msc in analytical sciences - uvahigh performance liquid chromatography with precolumn...

MSc in Analytical Sciences

Amino acid analysis: comparison and validation of gas

chromatography with mass spectrometric detection and

liquid chromatography with triple quadrupole mass

spectrometric detection. Application in mouse

embryonic fibroblast NIH-3T3 cells.

Master Research Thesis

Alexandra Koukou

2013-2014

1 | 132

2 | 132

MSc Chemistry

Analytical Sciences

Master Thesis

Amino acid analysis: comparison and validation of gas

chromatography with mass spectrometric detection and

liquid chromatography with triple quadrupole mass

spectrometric detection. Application in mouse embryonic

fibroblast NIH-3T3 cells.

by

Alexandra Koukou

June 2014

Supervisor:

dr H. Lingeman

Daily Supervisor:

P. Krumpochova

Vrije Universiteit Amsterdam

3 | 132

Abstract

The comprehension of metabolic profiling plays an important role in enhancement of

knowledge about prognosis and diagnosis of disease. Amino acids are one of the mainly

building blocks of cells among carbohydrates, lipids and nucleic acids. Therefore, the

fluctuating blood concentration of amino acids may provide mechanistic insights into diseases.

This thesis describes two methods for the determination of amino acids in standard solutions.

One with gas chromatography (GC) coupled with mass spectrometric detection (MS) and one

with liquid chromatography (LC) coupled with triple quadrupole (QQQ) mass spectrometric

detection (MS). In both methods the sample pretreatment and derivatization steps were

performed by Phenomenex EZ faast amino acid analysis kit for gas and liquid chromatographic

analysis respectively and last 7 minutes per sample.

For the GC-MS method three different internal standards were tested; Norvaline, amino acids

labeled with 13C and amino acids labeled with 13C/15N. The detection limit was ranging between

0.1 and 2.4 μM in the selected ion monitoring mode. The analysis time was approximately 7

minutes per sample.

For the LC-MS method no internal standard was used. The detection limit was 0.25 μM in the

multiple reaction monitoring mode. The analysis time was approximately 25 minutes per

sample.

Finally, the GC-MS method with amino acids labelled with 13C/15N was applied on mouse

embryonic fibroblast NIH-3T3 cells in order to study the cancer mechanism.

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Acknowledgements

First and foremost, I would like to thank Petra Krumpochova, my daily supervisor, for made

me feel so welcome from the first moment, for believing in me and for all the time she invested

in my project.

Secondly, I want to thank Ben Bruyneel for being always next to me when I needed his help,

answering the questions that I had.

Another important contribution in the attainment of my project was my cooperation with Azra

Mujic-Delic, who providing me with biological samples.

Furthermore, I would like to thank Dr. Henk Lingeman for being my supervisor and for

providing me with lot of useful ideas and feedback. I am also grateful about Prof. Wilfried

Niessen and Prof. Manfred Wuhrer, who contributed with their own way in this project being

always willing to share their knowledge.

Last but not least, big thanks to everyone in the group of Bioanalytical Chemistry of VU

University. You listened to me and you advised me about both relevant and irrelevant subjects

concerning my project.

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Contents

Abstract ................................................................................................................................................... 3

Acknowledgements ................................................................................................................................. 4

Abbreviations .......................................................................................................................................... 7

1. Theoretical ......................................................................................................................................... 10

1.1 Introduction ............................................................................................................................. 10

1.2 Historical Background ............................................................................................................ 12

1.3 A novel breakthrough approach .............................................................................................. 14

1.4 Derivatization .......................................................................................................................... 15

1.5 Types of Derivatization............................................................................................................ 15

1.6 Derivatization modes ............................................................................................................... 20

1.7 Chloroformate derivatization .................................................................................................. 23

2. Experimental ................................................................................................................................. 24

2.1 Chemicals ................................................................................................................................ 24

2.2 Kit components ........................................................................................................................ 25

2.3 Additional equipment............................................................................................................... 26

2.4 Storage and Stability ............................................................................................................... 27

2.5 Cleaning .................................................................................................................................. 27

2.6 Preparation of Eluting Medium ............................................................................................... 28

2.7 General Procedure for GC-MS experiments ........................................................................... 29

2.8 General Procedure for LC-MS experiments ............................................................................ 29

2.9 Instruments Settings ................................................................................................................ 30

2.10 Biological Samples ................................................................................................................ 31

3. GC/MS Results ................................................................................................................................... 32

3.1 Full-SCAN GC/MS .................................................................................................................. 32

3.2 Selected Ion Monitoring (SIM) for standards amino acids ..................................................... 35

3.3 Repeatability (within a day) .................................................................................................... 36

3.4 Intermediate Precision ............................................................................................................ 37

3.5 Calibration Curves .................................................................................................................. 38

3.6 Limit of Detection (LOD) and Linearity Range ....................................................................... 40

3.7 Amino acids labeled with 13C .................................................................................................. 41

3.8 Selected Ion Monitoring (SIM) for amino acids labeled with 13C ........................................... 42

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3.9 Amino acids labeled with 13C-15N ............................................................................................ 43

3.10 Selected Ion Monitoring (SIM) for amino acids labeled with ........................................... 44

13C-15N ........................................................................................................................................... 44

3.11 Biological Samples ................................................................................................................ 45

4. LC/MS Results .................................................................................................................................... 49

4.1 Manual Tuning for Probe and Turbo Ionspray Gas................................................................ 49

4.2 Flow injection analysis (FIA) .................................................................................................. 49

4.3 Full-SCAN LC/MS ................................................................................................................... 51

4.4 Multiple Reaction Monitoring (MRM) .................................................................................... 53

4.5 Calibration Curves .................................................................................................................. 57

4.6 Relative Standard Deviation (RSD %) .................................................................................... 59

4.7 Limit of Detection (LOD) and Linearity Range ....................................................................... 60

5. Conclusion and Perspectives ............................................................................................................. 61

References ............................................................................................................................................. 63

Appendix I: Calibration Curves of the amino acids using GC/MS .......................................................... 66

Appendix II: Average mass spectra of standard and label amino acids. Highlighted ions were used as

target ions to a single ion monitoring mode for labeled 13C amino acids. ............................................ 75

Appendix III: Overlapping target ions’ chromatograms for the labeled 12C/13C SIM method. Ions with

highest abundance were chosen for further quantification. ................................................................ 83

Appendix IV: Average mass of standard and label amino acids. Highlighted ions were used as target

ions to a single ion monitoring mode for ladeled 13C-15N amino acids. ................................................ 92

Appendix V: Overlapping target ions’ chromatograms for the labeled 12C /13C -14N /15N SIM method.

Ions with highest abundance were chosen for further quantification. .............................................. 101

Appendix VI: Optimization of probe and heater gas flow ................................................................... 110

Appendix VII: Verification of compound/source-dependent parameters .......................................... 113

Appendix VIII: Determination of parent and product ions.................................................................. 116

Appendix IX: Calibration Curves of the amino acids using LC/MS ...................................................... 125

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Abbreviations

3T3 : 3 day transfer, inoculum 3 x 105 cells

AA : Amino acids

AAA : a-Aminoadipic acid

ABA : a-Aminobutyric acid

aILE : allo-isoleucine

ALA : Alanine

ASN : Asparagine

ASP : Aspartic acid

ATP : Adenosine triphosphate

BGE : Background electrolyte

CAD : Charged aerosol detection gas

C-C : Cysteine-cysteine

CDs : Cyclodextrins

CE : Capillary electrophoresis

CE : Collision energy

CO2 : Carbon dioxide

CoA : Coenzyme A

CTAB : Cetyltrimethylammonium bromide

CUR : Curtain gas

CXP : Collision cell exit potential

DMAB : 4-(N, N’-dimethylamino)-benzoic acid

DMAP : 4-dimethylaminopyridine

DMEM : Dulbecco’s Modified Eagle Medium

DP : Declustering potential

DTAB : Dodecyltrimethylammonium bromide

ECACC : European collection of cell cultures

EP : Entrance potential

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FIA : Flow injection analysis

FP : Focusing potential

GLN : Glutamine

GLU : Glutamic acid

GLY : Glycine

GS/MS : Gas chromatography coupled with spectrometric detection

H : Probe’s horizontal position

HCl : Hydrogen chloride

HGF : Heater gas flow

HIS : Histidine

HPLC : High performance liquid chromatography

HYP : Hydroxyproline

ILE : Isoleucine

IS : Ionspray voltage

L : Probe’s lateral position

LC/MS : Liquid chromatography coupled with spectrometric detection

LEU : Leucine

LLOL : Lower limit of Linearity

LOD : Limit of detection

LYS : Lysine

MET : Methionine

MRM : Multiple reaction monitoring

NEB : Nebulizer gas

OPA : ortho-Phthalaldehyde

ORN : Ornithine

PAS : p-aminosalicylic acid

PBS : Phosphate Buffered Saline

PFTBA : Perflurotributylanime

PHA : Phenylalanine hydroxylase

PHE : Phenylalanine

PMSF : Phenyl methyl sulfonyl flouride

PRO : Proline

RIPA : Radioimmunoprecipitation assay

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RSD % : Percentage relative standard deviation

SAR : Sarcosine

SDS : Sodium deoxycholate

SER : Serine

SIM : Selected ion monitoring

SPE : Solid phase extraction

TEM : Temperature

THR : Threonine

TRP : Tryptophan

TYR : Tyrosine

UV : Ultraviolet

VAL : Valine

βAIB : β-Aminoisobutyric acid

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1. Theoretical

1.1 Introduction

Over the past few decades numerous sample preparation, separation and detection techniques

have been developed and have been applied in the bioanalytical field. The most challenging

class of that field is the amino acids for two reasons. Firstly, amino acids have large differences

in their chemical structures so their separation

becomes too difficult since they range from non-polar

too highly polar amino acids. Secondly, amino acids

play an important role in human metabolism and the

metabolic profiling. Not only they are cell signaling

molecules, but they also have non-protein

functions.[1-2] In the body amino acids can be utilized

as an energy source for major organs such as brain,

liver, muscle and adipose tissue in most organisms.

Catabolism of amino acids consists of two steps. In

the first step the a-amino group is removed and

converted into urea. In the second step the remaining

carbon skeleton is converted into pyruvate,

oxaloacetate, fumarate, succinyl CoA, a-keto-

glutarate, acetyl CoA or acetoacetyl CoA which are

further utilized as energy source in different metabolic

pathways. Amino acids as a source of energy are divided in two groups: ketogenic amino acids

that can be degraded into acetyl CoA or acetoacetyl CoA and form ketone bodies; and

glucogenic amino acids that can be degraded into pyruvate, oxaloacetate, fumarate, succinyl

CoA or a-keto-glutarate and they are resulting to glucose formation. Of the twenty fundamental

amino acids, tryptophan, phenylalanine, tyrosine and isoleucine are both ketogenic and

glucogenic. Lysine and Leucine are only ketogenic and the rest are only glucogenic. [3-4]

Tyrosine and tryptophan are used as precursors for the catecholamines (dopamine,

norepinephrine and epinephrine) and the monoamine serotonin respectively. [5] Serine and

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alanine are utilized in the first step of ceramide de novo synthesis. [6] Glycine, Glutamine,

Aspartic acid and Glutamic acid take a part in nucleotide catabolism. [4] Moreover, in clinical

chemistry the increased amino acid levels in plasma are used as biomarkers for inborn errors of

metabolism. For example, high concentration of phenylalanine indicates that the newborns

suffer from phenylketonuria, a disease which characterized by a mutation in the gene

phenylalanine hydroxylase (PHA), prevents the transmutation of phenylalanine to tyrosine and

causes mental retardation. [7] It can be easily concluded that the quantitative analysis of amino

acids is required for the understanding of chemical reactions that happened into human body

and the prevention of human diseases.

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1.2 Historical Background

Ion-exchange chromatography with postcolumn ninhydrin detection

In 1958, Moore, Stein and Spackman introduced an automatic amino acid analyzer based on

ion exchange column chromatography after postcolumn ninhydrin derivatization.

A Li- or Na- based cation exchange system is used for complex or simpler physiological

samples respectively. The proper pH, temperature and cation strength lead to the desirable

separation. When ninhydrin reacts with the amino acid the reagent has purple color, shows an

absorption at 570nm and the detection limit is around 10 pmol for the majority of them. In case

of imino acids like proline the reagent has yellow color, shows an absorption at 440 nm and the

detection limit is 50 pmol. The linearity range is obtained between 20-500 pmol. The analysis

time ranges from 60 to 120 minutes.[8-9]

High performance liquid chromatography with precolumn Ortho-phthalaldehyde

derivatization and fluorometric detection

Roth, in 1972, used strongly acidic cation exchange column followed by postcolumn oxidation

with sodium hypochlorite. As precolumn derivatization used ortho-Phthalaldehyde (OPA) and

thiol compound, such as N-acetyl-L-cysteine and 2-mercaptoethanol. Once again the proper pH

and cation strength lead to the desirable separation. OPA reacts with primary amines in the

presence of thiol compound and forms fluorescent isoindole products but it does not react with

secondary amines, such as proline. In the last case, the reaction is accomplished because of the

oxidation with sodium hypochlorite. The reagent that pass through the fluorometric detector

has an excitation wavelength at 348 nm and an emission wavelength at 450nm. The detection

limit is around 10 pmol and the linearity range is obtained between 10 pmol-10 nmol. The

analysis time ranges from 40 to 70 minutes.[10]

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Gas chromatography with ethyl chloroformate derivatization and flame ionization

detection

In 1990, Hušek introduced a method for the simultaneously analysis of 20 protein amino acids.

