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Planta MedicaJournal of Medicinal Plant and Natural Product Research

www.thieme.de/fz/plantamedica l www.thieme-connect.com/ejournals

bAbstract!

This study describes the first analyticalmethod forthe determination of four triterpene saponins (ar-disicrenoside B, ardisiacrispinA, 3-O-α-L-rhamno-pyranosyl-(1→ 2)-β-D-glucopyranosyl-(1→ 4)-α-L-arabinopynanosyl cyclamiretin A and ardisi-mamilloside H) and three alkenated-phenolics(irisresorcinol, belamcandol B, and demethyl-belamcandaquinone B) from the leaves, leaves/stems, and roots of Labisia pumila using anHPLC‑UV-ELSD method. The separation wasachieved using a reversed-phase (C-18) column,PDA and ELS detection, and a water/acetonitrilegradient as the mobile phase. The major triterpe-noid (ardisiacrispin A) and irisresorcinol com-

pounds were detected at a concentration as lowas 10.0 and 0.2 µg/mL, respectively. Analysis ofvarious samples showed considerable variationof 0.11–2.46% for the major triterpenoid com-pound, ardisiacrispin A. LC-mass spectrometrymethod coupled with electrospray ionization(ESI) is described for the identification of com-pounds in plant samples. This method involvedthe use of the [M + Na]+ and [M + NH4]+ ions forcompounds 1–4 in the positive ion mode with ex-tractive ion chromatogram (EIC).

Supporting information available online athttp://www.thieme-connect.de/ejournals/toc/plantamedica

Quantitative Determination of Triperpene Saponinsand Alkenated-Phenolics from Labisia pumila Using anLC‑UV/ELSD Method and Confirmation by LC‑ESI‑TOF

Authors Bharathi Avula1, Yan-Hong Wang1, Zulfiqar Ali1, Troy J. Smillie1, Ikhlas A. Khan1,2

Affiliations 1 National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences,University of Mississippi, University, MS, USA

2 Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS, USA

Key wordsl" Labisia pumilal" Myrsinaceael" LC‑UV‑ELSDl" LC‑MS

received Dec. 22, 2010revised March 30, 2011accepted April 1, 2011

BibliographyDOI http://dx.doi.org/10.1055/s-0030-1271037Published online May 17, 2011Planta Med 2011; 77:1742–1748 © Georg ThiemeVerlag KG Stuttgart · New York ·ISSN 0032‑0943

CorrespondenceProf. Dr. Ikhlas A. KhanNational Centerfor Natural Products ResearchResearch Instituteof Pharmaceutical SciencesThe University of MississippiP.O. Box 1848University, MS 38677USAPhone: + 16629157821Fax: + 16629157989ikhan@olemiss.edu

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Introduction!

Labisia pumila (Myrsinaceae) is popularly knownin Malaysia as Kacip Fatimah and has been tradi-tionally used by the Malay women for many gen-erations as a herbal medicine for the inductionand facilitation of childbirth, treatment of men-strual disorders, and as a postpartum medicine[1–7]. There are three known varieties of Labisiapumila, which are locally known as Kacip Fati-mah: Labisia pumila var. alata, Labisia pumila var.pumila, and Labisia pumila var. lanceolata [3–5].The roots or whole plant is usually boiled, andthe water decoction is taken as a drink. The re-sults of a previous study showed uterotrophic ef-fects and the regulation of body weight [8] forwater extracts of L. pumila var. alata. The reportedfindings indicated that water-based extracts of L.pumila did not display any significant reproduc-tive toxicity or complication during pregnancyand delivery in rats [1]. In spite of the wide usageof this plant as a traditional herb, no thoroughphytochemical investigationwas previously avail-able. Recently our group isolated triterpene sapo-

tion of… Planta Med 2011; 77: 1742–1748

nins and alkenated phenolics from Labisia pumila[9] which have been used to develop the methoddescribed in this article.Until now no method has been developed for thephytochemical analysis of L. pumila. This articleprovides a simple, precise LC‑UV/ELSD analyticalmethod that was developed for the simultaneousdetection and quantification of ardisicrenoside B(1), ardisiacrispin A (2), 3-O-α-L-rhamnopyrano-syl-(1→ 2)-β-D-glucopyranosyl-(1→ 4)-α-L-arabi-nopynanosyl cyclamiretin A (3), and ardisima-milloside H (4), and three alkenated-phenolics[irisresorcinol (5), belamcandol B (6), demethyl-belamcandaquinone B (7)] (l" Fig. 1) from L. pu-mila. Four triterpene saponins (1–4) were ana-lyzed by an LC-ELSD method and three alken-ated-phenolics by an LC‑UV method at 210 nm(5–7). The compounds were numbered accordingto their order of elution. In the present work, thedeveloped method can also be used for chemicalfingerprinting analysis of Kacip Fatimah and issuitable for the quality control of various com-mercial samples. The LC‑ESI‑TOF analysis was

Fig. 1 Structure of chemical constituents ofLabisia pumila.

