DIFFERENTIATION OF GERMAN AND ROMAN CHAMOMILES
BY COMPREHENSIVE CHEMICAL FINGERPRINTING USING ULTRA
PERFORMANCE SUPERCRITICAL FLUID CHROMATOGRAPHY
COUPLED WITH MASS SPECTROMETRY
Shinnosuke Horie1, Motoji Oshikata1, Michael D Jones2, Bharathi Avula3, Kate Yu2 Yan-Hong Wang3, Mei Wang3, dominic moore2, Warren Potts2, and Ikhlas Khan3
1
NihonWaters, Tokyo, Japan; 2Waters Corporation, Milford, MA; 3University of Mississippi, Oxford, MS
INTRODUCTION
COMPARATIVE ANALYSIS
Natural product ingredient profiling is a challenging task due to
sample complexity. The analyte diversity requires the utility of
multiple analytical techniques to provide orthogonality which can
best provide comprehensive knowledge of the analyte composition.
Presently, a variety of analytical techniques exist (such as HPTLC,
UPLC, GC, NMR etc) which cover a range of chemical space.
Anthemis nobilis
(Roman Chamomile)
Roman Chamomile
A.
UPLC Reversed Phase
UV 350 nm
0.88
2
1. apigenin-7-O-
3
0.66
0.16
glucoside
2. chamaemeloside
3. apigenin
AU
0.44
0.04
0.22
Matricaria recutita
(German Chamomile)
0.00
0.00
0.00
3.20
6.40
9.60
12.80
16.00
Minutes
19.20
22.40
25.60
28.80
0.00
32.00
3.20
6.40
9.60
12.80
16.00
Minutes
0.20
3
B.
UPC2
UV 350 nm
2
0.10
1
0.05
EXPERIMENTAL
Sample Preparation
The Chamomile samples were extracted with methanol. Dry plant samples
(0.5 g) were sonicated in 2.0 mL of methanol for 30 min followed by
centrifugation for 15 min at 4000 rpm. The supernatant was transferred to
a 10 mL volumetric flask. The procedure was repeated four times and the
respective supernatants were combined. The final volume was adjusted to
10 mL with methanol and mixed thoroughly. Prior to analysis, an adequate
volume (ca. 2 mL) was passed through a 0.2 µm nylon membrane filter.
Method Development Scheme
An iterative screening of columns, modifiers, and additives were explored
with a series of goals to optimize for separation characteristics focused on:
1. Resolution of peaks
2. Overall chromatographic peak shape
3. Selectivity effects
Method Conditions are described with each figure.
The orthogonality of supercritical
fluid chromatography and reversed
phase chromatography for the
chamomile extracts were assessed.
The fingerprint of the chemical
profiles were compared to determine
added benefits between the
techniques. The primary goal was to
determine retention time differences
between the chromatographic
profiles facilitate by MS tracking.
The comparison of peak shape,
sensitivity and peak capacity were
not objectives at this time. The
chamomile samples were injected on
an ACQUITY UPLC system configured
to a ACQUITY SQD. The column used
was ACQUITY HSS T3 2.1mm x
100mm; 1.7µm. The samples were
eluted with a 30 minute gradient
flowing at 0.5mL/min. The gradient
profile was 5-50% over 30 minutes
and ramped to 100% B for two
minutes to elute any hydrophobic
peaks. Retention times and masses
were recorded to be confirmed and
elucidated later by accurate mass
Time of Flight MS and MSe.
0.018
AU
AU
0.15
0.00
2.20
4.40
6.60
8.80
11.00
Minutes
13.20
15.40
17.60
19.80
22.00
0.2% TEA in MeOH
MeOH:ACN:IPA with
0.1% TFA
MeOH:ACN with
0.1% TFA
0.1% Phosphic
acid in MeOH
1.6x106
MeOH:ACN:Water
with 0.1% formic acid
Isopropanol
AU
A.
320000
1.2x106
Acetonitrile
19.80
22.00
B.
240000
8.0x105
4.0x105
160000
80000
0
5.28
5.94
6.60
7.26
7.92
Minutes
8.58
9.24
9.90
10.56
11.60 11.80 12.00 12.20 12.40 12.60 12.80 13.00 13.20 13.40 13.60 13.80 14.00 14.20 14.40 14.60 14.80 15.00 15.20 15.40
Minutes
Figure 6. XIC of m/z=475. An additional peak was found in the UPC2
trace (A) when compared to the UPLC-RP (B) trace.
