1. .,. 1 - ~.Availableonline at www.sci'enccdircct.com.'8CI8NC.dOlR8CT8ANALmCA '..~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 20069 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 200-800 and 40-160 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 method. @ 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 (0.03-1.0 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. 0.03-0.25 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) [ 13.141, derivative spectrophotometry._' C I or l. 98831 " " 5°0. '..98831""' 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~') -