed in Table 4.5. The

procedure for esterification[67] is described in Section 2.2.3. Performing the

esterification reaction on methanol extract of the oil is to compare the product

analysis using both GC-MS and FTICR-MS.

255.1568

291.2295

319.2597

337.2729

395.2756

445.3122

471.2924504.3099

532.3405

587.5014615.5254

250 300 350 400 450 500 550 600 650 m/z

Figure 4.10- Positive-ion FTICR mass spectrum of esterified methanol extract of macadamia nut oil.


Table 4.5. Assignment of the mass spectral peaks (>1%) in the positive-ion ESI-FTICR mass spectrum of the esterified methanol extract of processed macadamia oil shown in Fig. 4.10.

Observed Mass m/z

Exact Mass m/z

Compound Class Assigned Sodium Adduct Compound*

Normalized Intensity

% 291.2295 291.2295 FAME Methyl palmitoleate- C16:1 23.7 293.2448 293.2451 FAME Methyl palmitate- C16:0 2.2 305.2447 305.2451 FAME Methyl heptadecenoate- C17:1 1.4 307.2610 307.2608 FAME Methyl heptadecanoate- C17:0 1.0 315.2288 315.2294 FAME Methyl linolenate- C18:3 4.8 317.2448 317.2451 FAME Methyl linoleate- C18:2 22.1 319.2597 319.2608 FAME Methyl oleate- C18:1 100.0 321.2770 321.2764 FAME Methyl stearate- C18:0 1.9 335.2558 335.2557 FAME Methyl hydroxyoleate- C18:1 6.6 337.2729 337.2713 FAME Methyl hydroxy stearate- C18:0 11.4 347.2922 347.2920 FAME Methyl eicosenoate- C20:1 3.7 363.2504 363.2505 FAME## Methyl dihydroxy nonadecdienoate- C19:2 3.6 381.2608 381.2611 MAG Glycerol hydroxy heptadecenoate 9.3 395.2756 395.2768 MAG Glycerol hydroxyoleate 75.5 445.3122 445.3136 MAG Glycerol dihydroxy stearate + methanol 55.5

* The hydroxy-compounds alternately may be assigned to peroxy substituents. See text for further discussion. ## Could be MAG, glycerol heptadecdienoate.

Table 4.5 lists the assignments of the mass spectral peaks in the FTICR mass

spectrum of esterified methanol extract of macadamia oil.

Twelve FAME sodium adducts are assigned to the peaks in the esterified methanol

extract of macadamia oil spectrum in Table 4.5. Comparison of Table 4.5 (esterified

methanol extract of macadamia oil) and Table 4.2 (methanol extract of macadamia

oil) shows a higher relative intensity of oleic FAME in Table 4.5. This is consistent

with observing an intense peak for glycerol dioleate in Table 4.2 that will release

more oleate FAME during esterification process, resulting in an increase in the

relative intensity of methyl oleate in Table 4.5.


Other FA sodium adducts assigned in the methanol extract of the oil in Table 4.2

such as heptadecenoic acid and eicosenoic acid also appear in the assignments of the

esterified methanol extract of the oil as FAME sodium adducts in Table 4.5.

4.7- A Comparison of the Fatty Acids Observed in the Positive-ion ESI FTICR Mass Spectra of the Neat, Methanol Extract, Hydrolysed, Esterified and Esterified Methanol Extract of Processed Macadamia oil

In this section, a summary of the occurrence of the common fatty acids (i.e.

unsubstituted), either as free fatty acids or as substituents on the acylglycerols, in the

processed macadamia oil is discussed.

Figure 4.11 shows a comparison of the fatty acid regions (m/z 175-450) positive-ion

ESI FTICR mass spectra of the (a) neat, (b) methanol extract, (c) hydrolysed, and (d)

esterified processed macadamia oil.

Figure 4.12 shows a graphical comparison of the unsubstituted FA or FA derivative

adducts assigned for the methanol extract (Table 4.2), the hydrolysed (Table 4.3), the

esterified (Table 4.4) and the esterified methanol extract (Table 4.5) of the processed

macadamia nut oil. For this figure, the FAs that show evidence of hydration, e.g.

NaC18H35O3+ are not included. Unlike the intensity values in the tables in this

chapter, the peak intensities in Figure 4.12 have been normalised to 100% for the FA

acid sodium adducts displayed. Table 4.6 lists the percentages of the fatty acid

components used in Figure 4.12.


As discussed in Section 4.2, there are no (or trace levels only) unsubstituted free FA

adducts observed in the positive-ion ESI FTICR mass spectrum of the neat processed

macadamia oil. Free FAs are indeed present in the neat oil according to the negative-

ion results presented next in Chapter 5, but these are not observed as adducts in

Figure 4.11(a) presumably because of charge suppression of the minor components

by the large number of TAG ions observed in Figure 4.1. Also, when these

experiments were undertaken no attempt was made to dope the sample with

additional sodium cations. Since the oil is made up of >98% TAGs, it is also possible

the free FAs do not compete effectively with the larger number of TAG molecules

for the adventitious sodium cations available present in the oil to form the adduct

ions.


181.7591

265.2526

319.2249339.2910

353.2617

393.2609

411.2706

m/z

277.2114

305.2418

319.2588351.2479

379.2776

395.2569413.2616

m/z

231.1143

277.2140

305.2453

319.2217 351.2521 413.2671

m/z

277.1132

291.2290

319.2599

351.2507379.2797

395.2758

413.2674175 200 225 250 275 300 325 350 375 400 425 m/z

(a)

(b)

(c)

(d)

Figure 4.11- A comparison of the ESI FTICR mass spectra of the FA region of (a) neat macadamia oil, (b) methanol extract of macadamia oil, (c) hydrolysed macadamia oil and (d) esterified macadamia oil.


C15:1

C16:0

C16:1

C17:0

C17:1

C18:0

C18:1

C18:2

C18:3

C19:1

C20:0

C20:1

C20:4

0

5

10

15

20

25

30

35

40

45

50

55

60

65

A Comparison of the Fatty Acid Components Observed in Positive-ion ESI-FTICR Mass Spectrometry Experiments of Processed Macadamia Oil

Peak

Rel

ativ

e In

tens

ity %

Fatty Acid

+ESI-MS Extract+ESI-MS Esterified Oil+ESI-MS Esterified Extract+ESI-MS Hydrolysed Oil

C15:1

C16:0

C16:1

C17:0

C17:1

C18:0

C18:1

C18:2

C18:3

C19:1

C20:0

C20:1

C20:4

0

5

10

15

20

25

30

35

40

45

50

55

60

65

C15:1

C16:0

C16:1

C17:0

C17:1

C18:0

C18:1

C18:2

C18:3

C19:1

C20:0

C20:1

C20:4

0

5

10

15

20

25

30

35

40

45

50

55

60

65

A Comparison of the Fatty Acid Components Observed in Positive-ion ESI-FTICR Mass Spectrometry Experiments of Processed Macadamia Oil

Peak

Rel

ativ

e In

tens

ity %

Fatty Acid



Figure 4.12- A graphical comparison of the unsubstituted FA anions observed in the positive-ion FTICR mass spectra of the neat (Table 4.1), the methanol extract (Table 4.2), hydrolysed (Table 4.3) and the esterified (Table 4.4) processed macadamia nut oil.


Table 4.6- A comparison of the fatty acid components observed in the positive-ion ESI-FT-ICR mass spectrometry experiments.

Fatty Acid Component

ESI FTICR-MS Neat oil

Positive-ion

ESI FTICR-MS Methanol Extract

Positive-ion

ESI FTICR-MS Esterified oil Positive-ion

ESI FTICR-MS Esterified Extract

Positive-ion

ESI FTICR-MS Hydrolysed Oil

Positive-ion

Literature GC-MS

Fatty Acids/ % C14:0 -* - - - - - C14:1 - - - - - - C15:0 - - - - - - C15:1 - - - - - - C16:0 - 12.2 3.0 1.4 5.2 7.4 C16:1 - 24.1 20.2 14.7 17.2 18.4 C17:0 - 2.2 1.6 0.6 - - C17:1 - - 3.4 0.9 - - C18:0 - 3.3 - 1.2 1.6 2.8 C18:1 - 39.5 61.7 62.2 61.1 64.9 C18:2 - 5.9 6.7 13.7 7.9 1.5 C18:3 - - - 3.0 1.6 - C19:1 - 8.7 - - 3.2 - C20:0 - - - - - - C20:1 - 4.2 3.6 2.3 2.3 2.3 C20:4 - - - - - 1.9

* Less than the limit of detection. Only fatty acid components are used in calculations and comparisons; fatty acids containing additional oxygen atoms are not listed. Peak intensities % is used for FTICR-MS and peak area % is used for literature Cavaletto[196] GC-MS results.


Figure 4.11(b), the positive-ion spectrum of the methanol extract of the oil, shows a

dramatic improvement in S/N ratio over that of the neat oil for the free FAs as a

consequence of the removal of the majority of the TAG molecules in the methanol

extraction process. This extraction effectively concentrates the minor component free

FAs in the sample and effectively removes the ion suppression created by the larger

number of TAG ions.

The ratio of the relative intensities of the peaks assigned to palmitoleic/oleic acids

(m/z 277.2114 and 305.2418) in the methanol extract of the oil is approximately

0.6:1.0, while this ratio in the hydrolysed oil and esterified oil spectra, 4.11(b) and

4.11(c) respectively, appears to be approximately 0.25:1.0. The major source of free

FAs in hydrolysate and FAMEs in esterified oil samples are the TAG molecules and

to a lesser extent the MAGs and DAGs. These FAs originating from the

acylglycerides are commonly what are quoted and indeed measured in the more

standard GC-MS analyses of plant oils as the fatty acid composition of the oil.

However, in the case of the spectrum 4.11(b), the methanol extract of the oil, the

source of the free FAs are the biological reactions in the macadamia nuts and any

remaining active enzymes in the oil after processing, the effect of atmospheric

oxygen and auto-oxidation as well as the various oil temperatures used in the

processing and indeed the processing procedure itself. All of these factors may result

in the difference between the free FA ratio and the FA ratio measured using more

conventional method for the oil. It is the presence in the macadamia oil of these

“free” fatty acids, as distinct from the “bound” fatty acids in the acylglycerides that

contributes to the overall acidity and in some cases “character” of the oil.


Figure 4.12 and Table 4.6 summarizes several interesting results from the positive-

ion ESI FTICR MS experiments discussed previously in this chapter. If one can

directly correlate the cation concentrations with those of the acids in the oil then it is

apparent that the C18:1 (oleic) and C16:1 (palmitoleic) with a smaller amount of

C18:2 (linoleic) acids are the most common acids both in the acylglycerides at

around 62%, 17% and 9% respectively. The free FA concentrations in the methanol

extract show a similar trend. In the latter case though there are increased

concentrations of the C19:1 (9%) and C16:0 (palmitic, 12%) observed. Palmitic acid

is observed in the hydrolysed and esterified oils but at a much lower concentration of

around 3%.

Interestingly we might expect that the FA concentrations deduced from the methanol

extract and the esterified methanol extract of the oil to show a similar FA profile

since the esterification reaction should essentially transform all of the FAs in the oil

extract into FA esters. The observation that they are indeed different and that the

latter more closely follows the trend of the hydrolysed neat oil and the esterified neat

oil indicates that derivatisation of the oil may lead to changes in the perceived oil

profile. The changes could occur because of the acylglycerides that have been

hydrated or the ones soluble in methanol thus being extracted along with the free

fatty acids and then undergoing hydrolysis in the transesterification reaction. It may

also to a lesser extent reflect the different sodium affinities of an ester versus a fatty

acid in the ESI spray process. The presence of DAGs and to lesser extent TAGs in

Figure 4.2 supports the former hypothesis.

Figure 4.12 highlights the presence of the other minor fatty acids present in the oil

such as the C20:1 and C20:4 as well as some of the odd numbered fatty acids C17:0


and C17:1. It is unexpected that the C18:3 (linolenic acid) is not observed in the

methanol extract and the esterified oil but is detected in the esterified extract and the

hydrolysed oil.

One interesting result which will be discussed again in Chapter 6 is the interesting

correlation between the observed profile of the major FA components in the oil and

that observed previously in GC-MS experiment by Cavaletto in 1980.[196] In their

results, they report concentrations for the major FA components in macadamia oil

which closely matches our results for the analysis of the acylglyceride FAs in our

processed macadamia oil. We observe 62%: 17%: 3% for the FAs C18:1, C16:1:

C16:0 acids compared to Cavaletto’s result of 65%: 18%: 7% respectively.

In brief, Figure 4.11 demonstrates that:

- Free FAs in the neat oil are not observed due to their low relative concentration

and high cloud effect of TAG positive ions in the FTICR cell that causes the ion-

suppression of the minor peaks of free FAs. To minimize ion suppression and to

improve the signal to noise ratio of minor constituents of the oil, solvent

extraction and chromatographic separations are carried out. Spectrum (b) in

Figure 4.11 shows a dramatic improvement in signal intensity of free FAs in the

methanol extract of the oil due to the removal of the majority of the TAG

molecules in the extraction process.

- The ratio of the relative intensities of the peaks assigned to palmitoleic/oleic

acids (m/z 277.2114 and 305.2418) in the methanol extract of the oil is

approximately 0.6:1.0, while this ratio in the hydrolysed oil and esterified oil

spectra, (b) and (c) respectively, appears to be approximately 0.25:1.0. The


major source of free FAs in the hydrolysed oil and FAMEs in esterified oil

are TAG molecules that comprise about 98% of the macadamia oil. However,

in the case of the spectrum (b) of the methanol extract of the oil, the source of

free FAs is the biological reactions, effect of atmospheric oxygen, light and

ambient temperature which result in a different ratio of relative intensities of

the assigned peaks.

- The signal to noise ratio of the peaks in the FA region of spectrum (a) is

significantly lower than that of the other three spectra in Figure 4.11. In

addition, peaks assigned to two major FAs oleic and palmitoleic acids are

observed in the methanol extract spectrum (b) but they are not observed in the

neat oil spectrum (a) due to the ion suppression effect of the TAG ions in the

FTICR cell. As a result, the observed peaks in the FA region of the neat oil

spectrum (a) basically arise from the background peaks due to the high signal

amplification applied by the Bruker software in FA region of spectrum (a).


Chapter 5

5. Negative-ion ESI FTICR-MS of Processed Macadamia Oil


5.1- Introduction

In this chapter the results of the negative-ion ESI FTICR mass spectrometry

experiments on the processed macadamia nut oil samples are presented and

discussed. In particular the neat processed macadamia nut oil, the methanol extract of

the oil and hydrolysed processed macadamia nut oil are examined using this

technique.

In the negative-ion electrospray ionisation of the macadamia nut oil samples, the

singly-charged negative ions are formed by the deprotonation of acidic species such

as fatty acids and alcohols by transferring a proton to the solvent molecules; in this

case, methanol. MAGs and DAGs generate negative ions in the ESI source by

deprotonation of the unsubstituted hydroxyl group on glycerol.

Unsubstituted TAG molecules do not produce negative ions in ESI mass

spectrometry due to the fact that they do not possess an ionisable proton. However,

numerous peaks are observed in the TAG region of the negative-ion FTICR mass

spectra of the neat macadamia oil samples and these peaks are assigned to TAG

molecules containing additional oxygen bearing functional groups such as hydroxyl

groups or hydroperoxy group on the acyl substituents. These ions are observed

because they are produced from substituted TAG molecules that contain a detachable

proton.


5.2- Negative-ion ESI FTICR Mass Spectra of Neat Processed Macadamia Oil

5.2.1- Introduction

In this section the results of negative-ion FTICR-MS analysis of neat processed

macadamia nut oil are presented and discussed. Peaks are assigned in three regions

of the spectra including free fatty acids and MAGs region (m/z 150-400), DAGs

region (m/z 500-750) and TAGs region (m/z 800-1000).

Figure 5.1 shows negative-ion ESI FTICR mass spectrum of the neat processed

macadamia nut oil. In this chapter only the peaks with intensities greater than 2% of

the base peak in each FTICR spectrum are discussed, unless otherwise stated.

The spectra presented in this chapter are also contained in digital form in the attached

DVD Appendix.

More than 120 peaks are distinguished in three mass regions in the FTICR mass

spectrum of neat macadamia oil in Figure 5.1. The region m/z 150-400 contains

peaks associated with anions of free FAs and MAGs. The region m/z 500-750

contains peaks assigned to the anions of FA dimers, DAGs and also DAGs with

additional oxygen bearing functional groups. The region m/z 800-1000 contains

peaks assigned to the anions of TAGs with additional oxygen bearing functional

groups such as hydroxyl functional group.


157.1232

187.0977

227.2016

255.2327

281.2486

311.1687

537.4880 627.4762655.5031

682.5263 875.7683901.7849

931.7126

200 300 400 500 600 700 800 900 1000m/z

847.7396

212.0753

339.1999

325.1841

Figure 5.1- Negative-ion ESI FTICR mass spectrum of neat processed macadamia nut oil.

5.2.2- Free Fatty Acids and Monoacylglycerol Region, m/z 150-400

Figure 5.2 shows an expanded fatty acid region (m/z 150-400) of the negative-ion

ESI FTICR mass spectrum of neat processed macadamia oil shown in Figure 5.1.

Table 5.1 lists the assignment of the associated mass spectral peaks in Figure 5.2.

The majority of the anions produced from the neat processed macadamia oil in this

region are associated with FAs and dicarboxylic fatty acids. The base peak in Figure

5.2 is at m/z 255.2327 and is assigned to C16H31O2¯ which is expected to be the

palmitate anion, C16:0 (<1 ppm). Another nearby peak at m/z 253.2173 (45%) is

FA Region

DAG Region TAG Region


assigned to C16H29O2¯ which is expected to be the palmitoleate anion, C16:1 (<0.1

ppm).

157.1232

173.0819187.0977

199.1702

227.2016241.2172

255.2327

269.2485

281.2486

297.1532

311.1687325.1841

339.1999

150 175 200 225 250 275 300 325 350 375 400m/z

212.0753

Figure 5.2- Fatty acid region of the negative-ion ESI FTICR mass spectrum of neat processed macadamia nut oil in Figure 5.1, m/z 150-400.

A related series of C18 anions is observed at m/z 283.2642 (17%), 281.2486 (74%)

and 279.2329 (6%). These are assigned to C18H35O2¯ (0.1 ppm), C18H33O2¯ (0.2

ppm) and C18H31O2¯ (<0.1 ppm) respectively. Based on the GC and HPLC results in

Chapters 6 and 7 respectively, we expect these anions to be stearate (C18:0), oleate

(C18:1) and linoleate (C18:2) respectively.

The peaks at nominal masses m/z 311, 325 and 339 might be considered to arise

from the next members of the homologous series C16 : C18 : C20 : C22 : C24,

however high resolution mass spectrometry throws some doubt on this assignment.

C20H39O2¯ has a mass of m/z 311.2956 that is 406 ppm above the observed mass at

m/z 311.1687. Similarly C21H41O2¯ has a mass of m/z 325.3112 that is 399 ppm


above the observed mass at 325.1841 and C22H43O2¯ has a mass of m/z 339.3269

which is 373 ppm above the observed mass at 339.1999. The existence of sulphur in

plant oils is reported by Wu et al. using the FTICR-MS technique,[106] therefore, we

assign these peaks to C17H27O3S¯ (<0.1 ppm), C18H29O3S¯ (0.7 ppm) and

C19H31O2S¯ (0.2 ppm) respectively.

Table 5.1- Assignment of the mass spectral peaks (>2%) in the expanded fatty acid region (m/z 150-400) of the negative-ion ESI FTICR mass spectrum of processed macadamia nut oil shown in Figure 5.2.