Specifically, he combined the rapid gas chromatography (analysis time 10 minutes) with a short

derivatization technique with chloroformate (20 minutes). He could even achieved shorter

analysis time without sacrificing the resolution, using shorter and narrower bore capillaries.

Ethyl chloroformate reacts with aqueous amino acid solutions and forms N (O, S)-

ethoxycarbonyl ethyl esters. Masami, Yamamoto and Kiyama used isobutyl chloroformate

instead of ethyl chloroformate but one extra step was necessary for the esterification of the

carboxylic group.[1,11-12]

Capillary zone electrophoresis with indirect absorbance detection

Kuhr and Yeung, in 1988, introduced a quite fast detection technique without derivatization

step. Fluorophore or chromophore presence in the background electrolyte (BGE) was used for

the indirect detection of the amino acids.[13] 18-20 amino acids were separated in 20-40

minutes using PAS and DMAB (10 mM) as a suitable background electrolytes, the pH in a

range <10.3-11.2>; concentration of Mg2+ = 0.05 mM; DTAB= 0.25 mM or CTAB

0.05mM.[14] However, with one single run lots amino acids cannot be efficiently separated

from the baseline whereas Leu and Ile could not be separated at all. Seven years later, Lee and

Lin tried to add cyclodextrins (CDs) to BGE, which contribute to better selectivity of CE. As a

result, in 35 minutes they managed to separate all the twenty amino acids, except from Leu and

Ile (pH 11 using; BGE PAS (10 mM); a-CD (20 mM)). Variants in the BGE can lead to better

resolution but longer analysis time and the substitution of a-CD to β-CD can lead to Leu and

Ile separation but inferior results.[15]

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1.3 A novel breakthrough approach

It can be agreed that amino acids are the “Achilles heel” on the separation analysis. During the

last decades lot of techniques were introduced, but none of them could dominate. Among the

techniques that are mentioned above, the first had low sensitivity, poor resolution, high costs

and long analysis time. The second had the same drawbacks but better sensitivity. The third had

a quite good analysis time but there were problems with the derivatization steps. And the last

one did not require derivatization however the separation was not really efficiently. All these

drawbacks can be overcome with the EZ-faast kit that Phenomenex introduced for simple and

fast sample preparation (7 minutes) and simultaneously determination of 384 amino acids.

The procedure starts with a solid phase extraction as a clean-up step during which the amino

acids and be extracted from the complex physiological fluids really fast. Afterwards,

derivatization occurs, emulsify is formed and the upper organic phase is collected and analyzed

on GC/MS or LC/MS system. In total, the sample preparation and analysis time for GC/MS is

around 13 minutes and for LC/MS around 32 minutes.[16-17]

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1.4 Derivatization

Derivatization can be defined as a technique that modifies a compound, which is not suitable

for the specific analytical procedure, to a product (derivative) with more suitable analytical

properties as the original compound.[18]

The use of this technique can improve the following:

i. Suitability (e.g. solubility, polarity, volatility):

In high performance liquid chromatography the main problem is the existence of

insoluble compounds in the mobile phase whereas in gas chromatography the existence

of nonvolatile compounds.

ii. Efficiency (e.g. polarity):

In many cases compounds interact with each other’s or with the column, causing

difficulties in the identification due to bad resolution and asymmetry of the peaks.

iii. Detectability (e.g. atomicity):

The analysis of smaller amounts of material requires an extension in the range of their

detectability.[19-21]

1.5 Types of Derivatization

There are three general types of derivatization that convert functional groups with active

hydrogens (-OH, -COOH, -NH, -NH2, and -SH groups) to derivatized products that can be

detected.

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Silylation

The silylation mechanism involves the replacement of the active hydrogens by substituted silyl

groups through a nucleophilic SN2 reaction.

where, X=halogen [21]

The most common reagents that are used for silylation derivatization are in the table below.[22]

Abbreviation Name Structure

BSA Bis-trimethylsilylacetamide

BSTFA Bis-trimethylsilyltrifluoroacetamide

MSTFA N-methyl-

trimethylsilyltrifluoroacetamide

TMCS Trimethylchlorosilane

TMSI Trimethylsilylimidazole

HMDS Hexamethyldisilzane

Table 1.5.1: Silylating Reagents.

Silylation is normally used in combination with gas chromatography to enhance the volatility

of the analytes.

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Alkylation ( Arylation )

The alkylation mechanism involves the replacement of the active hydrogens by aliphatic or

aliphatic-aromatic groups. In case of acids, this procedure is known as esterification and

contributes to better chromatograms.

RCOOH + PhCH2X → RCOOCH2Ph + HX

where, X=halogen or alkyl group R

The most common reagents that are used for alkylation derivatization are in the table below.[18]


PFBBr Pentafluorobenzyl bromide

BF3 Boron trifluoride

TBH Tetrabutylammonium hydroxide

BB Benzylbromide

DMF Dimethylformamide

DAM Diazomethane

Table 1.5.2: Alkylating Reagents.

Alkylation is used in combination with gas chromatography to improve the volatility of the

solutes as well as in liquid chromatography to improve the detectability on the separation

efficiency. In the last case UV absorbing or mass spectrometry sensitive reagents are used.

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Acylation

The acylation mechanism involves the replacement of the active hydrogens by acyl groups.

Therefore, compounds with –OH,–NH and –SH can be converted to esters, amides and

thioesters respectively.

where, R= acyl group[23]

The most common reagents that are used for acylation derivatization are in the table below.[18]


MBTFA N-methyl-bistrifluoroacetamide

PFPOH Pentafluoropropanol CF3CF2CH2OH

PFBCI Pentafluorobenzoyl Chloride

HFBI Heptafluorobutyrylimidazole

TFAA Trifluoroacetoic Anhydride CF3OCOCOCF3

Table 1.5.3: Acylating Reagents.

With respect to acylation, the same statements can be made as for alkylation reaction. They

are also used in combination with gas chromatography and liquid chromatography.

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The advantages and disadvantages of each type of derivatization are mentioned at the Table

1.5.4

Type of

derivatization

Advantages Disadvantages

Silylation 1. Suitable for a wide range

of compounds

2. Great variety of reagents

3. Easy preparation

1. Moisture sensitive reagents

2. Only aprotic organic solvents

can be used

Alkylation 1. Stable derivatives

2. Great variety of reagents

3. Reactions in a wide pH

range

1. Only suitable for amines and

acidic hydroxyls

2. Toxic reagents

Acylation 1. Stable derivatives

2. Good sensitivity

3. Can activate carboxylic

acids before the

esterification

1. Difficult preparation

2. Dangerous, smelly and

moisture sensitive reagents

3. By-products have to be

removed before analysis

Table 1.5.4: Advantages and disadvantages among derivatization types.

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1.6 Derivatization modes

The reactions that were mentioned in the above paragraph can be performed in three different

modes:[24-25]

Pre-column Mode

The compounds are derivatized before the analytical separation, usually manually but can also

be automated. After the derivatization the products are separated and detected.

The main advantages of pre-column derivatization are the freedom in choosing the most

optimal reaction conditions and the limited reagent consumption. The latter enables the use of

more expensive reagents and therefore improves the sensitivity because of the lower

background levels. However, the directly mixing of the reagent with the sample and the

resulting excess of derivatization reagent can be easily presented matrix effect.

Scheme 1.6.1: Pre-column derivatization.

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Post-column Mode

The compounds are derivatized after the separation and before the detection with the aid of a

second pump, so the reaction is fully automated. The automation is the main advantage of post-

column derivatization since it making the method reproducible and suitable for quantitative

analysis. Moreover, in comparison with pre-column derivatization there is less chance to appear

matrix effects because the components of the sample are separated earlier and there is no

directly mixing with the reagent. On the other hand, the improvement of selectivity is highly

unlikely due to the necessity of high and constant amount of reagent. An important requirement

on this mode is that the detection properties of the reagent have to be different compared with

the detection properties of the derivative.

Scheme 1.6.2: Post-column derivatization.

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On-column Mode

The less frequently used approach is the On-column mode. In this case the derivatization is

performed during separation. Particularly, the front-end of the capillary is used as a reaction

chamber. The main advantage is the increase in speed since it can be a whole automated

procedure. Moreover, the sample dilution is minimal, making this mode suitable for single-cell

analysis. On the other hand, the number of suitable reactions and procedures is rather limited

because the mixing of the analyte and the reagent plugs is obtained due to the difference of the

electrophoretic mobilities.[26]

Scheme 1.6.3: On-column derivatization.

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1.7 Chloroformate derivatization

The method that is used for the purpose of this project is based on a chloroformate derivatization

reagent, which can modify at the same time the amino and carboxylic groups of the amino acids

forming highly stable derivatives. It is a pre-column mode derivatization, where alkylation

occurs at the carboxylic group and acylation at the amino group.

The importance of this derivatization reagent can be concluded on the following:

The esterification of the carboxylic group occurs simultaneously with the protection of

the amino group. So, it is a single step reaction.

The extraction step can be avoided since the reaction can be performed in an aqueous

medium.

The reaction can be done at room temperature and mild acidic or basic conditions,

therefore no racemization phenomena can be occurred.

The reagent is accessible, cheap and easy to handle.

The reaction is extremely fast (approximately 60s) under continuously stirring.[27]

Therefore, it can be concluded that multiple derivatization represents a simple, useful and

effective technique for amino acid analysis and gave the “tinder” to search for new

derivatization reagents. Another example of this kind of derivatization is the derivatization with

diazomethane followed by acetic anhydride in the presence of 4-dimethylaminopyridine

(DMAP) and pyridine lead to derivatives β-amino acids.[28]

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2.Experimental

2.1 Chemicals

Amino acids were purchased from the Sigma-Aldrich (Stock No.AA-S-18). The concentration

of each amino acid is 2.5 μmoles per mL in 0.1 N HCl, except L-cystine, whose concentration

is 1.25 μmoles per mL.

COMPONENT MOL. Wt. μMoles/mL

L-Alanine 89.09 2.50

Ammonium chloride 53.49 2.50

L-Arginine 174.2 2.50

L-Aspartic acid 133.1 2.50

L-Cystine 240.3 1.25

L-Glutamic acid 147.1 2.50

Glycine 75.07 2.50

L-Histidine 155.2 2.50

L-Isoleucine 131.2 2.50

L-Leucine 131.2 2.50

L-Lysine 146.2 2.50

L-Methionine 149.2 2.50

L-Phenylalanine 165.2 2.50

L-Proline 115.1 2.50

L-Serine 105.1 2.50

L-Threonine 119.1 2.50

L-Tyrosine 181.2 2.50

L-Valine 117.2 2.50

Table 2.1.1: Stock No. AA-S-18.

However, the above standard did not include three proteinogenic amino acids. For those three

standard solutions were made, 2.5 μmoles per mL in water.

Component Supplier Mol. Wt. μMoles/mL

L-Glutamine 99% Sigma 146.14 2.50

L-Asparagine 98% Sigma 132.12 2.50

L-Tryptophan 98% Sigma/Bachem/99%

Merck

204.23 2.50

Table 2.1.2: Proteinogenic amino acids.

Moreover, MilliQ water, methanol (Baker HPLC grade) and ammonium formate, formic acid

ammonium salt (97% Aldrich/Sigma) were used for the HPLC experiments.

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2.2 Kit components

Kit components and materials are listed in Table 2.2.1 and Table 2.2.2.

Reagent Properties Ingredients Volume

Reagent 1 Internal Standard

Solution

Norvaline 0.2mM

N-propanol 10%

50 ml

Reagent 2 Washing Solution N-propanol 90 ml

Reagent 3A Eluting Medium

Component I

Sodium Hydroxide 60 ml

Reagent 3B Eluting Medium

Component II

N-propanol 40 ml

Reagent 4 Organic Solution I Chloroform 4 vials, 6 ml each

Reagent 5 Organic Solution II Isooctane 50 ml

Reagent 6 Re-dissolution Solvent Isooctane 80%

Chloroform 20%

50 ml

SD 1,2 & 3[1] Amino Acid Standard

Mixtures

Mix of amino acids 2 vials of each SD,2 ml each

Table 2.2.1: Reagents included at the EZ-faast kit.

