SIMPLE STABILITY-INDICATING VALIDATED HPLC METHOD FOR DIOSGENIN IN COSMECEUTICAL FORMULATIONS WITH LONG TERM STABILITY APPLICATION FOR LIGHT AND HEAT

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1 Acdemic Sciences Interntionl Journl of Phrmcy nd Phrmceuticl Sciences ISSN Vol 3, Suppl 5, 2011 Reserch Article SIMPLE STABILITY-INDICATING VALIDATED HPLC METHOD FOR DIOSGENIN IN COSMECEUTICAL FORMULATIONS WITH LONG TERM STABILITY APPLICATION FOR LIGHT AND HEAT WANG-TAEK HWANG, YOUNG-SOO KIM, CHAN-KI HONG, JONGSUNG LEE *, DOEKHOON PARK Biospectrum Life Science Institute, Eines Pltz 11th FL, Sngdewon Dong, Seoungnm City, Gyunggi Do, Republic of Kore. Emil: ABSTRACT Received: 4 Aug 2011, Revised nd Accepted: 30 Sep 2011 A rpid, simple, precise nd ccurte stbility-indicting nlyticl HPLC method ws developed nd vlidted for determintion of diosgenin in cosmeceuticl formultions. Modified extrction with suggested nlyticl method resulted in ssurnce of full recovery in ll types of formultions. In this study, Lun-C 18 (4.6 mm x 150 mm, 5 μm prticle size) column ws employed using mixture of distilled wter nd cetonitrile (15:85, v/v) s mobile phse with wvelength of 210 nm. Method vlidtion ws conducted bsed on ICH nd USP guidelines. For selectivity test, this method ws shown to seprte well with numerous interferences tht were commonly employed s formultion excipients. An dequte linerity reltionship (r 2 =0.999) ws obtined for diosgenin concentrtions rnging from 0.05 to 1.0 mg ml -1. For ll formultions, the ccurcy percentge rnged from to % nd the precision ws lower thn 2%. These cceptnce criteri indicted stisfctory vlidtion results. Moreover, the proposed method ws further pplied for long-term nd ccelerted stbility experiments in ll formultions in forced stress conditions of light nd het. Keywords: Diosgenin, Excipients, Tetrhydrofurn, Long-term stbility, Accelerted stbility * INTRODUCTION Diosgenin (25R-Spriost-5-en-3β-ol) is n glycone of dioscin, which is steroidl sponin tht occurs bundntly in nturl plnts like wild yms, including Dioscore wild species 1-2. As steroidl metbolite, diosgenin hs been used erly s strting mteril for vrious synthetic steroidl drugs in the phrmceuticl industry. Moreover, its estrogenic effect on mmmlin glnds hs been demonstrted 3, nd it hs been employed s component of combined orl contrceptive pills (COCP) 4. It hs lso been shown to led to cholesterol reduction 5 nd to hve ntioxidnt bility 6. Published studies hve reported tht diosgenin is potent chemotherpeutic gent ginst severl cncers including osteosrcom nd colon crcinom 7-8. Recently, s n ctive substnce, diosgenin in bulk extrct of wild ym ws widely used in skin cre formuls ment to reduce the sign of ging in reltion with kertinocyte prolifertion 9. Also, Lee et l. demonstrted tht diosgenin hd n inhibitory effect ginst melnogenesis tht occurred through ctivtion of phosphtidylinositol-3-kinse pthwy (PI3K) signling 10, suggesting its possible ppliction for skin lightening effect. Severl methods hve been reported to be useful for determintion of diosgenin from vrious pproches, including HPTLC, HPLC, LC ESI-MS nd ELISA LC ESI-MS method ws the most sensitive but it cnnot be commonly employed