Simultaneous determination of zidovudine and lamivudine in human serum using HPLC with tandem mass spectrometry

A method employing high performance liquid chromatography (HPLC) with tande m mass spectrometry (MS) has been developed and validated for the simultane ous determination of clinically relevant levels of zidovudine (AZT) and lam ivudine (3TC) in human serum. The method incorporates a Fully automated ult rafiltration sample preparation step that replaces the solid-phase extracti on step typically used for HPLC with UV detection. The calibration range of the dual-analyte LC-MS/MS method is 2.5-2500 and 2.5-5000 ng ml(-1) for AZ T and 3TC, respectively, using 0.25 ml of human serum. The lower limit of q uantification was 2.5 ng ml(-1) for each analyte, with a chromatographic ru n time of approximately 6 min. Overall accuracy, expressed as bias, and int er- and intra-assay precision are < +/- 7 and < 10% for AZT, and < +/- 5 an d < 12.1% for 3TC over the full concentration ranges. A cross-validation st udy demonstrated that the LC-MS/MS method afforded equivalent results to es tablished methods consisting of a radioimmuno-assay for AZT and an HPLC-UV method for 3TC. Moreover, the LC-MS/MS was more sensitive, allowed markedly higher-throughput, and required smaller sample volumes (for 3TC only). The validated method has been used to support post-marketing clinical studies for Combivir(TM) - a combination tablet containing AZT and 3TC. (C) 2000 El sevier Science B.V. All rights reserved.