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performed for additional confirmation of compounds withinplant samples.

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bMaterials and Methods

!

Instrumentation and chromatographic conditionsLC‑UV analysis: The HPLC system consisted of a Waters Alliance2695 HPLC system, equipped with a 996 photodiode array detec-tor (Waters Corp.) and ELS detector, as well as a computerized da-ta station equipped with Waters Empower 2 software and a Se-dex (SEDERE) model 75 ELSD. A Luna C18 (2) column (150 × 4.6mm; 5 µ particle size) from Phenomenex was used as the station-ary phase and temperature maintained at 40°C. The column wasequipped with a 2-cm LC-18 guard column (Phenomenex). Themobile phase consisted of water (0.1% acetic acid) (A), acetoni-trile (0.1% acetic acid) (B) at a flow rate of 1.0mL/min, that wereapplied using the following gradient elution: 0min, 70% A: 30% Bincreased in the next 30min to 100% B employing a slightly con-cave gradient profile (Waters curve type 7). Each run was fol-lowed by a 5-min wash with 100% B and an equilibration periodof 15min. The ELS detector was set up with a probe temperatureof 40°C, at gain 11.0 and the nebulizer gas (nitrogen) adjusted to3.5 bar. The UV detection wavelength was 210 nm for alkenated-phenolics. Ten microliters of sample was injected, and peakswere assigned by spiking the samples with standard compoundsand comparison of the UV spectra with those of standards.

Liquid chromatography/mass spectrometry (LC‑ESI‑TOF)The liquid chromatograph used was an Agilent Series 1100 com-prised of the following modular components: quaternary pump,a vacuum solvent microdegasser, an autosampler with 100-well tray. The mass spectrometric analysis was performed by us-ing the LC‑ESI‑TOF (Model #G1969A; Agilent Technologies)equipped with an ESI source. All acquisitions were performedunder positive ionization mode with a capillary voltage of 3000

A

V. Nitrogen was used as the nebulizing gas (30 psig) as well asthe drying gas at 11 L/min at a temperature of 350°C. The volt-ages of PMT, fragmentor, and skimmer were set at 850 V, 100 V,and 60 V, respectively. Full scan mass spectra were acquired fromm/z 200–1300. Data acquisition and processing was done usingthe Analyst™ QS software (Agilent Technologies).Separation was achieved on a Luna C18 column; 150 × 4.6mmI.D.; 5 µm particle size (Phenomenex). The columnwas equippedwith a guard column (Supelco). The mobile phase consisted ofwater with 0.1% formic acid (A), and acetonitrile with 0.1% formicacid (B) at a flow rate of 0.5mL/min, with the following gradientelution: 0min, 75% A/25% B to 100% B over 15min. Each run wasfollowed by a 5-min wash with 100% B and an equilibration peri-od of 11min with 75% A/25% B. The total run time for analysiswas 15 minutes. Ten microliters of the sample was injected, andpeaks were assigned with respect to the mass of the compoundsand comparison of the retention times.

Chemicals and plant samplesThe standard compounds (1–7) were isolated at the NationalCenter for Natural Products Research (NCNPR); their identityand purity were confirmed by chromatographic (TLC, HPLC)methods, by the analysis of the spectroscopic data (IR, 1D- and2D-NMR, HR‑ESI‑MS), and comparison with published spectro-scopic data [9]. The % purity of these compounds was calculatedas 97.27%, 85.41%, 94.81%, 98.31%, 96.13%, 98.33%, and 96.15%for compounds 1–7, respectively. Acetonitrile and formic acidwere of HPLC grade, purchased from Fisher Scientific. Water forthe HPLC mobile phase was purified using a Milli-Q system(Millipore).Leaves of Labisia pumila [sample # 3809 LAPUM (LP-1), 3810 LA-PUM (LP-2), 8400 LAPUALM (LPA-1) (voucher specimen, Labisiapumila (Blume) Fern.-Vill. var. alata (Scheff.) Mez)] and roots ofLabisia pumila [sample # 3811 LAPUM (LP-3), 8400 LAPUALM(LPA-2) (voucher specimen, Labisia pumila (Blume) Fern.-Vill.var. alata (Scheff.) Mez)] were obtained from Holista Biotech

