Anal. Chem. 1999, 71, 1579-1584
Determination of Ginsenosides in Plant Extracts from Panax ginseng and Panax quinquefolius L. by LC/MS/MS Xiaomin Wang,*,† Takeo Sakuma,† Ebenezer Asafu-Adjaye,‡ and Gerald K. Shiu‡
Perkin-Elmer Sciex Instruments, 71 Four Valley Drive, Concord, Ontario, Canada L4K 4V8, and FDA Product Quality Research Laboratory, 4 Research Court, Rockville, Maryland 20850
An HPLC/MS/MS method has been developed for the characterization and quantification of ginsenosides contained in extracts of the root of Panax ginseng (Korean ginsengs) and Panax quinquefolius L. (American ginsengs). The [M + H]+ and [M + Na]+ ions were observed for ginsenoside standards (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1) and four different ginseng extracts. The glycosidic linkages, the core, and the attached sugar(s) of the ginsenosides can be determined from the collision-induced dissociation spectra from the protonated molecules. The relative distribution of these ginsenosides in each extract of American or Korean ginseng was established. Ginseng, Panax ginseng C. A. Meyer, is the root of the Araliaceous plant and is the most popular medicinal herb used in traditional Chinese medicine. It was recorded in the first-ever written documentation of traditional Chinese medicine, “Shen nung ben tsao jing” (The Holy Farmer’s Material Medica) ca. 25 A.D., as an “imperial” herb because of its nontoxic and rejuvenating properties.1 Ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, mRb1, mRb2, mRc and GLC-Rc, see Figure 1) are the major constituents of ginseng (Panax ginseng C. A. Meyer, which is commonly known as Korean ginseng; Panax quinquefolius L., is commonly known as American ginseng). Ginsenosides have been shown to affect various biological processes: e.g., tumor metastasis and antidiabetes effect,2 central nervous system,3 and prevention of the aging process.4 Several mass spectral analyses of ginsenosides have been published, involving electron impact (EI) mass spectrometry (GC/ MS),5 field desorption ionization (FD) mass spectrometry,6 252Cfplasma desorption,7 liquid secondary ion mass spectrometry,8 and †
Perkin-Elmer Sciex Instruments. FDA Product Quality Research Laboratory. (1) Sticher, O. Phytomedicines of Europe: Chemistry and Biological Activity, Oxford University Press: New York, 1997. (2) (a) Wu, J. Y. J. Immunol. 1992, 148, 1519. (b) Newman, M. J. J. Immunol. 1992, 148, 2357. (3) (a) Kim, H. S. Pharmacol. Biochem. Behav. 1996, 53, 185-190. (b) Tokuyama, S. Pharmacol. Biochem. Behav. 1996, 54, 671. (c) Kim, H. S. Planta Med. 1990, 56, 158-160. (4) Metori, K. Biol. Pharm. Bull. 1997, 20, 237-241. (5) Kim, B.-Y.; Lee, M. Y. Arch. Pharmacol. Res. 1992, 15, 328-332. (6) Schulten, H.-R.; Soldati, F. J Chromatogr. 1981, 212, 37-49. (7) Elkin, Y. N.; Makhankov, V. V. Acta Pharmacol. Sin. 1993, 14, 97-100. (8) Yanamoto, M.; Sugiyama, K. Shoyakugaku Zasahi 1992, 46, 394-396. ‡
10.1021/ac980890p CCC: $18.00 Published on Web 03/16/1999
© 1999 American Chemical Society
Figure 1. Structures of ginsenosides.
liquid chromatography/mass spectrometry (LC/MS).9 However, derivatized ginsenosides fragment extensively during the EI and produce no or very little molecular ions. All of these FD MS produced only abundant [M + Na]+ or [M + K]+ ions. This study reports the first LC/MS/MS analysis of ginsenosides by an ion spray (IS, nebulizer gas-assisted electrospray) technique. Protonated molecular cations and deprotonated anions of ginsenosides were observed along with sodiated molecular cations by LC/MS, and the product ions from these cations were further analyzed by LC/MS/MS. The glycosidic linkages, the core, and the attached sugar(s) of the ginsenosides can be determined from the collision-induced dissociation (CID) of [M + H]+. We quantified ginseng extracts by selected reaction monitoring (SRM). An excellent linear relationship between response and concentration was observed in the range 0 and 2500 ng/mL for ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1. The relative proportion of ginsenosides in each extract of American or Korean (9) van Breemen, R. B.; Huang, C.-R. Anal. Chem. 1995, 67, 3985-3989.
