Differentiation and Authentication of

Differentiation and Authentication of Panax ginseng,Panax quinquefolius,and Ginseng Products by Using HPLC/MS

T.W.D.Chan,?,?P.P.H.But,*,?,§S.W.Cheng,?I.M.Y.Kwok,?https://www.360docs.net/doc/c73828700.html,u,?and H.X.Xu?Chinese Medicinal Material Research Centre,Department of Chemistry,and Department of Biology,

The Chinese University of Hong Kong,Shatin,NT,Hong Kong

An LC/MS-based method is established for the dif-ferentiation and authentication of specimens and com-mercial samples of Panax ginseng(Oriental ginseng)and Panax quinquefolius(American ginseng).This method is based on the separation of ginsenosides present in the ginseng methanolic extracts using high-performance liq-uid chromatography(HPLC),followed by detection with electrospray mass spectrometry.Differentiation of ginse-nosides is achieved through simultaneous detection of intact ginsenoside molecular ions and the ions of their characteristic thermal degradation products.An important parameter used for differentiating P.ginseng and P. quinquefolius is the presence of ginsenoside Rf and24-(R)-pseudoginsenoside F11in the RICs of Oriental and American ginsengs,respectively.It is important to stress that ginsenoside Rf and24(R)-pseudoginsenoside F11, which possess the same molecular weight and were found to have similar retention times under most LC conditions, can be unambiguously distinguished in the present HPLC/ MS method.The method developed is robust,reliable, reproducible,and highly sensitive down to the nanogram level.

The roots of Panax ginseng C.A.Meyer(Oriental ginseng) and Panax quinquefolius L.(American ginseng)are highly treasured medicinal herbs in traditional Chinese medicines. Research studies have shown that they possess antistress,1CNS-stimulating,2antidiabetic,3and anticancer4properties.Ginseng products have become very popular and have attracted worldwide consumption.However,Oriental ginseng and American ginseng are known to have different properties and medicinal values.The red form of Oriental ginseng,produced by steaming,is“warm”and known to replenish the“vital energy”,whereas American ginseng is“cool”and is mainly used for reducing the“internal heat”and promoting the secretion of body fluids.5In the ginseng markets worldwide,American ginseng usually commands a much higher price than the sun-dried Oriental ginseng.Since the roots of these ginsengs are similar in appearance and many commercial ginseng products are in form of powder or shredded slices, identification of the origins of the ginseng products is not an easy task.Authentication of the two sources of ginseng and ginseng products based on chemical profiling has aroused much interest.

Ginsenosides(ginseng saponins)are known to be the bioactive components of ginsengs.According to the difference in the aglycone in these saponins,ginsenosides are classified into three types:the20(S)-protopanaxadiol type(e.g.,ginsenosides Rb1,Rc, Rb2,and Rd),the20(S)-protopanaxatriol type(e.g.,ginsenosides Rg1,Rf,and Re),and the oleanolic acid type(e.g.,ginsenoside Ro)(see Figure1a-c).Ginsenosides Rg1,Re,Ro,Rb1,Rc,Rb2, and Rd are present in both Oriental and American ginsengs in different proportions,whereas ginsenoside Rf can only be found in Oriental ginseng.6-8In contrast,American ginseng contains24-(R)-pseudoginsenoside F11,an ocotillol type triterpene(Figure1d), which is absent in Oriental ginseng.7-9

Many analytical approaches have been used to identify ginse-nosides in ginseng extracts.Among these methods,the use of high-performance liquid chromatography(HPLC)in conjunction with mass spectrometry(MS or MS/MS)appears to be most promising.In comparison with the use of gas chromatography (GC)coupled with electron impact mass spectrometry(EI-MS), the HPLC/MS approach can overcome the problems associated with ginsenoside derivatization and the low abundance of molec-

*Corresponding author.Tel:(852)2609-6140.Fax:(852)2603-5248. E-mail:paulbut@https://www.360docs.net/doc/c73828700.html,.hk.

?Chinese Medicinal Material Research Centre.

?Department of Chemistry.

§Department of Biology.