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Table 2 Intra- and inter-day precision of plant sample LP-1 assayed under op-timized conditions for compounds 1–7 using LC‑UV/ELSD method.

Com-

pounds

Intraday (n = 3) Inter-Day

(n = 9)Day 1 Day 2 Day 3

1 0.061 (1.86) 0.063 (2.20) 0.062 (0.91) 0.062 (1.60)

2 0.740 (0.19) 0.741 (0.13) 0.741 (0.10) 0.741 (0.04)

3 0.105 (0.62) 0.105 (0.55) 0.105 (0.49) 0.105 (0.55)

4 0.140 (0.67) 0.140 (0.12) 0.141 (0.38) 0.140 (0.21)

5 0.038 (0.79) 0.038 (1.21) 0.038 (1.58) 0.038 (0.30)

6 0.015 (1.26) 0.015 (1.14) 0.015 (0.62) 0.015 (0.53)

7 0.013 (0.61) 0.013 (3.65) 0.013 (2.20) 0.013 (1.01)

Values in mg/100mg of plant sample; relative standard deviation are given in paren-

theses

Table 1 Regression equation, correlation coefficient (r2), limit of detection(LOD), and limit of quantitation (LOQ) for triterpenes and alkenated-phenolicsfrom L. pumila by LC‑UV/ELSD method.

Analyte Regression equation r2 LOD (µg/mL) LOQ (µg/mL)

1 Y = 1.78e+ 000 x + 6.61e-001

0.9998 10 25

2 Y = 1.74e+ 000 x + 7.65e-001

0.9999 10 25

3 Y = 1.71e+ 000 x + 8.42e-001

0.9998 5 10

4 Y = 1.69e+ 000 x + 9.90e-001

0.9999 5 10

5 Y = 5.10e+ 004 x + 8.97e + 004

0.9999 0.2 0.5

6 Y = 9.35e+ 003 x + 4.85e + 003

0.9999 0.9 1.5

7 Y = 3.07e+ 004 x + 4.84e + 004

0.9999 0.5 1.5

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b

Sdn. Bhd. Leaves/stems [sample # 5002 LAPUM (LP-4), 5003 LA-PUM (LP-5)] and leaves of Labisia pumila [sample # 5004 LAPUM(LP-6)] were obtained from Tropical Botanics, Malaysian Herbs.All samples were identified by Dr. Vaishali Joshi, NCNPR, The Uni-versity of Mississippi, MS, USA. Commercial samples and a vouch-er specimen are deposited at the NCNPR, University of Mississip-pi, MS.

Standard solutionsIndividual stock solutions of standard compounds were preparedat a concentration of 1.0mg/mL in methanol. The calibrationcurves were prepared at five different concentration levels. Therange of the calibration curves was 25–250 µg/mL for compounds1–2 and 10–250 µg/mL for compounds 3–4 by LC-ELSD method,0.7–150 µg/mL for compounds 5–7 by LC‑UV method at 210 nm.l" Table 1 shows LC‑UV-ELSD calibration data, calculated limits ofdetection, and limits of quantification.

Sample preparationDry plant samples (500mg)were sonicated in 2.5mL ofmethanolfor 30min followed by centrifugation for 15min at 3500 rpm. Thesupernatant was transferred to a 10-mL volumetric flask. Theprocedure was repeated thrice and respective supernatants werecombined. The final volume was adjusted to 10.0mL with meth-anol and mixed thoroughly. Prior to injection, an adequate vol-ume (ca. 2mL) was passed through a 0.45-µm nylon membranefilter (Whatmann). The first 1.0mL was discarded and the re-maining volume was collected in an LC sample vial. Each samplesolution was injected in triplicate. Similarly, the plant sampleswere also prepared in water.