Supplies Quantity

Sorbent tips in racks 4 x 96

Sample preparation vials 4 x 100

Microdispenser, 20-100 µL 1

Syringe, 0.6 mL 10

Syringe 1.5 mL 10

ZB-AAA 10m x 0.25mm Amino Acid Analysis GC

Column

1

Autosampler vials with inserts 4 x 100

FocusLiners 5

User Manual 1

Table 2.2.2: Supplies included at the EZ-faast kit.

[1] Only Standard 1 and 2 were used

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SD1: 23 amino acids, 200 nmoles/mL each

AAA ASP GLY LEU PHE THR

ABA βAIB HIS LYS PRO TYR

aILE C-C HYP MET SAR VAL

ALA GLU ILE ORN SER

SD2: Complementary amino acids not stable in acidic solution, 200 nmoles/mL each

ASN GLN TRP

2.3 Additional equipment

Other required materials are listed in Table 2.3.1.

Materials Supplier

1000µL - 200 µL pipette Gilson Pipetman Pipettes

200µL - 50 µL Gilson Pipetman Pipettes

100 µL - 20 µL Gilson Pipetman Pipettes

20 µL - 2 µL Gilson Pipetman Pipettes

Pipette tips Ultratip Greiner Bio-One

Vortex VWR International

Ultrasonic VWR International

4 µL Clear Vial, Screw Top

Hole Cap PTFE/Silicone Septa

Supelco (Sigma Aldrich)

Container for proper waste disposal Afval Energie Bedrijf (Gemeente Amsterdam)

Septa Thermogreen LB-2 Septa for Shimadzu

(conditioned) (Supelco/Sigma Aldrich)

Precolumn RP Phenomenex C18 4 x 2.00mm

Zorbax Eclipse XDB-C18, Rapid

resolution HT, 4,6x50mm,1,8 micron

Column

Agilent Technologies

Table 2.3.1: Extra supplies that were necessary and they were not included at the EZ-faast kit.

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2.4 Storage and Stability

Reagents 2, 3a, 5 and 6 should be stored at room temperature. At the same conditions should

be also stored the methanol and the mixture of ammonium formate in water and methanol.

Whereas, the reagents 1, 3B and 4 should be stored at 4 o C. Last but not least, all the standards

solutions should be stored at -20 o C.

All the components have one year shelf life when they stored at recommended conditions.

2.5 Cleaning

A propanol: water mixture (1:2, v/v) should be used to flush the plastic syringes that are used

for the SPE cleaning steps.

In addition, the Drummond Dialamatic Microdispenser should be cleaned with isopropanol:

acetone mixture (1:1, v/v) at the end of the experiments.

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2.6 Preparation of Eluting Medium

The Eluting Medium should be prepared fresh each day before the beginning of the experiment.

The volume is proportional of the number of samples. A combination of three parts of reagent

3A with two parts of reagent 3B consist the Eluting Medium, which should be stored at room

temperature during the day and the remaining mixture should be discarded at the end of the day.

The following table shows the necessary volume of each medium regard to the number of

samples.

Number of Samples Reagent 3A (µL) Reagent 3B (µL)

2 300 200

4 600 400

7 900 600

12 1.5 1.0

14 1.8 1.2

19 2.4 1.6

24 3.0 2.0

29 3.6 2.4

34 4.2 2.8

39 4.8 3.2

44 5.4 3.6

49 6.0 4.0

Table 2.6.1: The ratio between Reagent 3A and 3B.

P a g e 29 | 132

2.7 General Procedure for GC-MS experiments

Firstly, 100µL of sample was mixed with Reagent 1 in order to adjust pH and add internal

standard -Norvaline. The mixture was slowly passed through sorbent tip and further on washed

with Reagent 2. Thereafter, 200µL of freshly prepared Eluting medium was used to elute

sample and sorbent from the tip. In the next step derivatization reagent (Reagent 4) was added

to the mixture and the sample was vigorously vortexed. In the last step 100µL of Reagent 5

containing isooctane was added to sample in order to separate different phases after vortexing.

Top organic phase was transferred to a fresh vial, evaporated under Nitrogen stream and re-

suspended in Isooctane chloroform mixture.

2.8 General Procedure for LC-MS experiments

The procedure is identical with the previous except of re-dissolving step where the sample was

re-suspended in 1:2 ammonium formate in water: ammonium formate in methanol instead of

Reagent 6.

P a g e 30 | 132

2.9 Instruments Settings

GC/MS

A QP2012 plus GC/MS (Shimadzu) is used with an automatic injector AOC-2Oi and an auto

sampler AOC 2Os. The column was ZB-AAA 10m x 0.25mm Amino Acid Analysis GC

Column.

For the GC helium is used as carrier gas at flow rate of 2ml/min, split injection ( 250 ° C ) and

the oven temperature program was 30 ° C/ min from 110 ° C to 320 ° C.

The MS ion source temperature was 240 ° C, the interface 320 ° C and the scan range was 45-

450 m/z. Finally, the MS was tuned and calibrated using Perflurotributylamine (PFTBA) every

day.

LC/MS

A PE Sciex API 3000 (AB SCIEX) triple quadrupole mass spectrometer coupled to an Agilent

1100 Series LC system. The latter consist of a G1322A Degasser, a G1376A CapPump, a

G1367A Wpals, a G1330B ALSTherm and a G13116A Colcom.

P a g e 31 | 132

2.10 Biological Samples

For the purpose of this experiment biological samples were performed by Azra Mujic-Delic.

The procedure that she follows is describing below and it has three stages:

Cell culture:

Mouse embryonic fibroblast NIH-3T3 cells, obtained from European collection of cell cultures

(ECACC), were cultured in the Dulbecco’s Modified Eagle Medium (DMEM) supplemented

with a 10% bovine serum, penicillin (50 IU/ml) and streptomycin (50 mg/ml) in 5% CO2

atmosphere at 37 °C. Stable clones of NIH 3T3 expressing US28 receptor or empty vector were

kept under a selective pressure of G418 (400 mg/ml) in the culture medium to ensure expression

of US28 in all growing cells.

Determination of amino acid uptake and excretion:

Cells at the density of 75.000/well were plated in a 6 well dish and incubated in growth

medium for 24hrs in 5% CO2 atmosphere at 37 °C. After 24hrs cell growth medium was

replaced with 0.5% bovine serum -containing medium to synchronize. After another 24hrs

growth medium was exchanged for medium with 10% bovine serum. Since the change of

medium supernatant and cell lysate were collected (time 0, 24, 48, 72 and 96 hours), amino

acid analysis in the growth media and protein concentration determination was followed. In

fact, 20L of extracellular media were used for further amino acid analysis.

Cell lysis and protein determination:

Cells were quickly washed with 0.5 ml of ice cold Phosphate Buffered Saline (PBS) and

treated with 100 μl of Radioimmunoprecipitation assay (RIPA) lysis buffer (PBS containing

Nonidet P-40 , 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS and 1mM PMSF

supplemented, 1 mM sodium orthovanadate with protease (Roche Applied Science,

Mannheim, Germany) inhibitor cocktails) on ice for 20 min. Cell extracts were clarified using

centrifugation (18,000 rpm/10 min/4°C). Protein content was determined using BCA Protein

Assay kit (Pierce).

P a g e 32 | 132

3. GC/MS Results

3.1 Full-SCAN GC/MS

In the first step sample containing standard amino acids was injected in order to collect data

about the target ions, the reference ions and the retention time. Based on the obtained data,

a selected ion monitoring method was created in order to increase method’s sensitivity.

Scheme 3.1.1: Chromatogram of standard derivatized amino acids using a MS SCAN mode.

Range adjusted at 45-450 m/z.

C=200μM/L

Split Ratio=30

Retention Time (min)

Time

Ab

un

dan

ce

P a g e 33 | 132

The target and the reference ions that are chosen for the determination of the amino acids are

given at the Table 3.1.1. whereas, the retention time that is used for the selected ion

monitoring (SIM) method is listed in the Table 3.1.2.

Amino Acids Target Ions (m/z) Reference Ions (m/z)

Alanine 130.1 70.1 88.0

Glycine 116.1 56.1 74.1

Valine 116.1 72.1 55.1

Leucine 172.2 86.1 69.1

Isoleucine 130.1 101.1 -

Threonine 101.1 74.0 56.1

Serine 60.1 146.1 74.1

Proline 70.1 156.1 114.1

Asparagine 69.1 155.1 113.1

Aspartic Acid 88.1 130.1 216.2

Methionine 61.1 101.1 56.1

Glutamic Acid 84.1 170.1 85.1

Phenylalanine 91.1 148.1 74.1

Glutamine 84.1 59.1 58.2

Lysine 170.2 84.1 128.1

Histidine 81.1 82.1 110.2

Tyrosine 107.1 164.1 74.0

Tryptophan 130.1 131.1 77.1

Table 3.1.1: Target and Reference Ions of each amino acid.

P a g e 34 | 132

Amino Acids Retention Time (min)

Alanine 1.103

Glycine 1.203

Valine 1.434

Leucine 1.616

Isoleucine 1.671

Threonine 1.879

Serine 1.920

Proline 1.992

Asparagine 2.095

Aspartic Acid 2.662

Methionine 2.687

Glutamic Acid 3.033

Phenylalanine 3.049

Glutamine 3.674

Lysine 4.369

Histidine 4.559

Tyrosine 4.848

Tryptophan 5.133

Table 3.1.2: Retention time of each amino acid.

P a g e 35 | 132

3.2 Selected Ion Monitoring (SIM) for standards amino acids

With regard of the above data a SIM method for standards amino acids was set up. The method

was split in 10 time periods, where each of the period monitors certain targeted ions and

reference ions. This step is done in order to increase the sensitivity of the method.

SIM for Standards

Start

Time

End

Time

Event

Time

Amino Acids Ions

1.05 1.33 0.06 Ala-Gly 130.1,70.1,88 116.1,56.1,74.1

1.33 1.81 0.09 Val-IS-Leu-Ile 116.1,72.1,55.1 158.2 172.2,86.1,69.1 130.1,101.1

1.81 2.41 0.12 Thr-Ser-Pro-Asn 101.1,74,56.1 60.1,146.1,74.1 70.1,156.1,114.1 69.1,155.1,113.1

2.41 2.89 0.06 Asp-Met 88.1,130.1,216.2 61.1,101.1,56.1

2.89 3.4 0.06 Glu-Phe 84.1,170.1,85.1 91.1,148.1,74.1

3.40 4.05 0.05 Gln 84.1,59.1,58.2

4.05 4.49 0.05 Lys 170.2,84.1,128.1

4.49 4.73 0.05 His 81.1,82.1,110.2

4.73 5.02 0.05 Tyr 107.1,164.1,74

5.02 5.25 0.05 Trp 130.1,131.1,77.1

Table 3.2.1: SIM method for standard amino acids.

The below chromatogram is a representative one.

Scheme 3.2.1: Chromatogram of standard derivatized amino acids on SIM mode. Range

adjusted at 45-450 m/z.


Ab

un

dan

ce (

a.u

.) C=0.01 μM/L

Split Ratio=30

P a g e 36 | 132

3.3 Repeatability (within a day)

AA/Concentration RSD%

0.1 μM 0.3 μM 0.6 μM 1.2 μM 2.4 μM 5 μM 10μM

Alanine 5.4 5.5 4.4 3.6 0.6 1.1 0.5

Glycine 3.5 8.6 3.2 1.6 0.6 3.4 0.8

Valine 5.7 5.4 2.4 1.6 1.4 0.6 0.4

Leucine 9.2 2.0 2.7 3.2 1.0 1.7 0.6

Isoleucine 9.8 3.9 5.3 2.1 1.2 1.1 0.4

Threonine 13.5 10.6 9.4 4.2 3.0 4.2 2.9

Serine 14.3 8.9 7.1 4.2 1.6 3.5 3.7

Proline 4.1 2.2 3.6 3.1 0.3 0.6 0.8

Asparagine - - 11.9 5.6 4.2 5.4 0.7

Aspartic Acid - - 3.7 7.1 14.2 2.0 2.7

Methionine - - - - - 3.5 1.4

Glutamic Acid - - - 10.3 1.5 2.3 1.2

Phenylalanine - - 3.7 7.3 4.8 0.5 1.2

Glutamine - - - - 6.8 2.6 3.0

Lysine - 0.9 13.0 2.0 1.7 2.0 3.1

Histidine - - - - 9.5 6.6 6.7

Tyrosine - 3.3 3.3 3.5 3.5 2.9 1.7

Tryptophan - 11.1 6.0 4.6 3.5 3.1 2.6

Table 3.3.1: Relative standard deviation for all the amino acids in seven different

concentrations.

