Background:  NRTI are an important component of standard ARV regimens. All agents in this class must be activated by intracellular phosphorylation. Existing methods to measure intracellular NRTI phosphates are expensive, time consuming, and require large blood volumes. AMS is an extremely sensitive technique for measuring radio-labeled compounds in human cells, tissues, and body fluids.

Methods:  We used ¹⁴C-labeled zidovudine (ZDV) as a model drug to prove the feasibility of measuring ZDV-triphosphate (TP) in peripheral blood mononuclear cells (PBMC) in vivo using AMS. Six healthy volunteers received an oral dose of 300 mg ZDV combined with 100 μg (20 μCi) of ¹⁴C-labeled ZDV. Blood samples were collected from subjects before and 2 h and 6 h after administration of ZDV. ZDV-TP was extracted from PBMC and measured in split samples by both AMS and standard liquid chromatography/tandem mass spectrometry (LC/MS/MS).

Results:  Analysis of the AMS data indicated that ZDV was rapidly absorbed. ZDV-TP could be detected in PBMC lysates 2 h after administration of the ¹⁴C-labeled dose. The mean 2-h ZDV-TP concentration was 72.8 (range 22.5 to 159.4) fmol/10⁶ cells measured by AMS vs 70.3 (range 27.4 to 148.8) fmol/10⁶ cells measured by LC/MS/MS. The 6-h mean value of intracellular ZDV-TP was 40.5 (range 14.4 to 94.7) fmol/10⁶  cells by AMS vs 35.3 (range 20.3 to 71.5) fmol/10⁶ cells by LC/MS/MS. AMS correlated well with LC/MS analysis of the same samples (r = 0.97, p <0.001). The ratio of AMS to LC/MS/MS measurements was 1.03 (90%CI 0.92 to 1.17, p = 0.79). The estimated half-life of intracellular ZDV-TP in vivo was approximately 4 h by both AMS and LC/MS/MS. AMS was about 30,000-fold more sensitive than LC/MS/MS for measuring ZDV-TP.

Conclusions:  AMS analysis of the intracellular concentrations of ZDV-TP yielded excellent concordance with published data and with LC/MS/MS. Potential applications include in vivo synergy/antagonism studies, and rapid identification of active drug metabolites in early drug development. Given its greater sensitivity, AMS will require much smaller blood volumes than LC/MS/MS, and could allow more frequent sampling, measurement of intracellular TP in cell subsets such as CD4⁺ T cells, and studies in vulnerable populations including pediatric and anemic patients.