.,. 1 - ~.Availableonline at '..~CHIMICA...;1,;~ACTA EI.SEVlER1\l1alytic.I Chimicil 1\.:IiI 5/~ (~()()() ~J7-~~~ ;.\\(\\(\\(.c:t~c:"ierO:I'm1t1.:;lteaca Stability-indicatinghigh-performance thin-layer chromatographic determination of levonorgestrel and ethinyloestradiolin bulk drug and in low-dosage oral contraceptives Ali Reza Fakhari.a, Afshin Rajabi Khorrami b, Mojtaba Shamsip,urc,* " DepartmentofChemistI)',ShahidBeheshtiUni,'ersity,Tehran.Iran bDepartmentofChemistI)'.Faculty Science,ofIslatnicA:ad Universit.v.Karaj Branch,Karaj. Iran c:DepartmentofChemistI)'.Ra:i Uni,'ersity,Kermanshah.Iran Received JanuilryII2006:received revisedinfonn 20 April 2006:accepted May Availableonline 16May 2006 Abstract A stability-indicating high-perfonnance thin-layer chromatography (HPTLC) method wasdeveloped and validated for simultaneousdetennina- tion of steroidalhormones levonorgestrel and ethinyloestradiol both in bulk drug and in low-dosage oral contraceptives.Optimization of conditions for the spectrodensitometricprocedure was reached by eluting HPTLC silica gel platesin a 10 cm x 10 cm horizontal chamber,The solvent system consisted of hexane-chlorofonn-methanol (1.0:3.0:0,25, v/v/v). This system was found to give compact, dense and typical peaksfor both lev- onorgestrel (Rr = 0.65 :I: 0.03) and ethinyloestradiol (Rr = 0,43:I: 0.02). Densitometric analysisof the drugs was carried out in the reflectancemode at 225 nm by using a computer controlled densitometric scanner.The calibration curves of levonorgestreland ethinyloestradiol were linear in the range of and ng per spot, respectively. The method was validatedfor precision,robustnessand recovery. As the proposedmethod can effectively separatethe drugs from their degradation products, it canbe employed as a stability-indicating 2006 Elsevier B. V. All rights reserved. Key".ords:Oralcontraceptive;Levonorgestrel;Ethinyloestradiol;High-perfonnancethin-layerchromatographydetermination 1. Introductionproblem. Thus, low-dose oral contraceptives require sensitive and accurate analytical methods for simultaneous determina- Levonorgestrel,(- )-13f3-ethyl-17f3-hydroxy-18,19-dinor-tion of the small amounts of the estrogen in the presence of l7a-pregn-4-en-20-yn-3-one,is a syntheticprogestogenlarge amounts of progestogen. Some analytical methods such as female sex hormone and ethinyloestradiol, 19-nor-17a-pregna-colorimetry [1-3], UV-spectrophotometry [4-6] and fluorimetry 1.3,5( I 0)-trien-20-yne-3, 17f3-diol, is a semi synthetic steroid[7,8] have been developed for quantitative determination of rela- with an estrogenic effect. Their structures are shown in Fig. I.tively small amounts of steroid hormones in oral contraceptives. Combination of It; onorgestrel and ethinyloestradiol is usedHowever, most of these methods are sustainable to some inter- in pregnancy prevention in humans. In commonly used low-ferences and are unable to determine the drugs in the presence dosage oral contraceplives ethinyloestradiol is present at a veryof their degradation products and related impurities. low dosage level ( mg per tablet) in combination withFluorimetry [7,8] does not allow simultaneous determination )e\'onorgestrcl. which is present al a le\'el of 5-30 times that ofof honnones and gas chromatographic methods always need a the cthinylocstradiol.derivatization step prior to separation [9.10], R.ldio.immunoas-.Th<; formulation of these steroids in tablets of low dosage,say methods do not have enough selectivity for the quantitative.i.e mg per tablet. presented a challenging analyticaldetermination of hormones [11.12]. Meanwhile. several tech- niques including reverse phase high-performance liquid chro- ;matography (RP-HPLC) [ , derivative spectrophotometry._' C I or l " " 5°0. ' ""' 503ll:i.illl.micellar~lt:ctrokincticchromat('grarh'j(~tEKC)(171, ~I,. orrc:"ponuII1Silutlor.IC..+".3X.+"-"...., /':'.//'lli/"dJ,.,,'..,.1,:"".;."., v..h,..,.:.';',.,.ir,'r,~11<1111,1l'hromatography-mass spectrometry lLC-MS) [I R--OI, \~I :ii"'III"lpurl..alld voltammetry L21) have been reported for the detemtination 11I.)O3.~670/S frontm"lIer f) ~OO6-,;ccEI,;cvier V, All risht~re~crvc:d.B..It,i: 10.IOI6/j,a.:".~()I)".O5.0~') -