Profi ling Hoodia Extracts by HPLC with Charged Aerosol Detection, Electrochemical Array Detection, and Principal Component Analysis
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Profiling Hoodia Extracts by HPLC with Charged Aerosol Detection, Electrochemical Array Detection, and Principal Component Analysis Ian Acworth, Bruce Bailey, Marc Plante, Qi Zhang, and David Thomas Thermo Fisher Scientific, Chelmsford, MA, USA
Thermo Fisher Scientific, Chelmsford, MA
Overview Methods
Purpose: To combine fast and sensitive HPLC methods with chemometric analysis HPLC with Charged Aerosol Detectio
to profile botanical extracts from the botanical supplement Hoodia gordonii.
HPLC System: Thermo Scientific
Methods: Hoodia extracts were analyzed by gradient reversed-phase high - DAD–3000RS D
performance liquid chromatography with either a charged aerosol detector or - Corona™ ultra R
photodiode array detector in series with an electrochemical array detector (the Nebulizer Temper
spectro-electro array). Chromatographic data were interrogated by chemometric Power function: 1
software to identify patterns related to differences in plant species, origin or processing. Data collection ra
Results: Unsupervised chemometric evaluation of the information rich data obtained Data Analysis: Thermo Scientific
from HPLC with charged aerosol detection and electrochemical array detection Data System softw
correctly identified differences in sample origin and processing.
Column: Thermo Scientific
Introduction Temp.:
2.6 µm
40 oC
Injection Vol.: 10 µL
Hoodigosides are oxypregnane steroidal glycosides abundant in Hoodia gordonii Flow Rate: 2.0 mL/min
and related plants native to the deserts of southwestern Africa. These plants, used Mobile Phase A: Deionized water
traditionally to ease hunger during long hunting expeditions, enjoy wide use today Mobile Phase B: Acetonitrile, Fishe
in dietary supplements purported to aid in appetite suppression and weight loss. Gradient: 40%B to 47%B in
Presented are two approaches to profile Hoodia related products. In the first approach,
HPLC with Spectro-Electro Array De
Hoodia extracts were analyzed by HPLC with charged aerosol detection. Eight
hoodigosides isolated from dried plant material are separated within 15 min on a solid HPLC System: Thermo Scientific
core C8 analytical column that delivers superb resolution with low backpressure. With - DAD–3000RS D
low-nanogram sensitivity, the charged aerosol detector responds uniformly to all non- - CoulArray™ Ele
volatile species including the target analytes, degradation products and impurities that 16-channel array
may not possess a chromophore.
Data Analysis: Thermo Scientific
In the second approach, Hoodia extracts were analyzed by HPLC with an online System 6.8 and C
electrochemical array detector that responds to redox active compounds such as
polyphenols. Each of the sixteen sensors in the electrochemical array detects analytes Column: Thermo Scientific
at a unique potential to yield picogram on-column sensitivity for diverse compounds 3 µm
that vary widely in redox properties. The chromatograms resulting from this Temperature: 35 oC
voltammetric approach often clearly resolve co-eluting compounds. Flow Rate: 0.65 mL/min
Injection Vol.: 10 µL
These two techniques provide complementary data that are used to quantify known Mobile Phase A: 20 mM Monobasic
compounds. The complex patterns of known and unknown compounds revealed by ` 0.2% tetrahydrofu
the detectors were also interrogated by pattern recognition software to support Mobile Phase B: 20 mM Monobasic
inferences on product quality, authenticity, adulteration, or origin. Limits of detection, 10% tetrahydrofur
linear and dynamic range, precision, and pattern recognition results are compared Mobile Phase C: Methanol, Optima
for the two approaches.
Gradient:
FIGURE 1. HPLC with charged aerosol detection identifies hoodigosides in a Time Flow %A
standard with precision and sensitivity; overlay of seven injections, 10 mg/L (min) (mL/min)
0 0.65 92
2 0.65 92
30 0.65 0
45 0.65 0
45 0.65 92
50 0.65 92
Sample Preparation
50 mg powder from a commercially ava
tube. One 1 mL methanol was added a
supernatant was transferred to a 5 mL
two times with fresh methanol and the
with methanol and mixing, an aliquot w
13,000 rpm for 10 min and the superna
Data Analysis
Principal components analysis (PCA) a
pattern differences in chromatograms r
time. Data were aligned by using the P
Windows 3.0 software and imported int
modeling software version 4.5 for PCA
2 Profiling Hoodia Extracts by HPLC with Charged Aerosol Detection, Electrochemical Array Detection, and Principal Component Analysistific, Chelmsford, MA
Methods Results
methods with chemometric analysis HPLC with Charged Aerosol Detection Chromatography
pplement Hoodia gordonii.