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Figure 2. Positive ion mass spectra of (1 ng/µL) ginsenoside Rb1, Rc, and Re.
ginseng was established.10 The detection limit for ginsenosides is ∼2 pg on column with a PE-Sciex API 3000. EXPERIMENTAL SECTION Rb1, Rc, and Re standards were obtained from Sigma Chemicals (St. Louis, MO) and Rb2, Rd, Rf, and Rg1 were obtained from Indofine Chemical Co. (Somerville, NJ). Roots of American ginseng came from (1) a Wisconsin ginseng farmer and (2) a farm north of Toronto (ON, Canada). Roots of Korean ginseng (one grown in Korea and the other grown in northeast China) were kindly provided by Professor Baoqin Wang of the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Finely pulverized ginseng powder was weighed (1.0 g), and transferred to a 125-mL conical flask. Methanol (Fisher, HPLC grade, 25 mL) was added, and the mixture was stirred gently with a magnetic stirrer for 1 h. The mixture was then ultrasonicated at 35 °C for 30 min. The supernatants were then separated after centrifugation at 12 000 rpm for 10 min. The four ginseng samples (two American, two Korean) were diluted 1000 times with a 1:1 (v/v) mixture of methanol and water. We used a 2.0 × 50 mm YMC ODS-AQ column for HPLC with a sample injection volume of 20 µL. The mobile phase consisted of (A) 95% water + 5% acetonitrile + 0.01% acetic acid and (B) 95% acetonitrile + 5% water + 0.01% acetic acid at a flow rate of 200 µL/min under linear gradient of 10-60% B over 18 min. Perkin-Elmer Sciex API 365 and 3000 triple-quadrupole mass (10) Wang, X.; Sakuma, T.; Asafu-Adjaye, E.; Shiu; G. K. Pharmacol. Res. 1997, 14, 591.
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Figure 3. CID spectra of 1 ng/µL ginsenoside Rc obtained from scanning [M + Na]+ (a) and [M + H]+ (b). Table 1. Fragment Ions Observed (m/z) in the CID Spectra from Ginsenoside Standards by Using [M + Na]+ as the Precursor Ions ginsenoside
precursor ion [M + Na]+
Rb1 Rb2 Rc Rd Re Rf Rg1
1131 1101 1101 969 969 823 823
X1a
789 789 643
X2a
C2b
789 789 789
365 335 335 349 365
C1b
203 203 203
a Where Xn is the ion of [Xn + Na - H], n ) 1, 2. b Where Cn is the ion of [Cn + Na + H], n ) 1, 2.
spectrometers, equipped with the ion spray (pneumatically assisted electrospray) and turbo ion spray probes were used. A highperformance liquid chromatographic (HPLC) system was controlled by a PE-Sciex MassChrom data system (version 1.0). The mass spectrometer was mass-calibrated with a poly(propylene) glycol solution. We acquired MS data of the standards by flow injection analysis (FIA) in the single-quadrupole, product ion, SRM, and precursor ion scan modes. The selection of collision energy for subsequent determination of active ingredients from ginseng extracts was made by examination of these product ion spectra.
Table 2. Fragment Ions Observed (m/z) in the CID Spectra from Ginsenoside Standards by Using [M + H]+ as the Precursor Ions ginsenoside
precursor ion [M + H]+
Rb1 Rb2 Rc Rd Re Rf Rg1
1109 1079 1079 947 947 801 801
Y1
X1
Y2
X2
947 899 947 767 767
929 929
767 767 767
621
603
785 737 785 587 587 441 441
619 407 405
X3
Y4
X4
B2
C2
B1
C1
605 605 605
443 443 443
325 325 325 325 309 325 325
343 295 295 343 327
163 163 163 163 147 163 163
181
443 457
423 423
RESULTS AND DISCUSSION IS MS Analysis of Ginsenosides. [M + H]+, [M + Na]+, [M - H]-, and [M + Na - H]- ions were observed by FIA experiments. The sodiated molecular ions [M + Na]+ for ginsenosides were observed by other workers, but no protonated molecules [M + H]+ were observed.9 Figure 2 shows the mass spectra obtained from ginsenoside (Rb1, Rc, Re) cations indicative of molecular weight, as either [M + H]+ or [M + Na]+. Because of the labile nature of the ginsenosides, a heated atmospheric pressure ionization (API) interface used elsewhere might have resulted in the decomposition of the protonated molecules in the interface region. In contrast, this experiment was done with the API interface at room temperature and with a low orifice potential (10 V). We observed the protonated molecules of ginsenosides. IS MS/MS Analysis of Ginsenosides. Figure 3 shows CID spectra obtained from [M + H]+ and [M + Na]+ of ginsenoside Rc. The most abundant ions are due to glycosidic bond cleavages. In contrast, the sodiated species produced only one glycosidic bond cleavage with limited structural information (Figure 3a). For example, the expected fragment ion arising from loss of the terminal glucose (-R2) from Rb1, Rb2, Rc, and Rd was observed at m/z 789 which is common to fragment X2 ions. The ions of the m/z 365, 335, 335, and 203 represent the [Glc - Glc + Na]+, [Glc - Ara(p) + Na]+, [Glc - Ara(f) + Na]+, and [Glc + Na]+ (which are the C1 and C2 ions) from Rb1, Rb2, Rc, and Rd, respectively. In Figure 3b, the peak at m/z 163 corresponds to the glucose residue (B1 ion), whereas the base peak at m/z 325 (B2 ion) belongs to the glucose chain (R1; see Figure 1). All the glycosidic bond cleavages have been obtained from the Rc CID spectrum. The peak at m/z 947 corresponds to [M + H]+ - 162 (Y1 ion), and the peak at m/z 785 corresponds to Y1 - 162 (Y2 ion). The ions of m/z 929, 767, 605, and 443 correspond to [M + H]+ H2O - 162 (X1 ion), X1 - 162 (X2 ion), X2 - 162 (X3 ion), and X3 - 162 (X4 ion), respectively. It is worth to note that peak at m/z 425 (X4 - H2O) corresponds to the core of the Rc molecule. Glycosidic cleavage and a ring-opening fragmentation of the protonated Rc led to the production of ion m/z 487.11 One can distinguish the Rb1 group (Rb1, Rb2, Rc, Rd, mRb2, mRc) and Rg1 group (Re, Rf, Rg1, glc-Rc) from two indicator ions, m/z 407 and 405 which represent the core molecules (protopanaxadiol and protopanaxatriol). Several water losses from the above ions are also evident. The CID spectra from [M + Na]+ and [M + H]+ of Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1 are summarized in Table 1 and Table 2, (11) Domon, B., Costello, C. E. Glycocojugate J. 1988, 5, 397-420.
Y3
343
149 181 165 181
Table 3. List of the Ion Pairs for Selected Reaction Monitoring Experiments
ginsenoside
precursor ion [M + H]+/ [M + Na]+ (m/z)
fragment ion (m/z)
retention time (min)
Rb1 Rb2 Rc Rd Re Rf Rg1
1109/1131 1079/1101 1079/1101 947/969 947/969 801/823 801/823
325/365 295/335 295/335 325/789 309/789 423/365 423/643
10.79 11.38 11.07 12.06 7.77 10.23 7.73
respectively. All the Xn and Yn (n ) 1-4) ions resulting from glycosidic bond cleavages and expected structural features of the ginsenoside molecules are indicated in the spectra clearly. The CID spectra for Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1 show an ion profile (Xn and Yn ions, n ) 1, 2, 3, 4) with mass intervals of 162 (or 180) amu, indicating the number of glucoses attached to the core molecule. The Bn and Cn (n ) 1, 2) ions indicate number of the glucose residues. A summary of the precursor and product ions and retention time for these ginsenosides is shown in the Table 3. IS LC/MS/MS Analysis of Ginsenosides. Positive ion IS LC/MS/MS analysis of standard ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1) and American ginseng extract are shown in Figures 4 and 5, respectively. Ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1) labeled in Figure 5 were identified by comparing precursor/ product ion pairs and retention times of the samples against those standards. Although most ginsenosides were detected in four ginseng extracts, their relative abundances were quite different. Figure 6 shows a comparison of the American and Korean ginsengs with the two pairs of the ginsenosides (Rg1/Rf, Rc/Rb2). These comparisons indicate that the relative abundances of these differences ginsenosides are significant enough to distinguish ginseng extracts of Korean and American species. However, we could not find obvious differences in the ginsenoside profiles of genetically same Korean ginseng grown in Korea or China. American ginseng grown in the United States and Canada did not show any major differences either. A much higher abundance of Rb1 group ginsenosides (Rb1, Rc, Rd) was found in the American ginseng extracts than in the Korean counterparts. In contrast, a higher abundance of Rg1 group ginsenosides (Rf, Rg1) were in Korean ginseng extracts than in the American counterparts. This finding in our LC/MS/MS experiments agrees well with HPLC results12 reported elsewhere. The differences in ginsenoside content were probably a result of genetic differences Analytical Chemistry, Vol. 71, No. 8, April 15, 1999
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Figure 4. Positive ion LC/MS/MS SRM trace of ginsenoside standards (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1; 71 ng/mL each) collected in 18 min. [M + H]+ was selected as the precursor ion. The ions of m/z 1109/325, 1079/295, 947/325, 947/309, and 801/423 were selected as the precursor/ fragment ion pair for monitoring Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1, respectively.
Figure 5. LC/MS/MS SRM chromatograms of American ginseng extract collected in 18 min.
between the two kinds of ginsengs, and these differences may explain their different biological activities. 1582 Analytical Chemistry, Vol. 71, No. 8, April 15, 1999
Calibrations for Quantitative Analysis. The most abundant fragment ion for each analyte was chosen for SRM quantitation.