Observed Mass m/z

Exact Mass m/z

Compound Class Assigned Anion Normalized

% 157.1232 157.1234 FA C9H17O2‾, C9:0 9.5 171.1388 171.1391 FA C10H19O2‾, C10:0 2.3 173.0819 173.0819 Di-FA* C8H13O4‾ 2.5 187.0977 187.0976 Di-FA* C9H15O4‾ 4.8 199.1702 199.1704 FA C12H23O2‾, C12:0 2.9 212.0753 Unknown - - 31.3 213.1860 213.1860 FA C13H25O2‾, C13:0 2.0 225.1858 225.1860 FA C14H25O2‾, C14:1 2.9 227.2016 227.2017 FA C14H27O2‾, C14:0 16.4 239.2018 239.2017 FA C15H27O2‾, C15:1 2.9 241.2172 241.2173 FA C15H29O2‾, C15:0 13.1 253.2173 253.2173 FA C16H29O2‾, C16:1 45.4 255.2327 255.2330 FA C16H31O2‾, C16:0 100.0 267.2329 267.2330 FA C17H31O2‾, C17:1 4.0 269.2125 269.2122 FA C16H29O3‾ 2.3 269.2485 269.2486 FA C17H33O2‾, C17:0 4.7 279.2329 279.2330 FA C18H31O2‾, C18:2 6.0 281.2486 281.2486 FA C18H33O2‾, C18:1 74.3 283.2642 283.2643 FA C18H35O2‾, C18:0 17.4 295.2277 295.2279 FA C18H31O3‾ 4.3 297.2431 297.2435 FA C18H33O3‾ 8.0 311.1687 311.1686 S-FA# C17H27O3S‾ 36.3 313.2388 313.2384 FA C18H33O4‾ 3.1 323.1680 323.1711 FA C14H27O8‾ 3.3 325.1841 325.1843 S-FA# C18H29O3S‾ 32.7 337.1836 337.1868 FA C15H29O8‾ 2.2 339.1999 339.1999 S-FA# C19H31O3S‾ 26.6

* Di-FA: Dicarboxylic fatty acid # S-FA: Sulphur containing fatty acid

Table 5.1 indicates that peaks observed at m/z 227.2016, 241.2172, 267.2329 and

269.2485 in Figure 5.2 are assigned to C14H27O2¯ (0.3 ppm), C15H29O2¯ (0.6 ppm),


C17H31O2¯ (0.1 ppm) and C17H33O2¯ (0.5 ppm) respectively. These fatty acids are not

observed in positive-ion FTICR mass spectrum of macadamia oil in Figure 4.1.

Dicarboxylic acids can form by oxidative cleavage of the double bonds of olefinic

hydrocarbons.[208] Two dicarboxylic acids are assigned in Table 5.1. The peak at m/z

173.0819 is assigned to C8H13O4¯ (<0.1 ppm) and the peak at m/z 187.0976 is

assigned to C9H15O4¯ (0.7 ppm).

Dicarboxylic acid species might be expected to form dianions (such as ¯OOC–R–

COO¯) because of the possible loss of two acidic protons from the two carboxylic

acid groups. However, no peaks were observed in Figure 5.2 that could be assigned

to the double-charge anions of dicarboxylic acids.

5.2.3- Diacylglycerol Region, m/z 500-750

Figure 5.3 shows an expanded view of m/z 500-750 region of the negative-ion

FTICR mass spectrum of neat processed macadamia oil in Figure 5.1. Table 5.2 lists

the assignment of the associated mass spectral peaks observed in Figure 5.3.


537.4880

563.5040

627.4762

655.5031

682.5263

500 525 550 575 600 625 650 675 700 725 750m/z

511.4740

Figure 5.3- DAG region of the negative-ion ESI FTICR mass spectrum of neat processed macadamia nut oil in Figure 5.1, m/z 500-750.

Table 5.2- Assignment of the mass spectral peaks (>0.2% of the base peak in Figure 5.1) in the expanded DAG region (m/z 500-750) of the negative-ion ESI FTICR mass spectrum of processed macadamia oil shown in Figure 5.3.

Observed Mass m/z

Exact Mass m/z

Compound Class Assigned Anion Normalized#

% 509.4566 509.4575 FA dimer C32H61O4‾ 1.8 511.4740 511.4732 FA dimer C32H63O4‾ 1.9 535.4734 535.4732 FA dimer C34H63O4‾ 1.9 537.4880 537.4888 FA dimer C34H65O4‾ 3.7 563.5040 563.5045 FA dimer C36H67O4‾ 2.9 565.5201 565.5201 FA dimer C36H69O4‾ 1.5 627.4762 627.4630 DAG C39H63O6‾ 3.6 629.4889 629.4787 DAG C39H65O6‾ 1.8 637.5016 637.4837 DAG C41H65O5‾ 1.4 655.5031 655.4943 DAG C41H67O6‾ 6.1 657.5103 657.5100 DAG C41H69O6‾ 2.1 665.5378 665.5362 DAG C40H73O7‾ 2.0 682.5263 682.5263 DAG C39H72O8N‾ 7.7 699.4532 699.4630 DAG C45H63O6‾ 0.2 701.4524 701.4787 DAG C45H65O6‾ 0.2

# Peak intensities normalized vs. base peak (palmitate anion, C16H31O2¯) in Figure 5.1.


5.2.3.1- Free Fatty Acid Dimers

Spectral complications in the ESI mass spectrometry analysis of mixtures of polar

compounds often arise from the formation of dimer ions (clusters) in the electrospray

process. During the spray process of the macadamia oil solutions that contain free

fatty acids, two fatty acids molecules can form a dimer in the gas phase via hydrogen

bonding around the hydrogen atom of one of the fatty acid molecules. In positive-ion

mode, fatty acid dimers with the Na+ attached are observed. In negative-ion mode,

[FA2-H+]¯ dimers are observed. These anions could form from loss of a proton from

one of the fatty acids in the FA2 dimer molecule. Alternatively, the anion could form

from the association of a [FA-H+]¯ with a FA.

In Table 5.2 six such clusters are assigned. The peak at m/z 509.4566 is assigned to

C32H61O4¯ (1.8 ppm), the peak at 511.4740 is assigned to C32H63O4¯ (1.6 ppm), the

peak at 535.4734 is assigned to C34H63O4¯ (0.4 ppm), the peak at 537.4880 is

assigned to C34H65O4¯ (1.6 ppm), the peak at m/z 563.5040 is assigned to C36H67O4¯

(0.9 ppm) and the peak at 565.5201 is assigned to C36H69O4¯ (4.8 ppm).

The peak observed at m/z 509.4566 in Figure 5.3 can be assigned to the anion cluster

formed by the combination of palmitoleic acid (C16H30O2) and palmitate anion

(C16H31O2¯) to give C32H61O4¯. Alternatively, a cluster anion of palmitic acid

(C16H32O2) and palmitoleate anion (C16H29O2¯) will produce the same anion.

The peak observed at m/z 511.4740 in Figure 5.3 can be assigned to the cluster anion

of palmitic acid (C16H32O2) and palmitate anion (C16H31O2¯) to give C32H63O4¯.


The peak observed at 535.4734 can be assigned to the anion cluster of oleic acid

(C18H34O2) and palmitoleate anion (C16H29O2¯) to give C34H63O4¯. Alternatively, the

combination of oleate anion (C18H33O2¯) and palmitoleic acid (C16H30O2) will

produce the same anion.

To assign chemical structures to these ions would require tandem mass spectrometry,

i.e. CID studies; however, we observe in Table 5.2 that the two most abundant free

fatty acids in the processed macadamia oil are palmitic (C16H32O2) and oleic

(C18H34O2) acids. An anion cluster formed from these two acids (C34H65O4¯) would

result in the peak observed at m/z 537.4880.

Figure 5.4 shows a comparison of the (a) experimental and (b) simulated mass

spectra of the C34H65O4¯ anion. The simulated spectrum was generated using the

Bruker software. The peak at m/z 537.4880 is chosen due to the fact that it shows the

highest intensity among the dimer peaks assigned in Table 5.2.

537.4880

537.4888

538.4923

539.4954

537 538 539 540 541 542 m/z

(a)

(b)

538.4926 539.4963

539.5066

540.5089

537.4880

537.4888

538.4923

539.4954

537 538 539 540 541 542 m/z

(a)

(b)

538.4926 539.4963

539.5066

540.5089

Figure 5.4- A comparison of the experimental (a) and simulated (b) isotopic distribution of the C34H65O4¯ fatty acid dimer anion.


The base peak at m/z 537.4880 is assigned to the 12C341H65

16O4¯ isotopic anion (1.4

ppm). The adjacent peak at m/z 538.4926 is assigned to the 13C112C33

1H6516O4¯

isotope (0.6 ppm). The third isotope peak at m/z 539.4963 is assigned to the

13C212C32

1H6516O4¯ isotope (1.6 ppm). It is interesting to note that whilst the peak at

m/z 537.4880 and 538.4926 show a good intensity correlation (1:0.4), the

experimental intensity of the peak at nominal mass m/z 539 is unusually high. A high

resolution inspection of this peak (not shown here) shows that the peak at nominal

mass m/z 539 is composed of two partially overlaying peaks. A smaller peak at m/z

539.4963 that is assigned to 13C312C31

1H6516O4¯ isotope (1.7 ppm) and a larger peak

at m/z 539.5066 that could be assigned to fatty acid dimer 12C341H67

16O4¯ (3.9 ppm).

The latter peak at m/z 539.5066 (<0.1%) could be assigned to an anion cluster of

palmitic acid (C16H32O2) and stearic acid (C18H36O2) to give C34H67O4¯ by releasing

a proton. The intensity of this latter peak is less than 1% of the base peak and is not

reported in Table 5.2.

The excellent isotope pattern match of the experimental peaks and the simulated

peaks in Figure 5.4 strengthens the assignments.

Similarly, the peak observed at m/z 563.5040 in Figure 5.3 can be assigned to the

anion cluster formed by the combination of oleic acid (C18H34O2) and oleate anion

(C18H33O2¯) to give C36H67O4¯.

The peak observed at m/z 565.5201 in Figure 5.3 can be assigned to the anion cluster

formed by the combination of oleic acid and stearic acid less a proton to produce

C36H69O4¯.


5.2.3.2- Diacylglycerols

The peak observed at m/z 627.4762 in Figure 5.3 is assigned to the DAG anion

(C39H63O6¯) containing additional oxygenated functional group. As the glycerol

group contains three carbon atoms, the remaining 36 carbon atoms could be of two

oleic acyl groups (C18:1). Other combinations of fatty acids can also be suggested

that will add up to 36 carbon atoms such as C20:1 with C16:1. As the most common

fatty acid in macadamia oil TAGs is known to be oleic acid, two C18:1 is the most

sensible assignment for fatty acids of this DAG anion. The location of the two oleic

acyl substituents on the glycerol chain could be on carbons 1 and 2 or on carbons 1

and 3. Without further tandem MS studies it is impossible to assign a conclusive

structure to this anion.

Similarly, DAGs containing additional oxygenated functional groups are observed at

m/z 629.4889, 655.5031, 657.5103, 665.5378, 682.5263, 699.4532 and 701.4524.

These molecules could bear an extra hydroxy group. Hydroxy acids form by

hydration of carbon double bonds. The oxidation of FAs and formation of hydroxy

FAs is discussed in detail by Hamilton et al.[209] and by Frankel.[210]

A related peak at m/z 629.4787 assigned to the DAG C39H65O6¯ (16 ppm) most

likely involves saturation of one of the double bonds of the fatty acid side chain of

the C39H63O6¯ DAG anion.

Similarly, the anion pair assigned to the peaks at m/z 655.5031 and 657.5103 also

differs by double bond saturation. These peaks are assigned to C41H67O6¯ (13 ppm)

and C41H69O6¯ (< 0.5 ppm) respectively.


ESI FTICR mass spectrometry differentiates the ions according to their masses and is

not capable of resolving isomers. Thus, further chromatographic separations,

chemical derivatisation and spectroscopic studies are necessary to identify more

conclusively the oxygen bearing functional groups on these molecules.


Figure 5.5 shows an expanded TAG region (m/z 800-1000) of the negative-ion ESI

FTICR mass spectrum of neat processed macadamia oil shown in Figure 5.1. Figure

5.5 (b) shows an expansion of the region m/z 870-882 of this region. Table 5.3 lists

the assignment of the mass spectral peaks observed in Figure 5.5.

847.7396

875.7683

901.7849

917.6892

931.7126

945.7158959.7272

800 820 840 860 880 900 920 940 960 980 1000m/z

847.7396

875.7683

901.7849

917.6892

931.7126

945.7158959.7272

800 820 840 860 880 900 920 940 960 980 1000m/z Figure 5.5- (a) TAG region of the negative-ion ESI FTICR mass spectrum of the neat processed macadamia nut oil in Figure 5.1, m/z 800-1000, (b) expanded peaks in the TAG region, m/z 870-882.

873.7573

875.7683

870 872 874 876 878 880 882m/z

873.7573

875.7683

870 872 874 876 878 880 882m/z

(b)

(a)


Table 5.3- Assignment of the mass spectral peaks (>2% of the base peak in Figure 5.1) in the expanded TAG region (m/z 800-1000) of the negative-ion ESI FTICR mass spectrum of processed macadamia nut oil shown in Figure 5.5.

Observed Mass m/z

Exact Mass m/z


% 847.7396 847.7396 TAG C53H99O7‾ 5.8 873.7573 873.7553 TAG C55H101O7‾ 6.1 875.7683 875.7709 TAG C55H103O7‾ 9.4 901.7849 901.7866 TAG C57H105O7‾ 6.3 917.6892 917.6876 TAG C58H93O8‾ 2.1 931.7126 931.7244 TAG C56H99O10‾ 4.0 945.7158 945.7036 TAG C53H101O13‾ 2.7 959.7272 959.7193 TAG C57H99O11‾ 2.5

In general, the peaks in TAG region appear less intense compared to the peaks in FA

and DAG regions. The most intense peak in this region is observed at m/z 875.7683

with an intensity of 9.4% of the base peak in Figure 5.1 (palmitate anion).

The peak at m/z 847.7396 is assigned to the TAG anion C53H99O7¯ (<0.1 ppm).

Removal of three carbon atoms for the glycerol backbone of the TAG leaves 50

carbon atoms for the fatty acid substituent groups. Given the abundance of palmitic

and oleic acid in these oils, a reasonable assignment for this anion is one which

contains two palmitic acids (C16:0) and one oleic acid (C18:1) side chain groups,

one of which contains an OH group. The OH group on the acyl chain could have

been generated by hydration reaction (addition of a water molecule) on the double

bond of a C16:1 acyl substituent to produce C16:0 with an OH substituent.

Figure 5.5 (b) shows an expansion of the region m/z 870-880 that contains two of the

most intense peaks in the TAG region of Figure 5.1.

The peak at m/z 873.7573 in Figure 5.5 (b) is assigned to the TAG anion C55H101O7¯

(2.3 ppm). This anion is likely to contain a glycerol substituted by three acyl groups


including two oleates and one palmitate, containing 52 carbon atoms in total (C18:1

+ C18:1 + C16:0) and an extra OH group that bears the negative charge by releasing

a proton.

The peak at m/z 875.7683 in Figure 5.5 (b) is assigned to the TAG anion C55H103O7¯

(2.9 ppm). This anion is likely to contain a glycerol backbone substituted by three

acyl groups including an oleate, a stearate and a palmitate, containing 52 carbon

atoms in total (C18:1 + C18:0 + C16:0) and an extra OH group that bears the

negative charge by releasing a proton.

Similarly, the peak at m/z 901.7849 is assigned to the TAG anion C57H105O7¯ (1.9

ppm). Again, a sensible assignment for the general structure of this TAG anion could

be two oleate and one stearate substituents (C18:1 + C18:1 + C18:0) on the glycerol

backbone and an extra OH group on one of the fatty acid side chain groups bearing

the negative charge.

The peak at m/z 917.6892 is assigned to C58H93O8¯ (1.7 ppm), this could correspond

to the TAG anion with two C18:2 and one C19:5 substituents. There are two hydroxy

groups on the fatty acid side chains.

The peak at m/z 931.7126 is assigned to C56H99O10¯ (13 ppm), the peak at m/z

945.7158 is assigned to C53H101O13¯ (9.5 ppm) and the peak at m/z 959.7272 is

assigned to C57H99O11¯ (8.2 ppm). These highly oxygenated TAG anions could be

clusters formed by the attachment of a number of methanol or glycerol molecules to

a TAG molecule. No further investigation to elucidate the structure of these TAG

anions was performed in this study because of the number of ions involved.


It is noticeable that all of the assigned anions in the TAG region of negative-ion

FTICR mass spectrum in Figure 5.5 contain at least one saturated fatty acid

substituent and one extra hydroxyl group. This observation supports the idea that the

hydroxyl group is produced by hydration reaction on a double bond on the fatty acid

side chain.

These highly oxygenated fatty acids are observed in negative-ion mode due to the

fact that they are likely to bear a hydroxyl group on the side chains that can release a

proton to generate a negative charge on the fatty acid chain. These compounds are

not observed in the GC-MS analysis of these samples. In GC-MS analysis of these

samples it is unlikely that such TAGs would have been identified due to the fact that

the samples are esterified prior to the GC-MS analysis that can dramatically change

the structure of these highly oxygenated compounds.

5.3- Negative-ion ESI FTICR Mass Spectra of the Methanol Extract of Processed Macadamia Oil

5.3.1- Introduction In this section the results of the negative-ion FTICR-MS analysis of the methanol

extract of processed macadamia nut oil are presented and discussed. Peaks are

associated and discussed in three regions including free fatty acids region (m/z 150-


400), DAGs and fatty acid dimers region (m/z 500-750) and TAGs region (m/z 800-

1000).

Figure 5.6 shows negative-ion ESI FTICR mass spectrum of the methanol extract of

processed macadamia nut oil, m/z 120-980, and selected peaks are assigned in Table

5.4. In this section only peaks with intensities >2% of the base peak are tabulated and

assigned.

As an overview, three mass regions are observed in Figure 5.6. The region m/z 150-

400 contains peaks associated with anions of free FAs. The region m/z 500-750

contains peaks assigned to charged FA dimers, DAGs and also DAGs with additional

oxygen bearing functional groups. The region m/z 800-1000 contains peaks assigned

to TAGs with additional oxygen bearing functional groups. Only selected peaks in

each mass region are assigned in Table 5.4 as a summary of the assignment of all

three regions. More comprehensive tables can be found in sections 5.3.2 to 5.3.4.

The concentration of TAGs is very low in the methanol extract of the oil due to the

fact that TAGs have a low solubility in methanol at 0 °C.[66] As a consequence, the

relative concentrations of the minor oil constituents and the DAGs are increased

dramatically in the extracted solution.


169.0875

253.2179

281.2493

391.2650

445.2677525.3814

563.5009

655.5151701.4695

901.7928

200 300 400 500 600 700 800 900 m/z

873.7773

Figure 5.6- Negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil.

FA Region

DAG Region TAG Region


Table 5.4- Assignment of selected mass spectral peaks (>2%) in the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil shown in Fig. 5.6.