Method 1: HPLC-Charged aerosol detection
HPLC System: Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLC system with a:
dient reversed-phase high - DAD–3000RS Diode Array Detector and a A mixture of eight hoodigosides eluted with bas
charged aerosol detector or - Corona™ ultra RS™ Charged Aerosol Detector: the Accucore C8 column under an aqueous/solv
ochemical array detector (the Nebulizer Temperature: 25 °C acetonitrile over 15 min (Figure 1). After separa
re interrogated by chemometric Power function: 1.00 washed for an additional 10 min with 95% aceto
s in plant species, origin or processing. Data collection rate: 20 Hz hydrophobic compounds that might be present i
of the information rich data obtained Data Analysis: Thermo Scientific™ Dionex™ Chromeleon™ Chromatography ample response can be seen in this overlay of s
lectrochemical array detection Data System software, 7.1 SR1 and diluent blank.
nd processing.
The chromatograms obtained from sample extra
Column: Thermo Scientific™ Accucore™ C8, 4.6 × 150 mm;
2.6 µm peaks eluting before, during and after the retent
Temp.: 40 oC standards. Figure 2 shows example chromatogr
Injection Vol.: 10 µL Hoodia gordonii mixed parts. In most cases, the
des abundant in Hoodia gordonii Flow Rate: 2.0 mL/min sufficiently to permit their accurate identification
western Africa. These plants, used Mobile Phase A: Deionized water mixtures. Some of the extracts included peaks
xpeditions, enjoy wide use today Mobile Phase B: Acetonitrile, Fisher Scientific™ Optima™ LCMS hoodigosides. Note that in many of the extracts
e suppression and weight loss. Gradient: 40%B to 47%B in 2.5 min; to 95%B in 15 min; hold 10 min. compounds that eluted between 15 and 25 min
related products. In the first approach, but were well detected by the charged aerosol d
HPLC with Spectro-Electro Array Detection obtained from standard injections ranging from
arged aerosol detection. Eight
re separated within 15 min on a solid The calibration curves spanned four orders of m
HPLC System: Thermo Scientific™ Dionex™ UltiMate™ 3000 RS system with a data and yielded a correlation coefficient of > 0.
solution with low backpressure. With - DAD–3000RS Diode Array Detector
tector responds uniformly to all non- - CoulArray™ Electrochemical Array Detector: FIGURE 2. HPLC-CAD chromatograms of me
radation products and impurities that 16-channel array from 0 to +900 mV versus Pd in +60 mV steps gordonii, Hoodia gordonii (from Namibia), an
Data Analysis: Thermo Scientific™ Dionex™ Chromeleon™ Chromatography Data
nalyzed by HPLC with an online System 6.8 and CoulArray 3.0 software.
edox active compounds such as
electrochemical array detects analytes Column: Thermo Scientific™ Acclaim™ C18, 3 × 150 mm;
n sensitivity for diverse compounds 3 µm
ograms resulting from this Temperature: 35 oC
uting compounds. Flow Rate: 0.65 mL/min
Injection Vol.: 10 µL
ata that are used to quantify known Mobile Phase A: 20 mM Monobasic sodium phosphate, 3% acetonitrile,
unknown compounds revealed by ` 0.2% tetrahydrofuran, pH 3.35
ecognition software to support Mobile Phase B: 20 mM Monobasic sodium phosphate, 50% acetonitrile,
ration, or origin. Limits of detection, 10% tetrahydrofuran, pH 3.45
recognition results are compared Mobile Phase C: Methanol, Optima LCMS
Gradient:
on identifies hoodigosides in a Time Flow %A %B %C Curve
ay of seven injections, 10 mg/L (min) (mL/min)
0 0.65 92 5 3 5
2 0.65 92 5 3 7 (concave)
30 0.65 0 97 3 7 (concave) FIGURE 3. PCA score plot of CAD data, only
for PCA analysis
45 0.65 0 97 3 5
45 0.65 92 5 3 5 Factor 2 (8.2%)
50 0.65 92 5 3 5
3126
Sample Preparation
50 mg powder from a commercially available capsule was placed into a 2 mL centrifuge Factor 3 (4.6%)
2925
tube. One 1 mL methanol was added and the mixture vortexed for 10 min. The HG4
supernatant was transferred to a 5 mL volumetric flask. Extraction was repeated
two times with fresh methanol and the extracts combined. After bringing to volume HG5
3558
2915
HG6
HG1
with methanol and mixing, an aliquot was centrifuged in a 1 mL centrifuge tube at 2821
2927
13,000 rpm for 10 min and the supernatant transferred to a glass HPLC autosampler vial.
