Studies On The Quality Control Method Of Raw Materials And Tablets And Identification Of Lamivudine And Its Metabolites In Rat Urine

Lamivudine is a new nucleoside anti-HIV drug.In this paper,the method of quality control of lamivudine raw materials and tablets and metabolism of lamivudine in rat were studied.1.RP-HPLC methods have been developed for the determination of the content and related substances of lamivudine raw materials and tablets.Separation of lamivudine and its related substances was performed on a Diamonsil C₁₈column(200mm×4.6mm,5μm).The mobile phase consisted of methanol -0.025 moL.L^-1ammonium acetate(pH 3.94±0.1)(10:90, v/v).The flow rate was 1.0 mL.min^-1and the detection wavelength was 277nm.Lamivudine was completely separated from impurities.Excipients did not interfere with the determination of lamivudine and related substances in lamivudine tablets.The linear range of lamivudine was 5-50μg-mL^-1and 10.0～150.0μg·mL^-1.The lowest limit of detection was 0.2 ng·mL^-1.The recoveries of lamivudine were 100.8%(RSD=0.38%,n=3),100.0%(RSD=0.32%,n=3),101.2 %(RSD=0.30%,n=3)at three different concentration levels,respectively.2.RP-HPLC methods have been developed for the determination of(+)-lamivudine in lamivudine raw materials and tablets.Separation of lamivudine enantiomers was performed on a CYCLOBOND I 2000 RSP column(250mm×4.6mm,5μm).The mobile phase consisted of methanol-0.05 moL·L^-1ammonium acetate(pH3.9±0.1)(5:95,v/v).The flow rate was 0.8 mL·min^-1and the detection wavelength was 270nm.(-)-lamivudine was completely separated from(+)-lamivudine.3.Headspace GC method has been established for the determination of eight kinds of residual organic solvents in lamivudine raw materials.A GDX-102 column was used.The carry gas was nitrogen.The programming temperature of column,the initial temperature was kept at 100℃for 2min,then the temperature was raised to 200℃at rate of 5℃/min and was maintained for 27min,The detector was Fid detector.The calibration curves of ethanol,dichloromethane, isopropanol,ethyl acetate,tetrahydrofuran,isopropyl ether,n-heptane and triethylamine were linear(r＞0.9940)within the ranges of 124.0～992.0μg/mL,30.6～489.61μg/mL,128.2～1025.8μg/mL,129.61～1036.6μg/mL,18.65～149.2μg/mL,125.8～1006.4μtg/mL,85.5～684.0μg/mL and 125.9～1007.4μg/mL,respectively.The detection limits of the eight solvents were 24.8,9.2, 12.8,3.9,2.8,0.38,1.71 and 25.19μg/mL,respectively.The average recoveries of dichlormethane,isopropanol and tetrahydrofuran were 99.0%(RSD=3.7%,n=6),98.8% (RSD=4.2%,n=6)and 100.6%(RSD=3.8%,n=6),respectively.4.The identification of lamivudine and its major metabolites in rat urine was performed.0-24 h urine samples of rat were collected after ig 500 mg/kg dose to a rat lamivudine and treated by protein precipitation with acetonitrile.Lamivudine and the metabolite were detected in the rat urine. The structure of lamivudine and its metabolite in urine was identified by direct comparison of the observed mass spectrum and the chromatographic retention time with these of the reference substance,or by elucidation of the cleavage of characteristic ions.The metabolite was the trans-sulfoxide lamivudine.