P a g e 37 | 132

3.4 Intermediate Precision

AA/Concentration 10 μM 50μM

Recovery % Recovery %

Peak Area Retention Time Peak Area Retention Time

Alanine 99 100 88 100

Glycine 108 100 98 100

Valine 99 100 95 100

Leucine 106 100 104 100

Isoleucine 99 100 99 100

Threonine 114 100 103 100

Serine 119 100 96 100

Proline 94 100 91 100

Asparagine 116 100 90 100

Aspartic Acid 117 100 98 100

Methionine 115 100 99 100

Glutamic Acid 129 100 67 100

Phenylalanine 107 100 103 100

Glutamine 126 100 95 100

Lysine 136 100 97 100

Histidine 121 100 120 100

Tyrosine 119 100 108 100

Tryptophan 128 100 146 100

Table 3.4.1: The percentage recovery of peak area and retention time in two representative

concentrations of all amino acids.

P a g e 38 | 132

3.5 Calibration Curves

For the purpose of this experiment six derivatized samples with standard amino acids are

injected three times each at GC/MS. The concentration was chosen, had two magnitude

range. Specifically, 0.1 μM, 1 μM, 5 μM, 10 μM, 25 μM and 50 μM. Norvaline was used

as internal standard. The experiment was performed using as SIM method the one was

described in paragraph 3.2 with split ratio 30 and injection volume 1 μL. In the next pages

you will find a representative graph including calibration equation and correlation

coefficient. The rest are available in Appendix I. Moreover, below the graph you will find

a table with the parameters of the calibration curve of each amino acid.

Alanine

Are

a R

atio

(1

2C

/No

rval

ine)

Concentration (µM) 0 10 20 30 40 Conc. Ratio

0.0

1.0

2.0

3.0

4.0

5.0

Area Ratio(x0.1)

Y = 1.021928e-002X - 4.121476e-003

R^2 = 0.9989563

R = 0.999478

P a g e 39 | 132

Amino Acids a β R2

Alanine 1.021928e-002 - 4.121476e-003 0.9989563

Glycine 7.586305e-003 4.077981e-003 0.9996499

Valine 9.995392e-003 - 5.121873e-003 0.9992979

Leucine 1.086172e-002 - 2.920057e-003

0.9983499

Isoleucine 1.006918e-002 - 5.515763e-003 0.9983963

Threonine 6.919126e-003 - 1.38511e-003 0.9933719

Serine 3.08389e-003 -5.75994e-004 0.9875481

Proline 1.591607e-002 - 1.142593e-002 0.9978341

Asparagine 5.979105e-003 - 4.470555e-003 0.9905939

Aspartic Acid 3.890992e-003 - 3.216384e-003 0.9905092

Methionine 4.30262e-003 - 2.062193e-002 0.9728832

Glutamic Acid 3.640853e-003 - 4.719553e-003 0.9849679

Phenylalanine 8.686568e-003 - 2.76655e-003

0.9966394

Glutamine 1.253481e-003 - 5.236893e-003 0.9781025

Lysine 3.009924e-003 -3.043628e-003 0.9922608

Histidine 1.552537e-003 - 6.532646e-003 0.9862679

Tyrosine 1.154867e-002 - 2.634247e-002 0.9795034

Tryptophan 1.372151e-002 - 2.566159e-002 0.9802783

Table 3.5.1: Parameters of the internal standard calibration curve, y= ax+ β.

P a g e 40 | 132

3.6 Limit of Detection (LOD) and Linearity Range

Amino Acids LOD (μM) Linearity Range (μM)

Alanine 0.1 0.3-200

Glycine 0.1 0.3-200

Valine 0.1 0.1-200

Leucine 0.1 0.1-200

Isoleucine 0.1 0.1-200

Threonine 0.1 0.1-200

Serine 0.1 0.1-200

Proline 0.3 0.3-200

Asparagine 1.2 1.2-200

Aspartic Acid 0.6 0.6-200

Methionine 1.2 1.2-200

Glutamic Acid 1.2 1.2-200

Phenylalanine 0.6 0.6-200

Glutamine 2.4 2.4-200

Lysine 0.3 0.3-200

Histidine 2.0 2.0-200

Tyrosine 0.3 0.3-200

Tryptophan 0.3 0.3-200

Table 3.6.1: Limit of detection and linearity limit of each amino acid.

P a g e 41 | 132

3.7 Amino acids labeled with 13C

In order to correct the matrix effect and the sample degradation, 13C labeled standard amino

acids were bought. During the validation procedure, 13C standards were added directly to the

samples before adding Reagent 1. SCAN MS mode was used to identify the ion fragments of

the label amino acids. All the spectra and chromatograms are available in Appendix II and

Appendix III respectively.

Scheme 3.7.1: Chromatogram of 13C labeled derivatized amino acids using a SCAN MS

mode. Range adjusted at 45-450 m/z.


Ab

un

dan

ce (

a.u

.)

P a g e 42 | 132

3.8 Selected Ion Monitoring (SIM) for amino acids labeled with 13C

With regard on the data that were collected from the spectra a new SIM method about label

amino acids was set up.

SIM for 12C/13C-14N

Start

Time

End

Time

Event

Time

Amino Acids Ions

12C 13C 12C 13C 12C 13C 12C 13C

1.05 1.33 0.06 Ala-Gly 130.1 132.1 116.1 117.1 102 104

1.33 1.81 0.09 Val-IS-Leu-Ile 116.1 120.1 158.2 172.2 177.2 130.1 135.1

1.81 2.41 0.12 Thr-Ser-Pro-Asn 101.1 103 60.1 62 70.1 74.1 69.1 72

2.41 2.89 0.06 Asp-Met 88.1 90 61.1 69 101.1 104 114.1 116

2.89 3.4 0.06 Glu-Phe 84.1 88 91.1 98

3.40 4.05 0.05 Gln 84.1 88

4.05 4.49 0.05 Lys 170.2 175.2

4.49 4.73 0.05 His 81.1 85

4.73 5.02 0.05 Tyr 107.1 114.1

5.02 5.25 0.05 Trp 130.1 139.1

Table 3.8.1: SIM method for labeled 13C amino acids.

Scheme 3.8.1: Chromatogram of labeled derivatized amino acids on SIM mode. Range



Ab

un

dan

ce (

a.u

.)

P a g e 43 | 132

3.9 Amino acids labeled with 13C-15N

Furthermore, amino acids labeled with 13C and 15N were added as internal standard. As a result,

instead of 100 μL of sample, 98 μL of sample and 2 μL of label were added. The rest of the

procedure followed precisely.

At the begging, SCAN MS mode was used to identify the ion fragments of the label amino

acids. The spectra and chromatograms are available in Appendix IV and Appendix V

respectively.

Scheme 3.9.1: Chromatogram of 13C-15N labeled derivatized amino acids using SCAN MS

mode. Range adjusted at 45-450 m/z.


Ab

un

dan

ce (

a.u

.)

P a g e 44 | 132

3.10 Selected Ion Monitoring (SIM) for amino acids labeled with 13C-15N

With regard on the data that were collected from the spectra a new SIM method about label

amino acids was set up

SIM for 12C/13C-15N

Start

Time

End

Time

Event

Time

Amino Acids Ions

12C 13C 12C 13C 12C 13C 12C 13C

1.05 1.33 0.06 Ala-Gly 130.1 132.1 116.1 117.1 102 104

1.33 1.81 0.09 Val-IS-Leu-Ile 116.1 120.1 158.2 172.2 177.2 130.1 135.1

1.81 2.41 0.12 Thr-Ser-Pro-Asn 101.1 103 60.1 62 70.1 74.1 69.1 72

2.41 2.89 0.06 Asp-Met 88.1 90 61.1 69 101.1 104 114.1 116

2.89 3.4 0.06 Glu-Phe 84.1 88 91.1 98

3.40 4.05 0.05 Gln 84.1 88

4.05 4.49 0.05 Lys 170.2 175.2

4.49 4.73 0.05 His 81.1 85

4.73 5.02 0.05 Tyr 107.1 114.1

5.02 5.25 0.05 Trp 130.1 139.1

Table 3.10.1: SIM method for labeled 13C-15N amino acids.

Scheme 3.10.1: Chromatogram of labeled derivatized amino acids on SIM mode. Range



Ab

un

dan

ce

(a.u

.)

P a g e 45 | 132

3.11 Biological Samples

Amino acid analysis in biological samples is considered as an important laboratory test for

the diagnosis of cancer mechanism. Specifically, it is considered that there are some amino

acids, whose difference in concentration implies cancer. But firstly, a brief reference to

Warburg effect is necessary.

Scheme 3.11.1: Warburg Effect.

Tumor Cells

+/-O2

Glucose

Pyruvate

Lactate 95%

Aerobic Glycolysis

(4ATP)

Warburg Effect

O2

Mitochondrial

CO2

5%

Normal Cells

-O2 +O

2

Glucose Glucose

Pyruvate Pyruvate

Lactate

Anaerobic Glycolysis

(2ATP)

O2

Mitochondrial

CO2

Oxidative Phosphorylation

(36ATP)

Lactate

P a g e 46 | 132

Normal cells obtain ATP as a source of energy through the mitochondrial oxidative

phosphorylation in the presence of oxygen instead of glycolysis, due to the higher yield of ATP.

In addition, the presence of oxygen results in the inhibition of glycolysis. On the other hand,

tumor cells can activate glycolysis even in the presence of adequate oxygen levels causing the

Warburg effect. [29]

The metabolic phenomenon where cancer cells rely on aerobic glycolysis instead of oxidative

phosphorylation, was described for the first time by Otto Warburg over 80 years ago and it was

named after him. Aerobic glycolysis is an inefficient way to generate ATP producing only 2

molecules of ATP per molecule glucose whereas oxidative phosphorylation generates 32 ATP

molecules per molecule glucose. The question immediately arises as to why cancer cells would

switch to less productive form of energy production. One hypothesis describes increased

aerobic glycolysis as an adaptation to hypoxic environment. Others argue that increased

glycolysis should facilitate uptake and incorporation of nutrients into the biomass. Although,

the exact mechanisms underlying the Warburg effect are unclear, the importance of increased

glycolysis in cancer cells has been experimentally demonstrated.

Consequently, it is accepted that glycolysis provides tumor cells with glucose to make ATP

intermediates such as nonessential amino acids, which serve as building blocks for tumor cells.

Specifically, glutamine is essential for cancer cell growth due to nutritional value as carbon and

nitrogen source. Moreover, it provided acid resistance. Therefore, the reduced concentration

implies Warburg effect. The reverse is happening to alanine, where the increased concentration

implies Warburg effect because pyruvate is converting to alanine. [30]

In our experiment we compared amino acids uptake and release in extracellular medium of two

cell lines, that differ in expression of viral receptor US28. Expression of such a receptor was

described to activate multiple signaling pathways with consequent cancer phenotype.

P a g e 47 | 132

Scheme 3.11.2: Glutamine concentration in the extracellular cell growth media in time. Red

bars show control cells MOCK, blue bars show cells US-28 expressing the viral receptor.

Glutamine is a source of energy side to a glucose and it was described in literature that certain

cancer lines are utilizing more glutamine compare to glucose. It was even described that

glutamine uptake is life depending and its depletion from growing media leads to a cell death.

Alanine usually serves in the cell metabolism as a sink for nitrogen in the presence of flux of

glutamine. Based on the huge uptake of glutamine, we assume that excretion of alanine would

take a place.

Scheme 3.11.3: Alanine excreting growth to extracellular growth media in time. Red bars show

control cells MOCK, blue bars show cells US-28 expressing the viral receptor.

0

50

100

150

200

250

medium 0 24 48 72 96

Co

nce

ntr

atio

n (

μM

)

Time points (hours)

GlutamineMOCK

US-28

0

10

20

30

40

50

60

medium 0 24 48 72 96

Co

nce

ntr

atio

n (

μM

)

Time points (hours)

AlanineMOCK

US-28

P a g e 48 | 132

The last graph represent isoleucine’s concentration in the extracellular growth media of two

different cell lines collected in different time points. The reduced concentration of isoleucine

shows that is utilized in very small amounts, therefore is not directly used as a source of energy

but only for the protein synthesis.

Scheme 3.11.3: Isoleucine excreting growth to extracellular growth media in time. Red bars

show control cells MOCK, blue bars show cells US-28 expressing the viral receptor.

To sum up, taking the above under consideration only glutamine is an important source of

energy. The rest amino acids, like isoleucine, show a small reduction on their concentration at

cells expressing US-28 receptor because they are utilized by the protein synthesis. For that

reason the rest graphs will not be provided.

Based on this analysis we do not see any significant differences in uptake or excretion amino

acids and we could conclude that metabolism of these cells does not differ. However, for real

conclusion more metabolites analysis would have to be performed.

0

10

20

30

40

50

60

70

medium 0 24 48 72 96

Co

nce

ntr

atio

n (

μM

)

Time points (hours)

IsoleucineMOCK

US-28

P a g e 49 | 132

4. LC/MS Results

4.1 Manual Tuning for Probe and Turbo Ionspray Gas

The first step on validation of a method using LC/MS is the tuning.

The initial conditions of probe’s horizontal position (H), probe’s lateral position (L) and heater

gas flow (HGF) were:

H=15

L=-5

HGF=6500

The values were chosen and measured for the optimization as well as the resulting graphs are

shown particularly in Appendix VI.