Validation procedureThe newly developed HPLC method was validated in terms ofprecision, accuracy, and linearity according to ICH guidelines[10]. The limit of detection (LOD) and limit of quantification(LOQ) were determined by injecting a series of dilute solutionswith known concentrations. Limit of detection (LOD) and limitof quantification (LOQ) were defined as the signal-to-noise ratioequal to 3 and 10, respectively. The accuracy of the assay methodwas evaluated in triplicate using two concentration levels. Intra-and inter-day variation of the assay was determined on 3 consec-utive days with 3 repetitions each.

Supporting informationUV spectra of compounds 5–7 are available as Supporting Infor-mation.

Results and Discussion!

Optimal chromatographic conditions were determined by usingdifferent mobile phases with various reversed-phase C18 col-umns. The different columns evaluated for HPLC were: Luna 5 µC18(2), Luna 5 µ C8(2), Lichrospher 5 RP18, Discovery C18, andGemini 5 µ C18 with the best results being obtained with a LunaC18(2) stationary phase from Phenomenex. Most of the columnmaterials [Discovery C18, Luna 5 µ C8(2), Lichrospher 5 RP18,and Gemini 5 µ C18] tested could not resolve compounds 3 and4. The best results were observed with the Luna C18 column(150mm× 4.6mm I.D., 5 µm) using water and acetonitrile, bothcontaining 0.1% acetic acid as the mobile phase. The mobilephase composition contained an acidic adjuvant to improve the

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peak symmetry of all compounds. The column was used at 40°Cwith a flow rate of 1.0mL/min and an injection volume of 10 µL.The five point calibration curve for compounds 5–7 showed a lin-ear correlation between concentration and peak area. l" Table 1shows the calibration data for all seven compounds, includingthe regression equation, correlation coefficient (r2 > 0.999), LOD,and LOQ. Using regression analysis, calibration curves for com-pounds 1–2 (25–250 µg/mL), compounds 3–4 (10–250 µg/mL),and compounds 5–7 (linear range from 0.7–150 µg/mL) was es-tablished, with UV signals following a linear response betweenconcentration and peak area. The ELSD response had an expo-nential relationship (log of response versus log of concentrationwas linear) and was a function of mass. The limits of detectionand limits of quantification for all seven compounds were foundto be in the range from 0.2–10 µg/mL and 0.5–25 µg/mL, respec-tively. All standards and samples were injected in triplicate.The specificity of the method was determined by injecting indi-vidual samples, wherein no interference was observed for any ofthe components. The chromatograms were checked for the ap-pearance of any extra peaks. The purity of the chromatographicpeaks was found to be satisfactory. The RSD of assay results ob-tained in the inter-day and intraday study (l" Table 2) was within5.0%. It was performed three times on three different days, andeach sample was injected in triplicate. Multiple injectionsshowed that the results are highly reproducible and showed alow standard error. The within-day RSD for the replicates were

Fig. 2 HPLC-ELSD chromatograms of a standardmixture and of the methanolic extracts of variousplant samples of L. pumila: ardisicrenoside B (1),ardisiacrispin A (2), 3-O-α-L-rhamnopyranosyl-(1→ 2)-β-D-glucopyranosyl-(1→ 4)-α-L-arabinopyna-nosyl cyclamiretin A (3), ardisimamilloside H (4)and three alkenated-phenolics, irisresorcinol (5),belamcandol B (6), and demethylbelamcanda-quinone B (7).

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bbetween 0.10 and 3.65% for compounds 1–7 using HPLC‑UV/

ELSD method, respectively. The RSD for the day-to-day replicateswere between 0.04–1.60% using the developed HPLC method forcompounds 1–7 (l" Table 2). The accuracy of the method was de-termined for the related compounds by spiking sample (LP-1)with a known amount of compounds 1–7. The plant sample (LP-1) was exhaustively extracted four times as described in the“sample preparation” section and dried then spiked with knownamounts of the standard compounds at two different concentra-tions, extracted again then analyzed under optimized conditions.The accuracy of the assay method was evaluated in triplicate attwo concentration levels, 25 and 50 µg/ml of standards in thesample. The percentage recovery in the samples ranged from97.0 to 104.5%.In an attempt to provide chemical markers for this herbal medi-cine, a phytochemical investigation of this plant was undertaken,leading to the identification of four triterpenes (1–4) and threealkenated-phenolics (5–7). The use of a PDA detector made itpossible to obtain the UV spectra for compounds 5–7 (Fig. 1S).All plant samples (commercial and authenticated) were analyzed