Observed Mass m/z

Exact Mass m/z


% 169.0875 169.0870 FA C9H13O3‾, C9:2 8.0 253.2179 253.2173 FA C16H29O2‾, C16:1 51.7 255.2335 255.233 FA C16H31O2‾, C16:2 21.2 279.2350 279.2330 FA C18H31O2‾, C18:2 7.4 281.2493 281.2486 FA C18H33O2‾, C18:1 100.0 283.2650 283.2643 FA C18H35O2‾, C18:0 4.6 391.2650 391.2643 Wax* C27H35O2‾ 11.3 535.4720 535.4732 FA Dimer C34H63O4‾ 1.9 563.5009 563.5045 FA Dimer C36H67O4‾ 3.3 609.5162 609.5100 DAG C37H69O6‾ 5.2 611.5250 611.5256 DAG C37H71O6‾ 3.2 627.4822 627.4871 DAG C36H67O8‾ 8.6 629.4869 629.4998 DAG C36H69O8‾ 3.1 637.5436 637.5413 DAG C39H73O6‾ 9.2 655.5151 655.4943 DAG C38H71O8‾ 10.5 657.5139 657.5100 DAG C38H73O8‾ 3.1 671.4377 671.4317 DAG C43H59O6‾ 4.3 673.4334 673.4321 DAG C39H61O9‾ 4.1 699.4664 699.4630 DAG C45H63O6‾ 7.3 701.4695 701.4787 DAG C45H65O6‾ 7.5 845.7322 845.724 TAG C53H97O7‾ 4.3 847.7458 847.7396 TAG C53H99O7‾ 3.8 873.7773 873.7917 TAG C56H105O6‾ 10.2 875.7690 875.7709 TAG C55H103O7‾ 5.7 901.7928 901.7855 TAG C57H105O7‾ 10.5

* See Section 1.1 for definition of waxes

5.3.2- Free Fatty Acids and Monoacylglycerol Region, m/z 150-400

Figure 5.7 shows an expanded view of the negative-ion FTICR mass spectrum of the

m/z 150-400 region of Figure 5.6 and Table 5.5 lists the corresponding assignment of

the mass spectral peaks in Figure 5.7.


157.0879

169.0875187.0977

212.0756227.2007

253.2179

281.2493

297.2445 325.1949 339.2095

391.2650

150 175 200 225 250 275 300 325 350 375 400 m/z Figure 5.7- FA region of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil in Figure 5.6, m/z 150-400.

Table 5.5- Assignment of the mass spectral peaks (>2%) in the expanded FA region (m/z 150-400) of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil shown in Figure 5.7.

Observed Mass m/z

Exact Mass m/z


% 169.0875 169.0870 FA C9H13O3‾, C9:2 8.0 171.1028 171.1027 FA C9H15O3‾, C9:1 3.4 187.0977 187.0976 Di-FA# C9H15O4‾ 4.3 227.2007 227.2017 FA C14H27O2‾, C14:0 3.9 253.2179 253.2173 FA C16H29O2‾, C16:1 51.7 255.2335 255.2330 FA C16H31O2‾, C16:0 21.2 279.2350 279.2330 FA C18H31O2‾, C18:2 7.4 281.2493 281.2486 FA C18H33O2‾, C18:1 100.0 283.2650 283.2643 FA C18H35O2‾, C18:0 4.6 295.2291 295.2279 FA C18H31O3‾, C18:2 3.9 297.2445 297.2431 FA C18H33O3‾, C18:1 4.6 311.1877 311.1864 FA C17H27O5‾, C17:3 3.6 313.2370 313.2384 FA C18H33O4‾, C18:1 2.3 325.2757 325.2748 FA C20H37O3‾, C20:1 2.0 391.2650 391.2643 Wax* C27H35O2‾ 11.3

# Di-FA: Dicarboxylic fatty acid * See Section 1.1 for definition of waxes


A comparison of the FA region of the ESI FTICR mass spectra of macadamia neat

oil and the methanol extract of the oil (Figures 5.2 and 5.6 respectively) shows that

extraction has increased the concentration of free FAs as larger number of peaks

assigned to free fatty acids are now observed, as well as increasing the S/N ratio.

Furthermore, the relative intensities of various free fatty acids also appear to change.

As an example, the relative intensity of the C16H29O2¯ / C18H33O2¯ peaks in Figure

5.2 is 61/100 (45/74) while this relative intensity is 52/100 in Figure 5.7.

The extraction appears to be working in different ways for different free FAs. As an

example, the absolute intensity of the peak at m/z 281.2493 (assigned to C18H33O2¯)

has increased by four times in the negative-ion extract spectrum compared to that in

the neat oil spectrum, while the absolute intensity of the peak at m/z 255.2440

(assigned to C16H31O2¯) shows a decrease in the methanol extract spectrum

compared to that in the neat oil spectrum. This difference in the relative

concentrations (and hence the relative negative-ion peak intensities) of the free FAs

in the oil and the methanol extract are likely to be due to the differences in the

solubility of these compounds in methanol at the extraction temperature. It is

expected that as the degree of unsaturation on the alkyl chain increases, the

corresponding solubility of the free fatty acid in methanol will increase at 0 °C.[66]

The most abundant free fatty acid observed in the methanol extract of the oil is at

m/z 281.2493 that is assigned to C18H33O2¯ (2 ppm). As stated in section 5.2.1 this is

expected to be the oleate anion (C18:1). The related peaks at m/z 279.2330 (7%) and

2832643 (5%) are assigned to C18H31O2¯ (2 ppm) and C18H35O2¯ (7 ppm) which are

the linoleate (C18:2) and stearate (C18:0) anions respectively.


The next most abundant peaks are at m/z 253.2179 (52%) and 255.2335 (21%).

These are assigned to C16H29O2¯ (2 ppm) the palmitoleate anion C16:1 and

C16H31O2¯ (2 ppm) the palmitate anion C16:0. Interestingly this is the reverse order

to what was observed in neat oil in Figure 5.2, where the C16:0/C16:1 intensity ratio

is observed to be 50/23 compared to 21/52 for the oil extract. As stated earlier, we

can only attribute this difference to the different solubilities of the fatty acids in

methanol at 0 °C used in the extraction procedure.

As observed in the FTICR mass spectrum of neat processed macadamia oil in Figure

5.2, short-chain fatty acids, di-carboxylic acids and dihydroxy FAs are assigned to

the peaks in Figure 5.7.

Two short-chain fatty acids and one dicarboxylic acid are assigned in Table 5.5.

These are at m/z 169.0875 (8%), m/z 171.1028 (3%) and m/z 187.0977 (4%) and are

assigned to C9H13O3¯ (C9:2, 3 ppm), C9H15O3¯ (C9:1, 0.6 ppm) and C9H15O4¯ (C9:0

dicarboxylic acid, 0.5 ppm). As mentioned earlier in Section 1.5, Steenhorst has

separated and reported short-chain hydroxy fatty acids as a product of oxidation of

acylglycerols.[177] The oxidation cleavage of the carbon-carbon double bond on the

fatty acid chains produces hydroxy fatty acids and dicarboxylic acids. Oleic acid is

the most abundant fatty acid in macadamia oil with a double bond at carbon 9 that

can produce C9 hydroxy fatty acids and C9 dicarboxylic acids in oxidation cleavage.

A number of fatty acids with odd numbers of carbon atoms are assigned to the peaks

in this region but these are not listed in Table 5.5 due to the fact that their intensities

are lower than 2% of the base peak. These fatty acids include C15H27O2¯ (C15:1, m/z

239.2020, 0.5 ppm), C15H29O2¯ (C15:0, m/z 241.2158, 0.3 ppm), C17H31O2¯ (C17:1,

m/z 267.2333, 0.7 ppm) and C17H33O2¯ (C17:0, m/z 269.2484, 0.4 ppm).


5.3.2.1- Kendrick Mass Defect (KMD) Values

As pointed out earlier in Section 1.6 the KMD values can be used to validate the

assignments of homologous series of compounds, in this case, fatty acids. In this

section we use the KMD values, in addition to high-resolution mass accuracy

measurements, to support the assignments of the homologous series of fatty acid

anions such as C15:1, C16:1, C17:1 and C18:1.

Table 5.6 lists the KMD values of the assigned anions in Table 5.5. A number of

fatty acid anions with an extra oxygen bearing functional group are assigned to the

peaks in this region and are included in Table 5.6.

Table 5.6- Calculated Kendrick mass defects for homologous series in the negative-ion ESI FTICR mass spectrum of methanol extract of macadamia nut oil.

Observed Mass m/z Assigned Anion Kendrick Mass Defect 157.0873 C8H13O3‾ 0.912 157.1237 C9H17O2‾ 0.948 171.1030 C9H15O3‾ 0.912 227.2021 C14H27O2‾ 0.948 239.2020 C15H27O2‾ 0.935 241.2158 C15H29O2‾ 0.947 253.2173 C16H29O2‾ 0.935 255.2330 C16H31O2‾ 0.949 269.2124 C16H29O3‾ 0.912 267.2333 C17H31O2‾ 0.935 269.2484 C17H33O2‾ 0.948 281.2486 C18H33O2‾ 0.935 283.2643 C18H35O2‾ 0.949 297.2437 C18H33O3‾ 0.912 309.2804 C20H37O2‾ 0.935 311.1693 C17H27O3S‾ 0.822 325.1851 C18H29O3S‾ 0.822 339.2013 C19H31O3S‾ 0.823


KMD values are expected to be similar for homologous series; for example, all fatty

acid anions with a saturated acyl chain show a KMD value of 0.948 ± 0.001 (such as

C9H17O2¯, C16H31O2¯ and C18H35O2¯), all free fatty acid anions with a

monounsaturated acyl chain show a KMD value of 0.935 ± 0.001 (such as

C15H27O2¯, C16H29O2¯ and C18H33O2¯), all fatty acid anions containing an additional

oxygen atom show a KMD value of 0.912 ± 0.001 (such as C8H13O3¯, C9H15O3¯,

C16H29O3¯ and C18H33O3¯) and all sulphur containing anions show a KMD value of

0.822 ± 0.001.

In Table 5.6 all of the members of each homologous series are shaded in the same

colour, for example, C15H27O2¯, C16H29O2¯, C17H31O2¯, C18H33O2¯ and C20H37O2¯

are mono-unsaturated fatty acid anions that show a KMD value of 0.935 and are

shaded in green colour. Similarly, C8H13O3¯, C9H15O3¯, C16H29O3¯ and C18H33O3¯

are assigned to fatty acid anions containing extra oxygen bearing functional groups

(such as hydroxyl group) and show a KMD value of 0.912 and are shaded in blue.

Similarly, C9H17O2¯, C14H27O2¯, C15H29O2¯, C16H31O2¯, C17H33O2¯ and C18H35O2¯

are assigned to saturated fatty acid anions with a KMD value of 0.948 ±0.001 and are

shaded in tan. Similarly, C17H27O3S¯, C18H29O3S¯ and C19H31O3S¯ are sulphur

containing anions that show a KMD value of 0.822 and are shaded in grey.

5.3.3- Diacylglycerol Region, m/z 500-750 Figure 5.8 shows an expanded view of the negative-ion FTICR mass spectrum of the

m/z 500-750 region of Figure 5.6 and Table 5.7 lists the assignment of selected mass

spectral peaks in this Figure.


511.3516

525.3814

535.4720

553.4007

563.5009

583.5034

599.4459

609.5162

627.4822

637.5436

645.4739

655.5151

671.4377

682.5405

691.4627

701.4695

717.4638

731.4594747.5145

500 525 550 575 600 625 650 675 700 725 750 m/z Figure 5.8- DAG region of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil in Figure 5.6, m/z 500-750.

Two FA dimers and twelve DAGs are assigned in Table 5.7. Further investigation is

needed to identify the functional groups and their positions on the DAG molecules,

but that is not undertaken in this work because this would be beyond the scope of the

present thesis.

KMD values are also listed in Table 5.7 as they are used to confirm various

assignments of homologous anion series in Figure 5.7. Similar homologous series are

shaded in same colour in Table 5.7.

A comparison of the DAG regions of the FTICR mass spectra of processed

macadamia oil shown in Figure 5.3 (Table 5.2) and the methanol extract of the oil

shown in Figure 5.8 (Table 5.7) reveals that, in general, all peaks have higher S/N

ratios and are more intense relative to the base peak in the extract spectrum.


Table 5.7- Assignment of the mass spectral peaks (>2%) in the expanded DAG region (m/z 500-750) of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil shown in Figure 5.8.

Observed Mass m/z

Exact Mass m/z

Compound Class

Assigned Anion

Normalized % KMD values

525.3814 525.3797 DAG C30H53O7‾ 4.4 0.797 535.4720 535.4732 FA Dimer C34H63O4‾ 1.9 0.875 563.5009 563.5045 FA Dimer C36H67O4‾ 3.3 0.875 609.5162 609.5100 DAG C37H69O6‾ 5.2 0.840 611.5250 611.5256 DAG C37H71O6‾ 3.2 0.847 627.4822 627.4871 DAG C36H67O8‾ 8.6 0.786 629.4969 629.4998 DAG C36H69O8‾ 3.1 0.789 637.5436 637.5413 DAG C39H73O6‾ 9.2 0.837 655.5151 655.4943 DAG C38H71O8‾ 10.5 0.783 657.5139 657.5100 DAG C38H73O8‾ 3.1 0.781 671.4377 671.4317 DAG C43H59O6‾ 4.3 0.694 673.4334 673.4321 DAG C39H61O9‾ 4.1 0.681 699.4664 699.4630 DAG C45H63O6‾ 7.3 0.692 701.4695 701.4787 DAG C45H65O6‾ 7.5 0.693

For example, the peak at m/z 699.4664 (C45H63O6¯) in the extract spectrum is 38

times higher in absolute intensity than the same peak in the neat oil spectrum. As in

the previous section, this observation illustrates the advantage of extracting minor

components from the TAG dominated oil prior to mass analysis if one is interested in

the composition of the minor components in the oil. It is noteworthy that such

components are not observed in a GC-MS analysis of such oils.

In Table 5.2, six fatty acid dimers are assigned, while in Table 5.7, only two fatty

acid dimers are assigned. This difference is due to the fact that the minimum reported

intensities in Table 5.7 are greater than 2% of the base peak, while in Table 5.2, the

minimum reported intensities are 0.2% of the base peak. The normalized intensities

of four of the assigned fatty acid dimers in Table 5.2 are lower than 2%. Two of the

assigned fatty acid dimers are reported in both Tables 5.2 and 5.7 include the peak at


m/z 535.4720 (2%) assigned to C34H63O4¯ (2 ppm) and the peak at m/z 563.5009

(3.3%) assigned to C36H67O4¯ (6 ppm). Since the most abundant fatty acids in

macadamia oil are oleic (C18:1), palmitic (C16:0) and palmitoleic (C16:1) acids, it is

expected that one C18:1 and one C16:1 can combine to produce the C34H63O4¯ dimer

anion. Similarly, it is expected that two C18:1 can combine to produce the C36H67O4¯

dimer anion. KMD values confirm that these latter two assignments belong to a

homologous series of dimers and possess same functional groups.

The peak at m/z 525.3814 (4%) is assigned to a DAG anion C30H53O7¯ (3 ppm). The

two fatty acid substituents on the glycerol could be C16 and C11. One of the fatty

acid substituents could be a short-chain hydroxy fatty acid.

Two peaks at m/z 609.5162 (5.2%) and 611.5250 (3.2%) are assigned to the DAG

anions C37H69O6¯ (10 ppm) and C37H71O6¯ (1 ppm) respectively. The peak at m/z

609.5162 could be assigned to a DAG anion that contains C18:1 and C16:0 side

chains, one of which has a hydroxyl group, possibly C16:0. The hydroxyl group

could have been produced in a hydration reaction of the double bond on a C16:1.

Alternatively, the two fatty acid substituents could be C18:0 and C16:1 and the

C18:0 could bear the hydroxyl group as a result of hydration reaction on the double

bond of C18:1. Further CID and MS/MS analysis is needed to elucidate the

substituents of this DAG molecule.

Branched side chains in TAG molecules, since TAGs are major components of

macadamia oil, are of interest because they may lead to different properties of the oil.

The detection and analysis of these groups and unsaturated carbon position can be

achieved by tandem MS and derivatisation. Careful HPLC and tandem MS could be

used to direct the acyl glycerol molecular ions to more completely define the exact


composition of the oil. The determination of the position of the side chains on

glycerol in acylglycerols are discussed in Section 1.1.3.1.

Similarly, the peak at m/z 611.5250 could be assigned to a DAG anion that contains

a C18:0 and a C16:0 side chains, one of which contains one hydroxyl substituent.

Given that the palmitic acid substituent is a major constituent of acylglycerols in

macadamia oil, the two fatty acid substituents on the glycerol in this TAG anion are

likely to be palmitic acid and hydroxy oleic acid acyl groups. The hydroxyl group on

the oleic acid acyl group could have been produced in hydration reaction on the

double bond of linoleic acid acyl substituent.

The anion at m/z 627.4822 in Table 5.7 could be assigned to two possible DAG

anion formulae, one of which contains a nitrogen atom, C39H65NO5¯ (7 ppm). The

second possible assignment is DAG anion C36H67O8¯ (3 ppm). Similarly, the peak at

m/z 629.4969 could be assigned to two DAG anions, one of which contains two

nitrogen atoms, C38H65N2O5¯ (3 ppm) and the other to C36H69O8¯ (5 ppm). To assess

the assignments, KMD values were calculated for the peaks at m/z 627.4822 (KMD=

0.782) and m/z 629.4869 (KMD= 0.782). The same KMD values suggest that these

two molecule anions belong to a homologous series. Consequently, the peak at m/z

627.4822 is assigned to DAG anion C36H67O8¯ and the peak at m/z 629.4869 is

assigned to DAG anion C36H69O8¯. The alternative nitrogen-bearing molecules have

different number of nitrogen atoms and cannot fit in same homologous series.

Similarly, the KMD value for the peak at m/z 655.5151 in Table 5.7 was selected as

0.783 for the DAG anion C38H71O8¯ (6 ppm). The DAG anion C41H67O6¯ (6 ppm)

which is another alternative assignment has a KMD value of 0.762 and was rejected.

Same calculations were carried out for the peak observed at m/z 657.5139 and the


KMD was found to be 0.783 for possible DAG anion C38H73O8¯ (6 ppm). The DAG

anion C41H69O6¯ (4 ppm) with a KMD value of 0.776 was rejected. These four latter

assigned anions at m/z 627.4822, 629.4869, 655.5151 and 657.5139 belong to the

same homologous series with a KMD value of 0.785 ± 0.004.

The peak at m/z 671.4377 is assigned to a DAG anion C43H59O6¯ (9 ppm) which

could be a glycerol with two C20H31O2¯ substituents, one of which has a hydroxyl

group substituent. Similarly, the peak at m/z 699.4664 is assigned to a DAG anion

C45H63O6¯ (5 ppm) that could be composed of a glycerol substituted with two

heneicosa hexaenoate (C21H29O2¯) substituents, one of which contains a hydroxyl

group. In a similar way, the peak at m/z 701.4695 is assigned to a DAG anion

C45H65O6¯ (13 ppm) that could contain a glycerol substituted with a heneicosa

hexaenoate (C21H29O2¯) and a heneicosa pentaenoate (C21H31O2¯), one of which

contains a hydroxyl group. The last three assigned anions at m/z 671.4377, 699.4664

and 701.4695 belong to a homologous series with a KMD= 0.693 ± 0.001.

As mentioned previously in Section 5.1, it is possible to observe DAG anions in

negative-ion mode because of the mobile proton on the hydroxyl substituent of the

glycerol. Such anions contain five oxygen atoms in the anion ionic formulae. It is

interesting that no such anions are observed in Table 5.7; in fact, most of the anions

observed contain six or eight oxygen atoms. No doubt the extensive hydration of the

unsaturated carbon double bonds on the acid side chain groups of the glycerol

enhances the ionisation efficiencies of the DAG molecules in the oil. It is noteworthy

that such species cannot be identified in the oil by the more traditional GC-MS

experiments. Whether such hydration of the acyl substituents occur naturally in the

oil or by the processing of the oil is not tested in this study.



Figure 5.9 shows the m/z 800-1000 expanded view of the negative-ion FTICR mass

spectrum of the methanol extract of the processed macadamia oil (Figure 5.6). Table

5.8 lists the assigned peaks and the associated KMD values in this spectrum.

An important point in Table 5.8 is that all of the peaks are assigned to TAG anions

with additional oxygen atoms compared to standard TAG anions. The assigned

anions in Table 5.8 contain seven oxygen atoms or more, whereas the common TAG

molecules have six oxygen atoms.