to nlyticl lbortories nd institutes due to requirement of unusul instrument nd experienced expert. One of them, HPLC is the most universl quntifiction method to ny lbortories for routine nlysis nd esily dptble even in complex mtrixes without disturbnce of excipients However, reported HPLC methods with extrction procedure were prcticlly not dptble in complex preprtions like commercil cosmetic formultions due to overlpping of diosgenin pek with formultion excipients nd showed limited recovery during extrction process. Also, to dte, there hve been no reports regrding HPLC determintion of diosgenin in formultions. Thus, the present study ws to develop simple, rpid, precise nd ccurte reversed phse-hplc for indiction of the stbility of diosgenin so tht it cn be esily dpted in nlyticl lbortories nd/or to vrious types of formultions. In this study, development nd vlidtion of the method used for nlysis of diosgenin were conducted ccording to the requirements of the ICH nd USP guidelines including specificity, linerity, ccurcy, precision, limit of detection nd limit of quntifiction without ny interference from complex excipients In the extrction process of diosgenin, incomplete solubiliztion occurred for ll formultions. However, diosgenin ws completely extrcted by employing THF, which is miscible polr protic strong solvent, s diluent of methnol. Finlly, the proposed method ws successfully pplied to stbility studies of ech formultion over six months of exposure to therml nd light stress conditions, suggesting tht diosgenin is stble substnce nd the method is pplicble to other stbility studies nd qulity control pplictions in the cosmeceuticl/phrmceuticl field. MATERIALS AND METHODS Mterils nd regents HPLC grde cetonitrile nd THF were purchsed from Fisher Science (USA). HPLC grde methnol ws purchsed from J.T. Bker (NJ, USA). A diosgenin stndrd ( 99%) ws purchsed from Sigm Aldrich. All regents were used without ny further purifiction. All cosmetic ingredients, which included disodium EDTA, glycerin, butylene glycol, sodium hyluronte, crbomer, cryltes/c10-30 lkyl crosspolymer, llntoin, sorbitol, methyl prben, ethnol, PEG-60 hydrogented cstor oil, phenyltrimethicone, lecithin, glyceryl sterte/peg-100 sterte, cyclomethicone, glyceryl sterte, propyl prben, polysorbte60, glyceryl sterte, sorbitn sesquiolete, steric cid, ceteryl lcohol, cetyl lcohol, cetylethylhexnote, minerl oil, dimethicone, cyclopensiloxne, nd propyl prben, were purchsed from Debong LS (Rep. of Kore). HPLC Instrument nd Condition A Wters HPLC system with 600 controller, 996 photodiode rry detector, 616 pump, nd 717 utosmpler ws used in this experiment. Dt cquisition ws chieved using the Wters Empower softwre. All chromtogrphic seprtions were conducted on Phenomenex Lun C 18 column (150 mm X 4.6 mm, 5 μm) t mbient temperture with detection t 210 nm. The mobile phse consisted of distilled wter nd cetonitrile (15:85, v/v). All HPLC experiments were conducted using n injection volume of 20 μl, n utosmpler nd totl run time of 30 min for ech smple. Preprtion of diosgenin supplemented commercil formultions Cosmetic formultions for the development, vlidtion nd ppliction studies were prepred s shown in Tble 1.

