Application of an intracellular LC-MS/MS assay for the determination of carbovir-triphosphate and remdesivir-triphosphate in peripheral blood mononuclear cells and dried blood spots.

Intracellular concentrations of antiviral nucleos(t)ide analogs are associated with a variety of clinical outcomes and toxicities, underscoring the need for novel quantification methods for commonly used antivirals in cellular matrices. Here, we extend a previously validated intracellular LC-MS/MS method to carbovir-triphosphate (CBV-TP) and remdesivir-TP (RDV-TP), the active anabolites of abacavir (ABC) and remdesivir (RDV), respectively, in peripheral blood mononuclear cells (PBMC) and dried blood spots (DBS). CBV-TP and RDV-TP concentrations in DBS were quantified using two 7 mm punches of spotted, dried whole blood extracted in 50:50 methanol:water solution (lysed intracellular matrix). Lysed PBMC samples were prepared in a 70:30 methanol:water solution. Phosphorylated intracellular analyte fractions were isolated from the lysed matrix using strong anion‑exchange solid‑phase extraction (SPE), enzymatically dephosphorylated to the parent drug, then desalted/concentrated with reversed phase SPE and quantified utilizing stable isotope labeled internal standards by LC‑MS/MS methods adapted from prior validated assays. Accuracy was within ±15% difference from nominal and precision was ≤ 15% coefficient of variation for all validation assessments for both CBV-TP and RDV-TP. The validated quantifiable range for CBV-TP was 5-2000 fmol/sample and 0.1-100 pmol/sample for RDV-TP. The methods were successfully applied to clinical research PBMC and DBS samples with reproducible results. Both analytes demonstrated accuracy and precision across tested concentration ranges and matrices, meeting regulatory criteria outlined in current bioanalytical guidance. These methods support novel intracellular data for CBV-TP and RDV-TP in PBMC and DBS, which will facilitate wider use in antiviral pharmacology research.