The extra oxygen atom in the assigned anions in Table 5.8 is likely to be a hydroxy

group that is capable of releasing a proton and producing an anion. As stated

previously in Section 5.1, the common TAG molecules with three ester bonds (six

oxygen atoms) are unable to produce negative ions in ESI source due to the absence

of ionisable functional groups such as hydroxyl group.

Two most intense peaks assigned in the TAG region in Table 5.8 are at m/z 901.7868

and m/z 873.7559. The peak at m/z 901.7868 (10.6%) is assigned to C57H105O7¯ (0.2

ppm). Since the most abundant fatty acid substituent in the TAGs in macadamia oil is

C18:1, it is expected that this anion is composed of a glycerol with two C18:1

substituents and one C18:0 substituent. The C18:0 glycerol substituent could be the

one that contains the hydroxyl group that produces the negative charge by releasing

the proton. The hydroxyl group could have been generated by the hydration reaction

of a carbon double bond on a C18:1 substituent.


845.7311

873.7559

891.7726

901.7868

919.7962

800 820 840 860 880 900 920 940 960 980 1000m/z

(b)

845.7322

873.7773

891.7837

901.7928

919.8176

(a)

845.7311

873.7559

891.7726

901.7868

919.7962

800 820 840 860 880 900 920 940 960 980 1000m/z

(b)

845.7322

873.7773

891.7837

901.7928

919.8176

(a)

845.7322

873.7773

891.7837

901.7928

919.8176

(a)

Figure 5.9- TAG region of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil in Figure 5.6, m/z 800-1000, (a) prior to internal calibration and (b) after internal calibration was carried out.

Table 5.8- Assignment of the mass spectral peaks (>2%) and KMD values in the expanded TAG region (m/z 800-1000) of the negative-ion ESI FTICR mass spectrum of the methanol extract of processed macadamia nut oil shown in Figure 5.9.

Calibrated Mass m/z

Exact Mass m/z

Compound Class

Assigned Anion

Normalized %

KMD Value

845.7311 845.7240 TAG C53H97O7‾ 4.3 0.780 847.7437 847.7396 TAG C53H99O7‾ 3.8 0.793 873.7559 873.7553 TAG C55H101O7‾ 10.2 0.780 875.7681 875.7709 TAG C55H103O7‾ 5.7 0.793 891.7726 891.7658 TAG C55H103O8‾ 2.9 0.770 901.7868 901.7866 TAG C57H105O7‾ 10.6 0.780 919.7962 919.7971 TAG C57H107O8‾ 2.5 0.770

From this study it is not possible to determine the location of the various C18:1

substitutions on the glycerol backbone. Such issues are discussed in Chapter 8 under

Future Work.


The peak at m/z 873.7559 (10.2%) is assigned to C55H101O7¯ (0.7 ppm). This ion

could be composed of a glycerol with two C18:1 and one C16:0 substituents. One of

these substituents would also contain a hydroxyl group, probably produced by the

hydration of a carbon double bond. The oil contains both C16:1 and C18:1

substituents on glycerol in the TAG molecule, and so both of these could be the part

of the hydration reaction.

The peak at m/z 875.7681 (5.7%) is assigned to C55H103O7¯ (3 ppm) that is expected

to be composed of a glycerol with one C18:1, one C18:0 and one C16:0 acyl

substituent groups on the glycerol. It also contains a hydroxyl group necessary to

produce the anion by providing a mobile proton. Whether the hydration of the carbon

double bond is on a C18:1 or a C16:1 to produce the C18:0 or C16:0 is unknown.

The peak at m/z 845.7311 (4.3%) is assigned to C53H97O7¯ (0.8 ppm) which could be

a hydroxy glycerol with the substituents C16:0, C16:1 and a C18:1 The C16:0 could

be the side chain that contains the hydroxyl group that produces the negative charge

by releasing a proton. The hydroxyl group could have been generated by the

hydration reaction of a carbon double bond on a C16:1, palmitoleate substituent.

The peak at m/z 847.7437 (3.8%) is assigned to C53H99O7¯ (4.8 ppm) which could be

a hydroxyglyceride with two C16:0 and one C18:1 substituents. One of the C16:0

substituents could contain the hydroxy group that provides the negative charge. The

hydroxyl group could have been generated by the hydration reaction of a carbon

double bond on a C16:1 substituent.

The peak at m/z 891.7726 (2.9%) is assigned to C55H103O8¯ (7.6 ppm) which could

be a hydroxyglyceride with C16:0, C18:0 and C18:1 substituents. There are two


hydroxyl groups on this anion, possibly with one on the C16:0 and one on the C18:0

substituents and produced by hydration of carbon double bond on a C16:1 and a

C18:1 substituent in a TAG such as C16:1; C18:1; C18:1. Such TAGs are common

in the oil (see Table 5.8).

The peak at m/z 919.7962 (2.5%) is assigned to C57H107O8¯ (<1 ppm). It is expected

that this anion is composed of a glycerol with one C18:1 substituent and two C18:0

substituents. Each of the C18:0 substituents could contain one hydroxyl group and

one of the hydroxyl groups could provide the negative charge by releasing a proton.

This anion could have been produced by a carbon double bond hydration reaction of

trioleate, the most common TAG in macadamia oil.

A comparison of Table 5.3 (TAGs region of neat oil) with Table 5.8 (TAGs region of

the methanol extract of the oil) reveals that peaks assigned to the TAG molecules

with higher molecular masses (such as peaks at m/z 931.7126, 945.7158 and

959.7272) are assigned in Table 5.3 but not in Table 5.8. This could be due to the

differences in the solubilities of TAG compounds in methanol at the extraction

temperature. In addition, one TAG molecule with relatively lower molecular mass

(m/z 845.7311) is assigned in Table 5.8 but not in Table 5.3.

5.3.4.1- KMD Values of the Assignments in the TAG Region

Additional support for the assignments in Table 5.8 are the calculated KMD values

for the assigned peaks. In general, the KMD values of the assigned anion TAG peaks


can be categorized into three groups. There are three anions with KMD= 0.780, two

anions with KMD= 0.793 and two anions with KMD= 0.770.

All of the three assigned TAG anions with KMD= 0.780 have two unsaturated fatty

acid substituents and one saturated fatty acid substituent. In particular, the peak

assigned at m/z 845.7311 has two C18:1 and one C16:0 substituents, the peak

assigned at m/z 873.7559 has two C18:1 and one C16:0 substituents and the peak

assigned at m/z 901.7868 has two C18:1 and one C18:0 substituents.

Both of the assigned TAG anions with KMD= 0.793 have two saturated substituents

and one unsaturated substituent. In particular, the peak assigned at m/z 847.7437 has

two C16:0 substituents and one C18:1 substituent and the peak assigned at m/z

875.7681 has C18:0, C16:0 and C18:1 substituents.

Both of the assigned TAG anions with KMD= 0.770 have two hydroxy groups and

both have two saturated fatty acid substituents and one unsaturated fatty acid

substituent on the glycerol backbone. The peak assigned at m/z 891.7726 has one

C16:0, one C18:0 and one C18:1 substituents on glycerol and the peak assigned at

m/z 919.7962 has two C18:0 and one C18:1 substituents.

5.3.5- Stability of the Methanol Extract of Processed Macadamia Oil

In this section it was decided to examine the possible change in the methanol extract

of processed macadamia oil over a period of time (576 days) by examining the

negative-ion FTICR mass spectra of the methanol extract of the oil over this period.


The methanol extract of processed macadamia was stored under nitrogen gas

atmosphere in a freezer.

Figure 5.10 shows a comparison of the negative-ion ESI FTICR mass spectra of the

methanol extract of processed macadamia oil on two dates, 22/01/2003 and

19/08/2004.

161.9739

253.2173

281.2486

311.1687 391.2617477.3009 563.5056

599.4452627.4757

655.5071

901.7856

169.0875

253.2179

281.2493

391.2650445.2677

563.5009 655.5151701.4695 845.7514 901.7928

200 300 400 500 600 700 800 900m/z Figure 5.10- A comparison of the negative-ion ESI FTICR mass spectra of the methanol extract of macadamia oil on two dates: (a) 22/01/2003 and (b) 19/08/2004.

Although this is not a detailed study, the comparison reveals several useful points

regarding the ESI FTICR mass spectrometry of the methanol extract of processed

macadamia oil and the relative stability of the oil. It would appear that even though

the oil is stored at about -15 °C and kept under nitrogen gas atmosphere, there is still

some degradation occurring.

The most obvious change is the ratio of the C18:1/C16:1 peaks at m/z 281.2486 and

253.2173 respectively. In Figure 5.10 (a) this ratio is 19/5 where in Figure 5.10 (b) it

is 23/12. This is an increase in the relative intensity of the peak at m/z 253.2173 by

1.9 times in Figure 5.10 (b).

(a)

(b)


Similarly, the fatty acid peak at m/z 311.1687 also appears to have diminished in

relative intensity. The ratio of the peak at m/z 281.2486 to the peak at 311.1687 in

Figure 5.10 (a) is 19/3 while this ratio in Figure 5.10 (b) is 23/0.8. This is a decrease

in the relative intensity of the peak at 311.1687 by 4 times in Figure 5.10 (b).

It should be noted that there are slight differences in the m/z values of the various

ions in Figures 5.10 (a) and (b). For example m/z 281.2468 and 281.2499. This arises

because only external calibration was used to mass calibrate the spectra. These

relative errors are taken into account here in comparing the spectra.

Furthermore the DAG and TAG regions show diminished relative ion intensities in

Figure 5.10 (b) compared to Figure 5.10 (a). For example, the relative intensity of the

peak at m/z 281.2486 to the peak at 655.5071 is 1.9 in Figure 5.10 (a), while this

ratio is 9.5 in Figure 5.10(b). This is 5 times decrease in the intensity of the peak at

655.5071 which is assigned to a DAG anion C38H71O8¯ (6 ppm). In the TAG region,

the relative intensity of the peak at m/z 281.2486 to the peak at m/z 901.7856 is 5.8

in Figure 5.10 (a), while this ratio is 9.5 in Figure 5.10 (b). This is 1.6 times decrease

in the intensity of the peak at 901.7856 which is assigned to the TAG anion

C57H105O7¯.

In general, even under storage of the oil under nitrogen gas in a freezer there is still

some activity in the oil chemistry. In particular, it appears that the oxygenated DAGs

and TAGs decrease in concentration producing increased quantities of free fatty

acids with an increase in the relative concentration of the C16:1 (palmitoleic acid) it

is possible that even after all the processing that occurred in the oil production there

are still some enzymes present in the oil which lead to its reactivity.


A general conclusion in this section is that the methanol extract of macadamia oil

shows a reasonably good stability; the changes in the spectrum are minimal after

about 19 months. Few peaks have diminished in the TAG and DAG region and few

peaks have diminished in the fatty acid region. There are few peaks that are not

common in both spectra, such as m/z 212.0748 and m/z 701.4695, the source of

which is not clearly known, but could be due to a trace-level contamination in the

solvent used for sample preparations on two different dates. Furthermore, there are

some differences in the intensities of the free fatty acids that can be a result of the

enzymatic activities in the sample.

5.4- Negative-ion ESI FTICR-MS of the Hydrolysed Processed Macadamia Oil

5.4.1- Introduction

In this section the results of negative-ion FTICR-MS analysis of hydrolysed

processed macadamia nut oil are presented and discussed. Peaks are discussed in two

regions including free FAs region (m/z 150-400) and FA dimers region (m/z 500-

750).

The hydrolysis of the processed macadamia oil releases all of the acyl substituents as

fatty acids. This reaction allows analytical experiments to be undertaken which

provide detailed information about the FA profile of the oil. The hydrolysis reaction

takes place in alkaline solution as described in section 2.2.2 and all of the acyl


substituents on the TAG, DAG and MAG molecules in the oil are released.

Therefore, the ESI FTICR mass spectrum of hydrolysed macadamia oil is expected

to contain fewer peaks compared to the FTICR mass spectrum of the neat macadamia

oil (Figure 5.1) and the methanol extract of the oil (Figure 5.6).

5.4.2- FTICR Mass Spectrum of Hydrolysed Processed Macadamia Oil

Figure 5.11 shows the ESI FTICR mass spectrum of hydrolysed processed

macadamia oil and Table 5.9 lists the assigned peaks in this Figure.

Figure 5.11 shows a base peak at m/z 281.2486 which is assigned to C18H33O2¯ (<0.1

ppm). This anion is expected to be the oleate anion as oleate is reported to be the

major acyl substituent on the TAG molecules in macadamia oil using GC-MS.[196]

Our GC-MS analysis of esterified macadamia oil confirms that in Chapter 6 also the

oleate substituent on the glycerol is the main constituent of macadamia oil (Section

6.1) comprising 59.6% of the total area of the GC-MS measured peaks.

Associated with this peak at m/z 281.2486 are two less intense peaks (approximately

4%) at m/z 279.2439 and 283.2640. These peaks are assigned to the anions

C18H31O2¯ (0.8 ppm) and C18H35O2¯ (1 ppm) which are most likely the linoleate

(C18:2) and stearate (C18:0) anions respectively, based on GC-MS results.


187.0975

253.2177

281.2486

357.2655

563.5039

100 200 300 400 500 600 700 800 900 1000m/z

535.4767

187.0975

253.2177

281.2486

357.2655

563.5039

100 200 300 400 500 600 700 800 900 1000m/z

535.4767

Figure 5.11- Negative-ion ESI FTICR mass spectrum of hydrolysed processed macadamia oil.

Table 5.9- Assignment of the mass spectral peaks (>2% of the base peak) in the negative-ion ESI FTICR mass spectrum of hydrolysed processed macadamia oil shown in Fig. 5.11.

Observed Mass m/z

Exact Mass m/z


% 187.0975 187.0976 Di-FA* C9H15O4 ‾ 2.7 227.2020 227.2017 FA C14H27O2‾, C14:0 2.1 253.2177 253.2173 FA C16H29O2‾, C16:1 34.3 255.2331 255.2330 FA C16H31O2‾, C16:0 14.1 269.2133 269.2122 FA C16H29O3‾ 4.5 279.2439 279.2330 FA C18H31O2‾, C18:2 4.5 281.2486 281.2486 FA C18H33O2‾, C18:1 100 283.2640 283.2643 FA C18H35O2‾, C18:0 4.1 295.2278 295.2279 FA C18H31O3‾ 2.9 297.2439 297.2435 FA C18H33O3‾ 9.9 309.2807 309.2799 FA C20H37O2‾, C20:1 3.3 311.2231 311.2228 FA C18H31O4‾ 3.7 535.4767 535.4732 FA dimer C34H63O5‾ 9.3 537.4843 537.4888 FA dimer C34H65O5‾ 5.6 563.5039 563.5045 FA dimer C36H67O4‾ 30.9 565.5071 565.5201 FA dimer C36H69O4‾ 4.2

* Di-FA: Dicarboxylic fatty acid (HOOC-R-COOH)

Two other peaks in this region of the spectrum at m/z 295.2278 and 297.2439 are

assigned to the C18 anions C18H31O3¯ (0.4 ppm) and C18H33O3¯ (1 ppm)

FA Region

FA Dimers Region


respectively. These anions could arise from linolenic (C18:3) and oleic (C18:1) acids

respectively where one of the double bonds has been hydrated.

A further C18 fatty acid is assigned to the peak at m/z 311.2231 as C18H31O4¯ (1

ppm). This C18 anion could arise from the hydration of two of the carbon double

bonds in the C18:4, stearidonic acid (moroctic acid, C18H28O2) effectively adding

two water molecules to the acid anion. Whether these anions showing evidence of

hydrated carbon double bonds are present in the original macadamia oil sample or

are generated in the hydrolysis reaction of the TAG molecules is not tested in this

study.

The second most intense peak (34%) in Figure 5.11 is at m/z 253.2177 is assigned to

the C16:1 anion C16H29O2¯ (1.6 ppm). This anion most likely arises from palmitoleic

acid. A related C16 peak at m/z 255.2331 is assigned to C16H31O2¯ (0.4 ppm) and

most likely arises from C16:0 palmitic acid. As a confirmation of this assignment,

palmitoleic acid is detected and assigned at a similar spectral percentage (32.7%) in

the esterified macadamia oil FTICR mass spectrum in positive-ion mode (Table 4.4).

Furthermore, our GC-MS analysis of esterified macadamia oil has measured the

relative intensity of palmitoleic acid / oleic acid as 20.6/59.6 that is 34.6%. This ratio

in negative-ion ESI FTICR mass spectrum of hydrolysed macadamia nut oil is

34/100 that is 34.0%. The results of two mass spectrometric experiments appear

consistent and strongly confirm each other.

One other related C16 peak is observed at m/z 269.2133. This peak is assigned to the

C16H29O3¯ (4 ppm) anion which as with the C18 series is associated with hydration

of a carbon double bond in a C16:3 acid.


Two other peaks are assigned to fatty acid anions in Figure 5.11. The first at m/z

227.2020 (2%) is assigned to C14H27O2¯ (2 ppm) and could arise from C14:0

myristic acid. The second peak at m/z 309.2807 (3%) is assigned to C20H37O2¯ (3

ppm) and could arise from C20:1 gadoleic acid.

The remaining peak in the FAs region (m/z 150-400) in Figure 5.11 is assigned to a

dicarboxylic acid. The dicarboxylic acid at m/z 187.0975 is assigned to C9H15O4¯

(0.5 ppm) and is most likely the HCO2-C7H14-CO2¯ anion.

Four odd numbered free FAs are observed in the negative-ion FTICR mass spectrum

of neat macadamia oil in Table 5.1; however, these FAs are not detected in the

FTICR mass spectrum of hydrolysed macadamia oil. This observation supports the

idea that the odd numbered free FAs in the neat macadamia oil are not released from

the TAG molecules, but they could be byproducts of possible biological or

biochemical activities in the oil by the microorganisms and fungi.[211,212]

In the FA dimer region (m/z 500-750) the most intense peak of the fatty acid dimers

is observed at m/z 563.5039 (30.9%) and is assigned to C36H67O4¯ (1 ppm). This

anion most likely forms from the combination of an oleic acid molecule with an

oleate anion (C18H34O2---C18H33O2¯).

Similarly, the peak at m/z 565.5071 (4.2%) is assigned to C36H69O4¯ (1 ppm) which

could be a combination of C18:1 and C18:0 acids less a proton.

The remaining two fatty acid dimers observed at m/z 535.4767 and 537.5039 are

assigned to the anions C34H63O4¯ (4 ppm) and C34H65O4¯ (8 ppm) respectively. The

latter anion could arise from the combination of oleic acid (C18:1) and palmitolenic


acid (C16:2) less a proton. These assignments are consistent with the observed

relative intensities of the fatty acids in Table 5.9.

As mentioned earlier (Section 4.1), fatty acids form dimers in gas phase. The dimers

are observed in negative-ion FTICR mass spectra of processed macadamia oil

samples. At high capillary-skimmer voltages, the FA dimer peaks disappear due to

an increase in the kinetic energy and collision induced dissociation of the dimer

anion species in the capillary-skimmer region. We have applied a relatively low

capillary-skimmer voltage in our FTICR-MS experiments to keep the peaks of the

dimers in our spectra for two main reasons:

1- Dimers are anions of interest with precisely known molecular masses in the

DAG region of the spectrum and are used as internal calibrants.

2- A low capillary-skimmer voltage ensures that the anions formed in the DAG

and TAG regions from the analysed samples are not removed from the

spectrum due to CID.

5.4.3- Comparison of FTICR Mass Spectra of Macadamia Oils

An interesting extension to this work as discussed in Chapter 8 as future works is to

examine the differences between processed and unprocessed (cold pressed)

macadamia oil, Preliminary studies were commenced in this present research but

only the results for this section are presented.