(1)(a)Grandhi,A.;Mujumdar,A.M.;Patwardhan,B.J.Ethnopharmacol.1994,

44(3),131-135.(b)Kumar,R.;Grover,S.K.;Divekar,H.M.;Gupta,A.

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Immunol.1992,148,2357-2362.

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Sin.1987,8,277-280.(b)Cott,J.Psychopharmacol.Bull.1995,31,(4), 745-751.(5)(a)Shanghai College of Traditional Chinese Medicine.Zhongcaoyaoxue

(Chinese Materia Medica);Commerce Press:Hong Kong,1975;pp511-515.(b)Shanghai College of Traditional Chinese Medicine.Zhongyao Linchuang Shouce(Clinical Manual of Chinese Medicine);Shanghai People’s Publishing House:Shanghai,1977;pp357-359.

(6)(a)Lang,W.S.;Lou,Z.C.;But,P.P.H.J.Chin.Pharm.Sci.1993,2(2),

133-143.(b)Chuang,W.C.;Wu,H.K.;Sheu,S.J.;Chiou,S.H.;Chang,

H.C.;Chen,Y.P.Planta Med.1995,61,459-465.

(7)Dou,D.-Q.;Hou,W.-B.;Chen,Y.-J.Planta Med.1998,64,585-586.

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(9)Chen,S.E.;Staba,E.J.;Taniyasu,S.;Kasai,R.;Tanaka,O.Planta Med.

1981,42(4),406-411.

Anal.Chem.2000,72,1281-1287

10.1021/ac990819z CCC:$19.00?2000American Chemical Society Analytical Chemistry,Vol.72,No.6,March15,20001281 Published on Web02/11/2000

ular ions.10It has also been reported that HPLC systems equipped with analytical columns containing materials ranging from reversed-phase silica 11and ion exchange packings 12to specialized carbo-hydrate-containing aminopropyl functional groups 13could separate intact,underivatized ginsenosides effectively from ginseng ex-tracts.The use of an on-line MS detector offers superiority over conventional UV absorbance detection in terms of specificity and sensitivity.14,15Among the various mass spectrometry approaches,

(10)(a)Kim,B.Y.;Lee,M.Y.;Cho,K.H.;Park,J.H.;Park,M.K.Arch.Pharm.

Res.1992,328-332.(b)Cui,J.-F.;Bjo ¨rkhem,I.;Eneroth,P .J.Chromatogr.,B 1997,689,349-355.

(11)(a)Kanazawa,H.;Nagata,Y.;Kurosaki,E.;Matsushima,Y.;Takai,N.J

Chromatogr.1993,632,79-85.(b)Court,W.A.;Hendel,J.G.;Elmi,J.J.Chromatogr.,A 1996,755,11-17.

(12)(a)Parr,M.K.;Parr,J.H.;Lee,M.Y.;Kim,S.J.;Park,I.J.J.Liq.Chromatogr.

1994,17,1171-1182.(b)Yamaguchi,H.;Kasai,R.;Matsura,H.;Tanaka,O.;Fuwa,T.Chem.Pharm.Bull.1988,36,3468-3473.

(13)(a)Paik,N.H.;Park,M.K.;Choi,K.J.;Cho,Y.H.Arch.Pharm.Res.1982,

5,7-12.(b)Yamaguchi,H.;Matsuura,H.;Kasai,R.;Tanaka,O.;Satake,M.;Kohda,H.;Izumi,H.;Nuno,M.;Katsuki,S.;Isoda,S.;Shoji,J.;Goto,K.Chem.Pharm.Bull.1988,36,4177-4181.(c)Park,M.K.;Park,J.H.;Han,S.B.;Shin,Y.G.;Park,I.H.J.Chromatogr.,A 1996,736,77-

81.

Figure 1.Structures of ginsenosides in forms of (a)20(S )-protopanaxadiol,(b)20(S )-protopanaxatriol,and (c)oleanolic acid and of pseudoginsenosides in form of (d)24(R )-ocotillol type triterpene.