2 Prk et l. Int J Phrm Phrm Sci, Vol 3, Suppl 5, Previously, ll pre-mixture solutions were mde t 75 C with dequte homogeniztion. Skin ws formulted by mixing the A nd B phses t 25 C with sufficient gittion followed by the ddition of triethnolmine, citric cid nd sodium citrte to djust the ph. Lotion nd crem were prepred by suspension of the A nd B phses t 75 C to chieve homogeneity in highly thickened solution, fter which triethnnolmine nd phenoxyethnol were dded under the sme conditions. All formultions contined 400 μg ml -1 of diosgenin in the dispersed stte. Tble 1: Formultion components for skin, lotion nd crem preprtions Phse Components Percentge in ech formultion (w/w) Skin Lotion Crem A Distilled wter Pre-mixture Pre-mixtureb Pre-mixturec B Pre-mixtured Pre-mixturee Pre-mixturef Diosgenin C Triethnolmine D Citric cid Sodium citrte Phenoxyethnol Disodium EDTA 0.03%, Glycerin 3.00%, Butylene glycol 3.00%, Sodium hyluornte 0.30%, Crbomer 0.10% Acryltes/C10-30 Alkyl Crosspolymer 0.10%) b (Allntoin 0.10%, Sorbitol 1.00%, Butylene glycol 2.00%, Crbomer 0.10%, Acryltes/C10-30 Alkyl Crosspolymer 0.10%, Methylprben 0.20%) c (Disodium EDTA 0.03%, Allntoin 0.10%, Sodium hyluornte 0.50%, Crbomer 0.20%, Methylprben 0.20%) d (Ethnol 5.00%, PEG-60 hydrogented cstor oil 0.40%, Phenyltrimethicone 0.30%, Methylprben 0.20%) e (Lecithin 0.50%, Ethnol 1.00%,Glyceryl sterte/peg-100 sterte 1.50%, Cyclomethicone 3.00%, Glyceryl sterte 0.50%, Propylprben 0.10%) f (Polysorbte %, Glyceryl sterte 0.80%, Sorbitn sesquiolete 0.30%, Glyceryl sterte/peg-100 sterte 2.00%, Steric cid 0.50%, Ceteryl lcohol 2.00%, Cetyl lcochol 1.50%, Cetylethylhexnote 1.00%, Minerl oil 3.00% Dimethicone, 0.30%, Cyclopentsiloxne 3.00%, Propylprben 0.10%) Preprtion of stndrd nd test solutions Diosgenin stndrd (Sigm, 99%) ws purchsed nd prepred. Briefly, 10 mg of diosgenin ws precisely weighed nd then dissolved in 10 ml of methnol mixed with tetrhydrofurn in rtio of 1 to 1 s stock solution. This 1 ml of stock solution ws subsequently diluted in 10 ml of the sme solvent bove s stndrd solution. For the test solution, 2.5 g of ech formultion were lso precisely weighed nd dissolved in 10 ml of methnol mixed with 50% THF for ll formultions. Both the stndrd nd test solution were filtered using 0.22 μm pore-sized syringe filter for further HPLC injection. All possible weighing errors were compensted by multiplying dilution fctor inversely. Vlidtion of the method All vlidtion processes were conducted bsed on the ICH nd USP guidelines To ssess the specificity nd selectivity of diosgenin in ech formultion, chromtogrphic run of test solutions prepred from skin, lotion nd crem formultions ws conducted using the developed method while simultneously identifying the diosgenin pek using photo-diode rry spectrum. A linerity study ws lso conducted over concentrtion rnge of μg ml-1, covering the nlyticl working concentrtion during chromtogrphic seprtion. Five concentrtions, 50, 100, 200, 500, nd 1000 μg ml -1, were selected nd prepred by dilution of the stock solution. Triplicte injections were then mde for ech concentrtion nd the obtined pek re ws used to plot the stndrd curve ginst the concentrtion of diosgenin. The ccurcy nd recovery were evluted by prepring plcebo of cosmetic skin, lotion nd crem formultions tht differed only in tht they hd diosgenin concentrtions of 320, 400 nd 480 μg ml-1 covering rnge of % of the climed concentrtion. Triplicte injections were mde for ech concentrtion nd recovery (%) in skin, lotion nd crem formultions nd the results were ssessed by comprison with stndrd