Figure 5.12 shows a comparison of the negative-ion ESI FTICR mass spectra of

hydrolysed (a) processed macadamia oil (Figure 5.11), (b) cold pressed batch 12 and

(c) cold press batch 13 macadamia oils.

The hydrolysis was carried out as discussed in Section 2.2.2 and cold pressing is

discussed in Section 1.2.6.

187.0975

253.2177

281.2486

297.2439357.2569

535.4767

563.5039

591.5321

253.2178

281.2484

309.2807535.4749

563.5027

591.5369

253.2176

281.2483

535.4761563.5045

100 200 300 400 500 600 m/z

187.0975

253.2177

281.2486

297.2439357.2569

535.4767

563.5039

591.5321

253.2178

281.2484

309.2807535.4749

563.5027

591.5369

253.2176

281.2483

535.4761563.5045

100 200 300 400 500 600 m/z Figure 5.12- Comparison of the negative-ion ESI FTICR mass spectrum of (a) hydrolysed processed macadamia oil, (b) hydrolysed cold pressed oil batch 12 and (c) hydrolysed cold pressed oil batch 13.

Figure 5.12 (b) and (c) show the same mass distribution of peaks assigned to FAs

and FA dimers as discussed in Section 5.4. For example, the peak at m/z 253, 281,

297 and 309 are assigned to the fatty acids C16:1, C18:1, C18-OH and C20:1. The

peaks at m/z 535, 563 and 591 are assigned to FA dimers of the common FAs present

(a)

(b)

(c)


in the oil. As might be expected from the different samples there are slight

differences in the relative intensities of these various ions.

Based on the work presented in this thesis thus far, it is not surprising these spectra

are similar. This is because the hydrolysis reaction effectively reduces the TAG

molecules to glycerol and fatty acids. To detect the differences between processing

an oil and cold pressing an oil one needs to compare the minor species present in the

oil such as the free FAs and MAGs and DAGs. As well, careful attention needs to be

paid to what actual processes are involved in the processing (Section X.Y) and cold

pressing (Section V.W) of the oil.

The experiments thus far, indicate that the methanol extraction of the oil concentrates

the minor components by removal of the TAGs which typically make up 95% of

macadamia oil. This is where one might expect to see differences between the

different oils.

Such a detailed study of the effect of oil preparation and even cultivar differences

and also oil rancidity would form the basis of another complete research project as

discussed in Chapter 8 under future work.

The industrial refining processes carried out on the macadamia oil have not altered

the total FA profile of the oil. In other words, the processes have only removed the

low-concentration ingredients of the oil such as free fatty acids and polyunsaturated

fatty acids.


5.5- A Summary of the Negative-ion ESI FTICR-MS of the Neat, Methanol Extract and Hydrolysed Processed Macadamia Oil

In this section, a summary of the occurrence of the common fatty acids either, as free

fatty acids or as substituents on the acylglycerols, in the processed macadamia oil is

discussed.

Figure 5.13 shows a comparison of the unsubstituted FA anions observed in

negative-ion FTICR-MS analysis of the neat (Table 5.1), the methanol extract of

(Table 5.4) and the hydrolysed (Table 5.9) processed macadamia nut oil. For this

figure, the anions that show evidence of hydration, e.g. C18H31O3¯ are not included.

Unlike the intensity values in the tables, the peak intensities in Figure 5.13 have been

normalised to 100% for the FA anions displayed.

Figure 5.13 highlights several interesting results from the negative-ion ESI FTICR-

MS experiments discussed previously in this chapter. If one can directly correlate the

anion concentrations with those of the acids then the hydrolysed oil, which contains

the fatty acids released from the acylglycerides, shows the highest concentration of

C18:1 oleic acid at 62% followed by the C16:1 palmitoleic acid at 21% and palmitic

acid at 9%. The presence of the C16 acids is important for the use of macadamia oil

in the cosmetic industry. The other minor acids (2 to 3%) such as the C14:0

(myristic), C18:0 (stearic) C18:2 (linoleic) and C20:1 (eicosenoic) are significant in

this experiment since for them to be observed they must be present in TAG side

chain groups in reasonable proportions. Their presences as such would help to give

the processed macadamia oil distinct chemical features.


C14:0

C14:1

C15:0

C15:1

C16:0

C16:1

C17:0

C17:1

C18:0

C18:1

C18:2

C18:3

C19:1

C20:0

C20:1

0

5

10

15

20

25

30

35

40

45

50

55

60

65

A Comparison of the Fatty Acid Components Observed in Negative-ion ESI-FTICR Mass Spectrometry Experiments of Processed Macadamia Oil

Peak

Rel

ativ

e In

tens

ity %

Fatty Acid

-ESI-MS Oil-ESI-MS Extract-ESI-MS Hydrolysed Oil

C14:0

C14:1

C15:0

C15:1

C16:0

C16:1

C17:0

C17:1

C18:0

C18:1

C18:2

C18:3

C19:1

C20:0

C20:1

0

5

10

15

20

25

30

35

40

45

50

55

60

65

A Comparison of the Fatty Acid Components Observed in Negative-ion ESI-FTICR Mass Spectrometry Experiments of Processed Macadamia Oil

Peak

Rel

ativ

e In

tens

ity %

Fatty Acid



Figure 5.13- A graphical comparison of the unsubstituted FA anions observed in negative-ion FTICR mass spectra of the neat (Table 5.1), the methanol extract (Table 5.4) and the hydrolysed (Table 5.9) processed macadamia nut oil.


It is equally striking in Figure 5.13 that the neat oil possesses a large concentration of

C16:0 (palmitic acid) at 35%, followed by 26% of C18:1 (oleic acid) and 16% of

C16:1 (palmitoleic acid). In the neat oil there are three other obvious components

with concentrations around 5 to 6%. These are the C14:0, C15:0 and C18:0 acids.

Also there are 5 other components with concentrations around 1 to 2% including the

C14:1, C15:1, C17:0, C17:1 and C18:2 acids.

It needs to be remembered that these acids are present in the processed oil as free

FAs and of course contribute to the overall acidity of the oil. In general, the free FAs

observed in the methanol oil extract tend to follow the concentrations of those

observed in the hydrolysed sample rather than those of the neat oil, given the slight

variations of intensity that are observed. This at first glance appears to be an unusual

result since the methanol extraction is expected to concentrate the free FAs in the

neat oil by removing the acylglycerides. The extraction does indeed improve the S/N

ratio of the experiment but it also appears to change the relative concentrations of the

free FAs in the oil sample. Our explanation for this unexpected result is that the

methanol extraction is more selective for the unsaturated free FAs resulting in a bias

in the free FA distribution in the methanol extract sampling of the processed

macadamia oil. The observed differences in fatty acid anion composition for

negative-ion ESI FTICR-MS experiments could also be due to the fact that FTICR-

MS is not a good quantitative tool as the errors in peak heights may vary between 5

to 15 percent, another source of observed differences could be due to the fact that the

averaging of the results for several experiments was not performed. However, the

agreement between the negative-ion ESI FTICR-MS results for the methanol extract

and the hydrolysed processed macadamia nut oil is reliable, whereas the results for


neat macadamia oil are totally different. Hence the results of the fatty acid anions for

the methanol extracted sample could be used to estimate the fatty acid composition

of the oil.

The TAGs account for >98% of the neat oil constituents. The similarity of the

hydrolysis and the esterification processes of neat macadamia oil is that the released

acyl substituents on the TAG molecules dominate the FTICR mass spectra. The

hydrolysate spectrum will contain free fatty acids and the esterified spectrum will

contain FAMEs. It is expected that the FA composition of the FTICR mass spectrum

of the hydrolysed neat oil in both positive and negative-ion modes show similarities

with the positive-ion FTICR mass spectrum of the esterified neat macadamia oil.

Figure 5.14 shows a comparison of the positive and negative-ion FTICR mass

spectra of hydrolysed neat macadamia oil and the positive-ion FTICR mass spectrum

of esterified neat macadamia oil. Table 5.10 lists the observed relative peak

intensities in the FTICR mass spectra of hydrolysed neat processed macadamia oil in

both positive and negative-ion modes and the positive-ion mode FTICR mass

spectrum of the esterified neat processed macadamia oil.

One final comparison can be made between the FAs observed in the hydrolysed

methanol extract negative-ion ESI FTICR spectrum and the literature positive-ion EI

GC-MS result obtained by Cavaletto in 1980.[196] This comparison shows that the six

common fatty acids in both experiments agree to within a few percent. For the most

common acids C16:0:C16:1:C18:1 the ratios are 9:21:62 for the FTICR MS and

7:18:65 for the GC-MS. However as should now be obvious, the negative-ion

FTICR MS provides considerably more detailed information with regard to the minor


and oxygenated compounds present in the oil than the GC-MS experiment.

Furthermore it is able to identify oxygenated MAGs, DAGs and TAGs in the oil.

In a comparison of the peak intensities in the positive-ion FTICR mass spectra of

esterified neat macadamia oil and the hydrolysed neat oil, the most intense peak in

both spectra is assigned to C18:1 with 61.1% and 61.7% for the hydrolysed and the

esterified oil respectively, followed by C16:1 with 17.2% and 20.2%. The peak

assigned to C18:2 shows a good consistency with 7.9% and 6.7% in hydrolysed and

esterified oil respectively, followed by the peaks assigned to C20:1 with 2.3% and

3.6%. The peaks assigned to C16:0 in both spectra show relative intensities of 5.2%

and 3.0% in hydrolysed and esterified oil respectively.

Comparison of Hydrolysed Oil (+ and -) and Esterified Oil (+) FTICR Mass Spectra

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

C16

:0

C16

:1

C17

:0

C17

:1

C18

:0

C18

:1

C18

:2

C18

:3

C19

:1

C20

:0

C20

:1

C20

:4

Fatty Acids

Rel

ativ

e Pe

ak In

tens

ity

Hydrol. +ve

Hydrol. -ve

Esterf . +ve

Figure 5.14- Comparison of the ESI FTICR mass spectrum of hydrolysed processed macadamia oil in positive and negative-ion modes with esterified neat oil in positive-ion mode


Table 5.10- Relative intensities of the assigned mass spectral peaks (>2% of the base peak) in the FTICR mass spectra of hydrolysed neat macadamia oil in both positive and negative-ion modes and the esterified oil in positive-ion mode.


ESI/FTICR-MS Hydrolysed Oil

Positive-ion

ESI/FTICR-MS Hydrolysed oil Negative-ion

ESI/FTICR-MS Esterified oil Positive-ion

C16:0 5.2 8.7 3.0 C16:1 17.2 21.1 20.2 C17:0 1.6 C17:1 3.4 C18:0 1.6 2.5 - C18:1 61.1 61.6 61.7 C18:2 7.9 2.8 6.7 C18:3 1.6 - - C19:1 3.2 - - C20:0 - - C20:1 2.3 2.0 3.6 C20:4 - - -

In the same way, we compare the relative intensities of the observed peaks in the

FTICR mass spectra of the hydrolysed neat macadamia oil in positive and negative-

ion modes. The most intense peak is assigned to C18:1 with 61.1% in positive and

61.6% in negative-ion modes, followed by the peak assigned to C16:1 with 17.2% in

positive and 21.1% in negative-ion modes. The peak assigned to C18:2 shows a

relative intensity of 7.9% in positive and 2.8% in negative-ion mode and the peak

assigned to C16:0 shows 5.2% in positive and 8.7% in negative-ion mode.


Chapter 6

6. Gas Chromatography-Mass Spectrometry of Processed

Macadamia Oil


6.1- Introduction

In this chapter the GC mass spectra of the esterified neat and methanol extract of the

processed macadamia oil are measured and discussed. A comparison of these results

with the positive-ion and negative-ion ESI FTICR mass spectra of the esterified and

hydrolysed neat processed macadamia oil is also presented.

Due to funding and time limitations only the processed macadamia oil was used for

GC-MS analyses.

Free fatty acids, DAGs and MAGs are not separated well in GC column due to the

high polarity of the acid molecules which binds them to the stationary phase thereby

resulting in poor separation efficiency. Also the TAG molecules have high boiling

points which means they need to be analysed in the GC column at high temperatures

(280 to 360 °C) which can result in the decomposition of temperature-sensitive

molecules in the sample and/or bleeding in the GC column, hence shorter column

life. The answer to this problem of sample polarity and volatility in this type of

analysis is to transesterify the acylglycerols thereby reducing the FA acyl side-chains

to fatty acid methyl esters which are less polar and possess lower boiling points.

It should be noted that it is possible to directly analyse free fatty acids in the oil

samples using special GC columns especially designed for this purpose. In general

such columns are not used routinely due to the complications in the associated mass

spectrometry arising from high temperature such as column bleeding.


The GC-MS results reported in this chapter are usually carried out on the basis of

retention indices, characteristic mass values and intensity ratio of mass peaks

compared to library spectra.

The GC-MS experiments reported in this chapter are to investigate the applicability

of the present ESI FTICR-MS experiments with those used in previous studies on

macadamia oil, in particular GC-MS. As well, we compare our measured oil

composition with other literature studies to provide an independent confirmation of

the actual fatty acid composition of the oil. For example, that the most common FA

in the oil is C18:1 (oleic acid). The chromatographic aspect of the GC-MS

experiment confirms this type of detail.

The GC-MS experiments were performed on a Hewlett-Packard HP 5890 Series II

with a J&W DB-Wax 60 m × 0.5 mm × 0.25 µm column and a VG QUATTRO mass

spectrometer that is discussed in section 2.3.3.

The 70 eV EI mass spectra were assigned from the GC retention indices and by

comparison of the GC-mass spectral m/z and peak intensity values with known

standards and library spectra.

The transesterification process for the oil has been discussed previously in Section

2.2.3.


6.2- GC-MS Analysis of Esterified Processed Macadamia Oil

Figure 6.1 illustrates the GC-MS total ion chromatogram (TIC) of the esterified

processed macadamia oil over a GC retention time of 80 min. The assignments of the

peaks in Figure 6.1 are listed in Table 6.1.

For the sake of space and since only standard well known FAMEs are identified in

the individual EI mass spectra corresponding to the peaks in Figure 6.1, these EI

mass spectra are not reported in this chapter.

Retention time (min)Retention time (min) Figure 6.1- GC-MS TIC of esterified processed macadamia oil.

Table 6.1- Assignment of the peaks in the GC-MS TIC of esterified processed macadamia oil.

Retention time (min) FAME Suggested by the Library Search Routine Peak Area %

52.75 Tetradecanoic acid, C14:0 1.40 59.75 Hexadecanoic acid, C16:0 10.92 60.67 Hexadecenoic acid, C16:1 21.50 66.15 Octadecanoic acid, C18:0 4.67 66.92 Octadecenoic acid, C18:1 58.41 68.08 Octadecdienoic acid, C18:2 1.93 72.68 Eicosanoic acid, C20:0 1.17 73.56 Eicosenoic acid, C20:1 1.18


The observed peak at 10.8 min in Figure 6.1 is assigned to residual toluene

(esterification solvent). The peak at 49.3 min is assigned to the antioxidant butylated

hydroxytoluene (BHT; 2,6-bis (1,1-dimethylethyl)-4-methylphenol) added to the

esterified oil to stabilize it. Since these materials are not part of the oil sample, they

are not included in Tables 6.1 and 6.2.

The remaining eight peaks in Figure 6.1 are assigned to one C14, two C18 and two

C20 FAMEs. The tetradecanoic ME C14:0, eicosanoic ME C20:0 and eicosenoic ME

C20:1 all represent minor components at peak area percentages of around 1 to 2%.

The two saturated FAMEs hexadecanoic (palmitic) ME, C16:0 and octadecanoic

(stearic) ME, C18:0 are observed at peak area percentages of 11% and 5%

respectively. The largest peak in the trace is assigned to octadecenoic (oleic) ME,

C18:1 at 58%.

6.3- GC-MS Analysis of the Esterified Methanol Extract of Macadamia Oil

Figure 6.2 shows the GC-MS TIC of the esterified methanol extract of processed

macadamia oil. The assignments of the peaks in Figure 6.2 are listed in Table 6.2.

For the sake of space and since only standard well known FAMEs are identified in

the individual EI mass spectra corresponding to the peaks in Figure 6.2, these EI

mass spectra are not reported in this section.


Retention time (min)Retention time (min) Figure 6.2- GC-MS TIC of esterified methanol extract of macadamia oil. Table 6.2- Assignment of the peaks in the GC-MS TIC of esterified methanol extract of processed macadamia oil.

Retention time (min) FAME Suggested by the Library Search Routine Peak Area % 52.66 Tetradecanoic, C14:0 0.73 59.55 Hexadecanoic, C16:0 8.38 60.45 Hexadecenoic, C16:1 26.80 63.15 Hydroxy octadecanoic#, C18:0 6.66 65.84 Heptadecanoic, C17:0 0.88 66.57 Octadecanoic, C18:0 1.98 66.70 Octadecenoic, C18:1 52.03 67.88 Octadecdienoic, C18:2 2.19 70.04 Octadectrienoic, C18:3 0.36

# See text for more discussion The observed peak at 49.3 min is assigned to the antioxidant BHT added to the

esterified oil. The remaining nine peaks in Figure 6.2 are assigned to one C14, two

C16, one C17, four C18 and one hydroxy C18 FAMEs. The tetradecanoic ME C14:0,

the heptadecanoic ME, C17:0, octadecanoic (stearic) ME, C18:0, octadecdienoic

(linoleic) ME, C18:2 and octadectrienoic ME, C18:3 (linolenic) ME, all represent

minor components at peak area percentages of around 1-2%. The two saturated MEs

hexadecanoic (palmitic) ME, C16:0 and hydroxy-octadecanoic (hydroxy-stearic)

ME, HO-C18:0 are observed at peak area percentages of 8% and 7% respectively.


The two largest peaks in the trace are assigned to octadecenoic (oleic) ME, C18:1 at

52% and hexadecenoic (palmitic) ME, C16:1 at 27%.

The EI mass spectrum observed for the peak in Figure 6.2 at a retention time of

63.15 min shows a good match with both that of hydroxy-octadecanoic as well as for

the hydroxy-FAME of this acid. The GC-MS library search routine suggests the

candidates are hydroxy stearic acid with a confidence level of 89% and hydroxy

stearic acid methyl ester with 87% confidence level. However, since a FA is not

expected to be observed in the esterified sample nor operate in the GC column, we

assign it to the FAME.

Interestingly, in the ESI FTICR mass spectrum of the esterified extract of macadamia

oil in Figure 4.10 methyl hydroxy stearate has been assigned with a relative intensity

of 11.4%.

6.4- A Summary of the Positive-ion and Negative-ion ESI FTICR-MS and GC-MS of the Hydrolysed and Esterified Neat Processed Macadamia Oil

In this section, a summary of the common fatty acid substituents on the acylglycerols

as determined by positive-ion and negative-ion ESI FTICR-MS and GC-MS of the

hydrolysed and esterified neat processed macadamia oil is presented and discussed.

We have deliberately chosen these above experiments and samples as they all

involve removal and identification of the FA side-chains from the acylglycerides; the

TAGs in particular. A comparison, with say the esterified extract of the oil, is not


necessarily expected to reflect the FA composition of the TAG molecules in the oil

sample and this is what is measured in the literature GC-MS experiment.[196]

Figure 6.3 shows a comparison of the FA ions observed in the positive-ion (Tables

4.3 & 4.4) and negative-ion (Table 5.9) ESI FTICR-MS as well as the GC-MS

analysis (Table 6.1) of the hydrolysed and esterified neat processed macadamia oil.

