Determination of Lamivudine, Zidovudine, and Nevirapine in capillary blood sampled on filter paper by LC

Abstract

A bioanalytical method for determination of lamivudine (3TC), zidovudine (AZT), and nevirapine (NVP) in 100 μL capillary blood applied onto sampling paper has been developed and validated. The antiretroviral drugs (ARV) were analyzed by reversed phase gradient liquid chromatography with UV detection. Separation was performed on a Zorbax SB C8 (250 X 4.6 mm) column with a two step gradient: (i) methanol–0.05 mol/L acetic acid-sodium acetate buffer (pH 3.95, 15:85 v/v) and (ii) methanol–0.05 mol/L acetic acid-sodium acetate buffer (pH 3.95, 50:50 v/v) with a flow rate of 1.0 mL/min. UV detection was performed at 260 nm. Total assay precisions were 6.3, 4.7, and 4.9% for 3TC at 0.34, 0.69, and 3.9 μg/mL, and 5.1, 5.5, and 3.2% for AZT at 0.40, 0.80, and 4.5 μg/mL. For NVP, total assay precisions were 5.2, 8.3, and 3.5% at 2.6, 4.5, and 8.8 μg/mL. Lower limit of quantifications (LLOQ) were 0.11 and 0.13 μg/mL for 3TC and AZT where the precisions were 2.0% for both the analytes. For NVP, LLOQ was 1.3 μg/mL where precision was 2.6%. Concentrations were determined for 10h for two subjects receiving standard twice daily antiretroviral therapy containing 3TC, AZT, and NVP. Maximum 3TC concentrations were 2.5 and 2.8 μg/mL for subject 1 and 2, respectively. For AZT, maximum concentrations were 1.8 and 1.1 μg/mL while being 15 and 9.6 μg/mL for NVP. Pre-dose trough concentration of NVP was 11 μg/mL for subject 1 and 9.6 μg/mL for subject 2.