1282Analytical Chemistry,Vol.72,No.6,March 15,2000

electrospray mass spectrometry(ESI-MS)is considered to be the best to couple with HPLC.Similar to other mass spectrometric techniques,ESI-MS was found to produce intense protonated or cationizated ginsenoside ions.14,15Application of electrospray tandem mass spectrometry(ESI-MS/MS)involving collision-induced dissociation(CID)of the selected ions has been shown to provide product ion spectra which are useful for structure determination.

At present,only a few papers have reported the use of HPLC/ ESI-MS and HPLC/ESI-MS/MS for differentiating Oriental and American ginsengs.Though the techniques were proved to be very powerful,further efforts devoted to method development are required.For instance,van Breeman et al.14pointed out that certain ginsenosides were detected only in the methanolic extract of Oriental ginseng.The assignment of the constituents,however, was based purely on the correction of a mass shift of m/z138 corresponding to the column bleed-off adducts.The results had not been confirmed by using authentic ginsenoside standards. Recently,Wang and coauthors15differentiated Oriental and American ginsengs by contrasting the relative abundances of ginsenosides present in both species.It was reported that the ginsenoside ratios Rg1/Rf and Rc/Rb2are higher for American ginseng than for Oriental ginseng.Nevertheless,it was well realized that American ginseng does not contain ginsenoside Rf6-8 but contains24(R)-pseudoginsenoside F11.7-9The problem is that ginsenoside Rf and24(R)-pseudoginsenoside F11share the same molecular weight and are found to elute at similar retention times under most LC conditions;false identification of Rf in American ginsengs is thus possible.

The objective of the present investigation is to develop a simple LC/ESI-MS technique for differentiation of Oriental and American ginsengs.The approach relies on the detection of indicative constituents besides comparing the differences in relative abun-dance of the ginsenosides common to both species.Positive identification of ginsenoside constituents was accompanied by the simultaneous detection of the sodiated ginsenoside molecular ions and the ions derived from in-source thermal degradation of the protonated ginsenoside molecular ions.The method developed was applied to the analysis of ginseng samples cultivated in China, Korea,Canada,and the United States.In addition,this method was also used to analyze commercial ginseng slices and ginseng teas.

EXPERIMENTAL SECTION

Standards of ginsenosides including Rg1,Re,Ro,Rf,Rb1,Rb2, Rc,Rd,and24(R)-pseudoginsenoside F11were kindly provided by Prof.Y.J.Chen of Shenyang Pharmaceutical University, Shenyang,China,and the Tobacco and Ginseng Monopoly,South Korea.Structures of these ginsenosides and the pseudoginseno-side are shown in Figure1.Samples of Oriental ginseng were obtained from China and Korea.Roots of American ginseng derived from cultivated samples in the United States,Canada,and China were gifts from the Wisconsin Ginseng Board.The solvents, acetonitrile and methanol,were of HPLC grade(Mallinckrodt, Paris,KY).Other reagents were of analytical grade.Deionized water(18M?)was prepared by passing distilled water through an ultrapure water system(Nanopure,Barnstead,Germany).

The ginseng sample was first homogenized.The powdered sample(300mg)was purified by extraction with10mL of chloroform in a microextractor under reflux for3h.The sample was then filtered,and the chloroform extract was discarded.The solid residue was further extracted with10mL of methanol under the same conditions.The methanolic extract was evaporated to dryness under vacuum.The residue of ginsenosides formed was redissolved in3.0mL of deionized water.The aqueous solution was applied to an SEP-PAK C18cartridge(Waters Associates).The ginsenosides were washed with10mL of water and then eluted with10mL of50%acetonitrile.The solution collected was filtered through a0.2μm filter(Millipore)prior to LC/MS analysis.For direct flow injection experiments,solutions of ginsenoside stan-dards were prepared at concentrations of1μg/μL.In each experiment,10μL of the sample solution was used.

A Hewlett-Packard1090liquid chromatograph system was interfaced to the mass spectrometer.An analytical column of46 mm×150mm packed with a3μm hydrophobic bonded C18phase (Adsorbosphere HS,Alltech Associates Inc.)was utilized.Gradient elution generated by the proportional mixing of two solvents was used.Solvent A consisted of50%acetonitrile and solvent

B contained10mM ammonium acetate,both in deionized water. Solvent gradient conditions were a linear change of solvent B from 70to50%in the first30min and to10%in the next30min.This solvent composition was maintained for another10min before returning to70%solvent B in2min.The flow rate was1.0mL/ min with the column kept at ambient temperature.For flow injection experiments,the HPL

C instrument delivered mobile phases of A and B in a1:1ratio at a flow rate of100μL/min.

