An LC-MS/MS method for determination of tenofovir (TFV) in human plasma following tenofovir alafenamide (TAF) administration: Development, validation, cross-validation, and use of formic acid as plasma TFV stabilizer.

ABSTRACT

Tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF) are both tenofovir (TFV) prodrugs, with the same active intracellular metabolite, TFV-diphosphate (TFV-DP). TAF delivers TFV-DP to target cells more efficiently and at lower doses than TDF, thereby substantially reducing systemic exposure to TFV, which results in improved bone and renal safety relative to TDF. As such, the method developed for the determination of TFV following TAF administration involved two key differences from determination of TFV following TDF administration. First, human plasma samples (500 µL) immediately upon collection were treated with 20% formic acid (40 µL) (plasma formic acid ratio of 1008) to minimize hydrolysis of TAF to TFV, and thereby avoided overestimation of TFV concentrations. Second, various TFV validation tests were conducted in the presence of TAF to mimic the high TAFTFV ratios in clinical samples collected within ~2 h after dosing. The method for determination of TFV was developed and validated at a US lab and followed FDA and EMA guidelines. To support global clinical studies of TAF, the method was cross-validated (one-way) between the US lab and a China lab and was successfully used for TFV determination in plasma samples from a clinical study that involved healthy Chinese subjects.