For the optimum conditions was chosen “The Golden Section” between the maximum intensity

of the peak and the less possible gas consumption:

L= -5

H=3

HGF=5000

4.2 Flow injection analysis (FIA)

Except of the probe and the turbo ionspray gas there are other parameters that should optimized

and they are related to each amino acid separately.

The EZfaast Kit has already provided us with a manual that gives us representatives values for

all the compound-dependent parameters and source dependent parameters as well.

However, it has to be checked if those parameters are suitable for the instrument that we are

using. Specifically, the compound-dependent parameters that need to be optimized are the

Declustering Potential (DP), the Focusing Potential (FP), the Entrance Potential (EP), the

Collision Cell Exit Potential (CXP) and the Collision Energy (CE). The Nebulizer Gas (NEB),

P a g e 50 | 132

the Curtain Gas (CUR), the Temperature (TEM) and the Ionspray Voltage (IS) are the source-

dependent parameters that have to be checked.

Two amino acids and the internal standard were checked and the given values were verified.

The data of those measurements can be found in Appendix VII. The below table sums up the

values of the compound-dependent parameters for each amino acid. The source-dependent

parameters will be given in the paragraph 4.4.

Amino Acids DP FP CE CXP EP

Glycine 21 130 13 8 10

Asparagine 60 250 15 10 10

Leucine 16 110 21 10 10

Glutamine 26 150 21 10 10

Threonine 40 200 17 10 10

Serine 25 140 17 8 10

Alanine 30 210 27 4 10

Methionine 21 140 17 12 10

Proline 26 160 19 10 10

Lysine 36 200 17 8 10

Aspartic Acid 30 110 19 14 10

Histidine 63 190 33 12 10

Norvaline 21 130 17 10 10

Glutamic Acid 26 150 21 10 10

Tryptophan 60 350 21 10 10

Tyrosine 60 300 43 8 10

Isoleucine 40 200 17 10 10

Phenylalanine 40 200 17 14 10

Table 4.2.1: Compound-dependent parameters based on EZ-faast manual.

P a g e 51 | 132

4.3 Full-SCAN LC/MS

In the first step sample containing standard amino acids was injected in order to check the

retention time and the precise mass of parents and products ions. In following tables are listed

all the masses. In Appendix VIII you will find the spectra that verify the choice of those masses.

In addition, table 4.3.3 shows the retention time of its amino acid in time order.

Parent Ions Amino acids m/z (manual) m/z (after scanning)

Glutamine 275.3 275.6

Serine 234.3 234.3

Asparagine 243.3 243.3

Leucine 260.2 260.4

Glycine 204.1 204.5

Threonine 248.3 248.7

Alanine 218.3 218.6

Proline 244.3 244.6

Methionine 278.3 278.4

Aspartic Acid 304.3 304.6

Histidine 370.2 370.3

Valine 246.3 246.6

Glutamic Acid 318.3 318.6

Isoleucine 260.3 260.5

Phenylalanine 294.3 294.5

Tyrosine 396.2 396.7

Lysine 361.2 361.7

Tryptophan 333.3 333.8

Table 4.3.1: Mass of parent ions based on the manual and after scanning.

P a g e 52 | 132

Product Ions Amino acids m/z (manual) m/z (after scanning)


Serine 146 146.2


Leucine 172.1 172.3

Glycine 144 144.2


Alanine 130.1 130.2

Proline 156.2 156.3




Valine 158.1 158.3




Tyrosine 136.2 136.4

Lysine 301.3 301.6


Table 4.3.2: Mass of product ions based on manual and after scanning.

Amino Acids Retention Time

Glutamine 1.25 Serine 1.40

Asparagine 1.44

Leucine 1.52

Glycine 1.58

Threonine 1.64

Alanine 2.23

Proline 2.83

Methionine 2.97

Aspartic Acid 3.38

Histidine 3.43

Lysine 3.48

Norvaline 3.57

Glutamic Acid 3.71

Tryptophan 3.95

Phenylalanine 4.68

Isoleucine 4.78

Tyrosine 6.78

Table 4.3.2: The retention time of each amino acid.

P a g e 53 | 132

4.4 Multiple Reaction Monitoring (MRM)

An MRM method was developed for the determination of the amino acids. This method was

divided in three time periods, each of them has a group of amino acids with their parent and

product ions that are allowed to pass through the triple quadrupole. The amino acids were

grouped on periods based on the time order of their appearance on the chromatogram and the

source-dependent parameters. The compound-dependent parameters can be defined for each

amino acid separately whereas the source-dependent parameters only for periods resulting on

compromises.

Period Start Time End Time Amino Acids Q1 Mass Q3 Mass

1st 0.000 1.995 Glycine 204.5 144.3


Leucine 260.3 172.3



Serine 234.2 146.2

2nd 0.1995 4.300 Alanine 218.6 88.4


Proline 244.5 156.2

Lysine 361.6 301.6



Norvaline 246.4 158.3



3rd 4.300 7.305 Tyrosine 396.6 136.4



Table 4.4.1: The distribution of the amino acids in the three periods.

P a g e 54 | 132

The compound-dependent parameters have already given at table 4.2.1 whereas the source-

dependent parameters of each period are given below.

Period Nebulizer

(NEB)

Curtain

Gas (CUR)

CAD Gas

(CAD)

Ionspray

Voltage (IS)

Temperature

(TEM)

Entrance

Potential (EP)

1st 12 12 10 1600 475 10

2nd 12 12 10 1600 475 10

3rd 10 10 10 1600 475 10

Table 4.4.1: Source-dependent parameters based on EZ-faast manual.

The following chromatograms indicate the distribution of the amino acids during the periods.

Scheme 4.4.1: Chromatogram of first period’s amino acids.

GLN

SER

ASN

LEU

GLY

THR

P a g e 55 | 132

Scheme 4.4.2: Chromatogram of second period’s amino acids.

Scheme 4.4.3: Chromatogram of third period’s amino acids.

ALA

PRO

MET

HIS

ASP

GLU

VAL

TYR

ILE

PHE

P a g e 56 | 132

The gradient program that was used to accomplish the necessary equilibrium, avoid the

retention shifting and the contamination of the column is describing below.

Time (min) Flow (ml/min) A% (10mM ammonium

formate in water)

B% (10nM ammonium

formate in methanol)

0 0.6 32 68

13 0.6 17 83

13.01 0.6 5 95

18 0.6 5 95

18.0 0.6 32 68

25 0.6 32 68

Table 4.4.2: The gradient program according to time.

Scheme 4.4.4: The fluctuations of the mobile phase.

60

65

70

75

80

85

90

95

100

0 5 10 15 20 25 30

B%

Gra

die

nt

Time (min)

P a g e 57 | 132

4.5 Calibration Curves

For the purpose of this experiment nine derivatized samples with standard amino acids are

injected three times each at LC/MS. The concentration was chosen, had three magnitude

range. Specifically, 0.25 μM, 0.5 μM, 1 μM, 3 μM, 5 μM, 10 μM, 25 μM, 50 μM and 100 μM.

Norvaline was used as internal standard. The experiment was performed using the MRM

method that was described in the above paragraph. In the next pages you will find a

representative graph including calibration equation and correlation coefficient. The rest are

available at Appendix IX. Moreover, below the graph you will find a table with the

parameters of the calibration curve of each amino acid.

Glycine

y = 51726x + 93576R² = 0.9984

0.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

5.00E+06

6.00E+06

0 20 40 60 80 100 120

Pe

ak A

rea

Concentration (μM)

P a g e 58 | 132

Amino Acids a β R2

Asparagine 128055 - 8810.4 0.9996

Leucine 505704 133475 0.9951

Glutamine 193378 74903 0.9846

Threonine 30962 55212

0.9979

Serine 83401 264814 0.9944

Alanine 20124 - 61986 0.9732

Methionine 217566 - 499397 0.9941

Proline 1E+06 210795 0.9918

Lysine 183816 705.56 0.9991

Aspartic Acid 247631 74509 0.9995

Histidine 992948 253136 0.9983

Glutamic Acid 320678 18095 0.9999

Tryptophan 16366 - 1337.6 0.9985

Tyrosine 238980 4586 0.9999

Isoleucine 1E+06 69562 0.9935

Tyrosine 1E+06 25739 0.9994

Table 4.5.1: Parameters of the internal standard calibration curve, y= ax+ β.

P a g e 59 | 132

4.6 Relative Standard Deviation (RSD %)

In order to check the precision and the repeatability of the method the relative standard

deviation was calculated for 8 different concentrations.

Relative Standard Deviation of Peak Area

Amino Acids Concentration

0,25μM 0,5 μM 1 μM 3 μM 5 μM 10 μM 50 μM 100 μM

Glycine 3.42 2.01 6.04 21.44 4.32 6.61 8.84 6.57

Asparagine 5.32 6.93 8.84 22.76 4.23 7.45 6.45 4.99

Leucine 3.94 0.61 16.14 25.37 9.31 11.69 7.16 4.53

Glutamine 4.95 4.99 5.65 20.83 5.61 5.63 8.14 4.60

Threonine 3.94 0.52 5.14 23.01 5.11 11.00 7,83 5.73

Serine 0.73 1.37 13.77 25.64 4.36 7.09 7.49 7.01

Alanine 8.44 3.09 10.52 18.80 3.65 2.35 9.40 3.27

Methionine 11.49 19.60 1.21 21.09 3.61 1.05 10.16 1.52

Proline 1.31 2.59 3.31 19.64 2.08 1.30 7.05 2.32

Lysine 5.53 1.54 4.97 22.87 1.51 5.91 7.56 2.97

Aspartic Acid 4.52 2.56 4.48 21.23 1.15 2.51 7.85 3.28

Histidine 0.54 6.16 4.53 22.56 0.81 1.32 9.53 4.60

Glutamic Acid 4.89 3.36 3.68 22.27 4.42 5.63 5.92 3.94

Tryptophan 8.49 11.06 9.90 23.49 9.09 13.66 1.18 4.84

Tyrosine 4.04 0.80 9.81 26.39 2.24 7.28 1.44 3.45

Isoleucine 4.26 2.24 3.22 19.12 2.87 1.16 7.19 2.50

Phenylalanine 2.81 5.09 3.66 20.97 3.04 1.60 9.86 2.39

Table 4.6.1: Relative Standard Deviation for all the amino acids.

P a g e 60 | 132

4.7 Limit of Detection (LOD) and Linearity Range

Amino Acids LOD (μM) LLOL(μM) Linearity Range

(μM) Glycine 0.25 0.25 0.25-100

Asparagine 0.25 0.25 0.25-10

Leucine 0.25 0.25 0.25-10

Glutamine 0.25 0.25 0.25-10

Threonine 0.25 0.25 0.25-100

Serine 0.25 0.25 0.25-100

Alanine 0.25 3 0.25-100

Methionine 0.25 1 0.25-100

Proline 0.25 0.25 0.25-5

Lysine 0.25 0.25 0.25-10

Aspartic Acid 0.25 0.25 0.25-10

Histidine 0.25 0.25 0.25-10

Glutamic Acid 0.25 0.25 0.25-10

Tryptophan 0.25 0.25 0.25-10

Tyrosine 0.25 0.25 0.25-10

Isoleucine 0.25 0.25 0.25-5

Phenylalanine 0.25 0.25 0.25-10

Table 4.7.1: Limit of detection and linearity limit of each amino acid.

P a g e 61 | 132

5. Conclusion and Perspectives

The Phenomenex EZ: faast amino acid kit was applied successfully for gas and liquid

chromatographic determination.

Specifically, the whole sample pretreatment and derivatization lasted 8 minutes per sample.

De-proteinization was not required since during the solid phase extraction amino acids

could be extracted from the complex physiological fluids really fast and proteins and salts

did not affect derivatization. The derivatized amino acids were stable for days at 4 o C.

Short analysis time ranged from 7 to 21 minutes for gas and liquid chromatography

respectively. The use of internal standard corrected the injection inaccuracy, whereas the

use of label standards corrected the matrix effects. Lastly, great baseline resolution of all

the amino acids was accomplished.

Both methods were validated since repeatability, stability, linearity and limit of detection

were suitable for simultaneously quantification of 18 amino acids with gas or liquid

chromatographic determination.

As a result, a comparison between these two chromatographic techniques made with GC-

MS outweigh to the follows:

Gas chromatography had shorter analysis time which is really important in metabolomics

research. In one hour GC-MS could analyze almost the double samples in comparison with

LC-MS. Likewise, with GC-MS was possible to run overnight since stability of samples

was validated and a shutdown system was available.

The Shimadzu software that was used for GC-MS was friendlier than the Analyst software

that was used for LC-MS. In addition, for GC-MS a library was available. Those two reason

made data analysis easier and faster.

On the other hand, LC-MS had lower limit of detection in case of some amino acids.