Electrospray mass spectra were acquired using an MAT TSQ 7000triple-stage quadrupole mass spectrometer(Finnigan,San Jose,CA).The needle potential was set at5kV.A heated stainless steel capillary was used as a bridge between the atmospheric pressure side and the first pumping stage.Desolvation occurred in this capillary.Nitrogen was used as the nebulizing gas at a pressure of50psi.Full-scan mass spectra were acquired by scanning the first quadrupole(Q1)over the range m/z300-2300 at a scan time of1.5s.Q2and Q3were set at the rf-only mode for transmission of all ions of the selected mass range.An electron multiplier was employed as the detector and was set at a potential of-1300V.

RESULTS AND DISCUSSION

Positive-Ion Mass Spectra of Ginsenoside Standards.In an attempt to obtain mass spectrometric-based information for differentiating different types of ginsenosides,direct flow injection experiments of ginsenoside standards were initially performed with a capillary temperature of175°C.In contrast to the results of earlier studies using252Cf-plasma desorption,16field desorption,17 laser desorption,18and liquid secondary ion mass spectrometry,19

(14)van Breemen,R.B.;Huang,C.-R.;Lu,Z.-Z.;Rimando,A.;Fong,H.H.S.;

Fitzloff,J.F.Anal.Chem.1995,67,3985-3989.

(15)Wang,X.;Sakuma,T.;Asafu-Adjaye,E.;Shiu,G.K.Anal.Chem.1999,71,

1579-1584.(16)Elkin,Y.N.;Makhankov,V.V.;Uvarova,N.L.;Bondarenko,P.V.;Zubarev,

R.A.;Knysh,A.N.Acta Pharmacol.Sin.1993,14,97-100.

(17)Schulten,H.R.;Soldati,F.J.Chromatogr.1981,212,37-49.

(18)Zhou,Y.;Song,F.R.;Liu,S.Y.;Li,X.G.Acta Chim.Sin.1998,56,298-

301.

(19)Yamamoto,M.;Sugiyama,K.;Ichio,Y.;Yokota,M.;Maeda,Y.;Senda,N.;

Shizukuishi,K.Shoyakugaku Zasshi1992,46,394-396.

Analytical Chemistry,Vol.72,No.6,March15,20001283

positive-ion mass spectra of all ginsenosides and 24(R )-pseudo-ginsenoside F 11featured signals indicating the production of [M +H]+and [M +NH 4]+molecular ions.No significant signals corresponding to the sodiated molecular ions were observed.Figure 2a shows the positive-ion flow injection mass spectrum of Rg 1.Apart from the abundant [M +H]+and [M +NH 4]+molecular ions,some low-mass ions corresponding to the thermal degradation products of the ginsenoside Rg 1(as proved subse-quently by using LC/MS analysis)were also observed.Initial attempts to utilize these low-mass signals for positive identification of the ginsenosides were hampered by their relatively low intensities and high variability.Figure 2b -d shows the positive-ion flow injection mass spectra of Rg 1at other capillary temper-atures.The intensities of the low-mass ions were substantially increased by elevating the temperature of the heated capillary.It is interesting to note that the [M +H]+and [M +NH 4]+ions were completely degraded at capillary temperatures above 250°C.In addition,signals corresponding to the [M +Na]+ions appeared to form at high capillary temperatures (>250°C).The production of ions of thermal degradation products simulta-neously with cationized molecular ions appears to be advanta-geous.Because the mass spectra obtained reveal common features in the fragmentation behavior of the ginsenosides,they are useful for the characterization and identification of these species.For example:for 20(S )-protopanaxadiol type ginsenosides,fragment ions at m /z 443,425,407were observed;for 20(S )-protopanaxatriol type ginsenosides,the corresponding fragment ions were ob-served m /z 441,423,405with an additional fragment at m /z 459;and for ginsenoside Ro,the oleanolic acid type ginsenoside,a single fragment at m /z 439was exhibited.For 24(R )-pseudo-ginsenoside F 11,fragment ions at m /z 475,457,439,421were observed.The spectral information is summarized in Table 1.Figure 3shows the mass spectra of ginsenosides Rb 2,Rf,and Ro and 24(R )-pseudoginsenoside F 11.One striking spectral feature of 24(R )-pseudoginsenoside F 11was the presence of abundant protonated molecular ions,[M +H]+,even at high capillary temperatures.

