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APPLICATION OF SUB-2µm PARTICLE CO2-BASED CHROMATOGRAPHY COUPLED TO MASS SPECTROMETRY FOR CHEMICAL PROFILING OF VARIOUS CHAMOMILES

Michael Jones1,2, Giorgis Isaac2, Bharathi Avula3, Yan-Hong Wang3, Kate Yu2, Troy J. Smillie3, Norman Smith1, Ikhlas A. Khan3,4

1King’s College London, Pharmaceutical Science Division, School of Biomedical and Health Sciences, London, SE1 9NH, UK., 2Waters Corporation, Milford, MA, USA., 3National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences,

The University of Mississippi, University, MS, USA., 4Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS, USA.

INTRODUCTION

Numerous preparations of chamomile have been developed, the

most popular being in the form of herbal tea and herbal infusions.

Chamomile can also be found in a variety of face creams, drinks,

hair dyes, shampoos, perfumes, ointments, and tinctures. As a

member of Asteraceae family, it is widely represented by two known

types viz German chamomile (Matricaria recutita) and Roman

chamomile (Anthemis nobilis). German chamomile in particular is

the most common type used for medicinal purposes.

The identification of chemical constituents to differentiate the two

types of Chamomiles (German and Roman) are achieved using the

UPC2/MS method previously developed and presented at HPLC

2012.1 The UPC2 methodology was then applied to profile 11

commercial chamomile tea samples extracted using supercritical

fluid extraction (SFE) instrumentation. The data was processed using

TransOmics software for Metabolomics and Lipidomics (TOIML).

Benefits of using TOIML are recognized by the deconvolution of the

accurate mass data and statistically analyzing the results via visual

aids such as principle component analysis (PCA), dendrograms, and

database searching. The overall goal of this work is to propose a

workflow for natural product profiling that would include sample

preparation, data analysis and data interpretation.

METHODS

Extraction Technique: The two authentic chamomile flower

types and each of the 11 commercial chamomile tea samples

were extracted separately by MV-10 SFE instrumentation using

5% methanol modifier for 15 minutes.

UPC2-UV/MS Analysis: All chromatographic separations

were carried out on an ACQUITY UPC2 system using an

ACQUITY UPC2 BEH 2-EP column (150 x 2.1mm, 1.7µm). The

mobile phase consisted of CO2(A), and MeOH:Isopropanol

(1:1) with 0.5 % formic acid (B). The flow rate was 1.7 mL/

min. The column and sample temperature were maintained at

50°C and 10°C, respectively. The effluent from the LC column

was directed into the ESI probe of the Xevo G2 Q-Tof mass

spectrometer (MS) . The MS conditions were optimized to

maximize sensitivity. The source temperature and the

desolvation temperature were maintained at 150°C and 350°C,

respectively. Each sample was injected in triplicate, randomly.

QC checks were performed after every 10 injections using a

composite sample mixture consisting of all sample extractions.

Scientific Findings

A total of 13 samples were analyzed; 2 authentic chamomile types and 11 commercial tea samples containing chamomile

were automatically extracted by SFE and analyzed by UPC2.

M. recutita and A. nobilis samples showed different chemical

fingerprinting chromatographically and statistically.

Reference standards of Cis/trans dicycloethers confirmed the

major constituents identified in M. recutita extract. Reference standards of apigenin and sesquiterpenes

confirmed the major constituents identified in the A. nobilis extract.

All commercial tea samples were determined to be related to German chamomile, yet distinctions were easily determined

by PCA

Benefits of Using Waters Solutions

Waters MV-10 SFE provided an automated extraction of multiple samples.

The organic extracts can be directly injected on the UPC2-MS system

The workflow proposed using SFE, UPC2- MS, and TOIML provided a flexible ease of use approach to profile natural

products from sample preparation through to data interpretation.

TranOmics for Metabolomic and Lipidomics (TOIML) provided an informatics solution needed to deconvolute and interpret

the complex data sets associated with natural product profiling

Authentic German vs. Authentic Roman Chamomile Analysis

Figure 7. PCA analysis of all sample extractions. As it can be

seen in the PCA plot the 9254 group is different from the rest

of the groups.

Figure 8. PCA plot after excluding 9254 from the analysis. Of

the 11 commercial chamomile samples, only 1 was

determined statistically very similar to that of the authentic

German chamomile flowers.

