Determination of zidovudine triphosphate intracellular concentrations in peripheral blood mononuclear cells from human immunodeficiency virus-infected individuals by tandem mass spectrometry

Nucleoside reverse transcriptase inhibitors (NRTIs) used against the human immunodeficiency virus (HIV) need to be activated intracellularly to their triphosphate moiety to inhibit HIV replication. Intracellular concentration s of these NRTI triphosphates, especially zidovudine triphosphate (ZDV-TP) are relatively low (low numbers of femtomoles per 10(6) cells) in HIV-infec ted patient peripheral blood mononuclear cells. Recently, several methods h ave used either high-performance liquid chromatography (HPLC) or solid-phas e extraction (SPE) coupled with radioimmunoassay to obtain in vivo measurem ents of ZDV-TP. The limit of detection (LOD) by these methods ranged from 2 0 to 200 fmol/10(6) cells. In this report, we describe the development of a method to determine intracellular ZDV-TP concentrations in HN-infected pat ients using SPE and HPLC with tandem mass spectrometry for analysis. The LO D by this method Is 4.0 fmol/10(6) cells with a linear concentration range of at least 4 orders of magnitude from 4.0 to 10,000 fmol/10(6) cells. In h ispanic HIV-infected patients, ZDV-TP was detectable even when the sampling time after drug administration was 15 h, Intracellular ZDV-TP concentratio ns in these patients ranged from 41 to 193 fmol/10(6) cells, The low LOD ob tained with this method will provide the opportunity for further in vivo ph armacokinetic studies of intracellular ZDV-TP in different HN-infected popu lations. Furthermore, this methodology could be used to perform simultaneou s detection of two or more NRTIs, such as ZDV-TP and lamivudine triphosphat e.