Roman Chamomile
Instrument Configuration
AU
Figure. It was important to maintain automated back pressure control,
maximum system pressure range for gradient flexibility, and maintain CO2 in
a supercritical state during method development. Below is a schematic of the
flow management used that worked best for this development process.
0.070
0.000
17.60
Interrogation of the MS data for both LC and SFC based techniques
displayed some differences when comparing isobaric separations indicated
by the XIC of selected masses, as seen in Figure 6.
MeOH:ACN with
0.1% TEA and
0.1% TFA
Methanol
0.105
0.00
15.40
The chromatograms of the two chamomile extract profiles are distinctly
different. The UV profiles at 350nm resulted in a difference of number of
peaks, intensity of major peaks, and differing spectral profile comparisons.
The mass spectra confirmed the changing selectivity of the flavonoid peaks
of interest for roman chamomile. The highly non-polar analytes that eluted
last in reversed phase were observed to elute very early in the UPC2
chromatographic trace, as expected. Experiments were performed at 100%
CO2 which provided greater retention of these highly nonpolar peaks
(data not shown). Masses with even numbers represent the presence of
alkaloids. Masses with odd numbers represent the polyphenolic compounds.
4.62
0.035
13.20
Figure 7. UPLC-RP (A) and UPC2 (B) results for
German Chamomile.
0.0
0.07
11.00
Minutes
German Chamomile
0.140
0.14
8.80
Roman Chamomile
Option 2
0.21
6.60
Intens ity
UPC2 HSS SB C18
Option 1
German Chamomile
4.40
Figure 5. Plot of retention times for masses found in
Roman Chamomile. The plot illustrates the orthogonality
of the two approaches
MeOH:ACN with
formic acid
0.05% TFA in
MeOH
ACQUITY Shield
2.20
DISCUSSION
UPC2 PFP
ACQUITY Cyano
32.00
0.012
0.00
Figure 6. Plot of retention times for each peak found in
German Chamomile. Aside from demonstrated orthogonality,
some retention mechanisms in this example are shown to be
behave in a non-linear behavior to that of RP-LC.
2g/L Ammonium
Formate in MeOH
UPC2 2-EP
28.80
Figure 4. UPLC-RP (A) and UPC2 (B) results for Roman
Chamomile. The three major flavonoid peaks of interest
are highlighted.
C0-SOLVENTS SYSTEMS
UPC2 BEH
25.60
0.000
0.00
Intensity
It was observed that two different method conditions provided the desired
peak shapes for the two chamomile samples, whereas Anthemis nobilis
(Roman Chamomile) peak shapes were the most affected by the co-solvent
system. The method development process was focused to address optimum
peak shape for the Roman Chamomile sample. Also, the UPC2-UV-MS method
was optimized for the identification of flavonoids, phenolic compounds and
coumarins from chamomile samples at 350nm. Injections were performed at
1µL to minimized overloading of the concentrated sample as observed at
greater injection volumes.
COLUMNS
22.40
0.006
METHOD DEVELOPMENT
Method development was performed utilizing a column and co-solvent
screening process. The dimensions of the columns during the screening
process were 3.0mm x 100mm; 1.7µm. A linear 12 minute gradient varied
from 5% to 35% co-solvent was used to elute the chamomile extract
injections. The back pressure was held isobarically at 1500psi with a column
temperature of 450C. The flow rate was 2mL/min. The analysis of chamomile
extracts or extracts of natural products with compounds with extract
components similar to those found in chamomile extract by packed column
SFC are rarely published. With little knowledge of predicting
chromatographic behavior, the co-solvents were chosen to explore various
retention mechanisms induced by protic and aprotic solvents, high pH and
low pH, and ion pairing interactions.
19.20
B.
UPC2
UV 350 nm
0.024
In this presentation, we use Chamomile as an application
example to investigate and explore the advantages and
limitations of a novel technology based on Supercritical fluid
chromatography with a goal of obtaining a comprehensive
ingredient profile for Chamomile and gaining chemical
understanding of the key ingredients that differentiated different
chamomile extracts.