Moreover, there are some amino acids like arginine, cysteine etc. that could not be

determined with GC-MS due to their thermal instability but with LC-MS their determination

was possible. However, those advantages do not offer anything to our study with mouse

embryonic fibroblast NIH-3T3 cells. Unlike, valine which is one of the essential amino

acids could not be detected with LC-MS. As a result, a GC-MS method with amino acids

labelled with 13C/15N was chosen to be applied on biological samples in order to study the

cancer mechanism.

P a g e 62 | 132

Based on the results it can be concluded that except of glutamine, whose concentration

differs a lot at cells expressing US-28 receptor, the rest of amino acids show a small

reduction and by extension the metabolism of those cells do not differ.

To sum up, this project will be ideally continued if a thorough metabolic analysis have been

performed. The number of the amino acids could be extended so the role of amino acids

such as sarcosine and ornithine in cancer mechanism could be more clearly. In addition, the

analysis of biological samples by LC-MS using labels might be useful as well. We believe

that this project was a step closer to understanding amino acids role in cancer mechanism

and raise the awareness for further research.

P a g e 63 | 132

References

[1] P. Hušek and P. Šimek, “Advances in Amino Acid Analysis The authors briefly review

current strategies of amino acid analysis and,” vol. 19, no. 9, pp. 986–999, 2001.

[2] H. Kaspar, “Amino acid analysis in biological fluids by GC-MS,” Fakultät für Chemie

und Pharmazie der Universität Regensburg, 2009.

[3] G. Wu, “Amino acids: metabolism, functions, and nutrition.,” Amino Acids, vol. 37, no.

1, pp. 1–17, May 2009.

[4] L. Stryer, Biochemistry (4th edition) by Lubert Stryer: W.H. Freeman & Company

9780716720096 Hardcover - A Trillion Books Inc. W.H. Freeman & Company, 1995,

p. 1064.

[5] J. D. Fernstrom and M. H. Fernstrom, “Tyrosine, Phenylalanine, and Catecholamine

Synthesis and Function in the Brain,” J. Nutr., vol. 137, no. 6, p. 1539S–1547, Jun.

2007.

[6] S. A. F. Morad and M. C. Cabot, “Ceramide-orchestrated signalling in cancer cells.,”

Nat. Rev. Cancer, vol. 13, no. 1, pp. 51–65, Jan. 2013.

[7] D. H. Chace and T. A. Kalas, “A biochemical perspective on the use of tandem mass

spectrometry for newborn screening and clinical testing.,” Clin. Biochem., vol. 38, no.

4, pp. 296–309, Apr. 2005.

[8] J. Csapó, C. Albert, K. Lóki, and Z. Csapó-Kiss, “Separation and determination of the

amino acids by ion exchange column chromatography applying postcolumn

derivatization.,” Acta Univ. Sapientiae - Aliment., vol. 1, pp. 5–29, 2008.

[9] Society of Japanese Pharmacopoeia, Japanese Parmacopoeia 15th Edition. The

Stationnery Office, 2007, p. 1779.

[10] A. Khaleduzzaman, Z. Khandaker, M. Khan, L. Banu, and M. Khan, “Evaluation Of A

High Performance Liquid Chromolography (Hplc) Mehtod For Amino Acid Analysis

In Feed With Precolumn Derivatization And Fluorescence Detection,” Bangladesh J.

Anim. Sci., vol. 37, no. 2, pp. 66–73, Feb. 2012.

[11] M. Makita, S. Yamamoto, and S. Kiyama, “Improved gas-liquid chromatographic

method for the determination of protein amino acids,” J. Chromatogr. A, vol. 237, no.

2, pp. 79–284, Mar. 1982.

[12] P. Hušek, “Amino acid derivatization and analysis in five minutes,” FEBS Lett., vol.

280, no. 2, pp. 354–356, Mar. 1991.

P a g e 64 | 132

[13] S. Oguri, “Electromigration methods for amino acids, biogenic amines and aromatic

amines.,” J. Chromatogr. B. Biomed. Sci. Appl., vol. 747, no. 1–2, pp. 1–19, Sep. 2000.

[14] Y.-H. Lee and T.-I. Lin, “Capillary electrophoretic determination of amino acids with

indirect absorbance detection,” J. Chromatogr. A, vol. 680, no. 1, pp. 287–297, Sep.

1994.

[15] Y.-H. Lee and T.-I. Lin, “Capillary electrophoretic determination of amino acids

improvement by cyclodextrin additives,” J. Chromatogr. A, vol. 716, no. 1–2, pp. 335–

346, Nov. 1995.

[16] Phenomenex, “EZ: faast USER ’ S MANUAL Free ( Physiological ) Amino Acid

Analysis by GC-MS,” .

[17] T. Farcas, J. E. Toulouee, and M. Li, “Fast and Easy Physiological Amino Acids

Analysis in 15 Minutes,” Medical/Biological, vol. 134, pp. 30–31, 2002.

[18] F. Orata, Advanced Gas Chromatography - Progress in Agricultural, Biomedical and

Industrial Applications. InTech, 2012, p. 460.

[19] H. Kataoka, “Derivatization reactions for the determination of amines by gas

chromatography and their applications in environmental analysis.,” J. Chromatogr. A,

vol. 733, no. 1–2, pp. 19–34, May 1996.

[20] A. E. Pierce, “GC Derivatization,” 2004.

[21] D. R. Knapp, Handbook of Analytical Derivatization Reactions. Wiley-Interscience,

1979, p. 768.

[22] A. Hulshoff and H. Lingeman, “Derivatization reactions in the gas-liquid

chromatographic analysis of drugs in biological fluids.,” J. Pharm. Biomed. Anal., vol.

2, no. 3–4, pp. 337–80, Jan. 1984.

[23] V. Zaikin and J. Halket, A Handbook of Derivatives for Mass Spectrometry. IM

Publications LLP, 2009, p. 543.

[24] T. Toyo’oka, Modern Derivatization Methods for Separation Science. John Wiley &

Sons, 1999, p. 312.

[25] Shimadzu Corporation, “Analytical Methods for Amino Acids.” [Online]. Available:

http://www.shimadzu.com/an/hplc/support/lib/lctalk/53/53intro.html. [Accessed: 04-

Mar-2014].

[26] H. A. Bardelmeijer, J. C. Waterval, H. Lingeman, R. van’t Hof, A. Bult, and W. J.

Underberg, “Pre-, on- and post-column derivatization in capillary electrophoresis.,”

Electrophoresis, vol. 18, no. 12–13, pp. 2214–27, Nov. 1997.

[27] M. G. Zampolli, G. Basaglia, F. Dondi, R. Sternberg, C. Szopa, and M. C.

Pietrogrande, “Gas chromatography-mass spectrometry analysis of amino acid

P a g e 65 | 132

enantiomers as methyl chloroformate derivatives: application to space analysis.,” J.

Chromatogr. A, vol. 1150, no. 1–2, pp. 162–72, May 2007.

[28] E. Forró, “New gas chromatographic method for the enantioseparation of beta-amino

acids by a rapid double derivatization technique.,” J. Chromatogr. A, vol. 1216, no. 6,

pp. 1025–9, Feb. 2009.

[29] M. G. Vander Heiden, L. C. Cantley, and C. B. Thompson, “Understanding the

Warburg effect: the metabolic requirements of cell proliferation.,” Science, vol. 324,

no. 5930, pp. 1029–33, May 2009.

[30] M. López-Lázaro, “The warburg effect: why and how do cancer cells activate

glycolysis in the presence of oxygen?,” Anticancer. Agents Med. Chem., vol. 8, no. 3,

pp. 305–12, Apr. 2008.

P a g e 66 | 132

Appendix I: Calibration Curves of the amino acids using GC/MS

Glycine

Valine

Are

a R

atio

(1

2C

/No

rval

ine)


0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0Area Ratio(x0.1)

Y = 7.586305e-003X + 4.077981e-003

R^2 = 0.9996499

R = 0.9998249

Are

a R

atio

(1

2C

/No

rval

ine)


0.0

1.0

2.0

3.0

4.0

5.0

Area Ratio(x0.1)

Y = 9.995392e-003X - 5.121873e-003

R^2 = 0.9992979

R = 0.9996489

P a g e 67 | 132

Leucine

Isoleucine

0 10 20 30 40 Conc. Ratio0.0

1.0

2.0

3.0

4.0

5.0

Area Ratio(x0.1)

Y = 1.086172e-002X - 2.920057e-003

R^2 = 0.9983499

R = 0.9991746

Concentration (µM)

Are

a R

atio

(1

2C

/No

rval

ine)

0 10 20 30 40 Conc. Ratio0.0

1.0

2.0

3.0

4.0

5.0

Area Ratio(x0.1)

Y = 1.006918e-002X - 5.515763e-003

R^2 = 0.9983963

R = 0.9991978

Concentration (μM)

Are

a R

atio

(1

2C

/No

rval

ine)

P a g e 68 | 132

Threonine

Serine

Concentration (µM)

0 10 20 30 40 Conc. Ratio0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Area Ratio(x0.1)

Y = 6.919126e-003X - 1.38511e-003

R^2 = 0.9933719

R = 0.9966804 Are

a R

atio

(1

2C

/No

rval

ine)

0 10 20 30 40 Conc. Ratio0.00

0.25

0.50

0.75

1.00

1.25

1.50

Area Ratio(x0.1)

Y = 3.08389e-003X - 5.75994e-004

R^2 = 0.9875481

R = 0.9937545

Concentration (µM)

Are

a R

atio

(1

2C

/No

rval

ine)

P a g e 69 | 132

Proline

Asparagine

0 10 20 30 40 Conc. Ratio0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

Area Ratio(x0.1)

Y = 1.591607e-002X - 1.142593e-002

R^2 = 0.9978341

R = 0.9989165

Are

a R

atio

(1

2C

/No

rval

ine)

Concentration (µM)

0 10 20 30 40 Conc. Ratio0.0

0.5

1.0

1.5

2.0

2.5

3.0

Area Ratio(x0.1)

Y = 5.979105e-003X - 4.470555e-003

R^2 = 0.9905939

R = 0.9952858

Concentration (µM)

Are

a R

atio

(1

2C

/No

rval

ine)

P a g e 70 | 132

Aspartic Acid

Methionine

0 10 20 30 40 Conc. Ratio0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

Area Ratio(x0.1)

Y = 3.890992e-003X - 3.216384e-003

R^2 = 0.9905092

R = 0.9952433

Concentration (µM)

Are

a R

atio

(1

2C

/No

rval

ine)

0 10 20 30 40 Conc. Ratio0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

Area Ratio(x0.1)

Y = 4.30262e-003X - 2.062193e-002

R^2 = 0.9728832

R = 0.9863484

Are

a R

atio

(1

2C

/No

rval

ine)

Concentration (µM)

P a g e 71 | 132

Glutamic Acid

Phenylalanine

0 10 20 30 40 Conc. Ratio0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

Area Ratio(x0.1)

Y = 3.640853e-003X - 4.719553e-003

R^2 = 0.9849679

R = 0.9924555

Concentration (µM)

Are

a R

atio

(1

2C

/No

rval

ine)

0 10 20 30 40 Conc. Ratio0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Area Ratio(x0.1)

Y = 8.686568e-003X - 2.76655e-003

R^2 = 0.9966394

R = 0.9983183

Concentration (µM)

Are

a R

atio

(1

2C

/No

rval

ine)

P a g e 72 | 132

Glutamine

Lysine

0 10 20 30 40 Conc. Ratio0.0

1.0

2.0

3.0

4.0

5.0

6.0

Area Ratio(x0.01)

Y = 1.253481e-003X - 5.236893e-003

R^2 = 0.9781025

R = 0.9889907 Are

a R

atio

(1

2C

/No

rval

ine)

Concentration (µM)

0 10 20 30 40 Conc. Ratio0.00

0.25

0.50

0.75

1.00

1.25

1.50

Area Ratio(x0.1)

Y = 3.009924e-003X - 3.043628e-003

R^2 = 0.9922608

R = 0.9961229

Concentration (µM)

Are

a R

atio

(1

2C

/No

rval

ine)

P a g e 73 | 132

Histidine

Tyrosine

0 10 20 30 40 Conc. Ratio0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

Area Ratio(x0.01)

Y = 1.552537e-003X - 6.532646e-003

R^2 = 0.9862679

R = 0.9931102

Concentration (µM)

Are

a R

atio

(1

2C

/No

rval

ine)

0 10 20 30 40 Conc. Ratio0.0

1.0

2.0

3.0

4.0

5.0

6.0Area Ratio(x0.1)

Y = 1.154867e-002X - 2.634247e-002

R^2 = 0.9795034

R = 0.9896986

Concentration (µM)

Are

a R

atio

(1

2C

/No

rval

ine)

P a g e 74 | 132

Tryptophan

0 10 20 30 40 Conc. Ratio0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

Area Ratio(x0.1)

Y = 1.372151e-002X - 2.566159e-002

R^2 = 0.9802783

R = 0.9900901 Are

a R

atio

(1

2C

/No

rval

ine)

Concentration (µM)

P a g e 75 | 132

Appendix II: Average mass spectra of standard and label amino

acids. Highlighted ions were used as target ions to a single ion

monitoring mode for labeled 13C amino acids.

