Unique methotrexate polyglutamates distributions in peripheral blood mononuclear cells of rheumatoid arthritis patients: Development and validation of a UPLC-MS/MS method

Methotrexate is pivotal in treating immune-mediated inflammatory diseases. Intracellularly, methotrexate is metabolized to methotrexate-polyglutamates (MTX-PG_1-7), comprising up to six additional glutamate moieties, crucial for cellular retention and therapeutic efficacy. Hitherto, quantification of MTX-PG_1-6 in peripheral blood mononuclear cells (PBMCs) from methotrexate-treated patients was challenging due to their low abundance in blood and matrix effects. We present a robust validated UPLC-MS/MS method to quantify individual MTX-PG_1-6 in PBMCs. Stable-isotope labelled internal standard mixture of MTX-PG_1-6 was added to 5 million PBMCs, followed by deproteinization with perchloric acid, and additional sample clean-up using solid phase extraction columns. MTX-PG_1-6 were detected and quantified using UPLC-MS/MS. The method was validated for lower limit of quantification (LLOQ), linearity, carryover, recovery, matrix effects, precision and stability. We assessed MTX-PG_1-6 in PBMCs derived from five methotrexate-treated rheumatoid arthritis patients. For all MTX-PG_1-6, LLOQs were < 1 fmol-MTX-PG_1-6/million cells with linearities R² > 0.995. The recoveries, carryover and stability were acceptable and no matrix effects were observed. The intraday and interday precision %CVs of quality controls ranged from 2.7 % to 11.4 % and 3.5-14.9 % respectively. Interday precision using nine PBMCs aliquots from a single MTX-treated patient aligned similarly (%CV <15 %). In patient-derived PBMC samples, MTX-PG₁ was the highest, with decreasing concentrations of MTX-PG₂ to MTX-PG₅. No signal for MTX-PG₆ was detected in the patient samples. We validated a new UPLC-MS/MS method to quantify MTX-PG_1-6 in PBMCs, thus facilitating PBMC-based therapeutic drug monitoring studies and understand associations between MTX-PG_1-6 concentration and therapy efficacy or adherence.