Table 6.3 lists the percentages of the fatty acid components used in Figure 6.3. These

results are also compared with the GC-MS measurement by Cavaletto on macadamia

oil reported in 1980.[196]


Comparison of FTICR of Hydrolysed Oil (+ and -), Esterified Oil (+)and GC-MS TIC of the Present Study and the Literature

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0C

14:0

C14

:1

C15

:0

C15

:1

C16

:0

C16

:1

C17

:0

C17

:1

C18

:0

C18

:1

C18

:2

C18

:3

C19

:1

C20

:0

C20

:1

C20

:4

Fatty Acids

Rel

ativ

e Pe

ak In

tens

ity %

Hydrolysed PositiveHydrolysed NegativeEsterfied PositiveGC-MS Present StudyGC-MS Literature

Figure 6.3- A comparison of the FA ions observed in the positive-ion (Tables 4.3 & 4.4) and negative-ion (Table 5.9) FTICR-MS as well as the GC-MS analysis (Table 6.1) of the hydrolysed and esterified neat processed macadamia nut oil. GC-MS literature values are from reference 190.


Table 6.3- A comparison of the FA ions observed in the positive-ion (Tables 4.3 & 4.4) and negative-ion (Table 5.9) FTICR-MS as well as the GC-MS analysis (Table 6.1) of the hydrolysed and esterified neat processed macadamia nut oil.


ESI/FTICR-MS Hydrolysed Oil

Positive-ion

ESI/FTICR-MS Hydrolysed oil Negative-ion

ESI/FTICR-MS Esterified oil Positive-ion

GC-MS Esterified oil‡

GC-MS Literature‡

C14:0 Not observed* 2.1 Not observed 1.4 Not observed C14:1 Not observed Not observed Not observed Not observed Not observed C15:0 Not observed Not observed Not observed Not observed Not observed C15:1 Not observed Not observed Not observed Not observed Not observed C16:0 5.2 8.7 3.0 10.9 7.4 C16:1 17.2 21.1 20.2 21.5 18.4 C17:0 Not observed Not observed 1.6 Not observed Not observed C17:1 Not observed Not observed 3.4 Not observed Not observed C18:0 1.6 2.5 Not observed 4.7 2.8 C18:1 61.1 61.6 61.7 58.4 64.9 C18:2 7.9 2.8 6.7 1.9 1.5 C18:3 1.6 Not observed Not observed Not observed Not observed C19:1 3.2 Not observed Not observed Not observed Not observed C20:0 Not observed Not observed Not observed 1.2 Not observed C20:1 2.3 2.0 3.6 Not observed 2.3 C20:4 Not observed Not observed Not observed Not observed 1.9

* Less than the limit of detection Peak intensities % is used for FTICR-MS and peak area % is used for GC-MS Lit. (Cavaletto, Ref. 190) results. ‡ Only fatty acid components are used in calculations and comparisons; fatty acids containing additional oxygen atom are not included.


For this figure, any ions that show evidence of hydration, e.g. C18H31O3¯ are not

included. Unlike the intensity values for the FAs presented the mass spectrometry

tables, the peak intensities in Figure 6.3 have been normalised to 100% for the

various FAs and FAMES.

Figure 6.3 highlights several interesting results from the ESI FTICR MS and GC-MS

experiments discussed previously in this and preceding chapters. If one can directly

correlate the ion concentrations with those of the corresponding acids in the oil then

there is an excellent correlation observed for the FA composition of the oil using

these experiments; all of which involve oil derivatisation. All five experiments show

the major FA component of the processed macadamia oil as oleic acid C18:1 at 62%

±2%, follow by palmitoleic acid C16:1 at 20% ±2% and palmitic acid C16:0 at 7%

±3% where the error is one standard deviation. This is quite a remarkable result

given the literature measurement was nearly 30 years ago.

The minor acids observed such as the C14:0 (myristic), C17:0, C17:1, C18:0 (stearic)

C18:2 (linoleic), C19:1 and C20:0 (eicosanoic), C20:1 (eicosenoic) and C20:4 are

significant in Figure 6.2 since for them to be observed they must be present in the

TAG molecules in reasonable concentrations. The minor acid components C18:0,

C18:2 and C20:1 are detected in several experiments but some acids, e.g. C17:1 and

C20:4 are only observed in a single mass spectrometry experiment.

A comparison of the percentage of the FAME components resulting from GC-MS

analysis of the esterified macadamia oil experiments (Table 6.1) with those obtained

in the ESI FTICR-MS experiment on the esterified macadamia oil (Table 4.4) shows

a remarkably good correlation. For example, the ESI FTICR-MS ratio of oleic /

palmitoleic methyl esters is 100:33 and in the GC-MS experiment it is 100:37


(58:22). This result gives confidence in the use of the high resolution ESI FTICR

mass spectrometer for the direct semi-quantitative analysis of the macadamia nut oil

(excluding isomers).

One important difference between the GC-MS and FTICR-MS results is that the

former does not indicate the presence of the FA components bearing one or more

additional oxygen components on the acyl chain of the acylglycerols or the free FAs.

To observe these compounds, a higher temperature GC column and/or derivatisation

using trimethylsilane would need to be incorporated into the GC-MS experiment.

Interestingly free FAs are readily observed in the ESI FTICR-MS experiments that

demonstrates the simplicity of the sample preparation in the latter technique for

detailed semi-quantitative analysis of such plant oils.

6.5- Conclusions

GC-MS provides useful information regarding the overall fatty acid composition of

the acylglycerols of the macadamia oil samples through esterification and analysis of

the neat oil. A similar experiment carried out on the methanol extract of the oil can

reveal information about the FA composition of the MAGs and DAGs in the oil.

Finally, a careful GC-MS examination of the methanol extract of the oil could reveal

limited information about the composition of the free FAs in the oil. The

chromatography and the availability of large mass spectral libraries is a major

advantage of this method as it allows the direct confirmation of FAME isomers and

hence the FAs present in the TAG molecules in the oil.


In general the results of this chapter demonstrate that high resolution ESI FTICR-MS

is a much more sensitive technique than GC-MS which allows the observation of all

the pristine trace components in the oil as well as the production of an accurate FA

profile of the oil when the correct oil samples are analysed. In the latter case, this

involves sampling the hydrolysed or esterified neat oil in either positive-ion or

negative-ion mode. Because the pristine oils can be analysed on ESI FTICR-MS

experiment whereas only the esterified oil can be examined by GC-MS, the former

technique reveals the presence of many more minor components in the macadamia

oil; In particular, the observation of free FAs, MAGs and DAGs and hydrated

derivatives of these materials.

The results reported in this chapter give confidence in the use of the high resolution

ESI FTICR mass spectrometer for the direct semi-quantitative analysis of the FAs in

macadamia nut oil and indeed other plant oils. All five experiments in Figure 6.3

show a very similar fatty acid profile for the processed macadamia oil and this is

quite a remarkable result given the GC-MS literature measurement was obtained

nearly 30 years ago.

However it should be remembered that the ESI FTIC-MS experiments as presented

to date do not distinguish FA isomers and for this chromatography is required. In

Chapter 7, off-line ESI FTICR-MS of HPLC fractions of the methanol extract of

processed macadamia oil is presented and discussed.


Chapter 7

7. Off-line ESI FTICR-MS of HPLC Fractions of the Methanol Extract

of Processed Macadamia Oil


7.1- Introduction

In this chapter the results of the HPLC separation and the associated FTICR mass

spectra of the methanol extract of the processed macadamia oil fractions are

presented and discussed.

As discussed earlier in section 1.5, previous studies have been carried out on the

application of HPLC in the separation and identification of the plant oil

ingredients,[79,179] particularly in combination with mass spectrometry.[213-215]

There are two major reasons behind the fraction collection experiment performed in

this present study.

First, online HPLC ESI FTICR-MS was not practical with the FTICR instrument

used. As Figure 2.11 shows, a FTICR-MS pulse sequence to obtain a broadband

mass spectrum takes about 5 seconds to complete. For this present analysis it was

found that an accumulation time in the hexapole ion guide of 3 to 4 seconds was

needed to obtain sufficient signal intensity for the analysis of the macadamia oil

samples. Furthermore, the scan is repeated 50 times to increase the signal/noise ratio

in the FT mass spectra. Overall, the accumulation of a spectrum could take up to 5

minutes. This time is too long for online HPLC ESI FTICR mass spectrometry

because during the 5 minute period, more than one compound might elute out of the

HPLC column and this is contrary to the degree of separation expected for this

experiment. The only practical option in this case was to collect the fractions and

analyse each fraction on the ESI FTICR mass spectrometer at a later time (off-line


mode). Newer instruments have been developed and used to perform online HPLC

FTICR-MS in the analysis of biochemical samples.[216]

Secondly, to overcome possible ion charge cloud effects in the FTICR cell the

methanol extract was fractionated to reduce the total number of ions with a wide

range of concentration (peak intensities) in the FTICR cell. The ion charge cloud

effect can result in a low signal to noise ratio for the low-concentration ions in the

FTICR cell (signal suppression).[193,217,218]

In addition, possible isomers could be isolated and collected using HPLC technique.

The isomers of unsaturated fatty acids and acylglycerols can arise from different

double bond locations on the FA chain or different substitution positions of FA

substituents on the glycerol molecule.

For the experiments described in this chapter, 90 fractions of one minute duration of

the methanol extract of processed macadamia oil were collected using a dedicated

fraction collector (Section 2.3.2). The Majority of the collected fractions were

subsequently analysed in the ESI FTICR mass spectrometer in both negative- and

positive-ion modes without dilution. The mass spectra obtained are discussed in

conjunction with the HPLC chromatograms in this chapter. A complete set of

positive- and negative-ion FTICR-MS data files can be found in Appendix DVD.

Note that the assignments in the tables are postulations and no further CID or

MS/MS analysis has been conducted to elucidate the chemical structures of the ions.


7.2- HPLC of the Methanol Extract of Processed Macadamia Oil

The HPLC chromatogram of the methanol extract of processed macadamia oil is

shown in Figure 7.1. The specific ion assignments for the peaks shown in Figure 7.1

are based on the ESI FTICR mass spectra discussed in more detail in section 7.3 to

7.7 and listed in Tables 7.1 to 7.6. The HPLC experiment was carried out seven times

to verify the reproducibility of the separation. The retention times were found to be

reproducible to 0.1%.

Considerable time was invested in the HPLC method development in an attempt to

separate the large number of compounds in plant oils. In fact this method enables

FFAs and MAGs to be separated from DAGs. This method allows oil samples from

different batches of the same oil to be clearly differentiated, but no chromatographic

technique will separate all the components in plant oil samples. In order to improve

the separation of compounds in plant oils several different sample preparation

methods and HPLC techniques would have to be used, even then it is not possible to

separate all of the compounds into single peaks.

Assuming that the eluted components possess similar solubilities in the mobile phase

and separation occurs exclusively according to the hydrophobicities, it is expected

that the relatively less hydrophobic molecules such as free FAs, highly unsaturated

esters and MAGs will elute earlier on a C18 reversed-phase column, followed by the

more hydrophobic DAGs and TAGs. A number of peaks, assigned to residual TAG

molecules, appear in the chromatogram at retention times 78-84 minutes; the

methanol extraction procedure (section 2.2.1) removes the majority of the TAGs

from the processed oil sample. On further examination of the peak shapes when the


chromatogram was expanded, it was observed that each major peak did contain in

general more than one chemical compound, in fact several compounds.

Three distinct regions are visible in Figure 7.1, containing free FAs and MAGs,

DAGs and TAGs. Each region is discussed in more detail in sections 7.3 to 7.7.

0 20 40 60 80Retention time (min)

29.33

54.4458.16

52.01

62.61

80.83

23.91



29.33

54.4458.16

52.01

62.61

80.83

23.91

Figure 7.1- HPLC chromatogram of the methanol extract of processed macadamia oil.

The first four eluted HPLC fractions are considered as blanks for the ESI FTICR

mass spectrometry analyses. The HPLC solvent system (methanol-water, 0.05% in

acetic acid) contains a number of compounds which contribute to the peaks in the

blank FTICR mass spectra (Figures 7.16 and 7.17). These background peaks are

excluded from the mass spectra of the macadamia oil samples. The positive-ion and

Glycerol Palmitoleate-oleate Glycerol dioleate

Oleic acid

Glycerol Oleate

Glycerol dipalmitoleate

Triacylglycerols Free fatty acids and MAGs

Diacylglycerols


negative-ion FTICR mass spectra of the blank fractions are shown in Figures 7.16

and 7.17 in Section 7.8.

7.3- ESI FTICR-MS of the HPLC Fractions 19 to 31 of the Methanol Extract of Processed Macadamia Oil

Figure 7.2 shows an expansion of the HPLC chromatogram of the methanol extract

of processed macadamia oil (Figure 7.1) at retention times 19 to 31 minutes.

20.90

22.51

23.90

25.02

29.30

Retention time (min)20 25 30

20.90

22.51

23.90

25.02

29.30

Retention time (min)20 25 30

Figure 7.2- Expansion of HPLC chromatogram of the methanol extract of processed macadamia nut oil (Figure 7.1) at retention times of 19 to 31 minutes.

The more intense peaks in Figure 7.2 are observed at retention times 20.9 minutes

(fractions 20 and 21), 23.9 minutes (fractions 23 and 24) and 29.3 minutes (fractions

29 and 30). Figure 7.3 shows the positive-ion FTICR mass spectra and Figure 7.4


shows the negative-ion FTICR mass spectra of the HPLC fractions 19 to 31

(retention times 19 to 31 minutes).

407.3152327.2286305.2468

379.2834

353.2668

277.2144 299.1956

377.2651

m/z270 280 290 300 310 320 330 340 350 360 370 380 390 400

Fraction 19

Fraction 31

Fraction 25

407.3152327.2286305.2468

379.2834

353.2668

277.2144 299.1956

377.2651

m/z270 280 290 300 310 320 330 340 350 360 370 380 390 400

Fraction 19

Fraction 31

Fraction 25

Figure 7.3- Positive-ion ESI FTICR mass spectra of the HPLC fractions 19 to 31 of the methanol extract of processed macadamia nut oil.


Fraction 31

281.2486

253.2173

Fraction 19

m/z250 300 350270260 290280240 320310 330 340 360

317.2264

Fraction 31

281.2486

253.2173

Fraction 19

m/z250 300 350270260 290280240 320310 330 340 360

m/z250 300 350270260 290280240 320310 330 340 360

317.2264

Figure 7.4- Negative-ion ESI FTICR mass spectra of the HPLC fractions 19 to 31 of the methanol extract of processed macadamia nut oil. Table 7.1 lists the assignments of the observed peaks in the positive-ion FTICR mass

spectra of HPLC fractions 19 to 31 and Table 7.2 lists the assignments of the

observed peaks in the negative-ion FTICR mass spectra of HPLC fractions 19 to 31.


Table 7.1. Assignment of the observed mass spectral peaks in the positive-ion ESI-FTICR mass spectra of the HPLC fractions 19 to 31 in Figure 7.3.

Fraction Observed Mass m/z

Assigned Sodium Adduct Cation

19 377.2655 C21H38O4 Glycerol linoleate 20 277.2127 C16H30O2 Palmitoleic acid 20 299.1956 C18H28O2 Octadecatetraenoic acid 21 277.2142 C16H30O2 Palmitoleic acid 21 353.2668 C19H38O4 Glycerol palmitate 21 379.2820 C21H40O4 Glycerol oleate 22 353.2665 C19H38O4 Glycerol palmitate 22 379.2809 C21H40O4 Glycerol oleate 23 379.2809 C21H40O4 Glycerol oleate 24 379.2823 C21H40O4 Glycerol oleate 25 379.2830 C21H40O4 Glycerol oleate 26 379.2834 C21H40O4 Glycerol oleate 27 305.2457 C18H34O2 Oleic acid 27 379.2840 C21H40O4 Glycerol oleate 28 305.2455 C18H34O2 Oleic acid 28 349.2928 C21H40O2 Heneicosenoic acid 28 361.2720 C21H38O3 Hydroxy heneicosadienoic acid 28 379.2840 C21H40O4 Glycerol oleate 29 305.2455 C18H34O2 Oleic acid 29 361.2726 C21H38O3 Hydroxy heneicosadienoic acid 29 381.2993 C21H42O4 Glycerol stearate 30 381.3001 C21H42O4 Glycerol stearate


Table 7.2. Assignment of the observed mass spectral peaks in the negative-ion ESI-FTICR mass spectra of the HPLC fractions 19 to 31 in Figure 7.4.


Assigned Anion

19 277.2169 C18H29O2‾, Linolenate, C18:3 20 253.2173 C16H29O2‾, Palmitoleate, C16:1 20 277.2177 C18H29O2‾, Linolenate, C18:3 21 253.2173 C16H29O2‾, Palmitoleate, C16:1 22 253.2173 C16H29O2‾, Palmitoleate, C16:1 22 279.2329 C18H31O2‾, Linoleate, C18:2 23 279.2341 C18H31O2‾, Linoleate, C18:2 24 279.2328 C18H31O2‾, Linoleate, C18:2 25 255.2329 C16H31O2‾, Palmitate, C16:0 26 255.2328 C16H31O2‾, Palmitate, C16:0 27 255.2329 C16H31O2‾, Palmitate, C16:0 27 281.2486 C18H33O2‾, Oleate, C18:1 28 255.2328 C16H31O2‾, Palmitate, C16:0 28 281.2486 C18H33O2‾, Oleate, C18:1 29 281.2486 C18H33O2‾, Oleate, C18:1 30 281.2486 C18H33O2‾, Oleate, C18:1

Figure 7.2 shows a peak at retention time 20.9 minutes. Analogous peaks are

observed in the positive-ion FTICR mass spectra of fractions 20 and 21 at m/z

277.2127 and m/z 277.2142 in Figure 7.3 that are assigned to the palmitoleic acid

sodium adduct cation in Table 7.1. In addition, analogous peaks are observed in the

negative-ion FTICR mass spectra of fractions 20 and 21 at m/z 253.2184 in Figure

7.4 that are assigned to palmitoleate anion in Table 7.2.

The peak observed at retention time 23.9 minutes in the expanded HPLC

chromatogram in Figure 7.2 shows an analogous peak in fractions 23 and 24 in the

positive-ion FTICR mass spectra in Figure 7.3 at m/z 379.2834 that is assigned to

glycerol oleate in Table 7.1.

The peak at retention time 29.3 minutes in the chromatogram Figure 7.2 shows an

analogous peak in the positive-ion FTICR mass spectrum of fraction 29 at m/z


305.2455 in Figure 7.3 that is assigned to oleic acid sodium adduct cation in Table

7.1. In addition, in the negative-ion FTICR mass spectra in Figure 7.4, a peak

appears in fractions 28 and 29 at m/z 281.2492 that is assigned to oleate anion in

Table 7.2.


Figure 7.5 shows an expansion of the high performance liquid chromatogram of the

methanol extract of processed macadamia oil (Figure 7.1) at retention times 32 to 50

minutes.

30 35 40 45 50

32.94

37.2738.85

42.6744.29

46.79

47.71

Retention time (min)

30 35 40 45 50

32.94

37.2738.85

42.6744.29

46.79

47.71

Retention time (min) Figure 7.5- Expansion of the HPLC chromatogram of the methanol extract of processed macadamia nut oil (Figure 7.1) at retention times 32 to 50 minutes.


7.4.1- Fractions 32 to 35

In the positive-ion FTICR mass spectra of the fractions 32 to 35 all the observed

peaks appear weaker than the background peaks.

In the negative-ion FTICR mass spectra of HPLC fractions 32 to 35, a weak peak

appears at m/z 283.2653 in fractions 34 and 35 that is assigned to stearic acid anion.

Figure 7.6 shows the negative-ion FTICR mass spectra of fractions 34 and 35.

223.0198 255.2330

283.2642

305.0243 325.1846 339.2007365.2676

223.0197255.2332

283.2642309.2804

319.2403 345.2577 377.0852

220 240 260 280 300 320 340 360 380m/z

(a)

(b)

223.0198 255.2330

283.2642

305.0243 325.1846 339.2007365.2676

223.0197255.2332

283.2642309.2804

319.2403 345.2577 377.0852

220 240 260 280 300 320 340 360 380m/z

(a)

(b)

Figure 7.6- Negative-ion ESI FTICR mass spectra of the HPLC fractions: (a) fraction 34 and (b) fraction 35 of the methanol extract of processed macadamia nut oil.

