Method Validation for Determination of Alkaloid Content in Goldenseal Root Powder
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1 476 WEBER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 3, 2003 DIETARY SUPPLEMENTS Method Vlidtion for Determintion of Alkloid Content in Goldensel Root Powder HOLLY A. WEBER, MATTHEW K. ZART, ANDREW E. HODGES, KELLIE D. WHITE, SARAH M. BARNES,LESLIE A. MOODY, ALICE P. CLARK, nd ROGER K. HARRIS Midwest Reserch Institute, 425 Volker Blvd, Knss City, MO J. DIANE OVERSTREET nd CYNTHIA S. SMITH Ntionl Institute of Environmentl Helth Sciences, 111 Alexnder Dr, Reserch Tringle Prk, NC A fst, prcticl mbient extrction methodology followed by isocrtic liquid chromtogrphy (LC) nlysis with UV detection ws vlidted for the determintion of berberine, hydrstine, nd cndine in goldensel (Hydrstis cndensis L.) root powder. The method ws lso vlidted for plmtine, mjor lkloid present in the possible biodulternts Coptis, Oregon grpe root, nd brberry brk. Alkloid stndrd solutions were liner over the evluted concentrtion rnges. The nlyticl method ws liner for lkloid extrction using g goldensel root powder/100 ml extrction solvent. Precision of the method ws demonstrted using 10 replicte extrctions of 0.5 g goldensel root powder, with percent reltive stndrd devition for ll 4 lkloids 1.6. Alkloid recovery ws determined by spiking ech lkloid into triplicte liquots of net goldensel root powder. Recoveries rnged from 92.3% for plmtine to 101.9% for hydrstine. Ruggedness of the method ws evluted by performing multiple nlyses of goldensel root powder from 3 suppliers over 2-yer period. The method ws lso used to nlyze Coptis root, Oregon grpe root, brberry brk, nd celndine herb, which re possible goldensel biodulternts. The resulting chromtogrphic profiles of the biodulternts were significntly different from tht of goldensel. The method ws directly trnsferred to LC with mss spectrometry, which ws used to confirm the presence of goldensel lkloids tetrhydroberberstine, berberstine, cndline, berberine, hydrstine, nd cndine, s well s lkloids from the biodulternts, including plmtine, jtrorrhizine, nd coptisine. Goldensel (Hydrstis cndensis L.) is slow-growing, perennil herbceous plnt in the Received My 30, Accepted by SG Februry 12, Corresponding uthor s e-mil: hweber@mrireserch.org. Rnunculcee fmily. It is ntive to the estern portion of North Americ nd ws used extensively by Ntive Americns s n herbl mediction nd clothing dye. Historiclly, goldensel s medicinl use centered on its stringent properties nd the tretment of vriety of conditions, including inflmmtion nd infection of the eyes, skin, nd mucous membrnes throughout the body. It is lso used for tretment of excess mucus in the respirtory system nd s tonic to increse ppetite nd stimulte digestion (1, 2). Extrcts of H. cndensis re known to possess ntibcteril (3, 4), ntituberculr (5), nd immunostimulnt (6) ctivities nd to inhibit cytochrome P450 3A4-medited metbolism of some substrtes (7). Potentil dverse rections from goldensel use include digestive disorders, mucous membrne irrittion, excittory sttes, nd hllucintions (8). It is believed tht goldensel s bioctivity is due to the presence of the mjor isoquinoline lkloids berberine, hydrstine, nd cndine (5; Figure 1), s well s other minor lkloid components (9 11). Goldensel hs become n incresingly populr dietry supplement nd is often sold in conjunction with Echince. In 2000, goldensel ws one of the 15 top-selling herbl supplements, with sles of $39 million (12). It is slow-growing plnt tht is hrvested fter 3 5 yers of growth. Becuse of its extended cultivtion time nd errors mde during hrvesting of wild-crfted roots, number of other berberine-contining plnts hve been substituted for goldensel, including goldthred (Coptis jponic), yellow root (Xnthorhiz simplicissim), Oregon grpe (Mhoni quifolium), celndine (Chelidonium mjus), nd brberry (Berberis vulgris). Of the forementioned plnts, only goldensel contins hydrstine nd cndine, wheres plmtine (Figure 1) is present in Coptis, Oregon grpe root, nd brberry brk, s well s in other berberine-contining plnts (13 17). Adultertion of goldensel in the mrketplce hs been clerly demonstrted by Wng et l. (18), who recently reported tht severl mrketed goldensel extrcts did not contin hydrstine. According to the recent monogrph published by the Americn Herbl Phrmcopei (19), goldensel preprtions should be from the roots nd rhizomes of H. cndensis, which should not contin <2.0% hydrstine nd 2.5% berberine, clculted on dry weight bsis.