solutions of diosgenin prepred dily. Repetbility nd intermedite precision were evluted by nlyzing prepred skin, lotion nd crem formultions contining 400 μg ml-1 of diosgenin. Repetbility ws ssessed bsed on the R.S.D. (%) of the pek re of diosgenin obtined from six replicte injections t 100% of the climed concentrtion. Intermedite precision ws conducted using the sme procedure s for the repetbility experiment; however, nlyses were conducted on two different dys nd evluted by R.S.D. (%). LOD nd LOQ were determined by direct clcultion bsed on the signl to noise rtio mesured during the chromtogrphic run. LOD nd LOQ were determined s the concentrtion t which the signl to noise rtio ws 3:1 nd 10:1, respectively. Specificlly, 50 μg ml-1 of the diosgenin stock solution ws diluted in seril units of 10 μg ml -1, fter which triplicte injections of the test solution nd methnol:thf (1:1) s blnk for noise ssessment were nlyzed. To ensure dequte sustinbility of performnce between the method developed here nd the HPLC instrument for dily nlysis, system suitbility test (SST) ws conducted during development nd vlidtion of the method. The following evlution prmeters were considered, cpcity fctor (K), symmetry (A), tiling (T), theoreticl pltes (N) nd R.S.D. (%) of retention time/pek re/pek height Dt from six replictes of injections were obtined nd clculted to determine the R.S.D. (%) vlue. Long term nd ccelerted stbility evlution The proposed method ws employed to observe the long-term nd ccelerted stbility of diosgenin in skin, lotion nd crem formultions to evlute the persistence of diosgenin by elpsed time bsed on the stbility testing guideline of ICH 21. For longterm study defined to monitor t 25 C, ech formultion contining 400 μg ml -1 of diosgenin ws kept t room temperture with/without rtificil shde provided by luminum foil for six month period. An ccelerted stbility study under forced 467

3 Prk et l. Int J Phrm Phrm Sci, Vol 3, Suppl 5, therml condition ws lso conducted using the sme procedure, but the smples were mintined t 50 C in n oven. At pproprite time intervls (0.5, 1, 2, 4 nd 6 months), ech smple ws withdrwn nd used to prepre test solution for determintion of the diosgenin to verify the degree of therml nd light induced influences. RESULTS AND DISCUSSION Method development The preceding optimiztion ws necessry before method vlidtion nd seprtion of diosgenin under vrious conditions, while SST test ws simultneously conducted for ll formultions. Fig. 1: The structure of diosgenin As shown in Fig. 1, the nlyte diosgenin only hs one conjugted double bond, indicting lck of specific chromospheres. Becuse of this property, diosgenin could not be detected, even t higher wvelengths. Accordingly, the detection rnge of diosgenin is limited to its low wvelength region (< 220 nm), which hs poor sensitivity due to bsorption competition with the mobile phse. However, when considering the signl to bseline rtio of the mobile phse, wvelength selection t 210 nm ws found to be pproprite for quntifiction. Evlution of the mobile phse reveled tht cetonitrile produced better bseline t 210 nm thn methnol nd ws more dvntgeous in tht it contributed low pressure to the chromtogrphic system. To improve the pek shpe, the ddition of n ion pring gent such s cetic cid, trifluorocetic cid nd buffer t different concentrtions ws conducted; however, considerble effects were not observed (dt not shown). Through vrition testing of the composition in wter nd cetonitrile bsed on previous report, 2, isocrtic elution using wter:cetonitrile (15:85) mobile phse showed ffordble