Data Analysis
Figure 3 shows the PCA score plot of the hoodi
Principal components analysis (PCA) and cluster analysis (HCA) were used to investigate aerosol detection. Capsule samples HG1 and H
pattern differences in chromatograms represented as detector response versus retention and sample vial 2915 containing Opuntia leptoc
time. Data were aligned by using the Pattern Recognition Wizard in CoulArray for primary difference between these clusters is ide
Windows 3.0 software and imported into Pirouette® Comprehensive Chemometrics (A description of the pattern recognition analysis
modeling software version 4.5 for PCA and HCA.
Thermo Scientific Poster Note • PN70540_E 03/13S 3Results Method 2: HPLC-Spectro-Electro Array
Chromatographic results:
n Chromatography
The Spectro-Electro Array makes use o
Method 1: HPLC-Charged aerosol detection data. While UV detection provides iden
™ Dionex™ UltiMate™ 3000 RSLC system with a:
ode Array Detector and a A mixture of eight hoodigosides eluted with baseline resolution within 10 min from components in a sample, EC array det
S™ Charged Aerosol Detector: the Accucore C8 column under an aqueous/solvent gradient ranging from 40–95% measuring compounds missed by UV.
ture: 25 °C acetonitrile over 15 min (Figure 1). After separation was achieved, the column was resolve compounds that co-elute chrom
00 washed for an additional 10 min with 95% acetonitrile to ensure complete elution of compound reacting across the array pr
e: 20 Hz hydrophobic compounds that might be present in sample extracts. Good precision and for analyte identification/authentication.
ample response can be seen in this overlay of seven injections of a 10 mg/L standard generating chemical fingerprints of sup
™ Dionex™ Chromeleon™ Chromatography
and diluent blank. compounds that are essential to flavor,
are, 7.1 SR1
the Spectro-Electro Array was optimize
The chromatograms obtained from sample extracts are very complex, with many components in the samples. Illustrative
™ Accucore™ C8, 4.6 × 150 mm;
peaks eluting before, during and after the retention time window of the hoodigoside obtained for a few of the samples analy
standards. Figure 2 shows example chromatograms obtained from extracts of dried these extracts contain many species th
Hoodia gordonii mixed parts. In most cases, the individual hoodigosides were resolved
sufficiently to permit their accurate identification and quantification in the sample
mixtures. Some of the extracts included peaks that coeluted with the standard FIGURE 3: HPLC-ECD Array chroma
Scientific™ Optima™ LCMS hoodigosides. Note that in many of the extracts there were several hydrophobic extract #3165
2.5 min; to 95%B in 15 min; hold 10 min. compounds that eluted between 15 and 25 min that are not well detected at 220 nm, 1.40
but were well detected by the charged aerosol detector. Calibration curves were
4-hydroxybenzaldehyde 9.76m
4-hydroxybenzyl alcohol 3.87m
1.20
ection obtained from standard injections ranging from 0.10 to 100 mg/L (not shown).
salicylic acid 15.3m
1.00
valillic acid 8.93m
Response (µA)
The calibration curves spanned four orders of magnitude, had a quadratic fit to the
[900 mV]
syringealdehyde 11.6m
0.80 [840 mV]
vanillin 11.0m
[780 mV]
™ Dionex™ UltiMate™ 3000 RS system with a
[720 mV]
syringic acid 9.22m
data and yielded a correlation coefficient of > 0.999 for each standard hoodigoside.