2 WEBER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 3, Figure 1. Overly of representtive chromtogrms for LC nlysis of mixed stndrd solution nd extrcts of net goldensel root powder from supplier B nd spiked goldensel root powder. Tble 1. Linerity of lkloid extrction from goldensel root powder from supplier B Weight of goldensel extrcted, g/100 ml Dilution, ml Finl goldensel concentrtion, mg/ml Plmtine in goldensel, weight % Berberine in goldensel, weight % Hydrstine in goldensel, weight % Cndine in goldensel, weight % / / / Averge results / / / / / / / / / Averge results Overll verge results b b b b b Correltion coefficient c c c c c n =3. b n = 12. c Determined by plotting re versus finl goldensel concentrtion (mg/ml).
3 478 WEBER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 3, 2003 Tble 2. Anlyticl system suitbility results Stndrd Theoreticl pltes (N) Tiling (T) Resolution (R),b Precision, % RSD c Plmtine NA 0.14 Berberine Hydrstine Cndine b c Obtined from single injection of mixed stndrd solution. Resolution between djcent lkloid peks. Obtined from 6 replicte injections of mixed stndrd solution. A number of nlyticl methods hve recently been reported for the determintion of berberine nd hydrstine content in goldensel root powder nd/or formultions (9, 10, 18 27). Mny of the reported methods were not vlidted, or included lengthy grdient elution liquid chromtogrphy (LC; 9, 10, 13, 21 23, 25). Other methods used phosphte buffers or ion-pir gents in the liquid chromtogrphic (LC) mobile phse, which prevents direct trnsfer of the method to LC/mss spectrometry (MS; 18 20, 24 28). Only 2 of the published methods nlyzed for plmtine, which would be present if goldensel were dulterted with Coptis or nother plmtine-contining species (13, 27); however, these methods were not vlidted. This report describes the development nd vlidtion of fst, prcticl nlysis for the determintion of 3 lkloids present in goldensel root powder. The method consists of 20 min, mbient, ultrsonic extrction of the root powder, followed by LC nlysis with UV detection. Berberine, hydrstine, nd cndine, s well s plmtine (present in Coptis, Oregon grpe root, nd brberry brk) re determined isocrticlly in 20 min. The method provides excellent precision for the routine determintion of lkloid content in goldensel root powder, s well s the flexibility of direct trnsfer to LC/MS for further identifiction. Becuse biouthentiction of the goldensel root rw mteril is required prior to the mnufcture of ny goldenselcontining product, the method ws lso used to nlyze the following common dulternts: Coptis (Coptis sp.) root, Oregon grpe (M. quifolium) root, celndine (C. mjus) herb, nd brberry (B. vulgris) brk. When these biodulternts were nlyzed by the vlidted method, the resulting chromtogrphic profiles differed significntly from tht of goldensel. In ddition, the method ws directly trnsferred to LC/MS, which confirmed the presence of goldensel lkloids tetrhydroberberstine, berberstine, cndline, berberine, hydrstine, nd cndine, s well s lkloids from the biodulternts, including plmtine, jtrorrhizine, nd coptisine. This work indicted tht the method cn be used not only to determine goldensel lkloid content, but lso s screen for berberine-contining biodulternts. METHOD Smples () Goldensel root powder (H. cndensis L.). Purchsed from 3 bulk commercil suppliers (A, B, nd C) nd stored t mbient temperture in the drk. (b) Botnicl dulternts. Coptis root powder, Oregon grpe (M. quifolium) root powder, celndine (C. mjus) herb, nd brberry (B. vulgris) brk powder purchsed from commercil supplier nd stored t mbient temperture in the drk. Tble 3. Stndrd curve results for plmtine, berberine, hydrstine, nd cndine Prmeter Plmtine Berberine Hydrstine Cndine Concentrtion rnge, g/ml % RSD of lowest stndrd Reltive error, % Averge % recovery, n = Correltion coefficient LOD, g/ml b LOQ, g/ml b b % RSD of lowest stndrd prepred in triplicte. Limits of detection were bsed on stndrd devition ( ) of lowest stndrd prepred in triplicte for which the % RSD 10%. LOD=3. LOQ=10.