resolution of excipients nd dequte retention time during chromtogrphic run time of 30 min. In previous study, 2,13 methnol ws used s diluent to extrct diosgenin from ech source without ny recovery problem. But, in our study, test solution extrcted by methnol did not chieve sufficient recovery for ll formultions due to disturbnce of complex excipients, indicting tht methnol is not n idel diluent for non-soluble diosgenin. Other extrcting gents nd dditives including ethnol, trifluorocetic cid, phosphte buffer, etc. were evluted to improve the solubility during the extrction of diosgenin. One of these, THF, ws employed t different rtios. Methnol mixed with THF t rtio of 1:1 showed the gretest extrction cpcity of ll formultions, indicting tht it is proper extrction solvent (Tble 2). Following this optimiztion, vlidtion of the method ws evluted. Tble 2: Recovery efficiency obtined by suspending vrious rtios of tetrhydrofurn to methnol when prepring smples from skin, lotion nd crem formultions Rtio of tetrhydrofurn Skin Lotion Crem to methnol (%) Recovery (%) ±SD Recovery (%) ±SD Recovery (%) ±SD ± ± ± ± ± ± ± ± ± ± ± ± Method Vlidtion System suitbility To ensure dequte performnce of the method, system suitbility prmeters of the nlyte pek were evluted using the ICH nd USP guidelines As shown in Tble 3, ll obtined prmeters fell in rnge of conventionlly ccepted criteri. The cpcity fctor ws within 2.2 < K < 6.8. In ddition, the vlue of symmetry nd tiling fctor ws lower thn 1.5 nd the theoreticl plte vlue for column efficiency ws All the R.S.D. vlues of the retention time/re/pek height were lower thn 2%, indicting tht the obtined vlue provided stisfctory results nd the system ws working properly. Tble 3: System suitbility prmeters of diosgenin stndrd Prmeters Vlue Cpcity fctor (K ) Asymmetry (A) 1.13 Tiling (T) 1.05 Theoreticl pltes (N) 5421 R. S. D. of retention time (%) R. S. D. of re (%) R. S. D. of pek height (%) (n=6) Specificity The proposed method ws evluted to verify the bility to seprte the diosgenin pek with possible interference of commonly used excipients including ntioxidnts, thickening gents, preservtives, ph regultors nd emollients. Chromtogrphic nlysis reveled good seprtion of the diosgenin pek nd no interference with the excipients in ny formultions, even in densely mixed lotion nd crem formultions (Fig. 2). Linerity Evlution of the linerity ws conducted t concentrtions rnging from 0.05 to 1.00 μg ml-1, which covered the working concentrtions. The correltion efficient vlue (r 2 =0.999) ws higher thn the cceptnce criteri of ccording to the USP guideline. Other relted prmeters for the regression equtions obtined by lest squres tretment of the results re shown in Tble 4. Accurcy To mesure the ccurcy of the proposed method, three known concentrtions of diosgenin were dded to ech formultion nd their clculted recovery ws compred to the ctul concentrtion (Tble 5). This clculted recovery vlue rnged from to %, indicting tht this method worked properly with excellent ccurcy in skin, lotion nd crem formultions s model cosmetic preprtions without ny disturbnce of excipients. Precision The precision of this method ws ssessed bsed on the R.S.D. (%) vlue obtined from intr- nd inter-dy nlysis of six replicte injections (Tble 6). All vlues for the intr- nd inter-dy precision study were lower thn 2%, confirming tht the precision of the method ws sufficient. In ddition, no significnt differences in repetbility mong formultion types were observed. 468