[660 mV]
ferulic acid 14.9m
sinapic acid 14.2m
0.60 [600 mV]
[540 mV]
ode Array Detector
[480 mV]
0.40 [420 mV]
[360 mV]
[300 mV]
trochemical Array Detector: FIGURE 2. HPLC-CAD chromatograms of methanolic extracts from Hoodia
[240 mV]
0.20 [180 mV]
[120 mV]
[60 mV]
om 0 to +900 mV versus Pd in +60 mV steps gordonii, Hoodia gordonii (from Namibia), and Opuntia leptocaulis
0.00 [0 mV]
0.00 10.00
™ Dionex™ Chromeleon™ Chromatography Data
oulArray 3.0 software.
™ Acclaim™ C18, 3 × 150 mm; FIGURE 4: HPLC-ECD Array chroma
capsule extract HG4
1.40
4-hydroxybenzyl alcohol 3.75m
1.20
sodium phosphate, 3% acetonitrile, 1.00
valillic acid 8.92m
Response (µA)
[900 mV]
an, pH 3.35
syringealdehyde 11.6m
0.80 [840 mV]
vanillin 11.0m
[780 mV]
[720 mV]
syringic acid 9.21m
[660 mV]
ferulic acid 14.9m
sodium phosphate, 50% acetonitrile,
0.60
sinapic acid 14.4m
[600 mV]
[540 mV]
[480 mV]
0.40 [420 mV]
n, pH 3.45
[360 mV]
[300 mV]
[240 mV]
0.20 [180 mV]
LCMS
[120 mV]
[60 mV]
0.00 [0 mV]
0.00 10.00
FIGURE 5: HPLC-ECD Array chroma
%B %C Curve tablet extract HG6
4-hydroxybenzyl alcohol 3.75m
5 3 5
4-hydroxybenzaldehyde
1.40
hydroxyphenyl acetic acid 7.79m
1.20
5 3 7 (concave)
salicylic acid 14.6m
1.00
FIGURE 3. PCA score plot of CAD data, only hoodigoside response were used
Response (µA)
97 3 7 (concave)
3,4-dihydroxybenzoic acid 4.54m
[900 mV]
0.80 [840 mV]
valillic acid 8.94m
[780 mV]
for PCA analysis
syringic acid 9.23m
[720 mV]
[660 mV]
ferulic acid 15.0m
0.60
sinapic acid 14.1m
[600 mV]
97 3 5 [540 mV]
[480 mV]
catechin 8.11m
MPG-D046
0.40 [420 mV]
[360 mV]
[300 mV]
5 3 5 Factor 2 (8.2%) [240 mV]
0.20 [180 mV]
[120 mV]
[60 mV]
0.00 [0 mV]
5 3 5
0.00 10.00
3126
Pattern Recognition Analysis
lable capsule was placed into a 2 mL centrifuge Factor 3 (4.6%)
2925
2926 Factor 1 (85.5%) Chromatographic data from either the c
d the mixture vortexed for 10 min. The HG4 2799 array detector were pre-processed to a
olumetric flask. Extraction was repeated recognition wizard in CoulArray 3.0 sof
xtracts combined. After bringing to volume HG5
3558
2915
HG6
HG1 for PCA and HCA. PCA is a mathemati
s centrifuged in a 1 mL centrifuge tube at 2821
2927
transformation to convert a set of obse
ant transferred to a glass HPLC autosampler vial. 3165
set of values of linearly uncorrelated va
3229
used as a tool in exploratory data analy
Figure 3 shows the PCA score plot of the hoodigosides data acquired by charged greatest variance by any projection of t
d cluster analysis (HCA) were used to investigate aerosol detection. Capsule samples HG1 and HG5, tablet samples HG-6 and 3558, (Factor 1), the second greatest varianc
presented as detector response versus retention and sample vial 2915 containing Opuntia leptocaulis form a distinct cluster. The and so on. Thus samples with different
ttern Recognition Wizard in CoulArray for primary difference between these clusters is identified as hoodigoside content. electrochemical profile are distinguishe
Pirouette® Comprehensive Chemometrics (A description of the pattern recognition analysis is provided below.) position in three- dimensional space. T
and HCA. to investigate authenticity and differenc
or capsule.
