Development and validation of a stability indicating method for seven novel derivatives of lamivudine with anti-HIV and anti-HBV activity in simulated gastric and intestinal fluids

Abstract

A simple micellar liquid chromatography (MLC) method has been developed and validated for use in stability indicating studies of lamivudine and its carbonate derivatives with proved activity against human immunodeficiency and hepatitis B viruses (HIV and HBV, respectively), in simulated gastric (SGF) and intestinal (SIF) fluids samples. The optimized method involves a C18 column thermostated at 30°C, UV detection at 272nm, a flow rate of 1.0mLmin−1 and a micellar mobile phase composed by 0.15M sodium dodecyl sulphate (SDS) – 4% (v/v) 1-butanol – 0.01M KH2PO4–Na2HPO4 (pH 7), using zidovudine (AZT) as internal standard. Validation under Food and Drug Administration (FDA) guideline of the analytical parameters include: linearity (r2>0.9996), LODs (1.6×10−7–6.9×10−6M) and LOQ (1×10−5M), intra (0.02–1.48%) and inter-day precision (0.04–1.66%) expressed as relative standard deviation (R.S.D.), and robustness parameters (less than 1.98%). Using this method, recoveries ranging from 92.9 to 119% were obtained for the eight substances. Thus, this method provides a simple, sensitive, accurate and precise assay for the determination of all compounds that can be readily adaptable to routine use by clinical laboratories with standard equipment. In addition, we evaluated the stability of carbonates of lamivudine in buffer pH 1.2 and 6.8; SGF (pH 1.2) and SIF one (pH 6.8), all as indicated in United States Pharmacopeia (USP) 32. Finally, this chromatographic method was applied to stability studies which resulted in all the compounds following a pseudo-first-order kinetics, and in the determination of its kinetic constant and half-life time.