4 Prk et l. Int J Phrm Phrm Sci, Vol 3, Suppl 5, Fig. 2: Chromtogrphic seprtion of diosgenin in (A) stndrd, (B) skin, (C) lotion nd (D) crem solution using proposed method. (1) Tetrhydrofurn (2) Diosgenin Tble 4: Sttisticl nlysis of regression dt nd limit of detection nd quntifiction Prmeters Vlue Regression eqution (Y) Clibrtion rnge μg ml-1 Slope (b) Stndrd devition of the slope (S b) Reltive stndrd devition of the slope % Confidence limit of the slopeb Intercept () Correltion coefficient (r) Stndrd error of the estimtion Limit of detection c Limit of quntifiction ~10 μg ml ~30 μg ml c -1 Y= + bc, where C is the concentrtion of diosgenin in μg ml -1 nd Y is the re of pek b 95% confidence limit c direct clcultion of signl to noise rtio -1 Tble 5: Accurcy of dt obtined from skin, lotion nd crem formultions in concentrtion rnge of 320 to 480 μg ml -1 of diosgenin. Type of % of imed Prepred Mesured Recovery (%) formultion concentrtion concentrtion concentrtion ± SD ± SD Skin ± ± ± ± ± ± Averge ± SD ± Lotion ± ± ± ± ± ± Averge ± SD ± Crem ± ± ± ± ± ± Averge ± SD ±

5 Prk et l. Int J Phrm Phrm Sci, Vol 3, Suppl 5, Tble 6: Precision dt in skin, lotion nd crem formultions Type of % of imed concentrtion Prepred Intr-dy Intr-dy formultion concentrtion Mesured R.S.D. (%) Mesured R.S.D. (%) b Skin Lotion Crem (n=6); b (n=12) Detection nd quntifiction limit LOD nd LOQ vlues were estimted by direct clcultion using the designted signl to noise rtio. As shown in Tble 6, the LOD ws found to pproximtely 10 μg ml -1 nd the LOQ ws bout 30 μg ml - 1. This inferior sensitivity resulted from the intrinsic low bsorbnce of diosgenin, which mde its spectrometric quntifiction difficult t levels below the LOD, necessitting nother pproch. Appliction of method for skin, lotion nd crem formultions Bsed on the ICH stbility testing guideline 21, long term experiment ws conducted t room temperture nd n ccelerted experiment ws conducted t 50 C using the proposed method. Therml nd light influences on diosgenin stbility were evluted in ech formultion over six month period. Chromtogrphic nlysis during the designted intervl reveled tht there ws no decrese in diosgenin concentrtion when compred to the initil period for ll formultions (Fig 3). These results show tht diosgenin is stble substnce ginst het nd light with no production of degrdtion compounds. In ddition, no effects of formultionl differences on diosgenin stbility were observed. Fig. 3: Appliction of the proposed method for evlution of long term stbility (25 C) in skin (A), lotion (B) nd crem (C) nd ccelerted stbility (50 C) in (D) skin, (E) lotion nd (F) crem with/without light (Men ± S.D., n=3) CONCLUSION Confirmtion of the stbility of the min ctive substnce in finished products is essentil in the re of cosmeceuticls nd phrmceuticls to ensure nd suggest the period of effectiveness, expirtion dte nd sfety to consumers. In this study, simple, rpid, ccurte nd precise stbilityindicting HPLC method ws developed nd vlidted for the nlysis of diosgenin in skin, lotion nd crem types of cosmetic formultions s model preprtions. During the smple preprtion step, diosgenin ws extrcted from ll formultions without loss nd with full recoveries, indicting suitble nlysis. This method cn be prcticlly pplied to other types of cosmeceuticl formultions, s well s phrmceuticl preprtions for other stbility studies nd routine qulity control pplictions s the need for diosgenin nlysis increses. 470