All major thermal degradation product ions appeared to arise from the loss of H 2O molecules from the deglycoslyated ginse-noside core moieties.For 20(S )-protopanaxadiol type

ginsenosides,

Figure 2.Positive-ion ESI-MS spectra of ginsenoside Rg 1at capillary temperatures of (a)175°C,(b)200°C,(c)250°C,and (d)300°C.Table 1.Major Ions (m /z )Observed in the Positive-Ion ESI-MS Spectra of the Ginsenoside Standards a ginsenoside

type

[M +H]+

[M +Na]+

other major ions

Rb 120(S )-protopanaxadiol ND b 1132.1407.4,425.4,443.3,569.5,587.6,605.5,749.7,784.8Rb 220(S )-protopanaxadiol ND 1102.0330.1,407.4,425.5,443.5,587.8,605.6,786.0Rc 20(S )-protopanaxadiol ND 1101.9407.4,425.4,443.5,587.5,605.7,790.5Ro oleanolic acid

ND 979.6439.2

Rd 20(S )-protopanaxadiol ND 969.9407.4,425.5,443.6,569.6,605.7,749.7,784.9

Re 20(S )-protopanaxatriol ND 969.7405.5,423.4,441.4,459.6,587.7,603.7,621.9,749.8,767.9Rf 20(S )-protopanaxatriol ND 823.9405.3,423.3,441.3,459.3,587.5

Rg 120(S )-protopanaxatriol ND 823.8405.2,423.2,441.2,459.2,603.3,621.5

F 11

24(R )-pseudoginsenoside

801.5

823.7

308.9,421.2,439.2,457.2,475.2,603.7,655.4,783.5

a

The temperature of the heated capillary was set at 300°C.b ND,not detected.

1284Analytical Chemistry,Vol.72,No.6,March 15,2000

the simultaneous loss of R 1and R 2groups yielded initially the core moiety at m /z 461.As a result of combinational loss of H 2O molecules derived from the three hydroxyl groups of the core moiety,fragment ions at m /z 443,425,407were produced.For 20(S )-protopanaxatriol type ginsenosides,the loss of R 1and R 2groups gave rise to a core moiety with four hydroxyl functional groups at m /z 477.The mass spectra therefore featured four characteristic ions,i.e.,those at m /z 459,441,423,405.For the oleanolic acid type ginsenosides,the corresponding core moiety arising from the loss of R 1and R 2contained only one hydroxyl group.Loss of a water molecule from this hydroxyl group produced a product ion at m /z 439.For the 24(R )-pseudoginse-noside F 11,the core moiety with four hydroxyl groups at m /z 493was first derived from the loss of the R group.Successive elimination of four H 2O molecules generated four product ions at m /z 475,457,439,421.

With in-source thermal degradation ESI-MS,ginsenosides of different types possessing identical molecular weight,e.g.Rg 1/Rf vs F 11(MW )800)and Re vs Rd (MW )946),could be easily recognized by their diagnostic product ions.However,for two pairs of ginsenosides,Rg 1/Rf (MW )800)and Rc/Rb 2(MW )1078),unambiguous identification could only rely on the observa-tion of other minor product ions such as those at m /z 603in Rg 1and m /z 330in Rb 2.However,with the use of on-line liquid chromatographic separation,their identifications could be achieved from their characteristic retention times.

In summary,the present ESI-MS method provided a reliable means to distinguish different types of ginsenosides.Both the molecular weight and structural information could be revealed in a single mass spectrum.The fragmentation patterns generated were characteristic of the ginsenoside types.Our results also suggested that the positive ESI-MS approach can be applied to identify the type and molecular weight of an unknown ginsenoside even if an authentic standard is not available.