4 Profiling Hoodia Extracts by HPLC with Charged Aerosol Detection, Electrochemical Array Detection, and Principal Component AnalysisMethod 2: HPLC-Spectro-Electro Array. FIGURE 6. PCA score plot for ECD array dat
Chromatographic results:
The Spectro-Electro Array makes use of both spectrophotometric and electrochemical
data. While UV detection provides identification and quantification of the major
eline resolution within 10 min from components in a sample, EC array detection is incredibly sensitive and is capable of Factor 2 (10.8%)
ent gradient ranging from 40–95% measuring compounds missed by UV. Furthermore, the EC array can voltammatrically 3229
ion was achieved, the column was resolve compounds that co-elute chromatographically, and the redox behavior of a 2915
nitrile to ensure complete elution of compound reacting across the array provides qualitative information that can be used
for analyte identification/authentication. The CoulArray is particularly suitable for
29
HG5
n sample extracts. Good precision and
even injections of a 10 mg/L standard generating chemical fingerprints of supplements containing endogenous electroactive
compounds that are essential to flavor, color and stability. The HPLC method used with 3558
the Spectro-Electro Array was optimized to resolve the earlier-eluting more polar Factor 3 (6.3%)
acts are very complex, with many components in the samples. Illustrative examples of HPLC-ECD Array chromatograms
ion time window of the hoodigoside obtained for a few of the samples analyzed are shown in Figure 3–5. As can be seen,
ams obtained from extracts of dried these extracts contain many species that are electrochemically active.
individual hoodigosides were resolved
and quantification in the sample
that coeluted with the standard FIGURE 3: HPLC-ECD Array chromatogram of Hoodia gordonii mixed parts HG1
HG6
there were several hydrophobic extract #3165
that are not well detected at 220 nm, 1.40
etector. Calibration curves were 4-hydroxybenzaldehyde 9.76m
A score plot from the HPLC-Charged aerosol d
4-hydroxybenzyl alcohol 3.87m
1.20
0.10 to 100 mg/L (not shown).
salicylic acid 15.3m
1.00
discussed in that section. A score plot from PC
luteolin 25.8m
valillic acid 8.93m
Response (µA)
agnitude, had a quadratic fit to the
[900 mV] syringealdehyde 11.6m
ethyl vanillin 16.2m
0.80 [840 mV]
data is shown in Figure 6. Samples 2821, 2799
cavarcrol 31.9m
vanillin 11.0m
[780 mV]
[720 mV]
syringic acid 9.22m
999 for each standard hoodigoside.
[660 mV]
ferulic acid 14.9m
sinapic acid 14.2m
0.60 [600 mV]
eugenol 29.1m
PCA analysis because of problems with the chr
[540 mV]
[480 mV]
0.40 [420 mV]
[360 mV]
major groups among the remaining samples. O
[300 mV]
thanolic extracts from Hoodia
[240 mV]
0.20 [180 mV]
[120 mV]
the non-hoodia species Opuntia leptocaulis. An
[60 mV]
0.00
nd Opuntia leptocaulis
[0 mV]
0.00 10.00 20.00
Retention time (minutes)
30.00
processed into capsules or tablets. The third se
to authentic Hoodia gordonii dried plant materia
unsupervised chemometric analysis correspon
FIGURE 4: HPLC-ECD Array chromatogram of Hoodia commercial product the samples, allowing us to verify the accuracy
capsule extract HG4 combined chromatographic/chemometric appro
more or less automated way to quickly screen
Conclusion
1.40
4-hydroxycoumarin 18.8m
4-hydroxybenzyl alcohol 3.75m
1.20
1.00
valillic acid 8.92m
Response (µA)
• Gradient HPLC with charged aerosol or ele
[900 mV]
syringealdehyde 11.6m
ethyl vanillin 16.8m
0.80 [840 mV]
vanillin 11.0m
32.1m
[780 mV]
31.9m
[720 mV]
syringic acid 9.21m
[660 mV]
ferulic acid 14.9m
approaches that generate information-rich
0.60
sinapic acid 14.4m
luteolin 25.8m
cavarcrol
[600 mV]
eugenol 29.1m
thymol
[540 mV]
[480 mV]
0.40 [420 mV]
reveal analytes overlooked by UV detection
quercetin 26.4m
[360 mV]
[300 mV]
[240 mV]
0.20 [180 mV]
[120 mV]
[60 mV]
0.00 [0 mV]
0.00 10.00 20.00 30.00
Retention time (minutes)
• Chemical profile data can be imported into
principal components analysis used to read
FIGURE 5: HPLC-ECD Array chromatogram of Hoodia commercial product
tablet extract HG6
• The combined chromatographic/chemomet
4-hydroxybenzyl alcohol 3.75m
to known test samples, but can be readily a
4-hydroxybenzaldehyde
1.40
4-hydroxycoumarin 18.3m
samples.