4 WEBER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 3, Tble 4. Precision of lkloid extrction from goldensel root powder from supplier B Goldensel dded, g Plmtine, % Berberine, % Hydrstine, % Cndine, % Alkloids, % totl Overll results (n = 10) RSD, % All results reported s % w/w. Regents () Alkloid stndrds. Berberine chloride dihydrte (berberine) nd -hydrstine hydrochloride (hydrstine; Sigm, St. Louis, MO); plmtine chloride hydrte (plmtine; Aldrich, Milwukee, WI); cndine (Apin Chemicls Ltd., Abingdon, UK). (b) Solvents. Acetonitrile, methnol, chloroform, nd wter (LC grde). (c) Chemicls. Ammonium cette (LC grde); triethylmine (TEA), phosphoric cid, nd cetic cid (ACS grde). (d) LC mobile phse. (1) Buffer solution (30mM cette, 14mM TEA, ph 4.85). Add 2.3 g mmonium cette nd 2 ml TEA to 1 L wter nd djust ph to 4.85 with cetic cid. (2) Mobile phse. Degs before use. Buffer cetonitrile ( , v/v). (e) Extrction solvent. Acetonitrile wter phosphoric cid ( , v/v/v). (f) Diluent. Acetonitrile wter ( , v/v). Apprtus () LC system. Wters 2690 Seprtions Module (Wters Corp., Milford, MA) equipped with solvent delivery system, utosmpler, controller, nd column heter. Operting conditions: isocrtic, 100% mobile phse; flow rte, 1.0 ml/min; injection volume, 10 L; column temperture, 30 C; UV (Wters 2487 dul bsorbnce detector) t 230 nm; dt system [TurboChrom TM (Perkin-Elmer, Wellesley, MA)]; Zorbx Eclipse XDB-C18, 80 Å, 3.5 m, mm nlyticl column fitted with Zorbx Eclipse XDB-C18 80 Å, 5 m, mm gurd column (Agilent Technologies, Plo Alto, CA). (b) LC/MS equipment Series II chromtogrph with pump, utosmpler, nd column heter (Hewlett-Pckrd, Plo Alto, CA). UV detector (Spectromonitor 3200, Thermo Seprtion Products, Sn Jose, CA) interfced with Quttro I (Micromss, Mnchester, UK) mss spectrometer; dt system, Micromss MssLynx softwre (version 2.2). LC operting conditions: previously described, with flow from UV detector split, with 0.2 ml/min directed to mss spectrometer. Mss spectrometer operting conditions: positive electrospry ioniztion (ESI) mode; mss rnge scnned, mu; source temperture, 150 C; cone voltge, 35 V; scn time, 2 s; nitrogen, 120 psi; nd nebulizer gs, 15 L/h. (c) Extrction equipment. Ultrsonic bth (Model 5510, Brnson, Dnbury, CT); wrist-ction shker (Model 75, Burrell, Pittsburgh, PA); centrifuge (Dmon/IEC, Needhm Heights, MA). Stndrd Curve Preprtion () Mixed stndrd stock solutions. (1) Berberine nd hydrstine stock solutions A nd B. Weigh 65 mg berberine chloride dihydrte nd 60 mg hydrstine hydrochloride into ech of 2 individul 100 ml flsks. (2) Plmtine nd cndine stock solutions C nd D. Weigh 12.5 mg plmtine chloride hydrte nd 12 mg cndine into ech of 2 individul 250 ml flsks. (3) Fill ech flsk hlf-wy with diluent, sonicte 5 min, dilute to mrk with diluent. (b) Stndrd solutions. Prepre triplicte stndrds t highest nd lowest concentrtions. Accurtely pipette 1.0 ml liquots of solutions B nd D into single 100 ml flsk. Accurtely pipette 1.0, 5.0, nd 10.0 ml liquots of solutions A nd C into respective 50 ml flsks. Accurtely pipette 2.5 nd 7.0 ml liquots of solutions B nd D into respective 50 ml flsks. Dilute flsks to volume with diluent. Correct ll concentrtions for wter, Cl, nd HCl content. Construct stndrd plots using bove LC conditions nd pproprite softwre.
