Quantitation of tenofovir and emtricitabine in dried blood spots (DBS) with LC-MS/MS

Abstract

A reversed-phase high performance liquid chromatographic (LC), tandem mass spectrometry (MS/MS) assay for the determination of tenofovir (TFV) and emtricitabine (FTC) in dried blood spots (DBS) from human whole blood was developed and validated. Whole blood samples were spotted, dried, and a 3mm punch was extracted with methanol for analysis by LC-MS/MS utilizing stable isotope labeled internal standards. The assay was validated over the range of 2.5-1000ng/mL for TFV and 2.5-5000ng/mL for FTC. The method was accurate (within ±15% of control) and precise (coefficient of variation ≤15%) for hematocrit concentrations ranging from 25% to 76%; using edge punches vs. center punches; and spot volumes of 10-50μL. Analytes were stable for five freeze/thaw cycles and up to 6 days at room temperature, whereas long-term storage required -20°C or -80°C. Comparison of TFV and FTC in DBS vs. plasma yielded r(2)≥0.96, indicating that DBS can be used as a plasma alternative for pharmacokinetic analyses in vivo.

Keywords: Analytical method; Antiretroviral therapy; Dried blood spot; LC–MS/MS; Nucleoside analog.