hydroxyphenyl acetic acid 7.79m
1.20
salicylic acid 14.6m
1.00
hoodigoside response were used
References
Response (µA)
3,4-dihydroxybenzoic acid 4.54m
[900 mV]
0.80 [840 mV]
valillic acid 8.94m
[780 mV]
hesperidin 18.7m
syringic acid 9.23m
[720 mV]
[660 mV]
ferulic acid 15.0m
0.60
sinapic acid 14.1m
[600 mV]
[540 mV]
[480 mV]
catechin 8.11m
0.40 [420 mV]
1. Avula, B.; Wang, Y.-H.; Pawar, R.S.; Shu
MPG-D046 [360 mV]
[300 mV]
[240 mV]
0.20 [180 mV]
A rapid method for chemical fingerprint a
[120 mV]
[60 mV]
0.00 [0 mV]
0.00 10.00 20.00 30.00
genera, and dietary supplements using U
Retention time (minutes) 2008, 48, 722–731.
Pattern Recognition Analysis 2. Gamache, P.; Acworth, I.; Lynch, M.; and
Detection for HPLC In the analysis of Jui
2926 Factor 1 (85.5%) Chromatographic data from either the charged aerosol detector or the Spectro-Electro Adulteration of Fruit Juice Beverages; Na
2799 array detector were pre-processed to align peak retention times by using the pattern USA, Inc.1995; Vol. 1, pp 120-144.
recognition wizard in CoulArray 3.0 software and then imported into Pirouette software
for PCA and HCA. PCA is a mathematical procedure that uses an orthogonal
transformation to convert a set of observations of possibly correlated variables into a Acknowledgements
set of values of linearly uncorrelated variables called principal components. It is mostly
3165
We would like to thank Drs. Ikhlas Khan and Bh
3229
used as a tool in exploratory data analysis and for making predictive models. The
Natural Products Research at The University o
gosides data acquired by charged greatest variance by any projection of the data is found in the first coordinate
the plant materials and hoodigoside standards
G5, tablet samples HG-6 and 3558, (Factor 1), the second greatest variance is found on the second coordinate (Factor 2),
aulis form a distinct cluster. The and so on. Thus samples with different patterns in their chromatographic or © 2013 Thermo Fisher Scientific Inc. All rights reserved. Piro
ntified as hoodigoside content. electrochemical profile are distinguished from each other based on their relative All other trademarks are the property of Thermo Fisher Scien
is provided below.) position in three- dimensional space. The relationships among these data were used intended to encourage use of these products in any manners
others.
to investigate authenticity and differences in processing, e.g., dried plant parts, tablet,
or capsule.
Thermo Scientific Poster Note • PN70540_E 03/13S 5FIGURE 6. PCA score plot for ECD array data from channels 1–16.
both spectrophotometric and electrochemical
ication and quantification of the major
ction is incredibly sensitive and is capable of Factor 2 (10.8%) 3165
urthermore, the EC array can voltammatrically 3229
atographically, and the redox behavior of a 2915
vides qualitative information that can be used
The CoulArray is particularly suitable for
2925
HG5
ements containing endogenous electroactive MPG
olor and stability. The HPLC method used with 3558 2926
Factor 1 (72.7%)
to resolve the earlier-eluting more polar Factor 3 (6.3%) 3126
2927
examples of HPLC-ECD Array chromatograms
ed are shown in Figure 3–5. As can be seen,
t are electrochemically active.