A

for the determination of triperpene saponins and alkenated-phe-nolics (l" Fig. 2 to Fig. 4). The identification of these compoundsin L. pumila samples was based on the retention times and com-parison of UV spectra with those of standards. Spiking samplewith standard compounds performed a further confirmation as-say. It was reported that the water decoction of the roots wereconsumed traditionally by Malay women for induction of labor,the water and methanolic-based extracts of roots, leaves/stems,and leaves were carried out for analysis of L. pumila samples.Roots contained the highest amounts of compounds 1–7, com-pared to leaves and leaves/stems samples. All samples showedthe presence of major compound 2, which is mainly responsiblefor utero-contraction activity [11].l" Table 3 shows the variationsin concentrations of triperpene saponins and alkenated-phe-nolics in all samples of L. pumila. Compound 2, a major triter-pene, was present in the range from 0.11–2.46% and 0–0.94% inextracts of L. pumila samples. The amounts of other triterpenes(compounds 1, 3, and 4) were in the range from 0–0.13%, 0–0.15%, and 0–0.14%, respectively, in all samples of L. pumila. Thecontent of 5–7 was present in levels from 0.04–0.47%, 0.02–

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Fig. 3 HPLC‑UV chromatograms of a standardmixture and of plant samples at 210 nm: ardisicre-noside B (1), ardisiacrispin A (2), 3-O-α-L-rhamno-pyranosyl-(1→ 2)-β-D-glucopyranosyl-(1→ 4)-α-L-arabinopynanosyl cyclamiretin A (3), ardisimamil-loside H (4), and three alkenated-phenolics, iris-resorcinol (5), belamcandol B (6), and demethyl-belamcandaquinone B (7).

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b0.85%, and 0.01–0.27%, respectively, in themethanolic extracts of

L. pumila samples. Compounds 1–4 were not detected in thewater extracts of authenticated samples from L. pumila leavesand compounds 6 and 7 were below the limits of quantificationlevels for these same samples. Compounds 5–7were in the rangeof DUL-0.09%, 0–0.14%, and DUL-0.03%, respectively, for all waterextracts of Labisia samples.LC‑MS provides a powerful qualitative technique for the selectivedetermination of molecular masses of analytes. This method in-volved the use of the [M + NH4]+ and [M + Na]+ ions in the positiveion mode with extractive ion monitoring (EIM). In the positiveion mode, the respective ammoniated species [M + NH4]+ at m/z1080.5954, 1078.5709, 916.5217, 928.5221 and sodiated species[M + Na]+ at m/z 1085.5508, 1083.5352, 921.4824, and 933.4824for compounds 1–4 were observed (l" Table 4).Further, the fragmentation patterns observed in the mass spec-trum were useful in the characterization of compounds. Com-pound 1 showed characteristic fragments at m/z 475.3717 [agly-cone + H]+, 457.3628 [aglycone + H‑H20]+, 439.3523 [aglycone +H-2H20]+, and 421.3420 [aglycone + H-3H20]+. Compounds 2

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and 3 showed characteristic fragments at m/z 455.35 [aglycone +H‑H20]+, 437.34 [aglycone + H-2H20]+, and 419.33 [aglycone + H-3H20]+ (l" Fig. 5). Compound 4 showed characteristic fragmentsat m/z 471.3459 [aglycone + H]+, 453.3348 [aglycone + H‑H20]+,435.3220 [aglycone + H-2H20]+, 423.3235 [aglycone + H-3H20]+.These ions were ascribed to the loss of sugar and water from thetriterpene skeleton (l" Table 4). The retention times and selectivemass spectra of plant samples exactly matched with the corre-sponding standard compounds. In conclusion, the newly devel-oped HPLC‑UV-ELSD method was found to be capable of the si-multaneous determination of triterpenes and alkenated-phe-nolics from the leaves, leaves/stems, and roots of L. pumila. Ardi-siacrispin A (compound 2) was found to be amajor compound forall samples analyzed. The developedmethod is simple, economic,and especially suitable for quality control analysis of commercialproducts.

bFig. 4 HPLC-ELSD chromatograms of a standardmixture and of water extracts of plant samples:ardisicrenoside B (1), ardisiacrispin A (2), 3-O-α-L-rhamnopyranosyl-(1→ 2)-β-D-glucopyranosyl-(1→ 4)-α-L-arabinopynanosyl cyclamiretin A (3),ardisimamilloside H (4), irisresorcinol (5), belam-candol B (6), and demethylbelamcandaquinone B(7).