5 480 WEBER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 3, 2003 Tble 5. Spike nd recovery results for plmtine nd berberine Amount of goldensel extrcted, g Determined plmtine, mg Theoreticl plmtine in spiked smple, mg Recovery, % Determined berberine, mg Theoreticl berberine in spiked smple, mg Recovery, % Overll results (n = 3) Sum of clculted ntive (mg) nd spiked lkloid (mg). Extrction Procedure () Generl extrction method. Use mortr nd pestle to grind smples to fine powder if needed. Weigh smple into 200 ml centrifuge bottle. Add 100 ml extrction solvent, sonicte t mbient temperture 5 min, shke on wrist-ction shker 10 min, nd centrifuge 5 min t 2200 rpm to clrify extrcts. Dilute s needed with diluent, filter [0.45 m polytetrfluoroethylene (PTFE)] liquots into utosmpler vils, nd nlyze using bove LC conditions. (b) Linerity of lkloid extrction. Prepre triplictes t high nd low concentrtions. Weigh g goldensel root powder into individul 200 ml centrifuge bottles s outlined in Tble 1. Extrct s previously described nd further dilute with diluent s outlined in Tble 1. (c) Precision of lkloid extrction. Weigh 0.5 g goldensel root powder into 10 individul 200 ml centrifuge bottles. Extrct s previously described nd dilute 5 ml of ech 25 ml extrct to with diluent before LC nlysis. (d) Recovery of lkloids. (1) Plmtine spiking solution. Weigh 11 mg plmtine chloride hydrte into 1 drm vil, dd 1 ml methnol, nd sonicte to dissolve. Correct concentrtion for wter nd Cl content. (2) Cndine spiking solution. Weigh 11 mg cndine into 1 drm vil, dd 1 ml chloroform, nd sonicte to dissolve. (e) Spiking of root powder. Weigh 0.5 g goldensel root powder into 3 individul 200 ml centrifuge bottles. Add 13 mg berberine chloride dihydrte nd 11 mg hydrstine hydrochloride directly to ech bottle. Add liquots of prepred spiking solutions to ech bottle in order to dd 0.75 mg plmtine nd 0.5 mg cndine. Mix ech liquot of spiked root powder nd ir-dry overnight t mbient conditions. Extrct s previously described nd dilute 5 ml of ech extrct to 25 ml with diluent before LC nlysis. (f) Ruggedness. Ruggedness of the vlidted method ws evluted by nlyzing goldensel root powder from 3 suppliers. The nlyses were performed by different nlysts, with c 1 yer between nlyses. Weigh triplicte 0.5 g liquots of ech lot of root powder into individul 200 ml centrifuge bottles. Extrct s previously described nd dilute 5 ml of ech extrct to 25 ml with diluent before LC nlysis. (g) Anlysis of biodulternts. Weigh triplicte 0.5 g liquots of ech powdered smple into individul 200 ml centrifuge bottles. Extrct s previously described nd dilute 5 ml of ech extrct to 25 ml with diluent before LC nlysis. Results nd Discussion Method Development The gol of the LC method development work ws to find fst, isocrtic method tht seprted plmtine, berberine, hydrstine, nd cndine, nd could lso be used for LC/MS nlyses. During development of the LC nlysis, severl column types, column dimensions, nd mobile phses were investigted. Buffer strengths, dditives (e.g., TEA), nd buffer ph rnges were lso investigted to improve resolution nd pek shpe of the 4 lkloids. The extrction of berberine nd hydrstine from goldensel root powder ws optimized by using n pproch reported by Anderson nd Burney (28). A number of extrction solvents consisting of vrious rtios of methnol wter, methnol wter cid, cetonitrile wter, nd cetonitrile wter cid were investigted. For screening