Toward Highly Sensitive and Reproducible LC–MS/MS Analysis of MK-8591 Phosphorylated Anabolites in Human Peripheral Blood Mononuclear Cells

Aim: MK-8591 (EFdA), a novel anti-HIV nucleoside analog, is converted to mono-, di- and tri-phosphates (MK-8591-MP, MK-8591-DP and MK-8591-TP) intracellularly, among which MK-8591-TP is the active pharmacological form. An ultrasensitive LC–MS/MS assay was required to measure MK-8591-DP and MK-8591-TP levels in human peripheral blood mononuclear cells (PBMCs). Sensitivity and reproducibility were major bottlenecks in these analyses. Materials and methods: Human PBMCs were isolated from blood and lysed with 70/30 methanol/RPMI-1640. An LC–MS/MS method was developed to simultaneously quantify MK-8591-DP and MK-8581-TP in PBMC lysates. Results: Low flow LC and dimethyl sulfoxide mediated signal enhancement enabled an extreme sensitivity with limit of quantitation at 0.1 ng/ml. Assay accuracy was 92.5–106% and precision was 0.7–12.1% for a linear curve range of 0.1–40 ng/ml. Matrix variability and interference liability were comprehensively evaluated. Conclusion: Our study findings and steps taken in addressing clinical sample issues help understand and overcome the challenges facing intracellular nucleotide analog analysis.