Development and validation of a LC‐MS/MS assay to quantify intracellular tenofovir‐diphosphate (TFV‐DP) and emtricitabine‐triphosphate (FTC‐TP)

Abstract

Introduction: Combination antiretroviral therapy has been associated with dramatic reductions in morbidity and mortality in HIVinfected patients. The key to successful HIV treatment is strict adherence to the prescribed regimen. The combination of tenofovir, emtricitabine and efavirenz co-formulated in a single tablet (Atripla) provides a 'one tablet once a day' regimen, making adherence easier for patients. Tenofovir and emtricitabine are converted intracellularly to active phosphate anabolites. Therefore, knowledge of intracellular pharmacokinetics should aid understanding of the virological response and toxicity of these agents. This intracellular methodology has been developed and applied to clinical trial samples. Method: Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by gradient centrifugation using ficoll. PBMC were counted and lysed (70% MeOH) giving a final cell density of 2x106 cells/mul. Lysate was centrifuged, supernatant (intracellular matrix), was spiked with drug anabolites at various concentrations to produce standard curves and quality controls (QC). Samples were extracted by protein precipitation (acetonitrile), evaporated to dryness, internal standard (IS) added, and reconstituted in 5 mM ammonuim formate. Weak anion exchange chromatography with an optimised step-wise gradient, interfaced with a triple quadrupole mass spectrometer operated in SRM with positive ionisation mode was used for quantification. Results: Analytes eluted within 12 minutes run-time with adequate separation. Calibration curves were validated over the following range TFV-DP =0.35-10.91 ng/mL, FTC-TP=0.38-103.17 ng/mL (r2 Values>0.99; linear 1/x). The lower limit of quantification was<20%, 20%, signal to noise was>5% and carryover<0.1%. The precision (relative standard deviation,% RSD) of the assays as determined from analysis of quality control samples was; TFV-DP=6.3-11% and FTCTP =6-18.6%, and accuracy was TFV-DP=97.5-100.8%, FTCTP =98-100.3%. Conclusions: A direct, highly sensitive intracellular anabolite assay has been developed and validated which elutes analytes and IS rapidly and has been applied to patient samples from a pivotal trial the results of which are crucial in our understanding of antiretroviral drug 'forgiveness'.