An LC-MS/MS assay for quantification of plasma biomarkers to assess transporter-mediated drug-drug interactions and biomarker baselines in healthy volunteers.

Endogenous molecules are proposed as biomarkers to assess drug transporter function, with changes in their concentrations reflecting altered transporter activity. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed, comprising a hydrophilic interaction liquid chromatography (HILIC) method and a reversed-phase liquid chromatography (RPLC) method both with MS/MS, using a single supernatant to quantify plasma biomarkers of clinically relevant drug transporters: coproporphyrin I (CPI), 4-pyridoxic acid (PDA), N1-methylnicotinamide (NMN), and 1-methyladenosine (m1A). Optimization resulted in a 5-min HILIC method for PDA, NMN, and m1A using a pH 9 mobile phase containing 10 mM ammonium acetate and 0.04% ammonium hydroxide to achieve acceptable peak shapes and retention, with 5% methanol added to the organic phase to maintain salt solubility. For CPI and CPIII, a 3.5-min RPLC method at pH 2.7 enabled baseline separation of the two isomers with a low gradient steepness of 0.032 at 60 °C. Protein precipitation provided lower limits of quantification of 0.18-4.00 ng/mL across the different biomarkers, while parallelism experiments confirmed 3 × charcoal-stripped K2EDTA human plasma as a suitable surrogate matrix. Fit-for-purpose validation demonstrated intra- and inter-assay accuracy and precision and confirmed assay selectivity. The final method was applied to plasma samples from healthy volunteers, resulting in a geomean of the baseline levels of 0.52 ng/mL for CPI, 5.5 ng/mL for PDA, 14.7 ng/mL for NMN, and 28.9 ng/mL for m1A, consistent with previously published data. The variation in the baseline levels was mainly described by sex for CPI and by the sex-age interaction for PDA.