LC/MS Analysis of a Mixture of Ginsenoside Standards.To demonstrate the identification of individual ginsenosides in a complex mixture,the ginsenoside standards were mixed and analyzed by HPLC/MS.Figure 4shows the positive-ion LC/MS traces of ginsenoside standards collected in 60min.A distinct difference between the present LC method and those that were previously reported 6a,15was the use of a rather gentle change of mobile phase composition,thus resulting in a longer time of elution.By sacrificing the time for analysis,we achieved a complete resolution of ginsenosides Rg 1/Re and Rf/F https://www.360docs.net/doc/c73828700.html,plete separation of 24(R )-pseudoginsenoside F 11and ginsenoside Rf was especially important,since the former component is the charac-teristic constituent of American ginseng while the latter is the characteristic constituent of Oriental ginseng.As shown in Figure 4,all ginsenosides were well resolved under the present LC conditions.Their respective mass spectra are similar to those obtained by direct flow injection analysis.Unlike the results obtained with the use of an analytical column containing amino-propyl functional groups on the silica-based stationary phase,no unexpected adduct ions from the column materials were ob-served.14Ginsenosides labeled on the reconstructed ion chro-matogram were identified on the basis of their retention times and the m /z values of the intact molecular ions and the thermal degradation product ions.Table 2summarizes the retention times and the set of ions for positive identification of individual ginsenosides.

Wang et al.15had previously reported the detection of ginse-noside Rf in the American ginsengs cultivated in both the United States and Canada by using LC/MS/MS with selected reaction monitoring (SRM)(i.e.,m /z 801f 423).However,it is

well-known

Figure 3.Positive-ion ESI-MS spectra of (a)ginsenoside Rb 2,(b)ginsenoside Rf,(c)24(R )-pseudoginsenoside F 11,and (d)ginsenoside Ro.The temperature of the heated capillary was set at 300°C.

Analytical Chemistry,Vol.72,No.6,March 15,2000

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that American ginsengs do not contain ginsenoside Rf but pseudoginsenoside F 11.The false-positive identification of Rf in their samples is intriguing.Although 24(R )-pseudoginsenoside F 11and ginsenoside Rf share the same molecular weight and are very often coeluted under most LC conditions,misrecognition of these components might happen if the assignments are based purely on the molecular masses and retention times,especially when authentic standards are not available for reference.However,under SRM conditions (i.e.,m /z 801f 423),such misrecognition would not be possible because of the absence of the CID fragment of F 11at m /z 423(data not included).Anyway,the present LC/MS method provides two independent types of parameters,i.e.,retention time and mass spectrometric information,for the positive identification of Rf,F 11,and other ginsenoside constituents.

LC/MS of Oriental and American Ginsengs.Twelve Ori-ental and American ginsengs,including some special reference ginseng standards purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing,China),were analyzed by the present LC/MS method.Figure 5shows some representative ion chromatograms (RICs).Consistent with the general findings,7ginsenoside Rf was found only in Oriental ginseng,whereas 24(R )-pseudoginsenoside F 11was found exclusively in American ginseng.In comparison with the com-monly used thin-layer chromatography (TLC)methods,the LC/MS method showed much superior quality in terms of sensitivity and specificity.It is also worthwhile to point out that 24(R )-pseudoginsenoside F 11does not contain any suitable chromophore identifiable by a UV absorbance detector and could not be detected by normal LC analysis.

Inspection of Figure 5and RICs of other ginseng samples showed that the ratios Rg 1/Re and Rb 2/Rc were also found to be specific for these two species of ginsengs.The Rg 1/Re ratio was always found to be much greater than 1in Oriental ginseng,whereas the same ratio was found to be smaller than 1in American ginseng.A more subtle difference was found in the Rb 2/Rc ratios,which approach 0.8and 0.2in Oriental ginseng and American ginseng,respectively.In addition,there was consistently a prominent peak eluted behind Rd (retention time:48.9min)for American ginseng.This signal peak was only present in extremely weak intensity for Oriental ginseng.Though the authentic standard was not available,some information on this component could be extracted from its characteristic thermal degradation products and its molecular weight.It was found to be a 20(S )-protopanaxadiol type ginsenoside having a molecular weight of 946;further identification of this component is currently under investigation.One of the major disadvantages of using relative peak height/area ratios for differentiating American and Oriental ginsengs is the dependence of the relative ginsenoside contents on the age of the ginseng and the physical parts of the ginseng being used.