Table 3 Content (%, w/w) of triterpenes and alkenated-phenolics from methanol and water extracts of L. pumila samples using an LC‑UV/ELSD method.

Com-

pound #

%

LP-1 LP-2 LP-3 LP-4 LP-5 LP-6 LPA-1 LPA-2

MeOH Water MeOH Water MeOH Water MeOH Water MeOH Water MeOH Water MeOH Water MeOH Water

1 0.06 ±1.21*

0.04 ±0.25

DUL ND 0.042 ±0.167

ND DUL DUL 0.038 ±0.264

DUL 0.128 ±1.56

DUL ND ND 0.07 ±0.67

ND

2 0.74 ±2.78

0.16 ±1.52

0.347 ±1.903

0.08 ±2.74

1.845 ±2.219

0.58 ±0.56

0.862 ±4.937

0.30 ±2.45

1.528 ±2.694

0.36 ±0.83

1.585 ±2.282

0.16 ±0.70

0.11 ±0.28

ND 2.46 ±3.95

0.94 ±1.41

3 0.11 ±0.66

0.10 ±1.74

ND ND 0.152 ±1.361

0.15 ±2.47

DUL DUL 0.076 ±1.621

0.08 ±1.78

ND ND ND ND DUL ND

4 0.14 ±2.34

0.09 ±2.03

ND ND 0.052 ±0.605

DUL DUL ND 0.052 ±0.880

DUL DUL DUL ND ND DUL ND

5 0.04 ±0.32

DUL 0.137 ±1.124

DUL 0.466 ±1.803

0.09 ±1.43

0.229 ±1.343

0.004 ±3.65

0.136 ±2.262

0.007 ±4.40

0.134 ±2.155

0.002 ±4.04

0.18 ±1.26

0.008 ±0.15

0.34 ±2.34

0.06 ±0.63

6 0.02 ±0.18

ND 0.179 ±1.845

DUL 0.848 ±1.013

0.14 ±0.39

0.101 ±0.979

0.002 ±4.15

0.109 ±0.652

0.006 ±1.04

0.026 ±0.377

ND 0.06 ±0.75

DUL 0.08 ±1.46

0.01 ±0.12

7 0.01 ±0.21

DUL 0.037 ±0.331

DUL 0.189 ±1.311

0.03 ±3.42

0.258 ±1.176

0.007 ±1.94

0.139 ±1.985

0.007 ±4.01

0.036 ±0.441

DUL 0.04 ±0.57

DUL 0.27 ±3.44

0.03 ±0.52

Weight(mg)

500.8 500.2 500.8 500.3 500.6 500.3 500.5 500.3 500.2 500.1 500.5 500.2 506.0 505.4 505.4 501.5

* Mean values (n = 3) ± standard deviations; ND = Not detected; DUL = Detected under limits of quantification

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bTable 4 Key fragment analysis of pure standard compounds 1–4 using LC‑ESI‑TOF method.

# Exact Mass [M + Na]+ [M + NH4]+ [M + H-sugars]+ [M + H-sugars-H2O]+ [M + H-sugars-2H2O]+ [M + H-sugars-3H2O]+

1 1062.5611 1085.5508 1080.5954 475.3717 457.3628 439.3523 421.3420

2 1060.5454 1083.5352 1078.5709 – 455.3473 437.3378 419.3260

3 898.4926 921.4824 916.5217 – 455.3482 437.3388 419.3292

4 910.4926 933.4824 928.5221 471.3459 453.3348 435.3220 423.3235

Fig. 5 Proposed fragment ions of compound 2.

1748 Original Papers

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Acknowledgements!

This research was funded in part by Holista Biotech Sdn Bhd, Ma-laysia and supported in part by “Science Based Authentication ofDietary Supplements” and “Botanical Dietary Supplement Re-search” funded by the Food and Drug Administration grant num-bers 5U01FD002071-09 and the United States Department ofAgriculture, Agricultural Research Service, Specific CooperativeAgreement No. 58-6408-2-0009. The authors would also like tothank Annette Ford for the extraction of plant samples.

Avula B et al. Quantitative Determination of… Planta Med 2011; 77: 1742–1748

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