chamo 9172 r3 - waters corporation · 2013-06-13 · commercial chamomile tea samples extracted...
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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