LC/MS of Commercial Ginseng Products.Twelve com-mercial ginseng products,including eight ginseng slices and four samples of ginseng tea granules,were analyzed by using the present LC/MS method.Among these products,two brands of ginseng slices and two brands of ginseng tea granules were found to contain ginsengs different from those described by the manufacturers.One brand of ginseng slices that claimed to be made from American ginseng actually contained Oriental ginseng.The opposite was true for the other brand of ginseng slices.For the ginseng tea granules,one brand of American ginseng tea granules was derived from Oriental ginseng,whereas another brand of American Ginseng tea granules was actually made from a mixture of both American and Oriental ginsengs.Figure 6shows the positive-ion LC/MS traces of ginsenosides isolated.Although the RICs show mainly contributions from the Oriental ginseng,SIM at m /z 439and 475has positively identified the presence of American ginseng.Similar studies using conventional TLC meth-ods would be very difficult,if not impossible.Close inspection of the RICs of Figure 6reveals another important limitation in methods that utilize relative peak height/area ratios for dif-ferentiation and authentication of ginseng products.Mixing of different ginsengs could actually alter the relative abundance

of

Figure 4.The LC/MS traces of the mixture of ginsenoside standards Rg 1,Re,Ro,Rf,Rb 1,Rc,Rb 2,Rd,and 24(R )-pseudoginsenoside F 11.

Table 2.Retention Times and the Set of Ions for Positive Identification of Individual Ginsenosides

m /z

ginsenoside

retention time (min)intact molecular ion

characteristic

thermal degradation ions

Rb 142.11132407,425,443Rb 244.41102407,425,443Rc 43.21102407,425,443Ro 26.6979439

Rd 47.1969407,425,443

Re 23.4969405,423,441,459Rf 38.5823405,423,441,459Rg 122.5823405,423,441,459F 11

37.4

801

421,439,457,475

1286Analytical Chemistry,Vol.72,No.6,March 15,2000

various ginsenosides in the final products and thus render the differentiation and authentication impossible.Results reported here indicate that Rf or F 11markers would be a better index for differentiating the origins and purities of ginseng products.

CONCLUSION

The present study demonstrated a simple HPLC/MS method for differentiation and authentication of Oriental ginseng,American ginseng,and commercial ginseng products.For ginseng speci-mens,the differentiation hinged on the detection of indicative ginsenosides (i.e.,Rf and F 11)in addition to contrasting the differences in relative abundance of various ginsenosides.For commercial ginseng products,such as ginseng slices and ginseng tea granules,authentication can only be performed by detection of ginsenoside Rf (for Oriental ginseng)and 24(R )-pseudoginse-noside F 11(for American ginseng).The limits of detection for the present HPLC/MS method were determined.All ginsenosides could be detected at the nanogram on-column level.In contrast to the employment of UV absorbance detection,where the detection limit is only 40μg,6a the LC/MS approach enormously increased detection sensitivity by over 1000times.The method developed is also straightforward and convenient and requires no expensive tandem equipment.ACKNOWLEDGMENT

Partial support from the Industrial Support Fund (Grants AF/181/97and AF/154/98)is gratefully acknowledged.We thank Prof.Y.J.Chen of Shenyang Pharmaceutical University,Shen-yang,China,and the Tobacco and Ginseng Monopoly,South Korea for the gifts of the ginsenosides.Thanks are also extended to the Wisconsin Ginseng Board for providing the ginseng specimens.

Received for review July 23,1999.Accepted December 14,1999.

AC990819Z

Figure 5.Reconstructed ion chromatograms for the LC/MS analyses of Oriental and American ginsengs

extracts.

Figure 6.The LC/MS traces of the extract of a brand of American ginseng tea granules.

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