The peak at m/z 283.2642 is assigned to stearic acid anion, C18H35O2¯ (C18:0), and

the peak at m/z 309.2804 is assigned to eicosenoic acid anion, C20H37O2¯ (C20:1).


Figure 7.7 shows the positive-ion FTICR mass spectra of the HPLC fractions 36 to

38 of the methanol extract of processed macadamia oil and Table 7.3 lists the


assignments of the observed peaks in the positive-ion FTICR mass spectra of HPLC

fractions 36 to 38.

603.4734 619.4605

643.4551

671.4958

705.5375

721.5297

601.4527

619.4606647.4999

673.5103

689.5142

705.5376 721.5298

601.4527

605.4836

629.4817 647.5010

673.5110

689.5150

705.5380721.5301

600 620 640 660 680 700 720 740m/z

(a)

(c)

(b)

603.4734 619.4605

643.4551

671.4958

705.5375

721.5297

601.4527

619.4606647.4999

673.5103

689.5142

705.5376 721.5298

601.4527

605.4836

629.4817 647.5010

673.5110

689.5150

705.5380721.5301

600 620 640 660 680 700 720 740m/z

603.4734 619.4605

643.4551

671.4958

705.5375

721.5297

601.4527

619.4606647.4999

673.5103

689.5142

705.5376 721.5298

601.4527

605.4836

629.4817 647.5010

673.5110

689.5150

705.5380721.5301

600 620 640 660 680 700 720 740m/z

(a)

(c)

(b)

Figure 7.7- Positive-ion ESI FTICR mass spectra of the HPLC fractions: (a) fraction 36, (b) fraction 37 and (c) fraction 38 of the methanol extract of processed macadamia nut oil.




36 619.4605 C35H64O7 Glycerol dihydroxy dipalmitoleate 36 671.4958 C39H68O7 Glycerol dihydroxy dilinoleate 37 601.4527 C35H62O6 Glycerol hydroxy palmitoleate palmitolenate 37 673.5063 C39H70O7 Glycerol dihydroxy oleate linoleate 37 705.5376 C40H74O8 Glycerol trihydroxy oleate nonadecenoate 38 605.4836 C35H66O6 Glycerol hydroxy palmitoleate palmitate


7.4.3- Fractions 38 to 40 Figure 7.8 shows the positive-ion FTICR mass spectra of the HPLC fractions 38 to


assignments of the observed peaks in Figure the positive-ion FTICR mass spectra of

HPLC fractions 38 to 40.

601.4563

605.4836

629.4817 647.5030

673.5110

689.5150

705.5334721.5311

605.4872

629.4836 647.5011

673.5119

689.5131

605.4874

627.4689631.5015

647.5023

655.5054

673.5119

689.5134

600 620 640 660 680 700 720 740m/z

(a)

(c)

(b)

601.4563

605.4836

629.4817 647.5030

673.5110

689.5150

705.5334721.5311

605.4872

629.4836 647.5011

673.5119

689.5131

605.4874

627.4689631.5015

647.5023

655.5054

673.5119

689.5134

600 620 640 660 680 700 720 740m/z

(a)

(c)

(b)

(a)

(c)

(b)

Figure 7.8- Positive-ion ESI FTICR mass spectra of the HPLC fractions, (a) fraction 38, (b) fraction 39 and (c) fraction 40 of the methanol extract of processed macadamia nut oil.



In the negative-ion FTICR mass spectra of HPLC fractions 38 to 40, only fraction 40

contains peaks with intensities above the background level. Figure 7.9 shows the

negative-ion FTICR mass spectrum of the HPLC fraction 40 of the methanol extract

of processed macadamia oil.

Table 7.5 lists the assignments of the observed peaks in the negative-ion FTICR

mass spectrum of HPLC fraction 40.

Table 7.5. Assignment of the observed mass spectral peaks in the negative-ion ESI-FTICR mass spectrum of the HPLC fraction 40 in Figure 7.9.



38 605.4836 C35H66O6 Glycerol hydroxy palmitoleate palmitate 38 629.4817 C37H66O6 Glycerol hydroxy oleate palmitate 38 689.5150 C43H70O5 Glycerol diarachidonate 39 673.5063 C39H70O7 Glycerol dihydroxy oleate linoleate 39 605.4836 C35H66O6 Glycerol hydroxy palmitoleate palmitate 40 605.4836 C35H66O6 Glycerol hydroxy palmitoleate palmitate 40 655.5054 C39H68O6 Glycerol hydroxy dilinoleate 40 671.4959 C39H68O7 Glycerol dihydroxy dilinoleate 40 673.5063 C39H70O7 Glycerol dihydroxy oleate linoleate


Assigned Negative Ion

40 617.4314 C37H61O7 Glycerol dihydroxy linolenate palmitolenate 40 683.4381 C41H63O8 Glycerol trihydroxy linolenate arachidonate


617.4314

683.4381

600 620 640 660 680 700 720 740m/z

617.4314

683.4381

600 620 640 660 680 700 720 740m/z

Figure 7.9- Negative-ion ESI FTICR mass spectrum of the HPLC fraction 40 of the methanol extract of processed macadamia oil.



48 of the methanol extract of processed macadamia oil.

661.5551

657.5162

607.5044

633.5147

673.5124

m/z600 650640630620610 670 690680660 700

659.5363

Fraction 44

Fraction 48

661.5551

657.5162

607.5044

633.5147

673.5124

m/z600 650640630620610 670 690680660 700

659.5363

661.5551

657.5162

607.5044

633.5147

673.5124

m/z600 650640630620610 670 690680660 700

m/z600 650640630620610 670 690680660 700

659.5363

Fraction 44

Fraction 48

Figure 7.10- Positive-ion ESI FTICR mass spectra of the HPLC fractions 44 to 48 of the methanol extract of processed macadamia oil.


Table 7.6 lists the assignments of the observed peaks in the positive-ion FTICR mass

spectra of HPLC fractions 44 to 48.


Figure 7.11 shows the negative-ion FTICR mass spectra of the HPLC fractions 44 to


assignments of the observed peaks in Figure 7.11.



44 629.4752 C37H66O6 Glycerol hydroxy oleate palmitate 44 657.5202 C39H70O6 Glycerol hydroxy oleate linoleate 44 673.5124 C39H70O7 Glycerol dihydroxy oleate linoleate 45 631.4987 C37H68O6 Glycerol hydroxy oleate palmitoleate 45 629.4752 C37H66O6 Glycerol hydroxy oleate palmitate 45 657.5199 C39H70O6 Glycerol hydroxy oleate linoleate 45 659.5360 C39H72O6 Glycerol hydroxy oleate oleate 45 673.5124 C39H70O7 Glycerol dihydroxy oleate linoleate 45 675.5281 C39H72O7 Glycerol dihydroxy dioleate 46 631.4987 C37H68O6 Glycerol hydroxy oleate palmitoleate 46 633.5185 C37H70O6 Glycerol hydroxy oleate palmitate 46 655.4984 C39H68O6 Glycerol hydroxy linoleate linoleate 46 657.5185 C39H70O6 Glycerol hydroxy oleate linoleate 46 659.5360 C39H72O6 Glycerol hydroxy dioleate 46 675.5244 C39H72O7 Glycerol dihydroxy dioleate 47 631.4987 C37H68O6 Glycerol hydroxy oleate palmitoleate 47 635.5287 C37H72O6 Glycerol hydroxy stearate palmitate 47 657.5162 C39H70O6 Glycerol hydroxy oleate linoleate 47 659.5218 C39H72O6 Glycerol hydroxy dioleate 47 661.5550 C39H74O6 Glycerol hydroxy oleate stearate 48 635.5304 C37H72O6 Glycerol hydroxy stearate palmitate 48 661.5550 C39H74O6 Glycerol hydroxy oleate stearate


673.5209

697.5669

647.4976

695.5555711.5540

645.4973

669.5363

680 690 700 710 720m/z670660650640

Fraction 44

Fraction 48

673.5209

697.5669

647.4976

695.5555711.5540

645.4973

669.5363

680 690 700 710 720m/z670660650640

Fraction 44

Fraction 48

Figure 7.11- Negative-ion ESI FTICR mass spectra of the HPLC fractions 44 to 48 of the methanol extract of processed macadamia oil.

Table 7.7. Assignment of the observed mass spectral peaks in the negative-ion ESI-FTICR mass spectra of the HPLC fractions 44 to 48 in Figure 7.11.


Assigned Anion

44 645.4973 C39H65O7 Glycerol dihydroxy linoleate linolenate 44 647.4976 C39H67O7 Glycerol dihydroxy dilinoleate 44 669.5363 C39H73O8 Glycerol hydroxy oleate dihydroxy stearate 45 645.4950 C39H65O7 Glycerol dihydroxy linoleate linolenate 45 669.5396 C39H73O8 Glycerol hydroxy oleate dihydroxy stearate 45 695.5555 C41H75O8 Glycerol trihydroxy arachidate linoleate 45 711.5540 C41H75O9 Glycerol tetrahydroxy arachidate linoleate 46 669.5312 C39H73O8 Glycerol hydroxy oleate dihydroxy stearate 46 695.5453 C41H75O8 Glycerol trihydroxy arachidate linoleate 47 673.5213 C41H69O7 Glycerol dihydroxy arachidonate oleate 47 697.5624 C41H77O8 Glycerol trihydroxy arachidate oleate 48 647.5013 C39H67O7 Glycerol dihydroxy dilinoleate 48 673.5209 C41H69O7 Glycerol dihydroxy arachidonate oleate 48 697.5669 C41H77O8 Glycerol trihydroxy arachidate oleate






6050 55

50.65

51.96

54.45 58.05


6050 55


6050 55 6050 55

50.65

51.96

54.45 58.05

Figure 7.12- Expansion of the HPLC chromatogram of the methanol extract of processed macadamia nut oil at retention times 50 to 60 minutes.

Four major peaks are observed in the HPLC chromatogram in Figure 7.12 at

retention times approximately 51, 52, 54 and 58 minutes.


60 corresponding to Figure 7.12 and Table 7.8 lists the assignments of the observed

peaks in Figure 7.13.


561.4554

661.5547

643.5359

615.5078

587.4741

689.5858

560 580 600 620 640 660 680 700m/z

Fraction 50

Fraction 60

561.4554

661.5547

643.5359

615.5078

587.4741

689.5858

560 580 600 620 640 660 680 700m/z

Fraction 50

Fraction 60

Figure 7.13- Positive-ion ESI FTICR mass spectra of the HPLC fractions 50 to 60 of the methanol extract of processed macadamia oil. Four distinct peaks are observed in the FTICR mass spectra in Figure 7.13 in

fractions 50, 51, 54 and 57 that is consistent with the observed peaks in the

chromatogram in Figure 7.12.

According to the assignments in Table 7.8, most of the observed peaks at retention

times 50 to 60 minutes are assigned to DAGs such as glycerol dioleate (m/z

643.5354) and few have been assigned to hydroxy DAGs such as Glycerol hydroxy

dioleate (m/z 659.5326).




A peak is observed in Figure 7.1 at retention time 62.6 minutes. Figure 7.14 shows

the positive-ion FTICR mass spectra of the HPLC fractions 60 to 63 of the methanol



50 561.4554 C33H62O5 Glycerol tetradecanoate palmitoleate 50 587.4738 C35H64O5 Glycerol dipalmitoleate 50 661.5547 C39H74O6 Glycerol hydroxy stearate oleate 51 561.4575 C33H62O5 Glycerol tetradecanoate palmitoleate 51 587.4741 C35H64O5 Glycerol dipalmitoleate 51 659.5363 C39H72O6 Glycerol hydroxy oleate oleate 52 587.4733 C35H64O5 Glycerol dipalmitoleate 52 613.4888 C37H66O5 Glycerol palmitoleate linoleate 52 639.5086 C39H68O5 Glycerol dilinoleate 52 659.5326 C39H72O6 Glycerol hydroxy oleate oleate 52 663.5629 C39H76O6 Glycerol hydroxy stearate stearate 52 689.5754 C41H78O6 Glycerol hydroxy oleate arachidate 53 589.4900 C35H66O5 Glycerol palmitate palmitoleate 53 615.5064 C37H68O5 Glycerol palmitoleate linoleate 53 639.5086 C39H68O5 Glycerol dilinoleate 54 589.4889 C35H66O5 Glycerol palmitate palmitoleate 54 615.5078 C37H68O5 Glycerol palmitoleate oleate 54 641.5207 C39H70O5 Glycerol oleate linoleate 55 615.5066 C37H68O5 Glycerol palmitoleate oleate 55 641.5191 C39H70O5 Glycerol oleate linoleate 56 617.5202 C37H70O5 Glycerol palmitate oleate 56 643.5354 C39H72O5 Glycerol dioleate 57 617.5226 C37H70O5 Glycerol palmitate oleate 57 643.5359 C39H72O5 Glycerol dioleate 58 617.5208 C37H70O5 Glycerol palmitate oleate 58 643.5355 C39H72O5 Glycerol dioleate 59 643.5340 C39H72O5 Glycerol dioleate 60 643.5337 C39H72O5 Glycerol dioleate


extract of processed macadamia oil. Table 7.9 lists the assignment of the observed

peak in Figure 7.14.

645.5562

671.5712

685.5493

643.5337

630 640 650 660 670 680 690 700m/z

Fraction 60

Fraction 63

645.5562

671.5712

685.5493

643.5337

630 640 650 660 670 680 690 700m/z

630 640 650 660 670 680 690 700m/z

Fraction 60

Fraction 63



In the negative-ion FTICR mass spectra of the HPLC fractions 60 to 63 of the

methanol extract of macadamia oil, only background peaks are observed.



60 643.5337 C39H72O5 Glycerol dioleate 61 645.5562 C39H74O5 Glycerol oleate stearate 61 671.5712 C41H76O5 Glycerol arachidate linoleate 61 685.5493 C41H74O6 Glycerol hydroxy eicosenoate linoleate 62 671.5712 C41H76O5 Glycerol arachidate linoleate 63 645.5562 C39H74O5 Glycerol oleate stearate 63 671.5712 C41H76O5 Glycerol arachidate linoleate






75 80 85

79.50

80.22

80.76

81.30

82.67


75 80 85

79.50

80.22

80.76

81.30

82.67

Figure 7.15- Expanded HPLC chromatogram of the methanol extract of processed macadamia nut oil at retention times 77 to 83 minutes.

Four major peaks are observed in the chromatogram in Figure 7.15 at retention times

approximately 79, 80 and 81 minutes. We examine the FTICR mass spectra of the

HPLC fractions 77 to 83 for possible corresponding peaks.




assignment of the observed peak in Figure 7.16.

851.7301 879.7475

923.7823

871.7582

897.7740

921.7902

m/z850 860 870 880 890 900 910 920 930

Fraction 77

Fraction 83

851.7301 879.7475

923.7823

871.7582

897.7740

921.7902

m/z850 860 870 880 890 900 910 920 930

m/z850 860 870 880 890 900 910 920 930850 860 870 880 890 900 910 920 930

Fraction 77

Fraction 83





77 871.7582 C53H100O7 Glycerol dipalmitate hydroxy oleate 77 897.7740 C55H102O7 Glycerol dioleate hydroxy palmitate 77 921.7902 C58H106O6 Glycerol dioleate nonadecenoate 78 871.7582 C53H100O7 Glycerol dipalmitate hydroxy oleate 78 897.7740 C55H102O7 Glycerol dioleate hydroxy palmitate 78 921.7902 C58H106O6 Glycerol dioleate nonadecenoate 79 897.7740 C55H102O7 Glycerol dioleate hydroxy palmitate 79 923.7823 C57H104O7 Glycerol dioleate hydroxy oleate


In the negative-ion FTICR mass spectra of the HPLC fractions 77 to 83, the intensity

of the observed peaks are lower than the background peaks.

All of the observed peaks in the positive-ion FTICR mass spectra of the HPLC

fractions 77 to 83 are assigned to TAG molecules in Table 7.10. Additionally, a

number of TAG molecules with additional oxygen bearing functional groups are

assigned to the peaks in the HPLC fractions 77 to 83.

7.8- Positive- and Negative-ion FTICR Mass Spectra of the HPLC Blank Fractions of the Methanol Extract of Processed Macadamia Oil Figure 7.17 shows the positive-ion FTICR mass spectrum of the HPLC blank

fraction.

268.9991301.1401

312.0576

344.9841

413.2671

441.2145455.2234

525.1366

550.6269

250 300 350 400 450 500 550 600m/z

268.9991301.1401

312.0576

344.9841

413.2671

441.2145455.2234

525.1366

550.6269

250 300 350 400 450 500 550 600m/z Figure 7.17- Positive-ion FTICR mass spectrum of the HPLC blank fraction. See text for more discussion.


The peak at m/z 413.2671 in the blank spectrum could be assigned to C24H38O4Na+

cation (2 ppm). The absolute intensity of the peak at m/z 413.2671 is about 1.2×107

which is considered a weak peak.

The background noise is visible in Figure 7.17 due to the fact that the peaks are weak

and the auto-scale function of the software has automatically expanded the intensities

to the full-scale.

Figure 7.18 shows the negative-ion FTICR mass spectrum of the HPLC blank

fraction.

223.0213

255.2343

283.2657

305.0247

387.0281463.0191

545.0138 627.0305

250 300 350 400 450 500 550 600 650m/z

223.0213

255.2343

283.2657

305.0247

387.0281463.0191

545.0138 627.0305

250 300 350 400 450 500 550 600 650m/z Figure 7.18- Negative-ion FTICR mass spectrum of the HPLC blank fraction. The intensity of the main peak at m/z 255.2343 is about 5×107 that is considered a weak peak.

The peak at 255.2343 could be assigned to C16H31O2¯ (5 ppm) that could be

palmitate anion. As the solvent contains 0.05% (v/v) acetic acid, the source of the

palmitate anion could be the impurities in the acetic acid.


7.9- General Discussion:

Table 7.11 lists a summary of the assignments of the major FTICR-MS peaks

observed in HPLC fractions of the methanol extract of processed macadamia oil in

both positive- and negative-ion modes. Since the HPLC peaks are broad, some of the

assigned peaks are observed in more than one fraction. Furthermore, as each fraction

contains one minute elution off the column, all of the fractions contain more than one

compound. It should be noted that due to the complexity of the chemical composition

of the oil samples, it is not plausible to separate all of the components in the oil

samples using reversed-phase HPLC. The lipid classes (such as FAs, MAGs, DAGs

and TAGs etc.) could be separated as groups. These groups could be separated and

analysed later using more sophisticated chromatography techniques. Time limitations

did not allow us to perform all of the possible separations in the present study.

Using acetic acid in the HPLC mobile phases suppresses the ionization of the free

FAs, hence assists in obtaining sharper peaks in the chromatograms. However, the

stationary phase of the Beckman C18 Ultrasphere reversed-phase column is not fully

end-capped, thus residual silanol groups are present on the surface of the stationary

phase material. These residual silanol groups will interact with the less hydrophobic

(more polar) molecules and cause significant peak broadening observed in this study.

In the negative-ion FTICR-MS, the anion is generated in a deprotonation reaction.

The proton is transferred from the neutral oil species to a solvent molecule in an

equilibrium reaction (affected by pH). As the HPLC solvent is 0.05% (v/v) in acetic

acid, the ionization equilibria of the weaker acids (such as hydroxyl groups on the

glycerol molecule) are pushed to the left. This suppresses the ionisation reactions of


the species with a proton on hydroxyl groups. As a result, in the negative-ion FTICR-

MS of the methanol extract of processed macadamia oil, only a few free fatty acids

are observed in Table 7.11.