purposes, extrction efficiency ws evluted by weighing triplicte 0.5 g liquots of goldensel root powder into individul 200 ml centrifuge bottles. The root powder ws extrcted with 100 ml liquots of extrction solvent. A 50 ml liquot of extrction solvent ws removed from ech bottle, with 10 ml reserved for LC nlysis. An dditionl 50 ml extrction solvent ws dded to ech bottle, the smples were re-extrcted, nd second 10 ml liquot ws removed for LC nlysis. The extrcts were nlyzed by LC, nd the weight percentges of berberine nd hydrstine were determined, correcting for the 50% dilution of the re-extrcted smples. The extrction efficiency of ech solvent system ws evluted by compring the weight percentges of berberine nd hydrstine obtined from the first nd second extrctions for ech liquot of root powder. Acetonitrile wter phosphoric cid ( , v/v/v) gve the highest recoveries of berberine nd hydrstine from goldensel root powder s well s extrction efficiencies 99%. Method Vlidtion A mixed stndrd solution, which contined plmtine, berberine, hydrstine, nd cndine, ws used to evlute the nlyticl system for precision, theoreticl pltes, pek tiling, nd resolution ccording to current USP guidelines (29) nd yielded cceptble results. For ech nlyte, precision ws
6 WEBER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 3, Tble 6. Spike nd recovery results for hydrstine nd cndine Amount of goldensel extrcted, g Determined hydrstine, mg Theoreticl hydrstine in spiked smple, mg Recovery, % Determined cndine, mg Theoreticl cndine in spiked smple, mg Recovery, % Overll results (n = 3) Sum of clculted ntive (mg) nd spiked lkloid (mg). <2.0%, tiling ws <1.5, resolution ws >2.3, nd theoreticl pltes were > (Tble 2; see Figure 1 for representtive chromtogrm). Results of the stndrd curve nlyses indicted liner response over the evluted concentrtion rnge of the 4 lkloids (Tble 3). Limits of detection nd quntittion were bsed on the stndrd devition of the lowest stndrd prepred in triplicte. The nlyticl method lso proved to be liner for lkloid extrction using g goldensel root powder with 100 ml extrction solvent (Tble 1). Goldensel root powder from supplier B ws used for method vlidtion. Precision of the method ws further evluted by using the verge results of 10 replicte extrctions of 0.5 g goldensel root powder. For ll 4 lkloids, the percent reltive stndrd devition for the 10 replictes ws 1.6. The verge (n = 10) weight percent ws determined for the 4 lkloids: plmtine, ; berberine, ; hydrstine, ; nd cndine, The verge totl weight percent of lkloids present in goldensel root powder ws (Tble 4). Recovery of plmtine, berberine, hydrstine, nd cndine ws determined by spiking ech lkloid into triplicte liquots of net goldensel root powder followed by extrction nd LC nlysis. Ech lkloid ws spiked t concentrtion equivlent to tht in the ntive goldensel root powder from supplier B (see bove for results). The recoveries (n = 3) were % for plmtine, % for berberine, % for hydrstine, nd % for cndine (Tbles 5 nd 6). Ruggedness of the method ws evluted by nlyzing goldensel root powder from 3 suppliers. Two nlysts nlyzed ech lot of mteril in triplicte; the second nlysis ws conducted pproximtely 1 yer fter the first nlysis. The results indicted tht the method ws suitble for dy-to-dy determintion of lkloid content in goldensel root powder (Tble 7). The mteril from supplier B ws the only nlyzed lot of goldensel root powder found to contin plmtine. The