ogram of Hoodia gordonii mixed parts HG1
HG6
A score plot from the HPLC-Charged aerosol detection data is shown in Figure 3 and
discussed in that section. A score plot from PCA analysis of the ECD array (CoulArray)
luteolin 25.8m
data is shown in Figure 6. Samples 2821, 2799 and HG4 were excluded as outliers before
cavarcrol 31.9m
eugenol 29.1m
PCA analysis because of problems with the chromatography. PCA readily identifies three
major groups among the remaining samples. One group contains only sample 2915, from
the non-hoodia species Opuntia leptocaulis. Another group corresponds to the samples
20.00
Retention time (minutes)
30.00
processed into capsules or tablets. The third set, within the black circle, corresponds
to authentic Hoodia gordonii dried plant material. In each case, the results from the
unsupervised chemometric analysis correspond to plainly observable characteristics of
ogram of Hoodia commercial product the samples, allowing us to verify the accuracy of the approach. The real power of this
combined chromatographic/chemometric approach is that it can be implemented in a
more or less automated way to quickly screen unknown samples.
Conclusion
4-hydroxycoumarin 18.8m
• Gradient HPLC with charged aerosol or electrochemical array detection are simple
32.1m
thymol 31.9m
approaches that generate information-rich chemical profile data. Both detectors can
luteolin 25.8m
cavarcrol
eugenol 29.1m
reveal analytes overlooked by UV detection alone.
quercetin 26.4m
20.00 30.00
Retention time (minutes)
• Chemical profile data can be imported into pattern recognition software and with
principal components analysis used to readily identify product authenticity.
ogram of Hoodia commercial product
• The combined chromatographic/chemometric approach was verified by applying it
to known test samples, but can be readily applied to quickly screen unknown
4-hydroxycoumarin 18.3m
samples.
References
hesperidin 18.7m
1. Avula, B.; Wang, Y.-H.; Pawar, R.S.; Shukla, Y.J.; Smillie, T.J.; Khan, I.A.
A rapid method for chemical fingerprint analysis of Hoodia species, related
20.00 30.00
genera, and dietary supplements using UPLC–UV–MS J. Pharm. Biomed. Anal.
Retention time (minutes) 2008, 48, 722–731.
2. Gamache, P.; Acworth, I.; Lynch, M.; and Matson, W. Coulometric Array
Detection for HPLC In the analysis of Juice Products in Methods to Detect
arged aerosol detector or the Spectro-Electro Adulteration of Fruit Juice Beverages; Nagy, S., Wade, R., Eds.; AGSCIENCE
gn peak retention times by using the pattern USA, Inc.1995; Vol. 1, pp 120-144.
ware and then imported into Pirouette software
al procedure that uses an orthogonal
ations of possibly correlated variables into a Acknowledgements
ables called principal components. It is mostly
We would like to thank Drs. Ikhlas Khan and Bharathi Avula of the National Center for
is and for making predictive models. The
Natural Products Research at The University of Mississippi, Oxford, MS, for providing
e data is found in the first coordinate
the plant materials and hoodigoside standards used in this study.
is found on the second coordinate (Factor 2),
atterns in their chromatographic or © 2013 Thermo Fisher Scientific Inc. All rights reserved. Pirouette is a registered trademark of Informetrix, Inc.
from each other based on their relative All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not
e relationships among these data were used intended to encourage use of these products in any manners that might infringe the intellectual property rights of
others.
s in processing, e.g., dried plant parts, tablet,
PO70540_E 03/13S
6 Profiling Hoodia Extracts by HPLC with Charged Aerosol Detection, Electrochemical Array Detection, and Principal Component Analysiswww.thermoscientific.com/dionex ©2013 Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization. Pirouette is a registered trademark of Informetrix, Inc. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of Thermo Fisher Scientific, Sunnyvale, CA others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult USA is ISO 9001:2008 Certified. your local sales representative for details. Australia +61 3 9757 4486 Denmark +45 70 23 62 60 Japan +81 6 6885 1213 Switzerland +41 62 205 9966 Austria +43 1 333 50 34 0 France +33 1 60 92 48 00 Korea +82 2 3420 8600 Taiwan +886 2 8751 6655 Belgium +32 53 73 42 41 Germany +49 6126 991 0 Netherlands +31 76 579 55 55 UK/Ireland +44 1442 233555 Brazil +55 11 3731 5140 India +91 22 2764 2735 Singapore +65 6289 1190 USA and Canada +847 295 7500 China +852 2428 3282 Italy +39 02 51 62 1267 Sweden +46 8 473 3380 PN70540_E 03/13S
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