In the positive-ion FTICR-MS, on the other hand, the attachment of sodium cation to

the non-bonding electrons on the oxygen atoms of free fatty acids and acylglycerols

generates the positive ion sodium adduct. As the attachment of sodium ion is a

simple dipole-dipole interaction and is not affected by the presence of acetic acid in

the solvent, positive ions are observed for free fatty acids and acylglycerols in Table

7.11.

According to Table 7.11, most of the compounds occur in more than one fraction. As

an example, the peak assigned to glycerol palmitate (m/z 353.2668) appears in

fractions 20 to 22 in Table 7.11. This is due to the fact that, in general, each peak

contains more than one compound, due to the isomers and co-eluting peaks[172]

(compounds with different chemical structure but with same retention times) in one

minute fraction collecting time.

Each fraction contains more than one compound due to the complex composition of

the macadamia oil samples. Some of the compounds have different structures but

similar retention times. In addition, some isomers are eluted at the same retention

times (fractions). For example, the peak assigned to glycerol oleate (m/z 379.2834)

appears in fractions 20 to 28 (19 to 28 minutes) in Table 7.11 that is an indication of

the existence of isomers. Several isomers of this compound could arise from different

double bond or side chain locations on the FA chain or different substitution sites on

carbon 1 or carbon 2 of the glycerol backbone (MAG with the substituent on carbon

3 is identical to the one with the substituent on carbon 1).


Table 7.11. Assignment of the major mass spectral peaks in the ESI-FTICR mass spectra of the HPLC fractions of the methanol extract of macadamia nut oil.

* See text for more discussion

The FTICR-MS analysis of the HPLC fractions 19 to 31 in positive-ion mode shows

five free FAs, MAGs and one hydroxy fatty acid (Table 7.1). In negative-ion mode

FTICR-MS of the fractions 19 to 31 shows only free fatty acids, mostly anions of

palmitic, palmitoleic, linolenic, linoleic and oleic acids (Table 7.2).

Observed in Fraction Numbers*

Assigned Compound

Assigned Positive-ion Mass

m/z

Assigned Negative-ion Mass

m/z Positive-

ion Negative-

ion

Palmitoleic acid C16:1 277.2144 253.2184 20, 21 20 to 22 Palmitic acid C16:0 279.2295 255.2341 - 25 to 28 Linolenic acid C18:3 301.2134 277.2169 19, 20 19, 20 Linoleic acid C18:2 303.2288 279.2340 22 to 24 22 to 24 Oleic acid C18:1 305.2468 281.2492 27, 28 27 to 32 Stearic acid C18:0 307.2627 283.2653 - 34, 35 Glycerol palmitoleate 351.2490 - 15 to 18 - Glycerol palmitate 353.2668 - 20 to 22 - Glycerol linoleate 377.2651 - 17 to 21 - Glycerol oleate 379.2834 - 20 to 28 - Glycerol eicosenoate 407.3145 - 29 to 32 - Glycerol heneicosapentaenoate 413.2668 - 27, 28 - Glycerol dipalmitoleate 587.4738 - 50 to 52 - Glycerol palmitoleate-palmitate 605.4872 - 39 to 41 - Glycerol palmitoleate-oleate 615.5078 - 53 to 55 - Glycerol palmitate-oleate 617.5202 - 56 to 58 - Glycerol hydroxy oleate-palmitoleate 631.4987 - 46, 47 - Glycerol hydroxy oleate-palmitate 633.5147 - 43 to 45 - Glycerol dioleate 643.5359 - 56 to 59 - Glycerol oleate-stearate 645.5562 - 61 to 63 - Glycerol linoleate-hydroxy oleate 657.5199 - 45 to 47 - Glycerol linoleate-hydroxy stearate 659.5360 - 45 to 47 - Glycerol oleate-hydroxy stearate 661.5534 - 47 to 50 - Glycerol eicosenoate-oleate 671.5712 - 61 to 63 - Glycerol hydroxy oleate-hydroxy linoleate 673.5119 - 39, 40 -

Glycerol palmitate-heptadecanoate- oleate 869.7438 - 69 to 72 - Glycerol hydroxy palmitoleate-oleate-linoleate 893.7718 - 68 to 70 -

Glycerol hydroxy palmitoleate-oleate-oleate 895.7503 - 72 to 76 -

Glycerol hydroxy palmitate-oleate-oleate 897.7740 - 77 to 79 - Glycerol hydroxy oleate dioleate 923.7823 - 79 -


The FTICR-MS analysis of the HPLC fractions 35 to 50 in both positive- and

negative-ion modes results in important points. First, most of the compounds

assigned to the FTICR peaks of fractions 35 to 50 (see Tables 7.2 to 7.6), contain

functional groups with additional oxygen atoms such as hydroxyl group.

Second, being able to separate a group of acylglycerols containing additional oxygen

atoms using HPLC is evidence that the redox reactions in the ESI chamber are not

the main origin of these compounds. It appears that the redox reactions in the ESI

source were not responsible for the observation of the acylglycerols with additional

oxygen atoms.

Third, as has been discussed elsewhere,[172] molecules with higher number of

hydroxy group are eluted faster on a C18 reversed-phase column due to their lower

hydrophobicity. As an example, the peak at m/z 711.5540 is observed in fraction 45

along with peaks at m/z 669.5312.

As explained in section 7.1, the separation in C18 column is based on the

hydrophobicity of the compounds being eluted. The less hydrophobic molecules

elute earlier and the more hydrophobic molecules elute later. This is evident in

Figure 7.11 and Table 7.7; peaks assigned to less hydrophobic molecules are

observed in earlier fractions and peaks assigned to more hydrophobic molecules are

observed in later fractions. As an example, the peak assigned to C33H62O5 (m/z

561.4554) appears in fraction 51, while the peak assigned to C39H72O5 (m/z

643.5337) is observed in fraction 60. Also, molecules containing hydroxyl groups

(such as C39H74O6, glycerol hydroxy stearate oleate, m/z 661.5547) elute faster on a non-

polar C18 column due to their lower hydrophobicity (more polar). Similar correlation

between HPLC retention times and the chemical structure of saturated, unsaturated


and oxygenated (hydroxy, keto and epoxy) fatty acids and their methyl esters on C18

reversed-phase HPLC column is reported by Lin et al.[172]

Similarly, as evident in Figure 7.14 and Table 7.9, DAG molecules are eluted in

accordance with their hydrophobicity. Less hydrophobic molecules (such as

C39H72O5 m/z 643.5337) are eluted in earlier fractions while more hydrophobic

molecules (such as C41H76O5 m/z 671.5712) are eluted in later fractions. Another

example is the peak assigned to C41H74O6 m/z 685.5493 that is eluted earlier in

fraction 61 due to the additional oxygen bearing functional group (such as hydroxy

group) that make the molecule less hydrophobic (more polar).

A comparison of the assigned peaks in the positive-ion FTICR mass spectrum of the

methanol extract of macadamia oil (Table 4.2) and the assigned peaks in the positive-

ion FTICR mass spectra of the HPLC fractions of the methanol extract of macadamia

oil (Table 7.11), shows that, as expected, most of the peaks observed in the FTICR

mass spectrum of the methanol extract of macadamia oil are observed in the FTICR

mass spectra of the HPLC fractions as well.

Few of the peaks that are assigned in Table 4.2 are not assigned in Table 7.11. This is

due to the fact that the minimum acceptable level of intensity of peaks in the FTICR

mass spectra of the HPLC fractions is elevated to exclude the peaks from the

background.

Fatty acids with odd number of carbon atoms have been observed in the methanol

extract of macadamia oil in low levels (see Table 4.2); however, they are not

assigned in Table 7.11 due to the fact that the intensities of the peaks assigned to


odd-numbered fatty acids are lower than the intensities of the peaks in the

background fractions.

A comparison between the assigned peaks in the negative-ion FTICR mass spectrum

of the methanol extract of macadamia oil (Table 5.5) with the assigned peaks in the

negative-ion FTICR mass spectra of the HPLC fractions of the methanol extract of

macadamia oil (Table 7.11) shows that, generally, in negative-ion mode, the peaks

generated by free fatty acids are strong. On the other hand, the peaks generated by

the acylglycerols in negative-ion mode are weak. In Table 7.11, none of the

acylglycerol peaks are assigned in negative-ion mode. This is a consequence of the

blank fraction peaks that are subtracted from the sample spectra. The minimum

acceptable intensity of the peaks was about 2% in the FTICR mass spectra of the

methanol extract of macadamia oil (Table 5.5), whereas it is elevated to about 10%

(absolute intensity of about 5×107) for the FTICR mass spectra of the HPLC

fractions (Table 7.11), resulting in no acylglycerol peaks being assigned, as their

peaks are weaker than 5×107.

In a previous study of HPLC of free FAs and FA methyl esters on the same C18

reversed-phase column used in the present study, Lin and coworkers have reported

the retention times for standard mixtures of free FAs and FA methyl esters in a range

of solvent systems and chromatographic conditions.[172]

Table 7.12 shows a comparison of the retention times of the free FAs obtained in

present study and the previous study by Lin and coworkers.[172]


Table 7.12. A comparison of the retention times of the HPLC analysis of the methanol extract of macadamia nut oil with standard FA solutions analysed on same column in a previous study.

The fraction numbers obtained in the present study are in excellent agreement with

the retention times reported in a study carried out on standard FA mixtures on a

similar column approximately 8 year earlier.[172] For example, the previously

reported retention times for oleic acid and palmitoleic acid are 27.1 and 20.6 minutes

respectively, whereas in Table 7.11 oleic acid is assigned to the peak observed in

fractions 27 and 28 minutes that includes timeframe of 26 to 28 minutes and

palmitoleic acid is assigned to the peaks observed in fractions 20 to 21 minutes that

includes the timeframe of 19 to 21 minutes.

It should be noticed that the fraction numbers in this present study do not reflect the

exact retention times, as the fractions are collected within a period of one minute.

The fatty acids elute in this period of time and their actual retention time could lay

anywhere within the measured fraction collection time frame.

Fatty Acid Retention Times Obtained in

Present Study (minutes)

Retention Times Reported in Previous Study

(minutes)

Palmitoleic acid C16:1 19 to 22 20.6 Palmitic acid C16:0 24 to 28 25.4 Linolenic acid C18:3 18 to 20 18.4 Linoleic acid C18:2 21 to 24 22.4 Oleic acid C18:1 26 to 28 27.1 Stearic acid C18:0 33 to 35 34.0


Chapter 8

8. Conclusions and Future Work


8.1- General Conclusion for this Study

The results from this study indicate that important analytical results can be obtained

by researchers in non-specialised Lipidomics laboratories. With the development of

new high resolution mass spectrometry instrumentation such as the “Orbitrap” one

might expect that studies such as the one reported in this thesis will become more

common.

In the food science and health industries, plant oils are viewed to be important. We

have shown in this study that our simple and straightforward analysis of such

material is readily achieved by ESI FTICR-MS. Research into biosynthesis and

metabolism of such seed oils will be stimulated by analytical results such as those

presented in this thesis.

This study shows that high-resolution ESI FTICR-MS can be used to postulate the

chemical composition of the various free fatty acids and mono-, di- and tri-

acylglycerols in plant oils. ESI FTICR-MS analysis of the methanol extract of plant

oil allows a profile of the free fatty acids in the oil to be readily and clearly produced

with minimal sample preparation. Methanol extract of plant oils could contain

various fatty acids that are not necessarily generated by simple hydrolysis of the

acylglycerols present in the oil. They could be generated through multistep biological

reactions carried out by enzymes such as lipoxygenase from microorganisms

including bacteria and fungi residing in or on the surface of the macadamia nut.

Furthermore, FTICR-MS analysis of the methanol extract of macadamia oil,

esterified oil, hydrolysed oil and the esterified methanol extract of the oil permits


clear assessments of the efficiency of extraction, esterification and hydrolysis

procedures respectively.

In addition, in the FTICR spectrum of the oil, various constituents of the oil are

observed in the same spectrum. These constituents include FAs, MAGs, DAGs and

TAGs. Although the intensities of the FTICR-MS peaks are not very well related to

the concentration of the species in the sample, the intensities of similar compounds in

macadamia oil are used to estimate the relative concentrations of the respective

species.

We observe for the first time, using this technique, the presence of fatty acids and

acylglycerols containing one or more additional oxygen atoms which may be in the

form of hydroxy, hydroperoxy, peroxy, oxo (ketone or aldehyde functionalities),

hydroxy and/or epoxy substituents in the macadamia oil. We also observe for the

first time the presence of a number of unusual free fatty acids, mono- and di-

acylglycerols containing an odd number of carbon atoms in the oil. The origin of the

odd-numbered free fatty acids could be the enzymes activities in the oil or the

industrial refining processes.

In this study, compound containing elements other than C, H and O are reported in

macadamia oil samples, including compounds containing nitrogen, phosphorus and

sulphur, using molecular formula generating routines in Bruker software that

calculates and suggests the possible molecular formulas combined with a particular

observed peak.

This study demonstrates the higher sensitivity of ESI FTICR-MS compared to GC-

MS for analysing low level compounds in plant oils. In FTICR-MS of plant oils


many more compounds are observed compared to GC-MS analysis. The simplicity of

the sample preparation when combined with the results from the high resolution high

mass accuracy ESI FTICR-MS experiments on the macadamia oil indicate that this

method is a powerful tool that can be used for the analysis of trace compounds in

lipids including plant oils and animal fats.

Compared to GC-MS technique, FTICR-MS requires simpler sample preparation

steps in the analysis of plant oils. The sample would need to be dissolved in preferred

solvent and introduced to the FTICR mass spectrometer using ESI source. However,

analyzing isomers in FTICR-MS needs application of a chromatographic separation

technique such as HPLC.

The continuous capillary-skimmer potential control feature enabled us to observe the

effect of the kinetic energy on the structure and the FTICR mass spectra of the

generated molecular ions. By varying the applied capillary-skimmer potential

difference, various fragments of the investigated molecules were generated. The

observed fragments provided additional information regarding the molecular formula

of the ions.

Positive-ion and negative-ion FTICR mass spectrometry produce valuable

complementary information in regard to the molecular formula of the chemical

content of the analysed lipid samples. The obtained information was used in

conjunction with the Kendrick mass tables to reject or accept the molecular formulas

proposed by the molecular formula generation routine of Bruker software.

HPLC fraction collection combined with FTICR-MS improves the sensitivity,

selectivity and signal to noise levels due to the lower number of compounds in each


HPLC fraction, resulting in lower ion-suppression and cloud effects in the FTICR

cell when analyzing fractions compared to the whole oil sample and the methanol

extract of macadamia oil.

The difficulties in the application of FTICR mass spectrometry in the analysis of

lipids include the inability of the technique to resolve isomers such as structural and

stereo-isomers.

8.2- Future Work

We show in this present work, for the first time, that off-line HPLC when combined

with ESI FTICR-MS is readily able to distinguish several isomeric fatty acids, mono-

and diacylglycerols in a methanol extract of the macadamia oil. To differentiate and

structurally characterize isomeric species some form of chromatography (CE, GC or

HPLC) and chemical derivatization must be used prior to any tandem CID or photo-

dissociation studies.

A possible development of application of FTICR-MS of plant oils is to produce high-

resolution libraries of the plant oil finger prints due to the high resolution and high

sensitivity of this technique. These libraries could be used in food and oil industry to

monitor the quality of plant oils and assess possible adulterations or contaminations.

Due to the applied hexapole delay in Bruker APEX II used in this study, the HPLC

separation and fraction collection was carried out in offline mode. New FTICR-MS

instruments with shorter hexapole delays or with different accumulation techniques


could be used to reduce the duration of each single analysis. This could lead to

application of online HPLC ESI FTICR-MS experiments.

A future project in this field could be the application and development of separation

techniques in conjunction with FTICR-MS technique to elucidate the structure of the

wide range of the compounds in plant oils including free fatty acids, fatty acid esters,

MAGs, DAGS, TAGs, waxes and trace level compounds in the oils. This could

involve the development of on-line HPLC-FTICR-MS techniques to aid in the

identification of chemical compounds in the oils. HPLC techniques with optimized

higher separation capabilities could be used to separate the isomers further apart.

Another wide range of future study could be aimed to separate acylglycerols present

in the oil and analyse the various acyl groups substituted on the glycerol backbone.

This includes identification of the acyl substituents and location of the acyl

substituents on the glycerol backbone (1,2 or 1,3 substitution) and steric

configuration of the chiral acylglycerols (R and S configuration), location of the

possible double bonds and branches on the acyl substituents.

A possible future application of high resolution FTICR mass spectrometry in this

field could be the online quality control of the trace level (potentially harmful)

byproducts produced during the refining processes in oil processing industries. The

final products could be routinely analysed for the level of undesirable compounds in

the oil such as trans fatty acids or oxidation products. The industrial oil processing

systems could then be optimised based on the feedbacks received from the high

resolution FTICR mass spectrometry laboratory. This application could lead to an

improvement in the quality of the oil processing industries that would enhance the


consumable products in terms of trace level potentially harmful compounds in a wide

range of food products.

The high resolution FTICR mass spectrometry could be applied in future study of the

products of the hydration reaction of carbon double bonds on the fatty acid chains of

acylglycerols. Such studies could investigate whether the hydroxyl groups are

produced in the hydration reaction on the carbon double bond or could be a

byproduct of the hydrolysis reaction we perform on the oil samples.

Another future study in the field of GC-MS analysis of plant oil samples could be

separation, derivatisation and analysis of the highly oxygenated acylglycerols that

can provide the food and oil industries with valuable information regarding the

reactions and products of plant oils oxidation and rancidity.

The oxidation of lipids plays an important role in the food industries, as the oxidation

of lipids can produce unpleasant compounds in the food product. By preparing

libraries of high resolution FTICR mass spectra of lipids and their oxidation

products, one can determine the level of oxidation and rancidity occurred in the lipid

sample in a particular set of conditions. This could lead to the design of optimized

and more environmentally friendly transport, storage and refrigeration systems that

could lead to higher quality products and could save the food industries time, energy

and money.

A potential future study would be the investigation of the possibility of oxidation-

reduction reactions occurring in the electrospray source which could be responsible

for some of the observed acylglycerol molecules with additional oxygen atoms or

oxygen bearing functional groups in this present study. Various separation and


spectrometric techniques such as GC, HPLC, CID, MS/MS and MSn, FTIR,

FTNMR, ESR and other methods could be used to elucidate the chemical structures

of these unusual compounds in the lipid samples such as animal fat and plant oils.

Applying higher magnetic fields such as 15 T FTICR-MS would increase the

resolution, mass accuracy and sensitivity (S/N ratio). This would enable high-

resolution (broadband) spectra to be obtained routinely. Achieving high resolution

(broadband) would enable one to observe the isotopes in more detail.

Further future work can include development of specialised instruments to carry out

all the necessary separations and spectrometric analyses of the lipids including plant

oils and animal fats. The development of Lipidomics methods could benefit the food

and health industries by investigating possible adulterations or contaminations of the

lipids prior to use.

In the field of organic and bioorganic research of lipids, assessment of the degree of

reaction completion, determination of the reaction products under a particular set of

physical conditions, effect of various catalysts on the kinetics, route and efficiency of

the reactions are among the possible applications of the FTICR mass spectrometry

technique. The optimum physical conditions and the most suitable catalysts to

achieve the highest efficiency of the organic synthesis and bioorganic reactions could

be applied using the information obtained in the FTICR-MS investigations.

Lipidomics techniques could be used in forensic and criminal investigations by

setting up a library of human lipids. Very high sensitivity and very low detection

limits of the FTICR-MS technique allows forensic and crime-scene investigators to

analyse human body fat left at the crime scene on a single human hair or in a finger


print. These evidences could be used in conjunction with proteomics, DNA analysis

and other forensic methods to provide additional information and evidence for

complicated crime scenes.


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