Alanine

Alanine 13C-12C

Glycine

Glycine 13C-12C

P a g e 76 | 132

Valine

Valine 13C-12C

Leucine

Leucine 13C-12C

P a g e 77 | 132

Isoleucine

Isoleucine 13C-12C

Threonine

Threonine 13C-12C

P a g e 78 | 132

Serine

Serine 13C-12C

Proline

Proline 13C-12C

P a g e 79 | 132

Methionine

Methionine 13C-12C

Glutamic Acid

Glutamic Acid 13C-12C

P a g e 80 | 132

Phenylalanine

Phenylalanine 13C-12C

Glutamine

Glutamine 13C-12C

P a g e 81 | 132

Lysine

Lysine 13C-12C

Histidine

Histidine 13C-12C

P a g e 82 | 132

Tyrosine

Tyrosine 13C-12C

Tryptophan

Tryptophan 13C-12C

P a g e 83 | 132

Appendix III: Overlapping target ions’ chromatograms for the

labeled 12C/13C SIM method. Ions with highest abundance were

chosen for further quantification.

Alanine

Glycine

1.11 1.12 1.13 1.14 1.15 1.16 1.17 0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

(x1,000,000)

104.00 (4.92) 102.00 (39.36) 117.10 (1.23) 116.10 (9.17) 132.10 (1.00) 130.10 (8.76) TIC


Ab

un

dan

ce

(a.u

.)

Ab

un

dan

ce

(a.u

.)

Retention Time (min) 1.21 1.2

2 1.23 1.24 1.25 1.26 1.27

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

(x1,000,000)

104.00 (4.92) 102.00 (39.36) 117.10 (1.23) 116.10 (9.17) 132.10 (1.00) 130.10 (8.76) TIC

P a g e 84 | 132

Valine

Leucine

1.41 1.42 1.43 1.44 1.45 1.46 1.47 0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

(x1,000,000)

135.10 (15.52) 130.10 (26.17) 177.20 (19.74) 172.20 (19.07) 72.10 (1.00) 120.10 (2.19) 116.10 (2.64) TIC


Ab

un

dan

ce (

a.u

.)

1.62 1.63 1.64 1.65 1.66 1.67 1.68 1.69 0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

(x1,000,000)

135.10 (15.52) 130.10 (26.17) 177.20 (19.74) 172.20 (19.07) 72.10 (1.00) 120.10 (2.19) 116.10 (2.64) TIC


Ab

un

dan

ce (

a.u

.)

P a g e 85 | 132

Isoleucine

Threonine

1.68 1.69 1.70 1.71 1.72 1.73 1.74 1.75 0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

(x1,000,000)

135.10 (15.52) 130.10 (26.17) 177.20 (19.74) 172.20 (19.07) 72.10 (1.00) 120.10 (2.19) 116.10 (2.64) TIC


Ab

un

dan

ce (

a.u

.)

Retention Time (min) 1.895 1.900 1.905 1.910 1.915 1.920 1.925 1.930 1.935 1.940

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

(x100,000)

72.00 (1.00) 69.10 (3.88) 74.10 (2.16) 70.10 (2.02) 62.00 (1.61) 60.10 (7.36) 103.00 (1.13) 101.10 (4.45) TIC

Ab

un

dan

ce (

a.u

.)

P a g e 86 | 132

Serine

Proline

1.935 1.940 1.945 1.950 1.955 1.960 1.965 1.970 1.975 1.980 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

(x100,000)

72.00 (1.00) 69.10 (3.88) 74.10 (2.16) 70.10 (2.02) 62.00 (1.61) 60.10 (7.36) 103.00 (1.13) 101.10 (4.45) TIC


Ab

un

dan

ce (

a.u

.)

2.010 2.015 2.020 2.025 2.030 2.035 2.040 2.045 2.050 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

(x100,000)

72.00 (1.00) 69.10 (3.88) 74.10 (2.16) 70.10 (2.02) 62.00 (1.61) 60.10 (7.36) 103.00 (1.13) 101.10 (4.45) TIC


Ab

un

dan

ce (

a.u

.)

P a g e 87 | 132

Asparagine

Aspartic Acid

2.10 2.11 2.12 2.13 2.14 2.15 2.16 2.17 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5 (x100,000)

72.00 (1.00) 69.10 (3.88) 74.10 (2.16) 70.10 (2.02) 62.00 (1.61) 60.10 (7.36) 103.00 (1.13) 101.10 (4.45) TIC


Ab

un

dan

ce (

a.u

.)

2.685 2.690 2.695 2.700 2.705 2.710 2.715 2.720 0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0 (x100,000)

116.00 (42.03) 114.10 (48.53) 104.00 (23.70) 101.10 (68.81) 69.00 (42.86) 61.10 (14.79) 91.00 (2.79) 88.10 (1.00) TIC


Ab

un

dan

ce (

a.u

.)

P a g e 88 | 132

Methionine

Glutamic Acid

2.710 2.715 2.720 2.725 2.730 2.735 2.740 2.745 0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0 (x100,000)

116.00 (42.03) 114.10 (48.53) 104.00 (23.70) 101.10 (68.81) 69.00 (42.86) 61.10 (14.79) 91.00 (2.79) 88.10 (1.00) TIC


Ab

un

dan

ce (

a.u

.)

3.055 3.060 3.065 3.070 3.075 3.080 3.085 3.090 3.095 3.100 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0 (x1,000,000)

98.00 (24.81) 91.10 (22.58) 88.00 (1.00) 84.10 (32.31) TIC


Ab

un

dan

ce (

a.u

.)

P a g e 89 | 132

Phenylalanine

Glutamine

3.075 3.080 3.085 3.090 3.095 3.100 3.105 3.110 3.115 3.120

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

(x1,000,000)

98.00 (24.81) 91.10 (22.58) 88.00 (1.00) 84.10 (32.31) TIC


Ab

un

dan

ce (

a.u

.)

3.700 3.705 3.710 3.715 3.720 3.725 3.730 3.735 3.740 3.745 3.750 3.755 3.760 3.765 3.770 0.00

0.25

0.50

0.75

1.00

1.25

1.50 (x1,000,000)

88.00 (1.00) 84.10 (20.33) TIC


Ab

un

dan

ce (

a.u

.)

P a g e 90 | 132

Lysine

Histidine

4.380 4.385 4.390 4.395 4.400 4.405 4.410 4.415 4.420 4.425 4.430 4.435 4.440 0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

(x100,000)

175.20 (1.00) 170.20 (6.77) TIC


Ab

un

dan

ce (

a.u

.)

4.500 4.525 4.550 4.575 4.600 4.625 4.650 4.675 4.700 4.725 0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75 (x100,000)

85.00 (1.00) 81.10 (3.99) TIC


Ab

un

dan

ce (

a.u

.)

P a g e 91 | 132

Tyrosine

Tryptophan

4.750 4.775 4.800 4.825 4.850 4.875 4.900 4.925 4.950 4.975 5.000 0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

(x100,000)

114.10 (1.00) 107.10 (1.51) TIC


Ab

un

dan

ce (

a.u

.)

5.13 5.14 5.15 5.16 5.17 5.18 5.19 5.20 5.21 5.22 0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00 (x100,000)

139.10 (1.39) 130.10 (1.00) TIC


Ab

un

dan

ce (

a.u

.)

P a g e 92 | 132

Appendix IV: Average mass of standard and label amino acids.

Highlighted ions were used as target ions to a single ion monitoring

mode for ladeled 13C-15N amino acids.

Alanine

Alanine 13C-12C/15N-14N

Glycine

Glycine 13C-12C/15N-14N

P a g e 93 | 132

Valine

Valine 13C-12C/15N-14N

Leucine

Leucine 13C-12C/15N-14N

P a g e 94 | 132

Isoleucine

Isoleucine 13C-12C/15N-14N

Threonine

Treonine 13C-12C/15N-14N

P a g e 95 | 132

Serine

Serine 13C-12C/15N-14N

Proline

Proline 13C-12C/15N-14N

P a g e 96 | 132

Asparagine

Asparagine 13C-12C/15N-14N

Aspartic Acid

Aspartic Acid 13C-12C/15N-14N

P a g e 97 | 132

69 104

Methionine

Methionine 13C-12C/15N-14N

Glutamic Acid

Glutamic Acid 13C-12C/15N-14N

P a g e 98 | 132

Phenylalanine

Phenylalanine 13C-12C/15N-14N

Glutamine

Glutamine 13C-12C/15N-14N

P a g e 99 | 132

Lysine

Lysine 13C-12C/15N-14N

Histidine

Histidine 13C-12C/15N-14N

P a g e 100 | 132

Tyrosine

Tyrosine 13C-12C/15N-14N

Tryptophan

Tryptophan 13C-12C/15N-14N

P a g e 101 | 132

Appendix V: Overlapping target ions’ chromatograms for the

labeled 12C /13C -14N /15N SIM method. Ions with highest abundance

were chosen for further quantification.

Alanine

Glycine

Retention Time (min) 1.080 1.085 1.090 1.095 1.100 1.105 1.110 1.115 1.120 1.125 1.130

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0 (x1,000,000)

118.10 (1.28) 116.10 (100.00) 133.10 (1.00) 130.10 (100.00) TIC

Ab

un

dan

ce

(a.u

.)

1.175 1.180 1.185 1.190 1.195 1.200 1.205 1.210 1.215 1.220 1.225 1.230 0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50 (x1,000,000)

118.10 (1.28) 116.10 (100.00) 133.10 (1.00) 130.10 (100.00) TIC


Ab

un

dan

ce

(a.u

.)

P a g e 102 | 132

Valine

Leucine

1.375 1.380 1.385 1.390 1.395 1.400 1.405 1.410 1.415 1.420 1.425 1.430 1.435 1.440 1.445 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0 (x1,000,000)

136.10 (2.02) 130.10 (100.00) 178.20 (1.05) 172.20 (100.00) 72.10 (1.00) 121.10 (1.66) 116.10 (2.41) TIC


Ab

un

dan

ce (

a.u

.)

Retention Time (min) 1.590 1.595 1.600 1.605 1.610 1.615 1.620 1.625 1.630 1.635 1.640 1.645

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5 (x1,000,000)

136.10 (2.02) 130.10 (100.00) 178.20 (1.05) 172.20 (100.00) 72.10 (1.00) 121.10 (1.66) 116.10 (2.41) TIC

Ab

un

dan

ce (

a.u

.)

P a g e 103 | 132

Isoleucine

Threonine

1.645 1.650 1.655 1.660 1.665 1.670 1.675 1.680 1.685 1.690 1.695 1.700

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5 (x1,000,000)

136.10 (2.02) 130.10 (100.00) 178.20 (1.05) 172.20 (100.00) 72.10 (1.00) 121.10 (1.66) 116.10 (2.41) TIC


Ab

un

dan

ce (

a.u

.)

Retention Time (min) 1.850 1.855 1.860 1.865 1.870 1.875 1.880 1.885 1.890 1.895 1.900 1.905

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4 (x1,000,000)

74.00 (1.00) 69.10 (72.40) 75.10 (1.00) 70.10 (58.21) 63.00 (3.15) 60.10 (5.16) 104.00 (1.00) 101.10 (13.28) TIC

Ab

un

dan

ce (

a.u

.)

P a g e 104 | 132

Serine

Proline

1.890 1.895 1.900 1.905 1.910 1.915 1.920 1.925 1.930 1.935 1.940 1.945

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

(x1,000,000)

74.00 (1.00) 69.10 (72.40) 75.10 (1.00) 70.10 (58.21) 63.00 (3.15) 60.10 (5.16) 104.00 (1.00) 101.10 (13.28) TIC


Ab

un

dan

ce (

a.u

.)

1.94 1.95 1.96 1.97 1.98 1.99 2.00 2.01 2.02 2.03 2.04 2.05 2.06 0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75 (x1,000,000)

74.00 (1.00) 69.10 (72.40) 75.10 (1.00) 70.10 (58.21) 63.00 (3.15) 60.10 (5.16) 104.00 (1.00) 101.10 (13.28) TIC


Ab

un

dan

ce (

a.u

.)

P a g e 105 | 132

Asparagine

Aspartic Acid

Retention Time (min) 2.05 2.06 2.07 2.08 2.09 2.10 2.11 2.12 2.13 2.14 2.15

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00 (x1,000,000)

74.00 (1.00) 69.10 (72.40) 75.10 (1.00) 70.10 (58.21) 63.00 (3.15) 60.10 (5.16) 104.00 (1.00) 101.10 (13.28) TIC

Ab

un

dan

ce (

a.u

.)

2.635 2.640 2.645 2.650 2.655 2.660 2.665 2.670 2.675 2.680 2.685

0.25

0.50

0.75

1.00

1.25

(x1,000,000)

63.00 (4.18) 61.10 (13.66) 92.00 (1.00) 88.10 (16.19) TIC


Ab

un

dan

ce (

a.u

.)

