Simultaneous determination of tenofovir alafenamide and its active metabolites tenofovir and tenofovir diphosphate in HBV-infected hepatocyte with a sensitive LC-MS/MS method

- A LC-MS/MS method was established for simultaneous determination of tenofovir prodrug tenofovir alafenamide (TAF) and its metabolites tenofovir (TFV) and TFV diphosphate (TFV-DP).
- Samples were collected by direct lyzing with 50% methanol, followed by further protein precipitation using pure methanol.
- Intracellular pharmacokinetics of TFV-DP, TFV and TAF were investigated in HepG2.2.15 cells. Tenofovir was generated quickly along with rapid elimination of TAF, and immediately further phosphorylated into active form TFV-DP largely.

Tenofovir (TFV), a first-line anti-viral agent, has been prepared as various forms of prodrugs for better bioavailability, lower systemic exposure and higher target cells loading of TFV to enhance efficacy and reduce toxicity. TFV undergoes intracellular phosphorylation to form TFV diphosphate (TFV-DP) in target cell to inhibit viral DNA replication. Hence, TFV-DP is the key active metabolite that exhibits anti-virus activity, its intracellular exposure and half-life determine the final activity. Therefore, simultaneous monitoring prodrug, TFV and TFV-DP in target cells will comprehensively evaluate TFV prodrugs, both considering the stability of ester prodrug, and the intracellular exposure of TFV-DP. Thus we intended to develop a convenient general analytical method, taking tenofovir alafenamide (TAF) as a representative of TFV prodrugs. A sensitive LC-MS/MS method was developed, and TAF, TFV and TFV-DP were separated on a XSelect HSS T3 column (4.6 mm × 150 mm, 3.5 μm, Waters) with gradient elution after protein precipitation. The method provided good linearity for all the compounds (2-500 nM for TFV and TAF; 20-5000 nM for TFV-DP) with the correlation coefficients (r) greater than 0.999. Intra- and inter-day accuracies (in terms of relative error, RE < 10.4%) and precisions (in terms of coefficient of variation, CV < 14.1%) satisfied the standard of validation. The matrix effect, recovery and stability were also within acceptable criteria. Finally, we investigated the intracellular pharmacokinetics of TAF and its active metabolites in HepG2.2.15 cells with this method.

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