0.060
0.44
Roman Chamomile
0.045
0.33
German Chamomile
AU
AU
0.030
0.22
0.015
0.11
0.000
0.00
0.00
0.08
1
Analytical methods can be used for plant comparison studies
including GC-MS, LC-UV-MS, and UPSFC/UV/MS. All have unique
capabilities for identification and authentication of plant samples
and commercial products. Use of all these techniques will be
helpful for chromatographic fingerprint analysis of chamomile
samples. Information obtained represents a comprehensive
qualitative approach for the purpose of species authentication,
quality control, ensuring the consistency and evaluation of their
related commercial products.
0.28
A.
UPLC Reversed Phase
UV 350 nm
0.12
AU
Chamomile is often used to relief symptoms of sleeplessness,
anxiety, and gastrointestinal conditions. The flowering tops of
the chamomile plant are used to make teas, liquid extracts etc.
Normally, Chamomile refers to either German chamomile or
Roman chamomile, which are from the same family (Asteraceae)
but belong to different genera. The main components described
in chamomile flowers belong to the classes of volatile derivatives
and flavonoid components.
German Chamomile
3.00
6.00
9.00
12.00
15.00
Minutes
18.00
21.00
24.00
27.00
0.00
30.00
0.056
AU
6.00
8.00
10.00
Minutes
12.00
14.00
16.00
18.00
20.00
The methodology using the UPC2 CSH PFP stationary phase with phosphoric acid as the modifier added to the co-solvent yielded satisfactory
peak shapes. Attempts to convert the methodology to a “MS Friendly”
mobile phase were unsuccessful. Formic acid, acetic acid, triflouroacetic
acid were all substituted for phosphoric acid, however severe tailing was
observed for the later eluting Roman Chamomile peaks.
0.042
0.028
0.014
0.000
0.00
4.00
Figure 3. UPC2 CSH PFP. 3.0mm x 100mm;1.7µm. 1.8mL/min at 40C.
1500psi backpressure. Gradient performed from 3 to 30% co-solvent.
Co-solvent was 90:10 MeOH:ACN with 0.05% phosphoric acid. λ=350nm.
Figure 1. UPC2 2-EP. 2.1 x 150mm;1.7µm. 1.2mL/min at 40C. 1500psi
backpressure. Gradient performed from 5 to 40% co-solvent. Co-solvent
was 90:10 MeOH:ACN with 0.05%H2O. λ=350nm.
The UPC2 column consisting
of 2-EP stationary phase and
co-solvent composition of
90:10 Methanol: Acetonitrile
with 0.05% water as an
additive provided the best
observed peak shape with
the least amount of column
tailing compared to the other
conditions.
2.00
3.00
6.00
9.00
12.00
15.00
Minutes
18.00
21.00
24.00
27.00
30.00
Unfortunately, as the
optimization progressed,
Figure 2. Peak deterioration over time.
the peak shape of the later
eluting peaks would deteriorate and excessive tailing would be observed.
It is undetermined if the observed tailing over time is column related or
rather an equilibrium that occurs over time with the stationary phase and
the mobile phase. The observations with the 2-EP stationary phase are
currently being investigated.
Investigation of the retention mechanisms was performed. Since phosphoric acid has chelating and hydrogen bonding properties, a series of
organic acids were explored to investigate other additives with chelation
properties. Oxalic acid, citric acid, tartaric acid, and succinic acid were
explored. The additives represent bi-dentate and tri-dentate properties.
It was observed that the additives with the tri-dentate properties maintained sharper peak shapes. Although these additives are not considered
MS friendly, the investigation has increased our understanding of the
chromatographic behavior of these class of compounds under supercritical
fluid conditions.
During development, it was observed the composition ratio of methanol
to acetonitrile controlled the selectivity of the peaks.
CONCLUSIONS
Method Development
• The method screening process resulted in two suitable stationary
phases. ACQUITY UPC2 CSH PFP with 90:10 (MeOH:ACN) with
0.05% phosphoric acid
• Selectivity of the main peaks of interest was controlled by the
composition ratio of acetonitrile and methanol. Tailing of the later
eluting peaks were controlled by addition of acids with tridentate
binding properties.
MS Comparative Analysis
• The UPC2 methodology proved to be orthogonal to the reversed
phase chromatography.
• UPC2 provides an approach to analyze the highly hydrophobic
analytes found in the extracts that seem to require a high percentage
of organic when using RP-LC.
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