Genuine GC

SFE Extracts Analysis of Commercial Chamomile Tea Samples

Figure 3: OPLS-DA plot of German vs. Roman chamomile

SFE Extracts.

Figure 6: Standardized abundance profile of selected features

illustrating good technical replicates. The trend plot indicates the major features that are up-regulated in German 9172

chamomiles.

Chamo_9172_R3

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00

%

0

100

01312013_033 1: TOF MS ES+ BPI

2.35e5

2.44229.1245

0.76473.3994

0.71473.3994

0.77473.3994

2.37229.1245

1.58359.2220

1.49292.2647

1.63227.1076

2.56229.1245

3.25383.1480

5.88385.1632

4.71396.20293.34

383.1480

3.51;399.1396

5.07277.1087

5.18383.1480

6.03385.1632

8.70401.15958.21

401.15556.88682.6335

12.66271.0594

01312013_039 1: TOF MS ES+ BPI

1.09e5

0.76473.3994

0.52393.2974

0.85293.1723

0.94277.1769

2.86543.4788

1.06201.0912 1.85

277.1769

6.93871.5746

6.85353.2302

3.70210.0919

Figure 2. Examples of MS ES+ chromatograms of German

and Roman chamomile extracts from SFE performed with 5%

addition of modifier

Figure 5. The up-regulated and down-regulated features in the

S-Plot (left) which distinguish the two chamomiles were extracted and charted in terms of average intensities (right).

trans-dicycloether

(Rt = 0.92 )cis-dicycloether

(Rt = 1.16 min)

Hydroxyisonobilin

(Rt = 6.17 min)Apigenin

(Rt = 11.86 min)

Figure 5: Reference standards of the above structures were

injected to verify the major constituents of dicycloethers from

German chamomile and sesquiterpenes from the Roman

chamomile.

Figure 9. The dendrogram in the TOIML software facilitated

data visualization. Similar masses could be found within

different sample extractions and tagged for searching by public

or custom databases.

Figure 4: The up-regulated and down-regulated features in

the S-Plot (left) which distinguish the most between the two chamomiles. This aids the determination of the major

contributing differentiating entities.

Why SFE for Natural Product Extraction? Classical liquid extraction methods can have drawbacks Limited selectivity Thermal degradation of heat-labile compounds

Oxidative degradation of highly unsaturated compounds Organic toxic solvents

Residual solvents

Government regulations on the use of organic solvent Advantages Supercritical-CO2 for extraction Lower temperature extraction conditions, typically 300C to 500C

Minimal degradation of thermo-labile molecules Highly selective Solvent power can be varied by control of pressure and

temperature Low viscosity aids rapid extraction

Negligible surface tension Utilization of non-toxic medium

No toxic residue

Isolation of extracted analytes from extraction medium is readily accomplished by pressure reduction

Workflow Overview

TIP: Muddle/grind the natural product prior to

filling the vessel. In this example, the flower

heads were ground to a semi-fine powder to

increase the efficiency of the supercritical fluid

extraction

Figure 1: Schematic of the

supercritical fluid

extraction device. The

Chamomile extraction

used 5% addition of co-

solvent (a.k.a. modifier)

Why UPC2 + Q-TOF MS + TOIML for Profiling? Natural product profiling typically involves complex chromatograms consisting of separations with related

compounds and isobaric species. An approach providing selectivity is KEY. Various extractions are typically required

and a streamlined analytical approach is desirable.

UPC² SIMPLIFIES the workflow, separates compounds with

structural SIMILARITY , and provides ORTHOGONALITY compared to RPLC.

Xevo G2 Q-ToF MS provides MSE data generation, whereas

low and high collision energy (CE) MS data can be collected within the same LC injection

TOIML provides a streamlined step-by-step informatics

workflow that will: Align the retention times of low and high CE MS data

Deconvolute the LC/MS data, adducts, and noise Provide peak picking capabilities

Visually indicate compound abundance Provide multivariate statistical analysis

Match against databases for component ID

REFERENCES: Jones et al; HPLC 2012 poster presentation titled “Sample

Comparison of Chamomile by Chemical Profiling Using UPC2/MS”; Anaheim, CA,

June 2012

Figure 10. The compounds list can be searched via public or

custom databases within the software. The compound list was

searched against a chamomile specific database. In this

figure, a nobilin-derived compound is found abundant in the

Roman species of chamomile (9245 ID); as expected, helping

to aid the validity of the database searching capability.

CONCLUSIONS