method ws lso used to nlyze Coptis root powder, Oregon grpe (M. quifolium) root powder, celndine (C. mjus) herb, nd brberry (B. vulgris) brk powder, 4 common goldensel biodulternts. The chromtogrphic profiles (Figure 2) of these herbs differ significntly from tht of goldensel. To ensure the method s fitness for use, it ws directly trnsferred to LC/MS. The flow from the UV detector ws split in order to direct 0.2 ml/min to the mss spectrometer. Aside from this prmeter, the method ws trnsferred without ltertion. The UV nd totl ion chromtogrms from the LC/MS nlysis re shown in Figure 3. LC/MS nlysis of goldensel root powder nd the 4 biodulternts led to the identifiction of goldensel lkloids tetrhydroberberstine, berberstine, cndline, berberine, hydrstine, nd cndine. The following lkloids present in extrcts of the 4 biodulternts were lso identified: mgnoflorine, chelidonine, homochelidonine, jtrorrhizine, coptisine, plmtine, protopine, snguinrine, Tble 7. Ruggedness of lkloid determintion in goldensel root powder Anlyst 1 b Anlyst 2 c Alkloid Supplier A Supplier B Supplier C Supplier A Supplier B Supplier C % Plmtine % Berberine % Hydrstine % Cndine All results reported s verge weight percentges (n = 3) long with corresponding stndrd devition. b Anlysis performed December c Anlysis performed December 2001.
7 482 WEBER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 3, 2003 plmtine, berberine, hydrstine, nd cndine, thus determining tht the mteril ws not pure goldensel. Conclusions Figure 2. Overly of representtive chromtogrms for LC nlysis of extrcts of net goldensel root powder nd the following common biodulternts: Coptis root powder, Oregon grpe root powder, brberry brk, nd celndine herb. chelerythrine, nd stylopine. LC/MS nlyses of goldensel root powder from suppliers A nd C confirmed the presence of hydrstinine, berberstine, tetrhydroberberstine, cndline, berberine, hydrstine, nd cndine. Plmtine ws not present in mteril from suppliers A nd C. LC/MS nlyses of goldensel root powder from supplier B confirmed the presence of hydrstinine, berberstine, tetrhydroberberstine, jtrorrhizine, coptisine, cndline, A rpid mbient extrction nd isocrtic LC method ws vlidted for the determintion of lkloids (berberine, hydrstine, nd cndine) in goldensel root powder. The method ws lso vlidted for the determintion of plmtine, n lkloid ntive to Coptis, Oregon grpe root, nd brberry brk, common biodulternts of goldensel products. The vlidted method, which provided excellent reproducibility, ws used for the routine nlysis of goldensel root powder nd for nlysis of 4 common goldensel biodulernts (Coptis, Oregon grpe root, brberry brk, nd celndine herb), which hve significntly different chromtogrphic profiles thn goldensel. The method ws trnsferred directly to LC/MS for further chrcteriztion nlyses. The presented method is sufficient for determintion of the predominnt lkloids in goldensel nd cn lso be used to determine the presence of common goldensel biodulternts. Acknowledgments This work ws supported by the Ntionl Institute of Environmentl Helth Sciences (NIEHS), Ntionl Institutes of Helth, under Contrct Nos. N01-ES nd N01-ES Figure 3. Totl ion nd UV chromtogrms from LC/MS nlysis of n extrct goldensel root powder from supplier B. Anlysis confirmed the presence of hydrstinine, jtrorrhizine, coptisine, berberstine, tetrhydroberberstine, cndline, plmtine, berberine, hydrstine, nd cndine in the root powder.
