Quantification of nirmatrelvir concentrations in rat plasma, cerebrospinal fluid, and peripheral blood mononuclear...

Highly sensitive LC-MS/MS methods were developed and validated to quantify nirmatrelvir (NMR). Although the plasma pharmacokinetics (PK) of NMR have been well characterized, its distribution into other biologically relevant compartments, such as cerebrospinal fluid (CSF), peripheral blood mononuclear cells (PBMCs), and tissues, remains poorly understood. Quantitative assessment of NMR across these matrices is essential for evaluating central nervous system and intracellular exposure. To address this gap, we developed and fully validated three independent LC-MS/MS assays for the quantification of NMR in rat plasma, CSF, and PBMC matrices following FDA bioanalytical method validation guidelines. Calibration ranges were 20-10,000 ng/mL for plasma, 1.00-250 ng/mL for CSF, and 0.100-5.00 ng/mL for PBMCs, using NMR-D9 as the internal standard. Matrix-specific extraction procedures were optimized to address physicochemical and protein-binding differences, including a methanolic ammonium hydroxide treatment to mitigate adsorption losses in CSF. Chromatographic separation was achieved on a C18 column with a 60:40:0.1 (v/v/v) acetonitrile:water:formic acid mobile phase, and detection was performed by mass spectrometry in positive multiple-reaction-monitoring mode. All assays demonstrated excellent linearity (r² > 0.99), precision and accuracy within acceptance criteria (< 15% CV and deviation, < 20% at the LLOQ), with no significant matrix interference or ion suppression. Stability testing confirmed consistent performance under short-term, freeze-thaw, and long-term conditions. These validated assays provide sensitive, selective, and reproducible quantification of NMR across multiple matrices and will facilitate preclinical pharmacokinetic studies aimed at understanding its distribution in sanctuary compartments relevant to SARS-CoV-2 infection.