6 Prk et l. Int J Phrm Phrm Sci, Vol 3, Suppl 5, ACKNOWLEDGMENT This reserch ws supported by grnt from the Koren Ministry of Knowledge nd Economy ( ). REFERENCES 1. Drpeu D, Suvire Y, Blnch HW, Wilke CR. Improvement of diosgenin yield from dioscore deltoide plnt cell cultures by use of non-trditionl hydrolysis method. Plnt Med 1986; 6: Yuqing Zhng, Linru Tng, Xun An, Erhong Fu, Chofn M. Modifiction of cellulose nd its ppliction to extrction of diosgenin from Dioscore zingiberensis C.H.Wright. Biochem Eng J 2009; 47: Ardhn, Ro AR, Kle RK. Diosgenin - growth stimultor of mmmry glnd of ovriectomized mouse. Ind J Exp Biol 1992; 30: Crl Djerssi. Steroid reserch t Syntex: the pill nd cortisone. Steroids 1992; 57: Kmisko T, Ogw H. Regultion of biliry cholesterol secretion is ssocited with bcg5 nd bcg8 expressions in the rts: effects of diosgenin nd ethinyl estrdiol. Heptol Res 2003; 26: In Suk Son, Ji Hyun Kim, Ho Yong Sohn, Kum Ho Son, Jong-sng Kim, Chong-suk Kwon. Antioxidnt nd Hypolipidemic effects of diosgenin, steroid sponin of ym (Dioscore spp.), on high-cholestrol fed rts. Biosci Biotech Bioch 2007; 71: Sndr Molic, Bertrnd Ligre, Cecile Corbiere, Arnud Binchi, Michel Duc, Krm Bordji,Jen L, Beneytout. A plnt steroid, diosgenin, induces poptosis, cell cycle rrest nd COX ctivity in osteosrcom cells. FEBS Lett 2001; 506: Feng Li, Prsn Priscill Fernndez, Permiyn Rjendrn, Km M. Hui, Gutm Sethi. Diosgenin, steroidl sponin, inhibits STAT3 signling pthwy leding to suppression of prolifertion nd chemosensitiztion of humn heptocellulr crcinom cells. Cncer Lett 2010; 292: Yyoi Td, Noko Knd, Akinori Hrtke, Megumi Tobiishi, Hideyo Uchiw, Shinichi Wtnbe. Novel effects of diosgenin on skin ging. Steroids 2009; 74: Lee J, Jung K, Kim YS, Prk D. Diosgenin inhibits melnogenesis through the ctivtion of Phosphtidylinositol-3-Kinse Pthwy (PI3K) signling. Life Sci 2007; 81: VB Kshrisgr, UA Deokte, VB Bhrkd, SS Kkdbdi. HPTLC method development nd vlidtion for the simultneous estimtion of diosgenin nd levodop in mrketed formultion. Asin J Res Chem 2008; 1: Wei J, Dong X. Determintion of diosgenin in Rhizom Pridis by high performnce liquid chromtogrphy. Se Pu 1999; 17: Debjni De, Brtti De. Elicittion of diosgenin production in dioscore floribund by ethylene-generting gent. Fitoterpi 2005; 76: Jime Nino, Diego A, Jimenez, Oscr M. Mosquer, Yned M. Corre. Diosgenin quntifiction by HPLC in dioscore polygonoides tuber collection from colombin flor. J Brz Chem Soc 2007; 18: Lin Xu.; Yueto Liu, Tie Wng, Yn Qi, Xu Hn, Youwei Xu, Jinyong Peng, Xinqing Tng.Development nd vlidtion of sensitive nd rpid non-queous LC ESI-MS/MS method for mesurement of diosgenin in the plsm of norml nd hyperlipidemic rts: A comprtive study. J Chr B 2009; 877: Li J, Yng D, Yu K, He J, Zhng Y. Determintion of diosgenin content in medicinl plnts with enzyme-linked immunosorbent ssy. Plnt Medic 2010; 76: Kpil S. Khndgle, Sntosh V. Gndhi, Pdmnbh B. Deshpnde, Nilesh V.Gikwd. A simple nd sensitive RP-HPLC method for simultneous estimtion of cefixime nd ofloxcin in combined tblet dosge form. Int J Phrm Phrm Sci 2011; 3: Sujn K, Gowri Snkr D, Bl Souri O, Swthi Rni G. Stbility indicting RP-HPLC method for the determintion of telmisrtn in pure nd phrmceuticl formultion.. Int J Phrm Phrm Sci 2011; 3: ICH, Vlidtion of Anlyticl Procedures Methodology Q2B, Interntionl Conference on Hrmoniztion, IFPMA, Genev; United Sttes Phrmcopoei, 31st ed. The United Sttes Phrmcopoei Convention, Rockville, USA; ICH, Stbility Testing of New Drug Substnces nd Products Q1A (R2), Interntionl Conference on Hrmoniztion, IFPMA, Genev; J.A. Admovics. Chromtogrphic Anlysis of Phrmceuticls, 2nd ed, Eston, Pennsylvni; IPC-USP 7th Annul Scientific Meeting, Revised USP System Suitbility Prmeters Hyderbd Interntionl Convention Center, Hyderbd, Indi;