8 WEBER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 3, References (1) Grieve, M. (1931) in A Modern Herbl, Vol. 1, Jonthn Cpe Ltd., London, UK, pp (2) Britton, J., & Kircher, T. (1998) in The Complete Book of Home Herbl Remedies, Firefly Books Ltd., Willowdle, Cnd, p. 123 (3) Knight, S.E. (1999) Bios 70, 3 10 (4) Sczzocchio, F., Comet, M.F., Tomssini, L., & Plmery, M. (2001) Plnt Med. 67, (5) Gentry, E.J., Jmpni, H.B., Keshrvrz-Shokri, A., Morton, M.D., Vnder Velde, D., Telikeplli, H., & Mitscher, L.A. (1998) J. Nt. Prod. 61, (6) Rehmn, J., Dillow, J.M., Crter, S.M., Chou, J., Le, B., & Misel, A.S. (1999) Immunol. Lett. 68, (7) Budzinski, J.W., Foster, B.C., Vndenhoek, S., & Arnson, J.T. (2000) Phytomedicine 7, (8) Thoms Fleming (Ed.) (2000) in PDR for Herbl Medicines, Medicl Economics Compny, Inc., Montvle, NJ, pp (9) Sturm, S., & Stuppner, H. (1998) Electrophoresis 19, (10) Leone, M.G., Comet, M.F., Plmery, M., & Sso, L. (1996) Phytother. Res. 10, S45 S46 (11) Gleffi, C., Comet, M.F., Tomssini, L., & Nicoletti, M. (1997) Plnt Med. 63, 194 (12) Blumenthl, M. (1999) Herblgrm 47, (13) Betz, J.M., Musser, S.M., & Lrkin, G.M. (1998) Differentition between Goldensel (Hydrstis cndensis L.) nd Possible Adulternts by LC/MS, Proceedings of the 39th Annul Meeting of the Americn Society of Phrmcognosy, Orlndo, FL, July 19 23, Americn Society of Phrmcognosy, Glendle, AZ (14) Glle, K., Müller-Jkic, B., Proebstle, A., Jurcic, K., Bldt, S., & Wgner, H. (1994) Phytomedicine 1, (15) Che, S.-H., Jeong, I.-H., Choi, D.-H., Oh, J.-W., & Ahn, Y.-J. (1999) J. Agric. Food Chem. 47, (16) Kim, J.P., Jung, M.Y., Kim, J.-P., & Kim, S.Y. (2000) J. Agric. Food Chem. 48, (17) Slvík, J., & Slvíková, L. (1995) Collect. Czech. Chem. Comm. 60, (18) Wng, M., Zhu, N., Jin, Y., Belkowitz, N., & Ho, C.-T. (2002) in Qulity Mngement of Nutrceuticls, C.-T. Ho & Q.Y. Zheng (Eds), ACS Symposium Series 803, Americn Chemicl Society, Wshington, DC, pp (19) Americn Herbl Phrmcopei TM nd Therpeutic Compendium (2001) Goldensel monogrph, Americn Herbl Phrmcopei TM, Snt Cruz, CA (20) Hermes, R.E., & Vldez, L.S. (1996) HPLC Method for the Determintion of Alkloids in Extrcts of Goldensel (Hydrstis cndensis), Book of Abstrcts, 211th Ntionl Meeting of the Americn Chemicl Society, New Orlens, LA, Mrch 24 28, ACS, Wshington, DC (21) Govindn, M., & Govindn, G. (2000) Fitoterpi 71, (22) Abourshed, E.A., & Khn, I.A. (2001) J. Phrm. Sci. 90, (23) Li, W., & Fitzloff, J.F. (2002) J. Phrm. Phrmcol. 54, (24) Schieffer, G.W., & Kohn, M. (2002) J. Liq. Chrom. Rel. Technol. 25, (25) Schieffer, G.W., & Pfeiffer, K. (2001) J. Liq. Chrom. Rel. Technol. 24, (26) Phrmcopeil Forum (2001) 27, (27) Weber, H.A., Zrt, M.K., Ferguson, S.L., Greves, J.G., Clrk, A.P, Hrris, R.K., Overstreet, D., & Smith, C. (2001) J. Liq. Chrom. Rel. Technol. 24, (28) Anderson, M.L., & Burney, D.P. (1998) J. AOAC Int. 81, (29) United Sttes Phrmcopei (2001) 24th Ed., United Sttes Phrmcopeil Convention, Inc., Rockville, MD, p. 1924
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