P a g e 106 | 132

Methionine

Glutamic Acid

Ab

un

dan

ce (

a.u

.)

2.670 2.672 2.675 2.678 2.680 2.683 2.685 2.688 2.690 2.692 2.695 2.697 2.700 2.703 2.705 0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

(x100,000)

63.00 (4.18) 61.10 (13.66) 92.00 (1.00) 88.10 (16.19) TIC


3.013 3.015 3.018 3.020 3.023 3.025 3.027 3.030 3.033 3.035 3.038 3.040 0.00

0.25

0.50

0.75

1.00

(x1,000,000)

98.00 (1.00) 91.10 (43.75) 89.00 (1.00) 84.10 (16.47) TIC


Ab

un

dan

ce (

a.u

.)

P a g e 107 | 132

Phenylalanine

Glutamine

Retention Time (min) 3.030 3.035 3.040 3.045 3.050 3.055 3.060 3.065 3.070 3.075

0.00

0.25

0.50

0.75

1.00

1.25

1.50

(x1,000,000)

98.00 (1.00) 91.10 (43.75) 89.00 (1.00) 84.10 (16.47) TIC

Ab

un

dan

ce (

a.u

.)

3.640 3.645 3.650 3.655 3.660 3.665 3.670 3.675 3.680 3.685 3.690 3.695 3.700 3.705 3.710 3.715 3.720 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0 (x100,000)

89.00 (1.00) 84.10 (30.05) TIC


Ab

un

dan

ce (

a.u

.)

P a g e 108 | 132

Lysine

Histidine

4.330 4.335 4.340 4.345 4.350 4.355 4.360 4.365 4.370 4.375 4.380 4.385 4.390 4.395 4.400 4.405 0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1 (x1,000,000)

176.20 (1.00) 170.20 (100.00) TIC


Ab

un

dan

ce (

a.u

.)

4.535 4.540 4.545 4.550 4.555 4.560 4.565 4.570 4.575 4.580 4.585 4.590 4.595 4.600 4.605 4.610 4.615

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

(x10,000)

87.00 (1.00) 81.10 (1.29) TIC


Ab

un

dan

ce

(a.u

.)

P a g e 109 | 132

Tyrosine

Tryptophan

4.830 4.835 4.840 4.845 4.850 4.855 4.860 4.865 4.870 4.875 0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0 (x100,000)

114.10 (1.00) 107.10 (100.00) TIC


Ab

un

dan

ce (

a.u

.)

5.110 5.115 5.120 5.125 5.130 5.135 5.140 5.145 5.150 5.155

0.25

0.50

0.75

1.00

1.25

(x1,000,000)

140.10 (1.00) 130.10 (100.00) TIC


Ab

un

dan

ce (

a.u

.)

P a g e 110 | 132

Appendix VI: Optimization of probe and heater gas flow

For the optimization of heater gas flow (HGF) and probe’s horizontal (H) and lateral (L)

position the following values were chosen and measured.

H= 15;13;11;9;7;5;3;1;15;4;3

L= -10;-8;-6;-4;-2;0;+2;+4;+6;-5

HGF=1000;2000;3000;4000;5000;6000;7000;8000

Each time one parameter was changed and the other two were remained stable.

H L HGF Time(min)

1 15 -5 6500 1,5

2 13 -5 6500 4,5

3 11 -5 6500 7,5

4 9 -5 6500 10,5

5 7 -5 6500 13,5

6 5 -5 6500 16,5

7 3 -5 6500 19,5

8 1 -5 6500 22,5

9 15 -5 6500 25,5

10 4 -5 6500 28,5

11 3 -5 6500 31,5

12 3 -10 6500 34,5

13 3 -8 6500 37,5

14 3 -6 6500 40,5

15 3 -4 6500 43,5

16 3 -2 6500 46,5

17 3 0 6500 49,5

18 3 +2 6500 52,5

19 3 +4 6500 55,5

20 3 -6 6500 58,5

21 3 -5 6500 1,5

22 3 -5 1000 4,5

23 3 -5 2000 7,5

24 3 -5 3000 10,5

25 3 -5 4000 13,5

26 3 -5 5000 16,5

27 3 -5 6000 19,5

28 3 -5 7000 22,5

29 3 -5 8000 25,5

P a g e 111 | 132

13

11

9

7

5 3

1

15 15

3 4

H optimization

-10

-8

-6 -4

-2

0

+4 +2

-6

L optimization

P a g e 112 | 132

-5

1000

2000 3000

4000

5000

6000 7000 8000

HGF optimization

Optimum

Conditions

P a g e 113 | 132

Appendix VII: Verification of compound/source-dependent

parameters

Alanine

Compound-dependent parameters:

Declustering Potential (DP): 15.0; 20.0; 25.0; 30.0; 45.0;

Focusing Potential (FP): 100.0; 150.0; 200.0; 250.0;

Entrance Potential (EP): 7.5; 10.0; 15.0;

Source-dependent parameters

Nebulizer Gas (NEB): 8.0; 10.0; 12.0; 14.0; 15.0;

Curtain Gas (CUR): 6.0; 8.0; 10.0; 12.0; 14.0;

Ionspray Voltage (IS): 1000.0; 1500.0; 2000.0; 3000.0; 4000.0;

Temperature (TEM): 250.0; 300.0; 350.0; 400.0; 450.0; 500.0; 550.0;

Parameters Optimized Chosen

DP 30 26

FP 200 210

EP 7.5 10

NEB 14 12

CUR 10 12

IS 1500 1900

TEM 550 475

P a g e 114 | 132

Aspartic Acid


Declustering Potential (DP): 10.0; 15.0; 20.0; 25.0; 30.0; 40.0;

Focusing Potential (FP): 100.0; 150.0; 200.0; 250.0;




Curtain Gas (CUR): 6.0; 8.0; 10.0; 12.0; 14.0;

Ionspray Voltage (IS): 1000.0; 1500.0; 2000.0; 2500.0; 3000.0; 4000.0;

Temperature (TEM): 250.0; 300.0; 350.0; 400.0; 450.0; 500.0; 550.0;


DP 30 16

FP 250 110

EP 15 10

NEB 12 12

CUR 10 12

IS 1500 1900

TEM 550 475

P a g e 115 | 132

Norvaline


Declustering Potential (DP): 10.0; 15.0; 20.0; 25.0; 30.0; 45.0;

Focusing Potential (FP): 50.0; 100.0; 150.0; 200.0; 250.0;




Curtain Gas (CUR): 6.0; 8.0; 10.0; 12.0; 14.0;

Ionspray Voltage (IS): 1000.0; 1500.0; 2000.0; 2500.0; 3000.0; 4000.0;

Temperature (TEM): 250.0; 300.0; 350.0; 400.0; 450.0; 500.0; 550.0;


DP 25 21

FP 100 130

EP 10 10

NEB 15 12

CUR 10 12

IS 1500 1900

TEM 500 475

P a g e 116 | 132

Appendix VIII: Determination of parent and product ions

Glutamine

Serine

Parent Ion: 12%

In Source Fragmentation: 4%

Parent Ion: 10%

Product Ion: 35%

P a g e 117 | 132

Asparagine

Leucine

Parent Ion: 50%


Parent Ion: 80%


P a g e 118 | 132

Glycine

Threonine

Parent Ion: 15%


Parent Ion: 52%


P a g e 119 | 132

Alanine

Proline

Parent Ion: 53%


Parent Ion: 15%


P a g e 120 | 132

Methionine

Aspartic Acid

Parent Ion: 20%


Parent Ion: 20%


P a g e 121 | 132

Histidine

Valine

Parent Ion: 100%


Parent Ion: 64%


P a g e 122 | 132

Glutamic Acid

Isoleucine

Parent Ion: 47%


Parent Ion: 40%


P a g e 123 | 132

Phenylalanine

Tyrosine

Parent Ion: 55%


Parent Ion: 100%


P a g e 124 | 132

Lysine

Tryptophan

Parent Ion: 55%


Parent Ion: 59%


P a g e 125 | 132

Appendix IX: Calibration Curves of the amino acids using LC/MS

Asparagine

Leucine

y = 128055x - 8810.4R² = 0.9996

0.00E+00

2.00E+05

4.00E+05

6.00E+05

8.00E+05

1.00E+06

1.20E+06

1.40E+06

0 2 4 6 8 10 12

Pe

ak A

rea

(a.u

.)

Concentration (μM)

y = 505704x + 133475R² = 0.9951

0.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

5.00E+06

6.00E+06

0 2 4 6 8 10 12

Pe

ak A

rea

(a.u

.)

Concentration (μM)

P a g e 126 | 132

Glutamine

Threonine

y = 193378x + 74903R² = 0.9846

0.00E+00

5.00E+05

1.00E+06

1.50E+06

2.00E+06

2.50E+06

0 2 4 6 8 10 12

Pe

ak A

rea

(a

.u.)

Concentration (μM)

y = 30962x + 55212R² = 0.9979

0.00E+00

5.00E+05

1.00E+06

1.50E+06

2.00E+06

2.50E+06

3.00E+06

3.50E+06

0 20 40 60 80 100 120

Pe

ak A

rea

(a.u

.)

Concentration (μM)

P a g e 127 | 132

Serine

Alanine

y = 83401x + 264814R² = 0.9944

0.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

5.00E+06

6.00E+06

7.00E+06

8.00E+06

9.00E+06

1.00E+07

0 20 40 60 80 100 120

Pe

ak A

rea

(a.u

.)

Concentration (μΜ)

y = 20124x - 61986R² = 0.9732

-1.00E+05

4.00E+05

9.00E+05

1.40E+06

1.90E+06

2.40E+06

0 20 40 60 80 100 120

Pe

ak A

rea

(a.u

.)

Concentration (μM)

P a g e 128 | 132

Methionine

Proline

y = 217566x - 499397R² = 0.9941

-1.00E+06

4.00E+06

9.00E+06

1.40E+07

1.90E+07

2.40E+07

0 20 40 60 80 100 120

Pe

ak A

rea

(a.u

.)

Concentration (μM)

y = 1E+06x + 210795R² = 0.9918

0.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

5.00E+06

6.00E+06

0 1 2 3 4 5 6

Pa

ek A

rea

(a.u

.)

Concentration (μM)

P a g e 129 | 132

Lysine

Aspartic Acid

y = 183816x + 705.56R² = 0.9991

0.00E+00

2.00E+05

4.00E+05

6.00E+05

8.00E+05

1.00E+06

1.20E+06

1.40E+06

1.60E+06

1.80E+06

2.00E+06

0 2 4 6 8 10 12

Pe

ak A

rea

(a

.u.)

Concentration (μM)

y = 247631x + 74509R² = 0.9995

0.00E+00

5.00E+05

1.00E+06

1.50E+06

2.00E+06

2.50E+06

3.00E+06

0 2 4 6 8 10 12

Pe

ak A

rea

(a.u

.)

Concentration (μM)

P a g e 130 | 132

Histidine

Glutamic Acid

y = 992948x + 253136R² = 0.9983

0.00E+00

2.00E+06

4.00E+06

6.00E+06

8.00E+06

1.00E+07

1.20E+07

0 2 4 6 8 10 12

Pe

ak A

rea

(a.u

.)

Concentration (μM)

y = 320678x + 18095R² = 0.9999

0.00E+00

5.00E+05

1.00E+06

1.50E+06

2.00E+06

2.50E+06

3.00E+06

3.50E+06

0 2 4 6 8 10 12

Pe

ak A

rea

(a.u

.)

Concentration (μM)

P a g e 131 | 132

Tryptophan

Tyrosine

y = 16366x - 1337.6R² = 0.9985

0.00E+00

2.00E+04

4.00E+04

6.00E+04

8.00E+04

1.00E+05

1.20E+05

1.40E+05

1.60E+05

1.80E+05

0 2 4 6 8 10 12

Pe

ak A

rea

(a.u

.)

Concentration (μM)

y = 238980x + 4586R² = 0.9999

0.00E+00

5.00E+05

1.00E+06

1.50E+06

2.00E+06

2.50E+06

3.00E+06

0 2 4 6 8 10 12

Pe

ak A

rea

(a.u

.)

Concentration (μM)

P a g e 132 | 132

Isoleucine

Phenylalanine

y = 1E+06x + 69562R² = 0.9935

0.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

5.00E+06

6.00E+06

7.00E+06

0 1 2 3 4 5 6

Pe

ak A

rea

(a

.u.)

Concentration (μM)

y = 1E+06x + 25739R² = 0.9994

0.00E+00

2.00E+06

4.00E+06

6.00E+06

8.00E+06

1.00E+07

1.20E+07

1.40E+07

0 2 4 6 8 10 12

Pe

ak A

rea

(a.u

.)

Concentration (μM)

msc in analytical sciences - uvahigh performance liquid chromatography with precolumn...

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