Figure 6. SRM chromatograms (m/z 801 f 423 and m/z 1079 f 295) for Korean and American ginseng extracts, respectively. Table 4. Quantitation of Ginsenosides by LC/MS/MS for American Ginseng sample
Figure 7. Calibration curve of Rb1. The ions of m/z 1109 and 325 were selected as the precursor ion and fragment ion, respectively. The calibration curve was constructed using a weighted linear regression of standard concentrations to measured peak area ratios (PE-Sciex MacQuan 1.4). The inset shows the signal-to-noise ratio at the lowest concentration of the calibration curve. This experiment has been done on the PE-Sciex API 365 in turbo ion spray (350 °C, 6 L/min).
I mean (n ) 3) SD % CV II mean (n ) 3) SD % CV III mean (n ) 3) SD % CV mean SD % CV
Rb1 (mg/g)
Rc (mg/g)
Re (mg/g)
30.8 3.3 10.7
6.4 0.8 12.1
24.9 2.0 8.1
27.8 4.8 17.3
5.5 1.07 19.6
16.8 2.4 14.4
34.0 2.4 7.2
6.7 0.5 6.7
22.1 1.8 8.4
30.9 3.1 10.0
6.2 0.6 10.0
21.3 4.1 19.3
Calibration curves were constructed for each of the reference standards (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1) in concentrations of 1.95, 3.91, 7.81, 15.6, 31.5, 62.5, 125, 250, 500, and 2500 ng/mL in duplicates. All calibration curves showed good linearity (coefficient of variation (CV) 0.997-0.999) over the concentration range. A typical calibration curve for Rb1 is shown in Figure 7. Quantitative Analysis of American Ginseng. Three aliquots of each crude extract were analyzed after the solid-phase extraction process. The ginsenosides Rb1, Rc, and Re were quantitatively
determined by means of the established calibration curves. As shown in Table 4, the reproducibilities (CV) among the solidphase extractions ranged from 7.2 to 17.3, 6.7 to 19.6, and 8.1 to 14.4% for ginsenosides Rb1, Rc, and Re, respectively. The overall reproducibility for analysis of Rb1 and Rc from American ginseng was 10% while higher variation (19.3%) was observed for Re. The authors believe that the variations should be improved by eliminating the solid-phase extraction step that was later found unnecessary for LC/MS/MS analysis.13 It is worth noting that internal standards should be introduced for both samples and calibration standards before the extraction; that will improve precision and accuracy for the quantitative analysis.
(12) Samukawa, K.; Yamashita, H. Chem. Pharm. Bull 1995, 43, 137-141.
(13) Wang, X.; Sakuma, T., unpublished experimental results, 1998.
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CONCLUSION The applicability of the LC/MS/MS method for ginsenoside characterization and quantitation has been illustrated with various ginseng samples. The method is sensitive (detection as little as 2 pg on the column, for API 3000) and reproducible (less than 10% CV). The [M + H]+ and [M + Na]+ ions were observed for each ginsenoside standard (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1) and the four different ginseng extracts. The glycosidic linkages, the core, and the attached sugar(s) of the ginsenosides can be determined from the CID experiments by using [M + H]+ as the precursor ions. The CID spectra of ginsenosides show great differences between the protonated and sodiated species. More structural information can be obtained from the CID spectra of protonated molecules than sodiated species. A distinctive distribution of the ginsenosides in each extract of American and Korean ginsengs was observed. Korean ginseng contains a much higher amount of the Rg1 group ginsenosides (Rf, Rg1) than the American ginseng does, while the opposite is true for the Rb1 group (Rb1, Rb2, Rc, Rd, mRb1, mRb2, mRc) in the American ginseng. We made quantitative (14) Wang, X.; Xue, Y.; Zhou, T.; and Sakuma, T. J. Food Drug Anal. 1997, 5, 337-346.
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determination of the ginsenosides from extracts of American ginseng by LC/MS/MS. We have shown the LC/MS/MS technology can provide a method to characterize and quantify complex herbal medicines, such as the ginsenosides, and the method greatly improves the accuracy and speed of the analysis when compared to the conventional HPLC method. The LC/MS/MS method has been previously demonstrated for the characterization of a traditional Chinese medicine.14 This approach can be utilized in clinical trials of herbal medicines for their efficacy and safety. ACKNOWLEDGMENT The authors thank Professor Baoqin Wang (at National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China)) and Mr. Simon Ko (KUB American Ginseng Corp., Marathon, WI) for kindly providing the ginseng samples.
Received for review October 12, 1998. Accepted February 8, 1999. AC980890P
