TY - JOUR
T1 - Unique methotrexate polyglutamates distributions in peripheral blood mononuclear cells of rheumatoid arthritis patients
T2 - Development and validation of a UPLC-MS/MS method
AU - Sundaresan, Janani
AU - Lin, Marry
AU - Jansen, Gerrit
AU - Hebing, Renske C F
AU - Bulatović-Ćalasan, Maja
AU - de Jonge, Robert
AU - Struys, Eduard A
AU - de Rotte, Maurits C F J
N1 - Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.
PY - 2025/4/8
Y1 - 2025/4/8
N2 - Methotrexate is pivotal in treating immune-mediated inflammatory diseases. Intracellularly, methotrexate is metabolized to methotrexate-polyglutamates (MTX-PG1-7), comprising up to six additional glutamate moieties, crucial for cellular retention and therapeutic efficacy. Hitherto, quantification of MTX-PG1-6 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-PG1-6 in PBMCs. Stable-isotope labelled internal standard mixture of MTX-PG1-6 was added to 5 million PBMCs, followed by deproteinization with perchloric acid, and additional sample clean-up using solid phase extraction columns. MTX-PG1-6 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-PG1-6 in PBMCs derived from five methotrexate-treated rheumatoid arthritis patients. For all MTX-PG1-6, LLOQs were < 1 fmol-MTX-PG1-6/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-PG1-6 in PBMCs, thus facilitating PBMC-based therapeutic drug monitoring studies and understand associations between MTX-PG1-6 concentration and therapy efficacy or adherence.
AB - Methotrexate is pivotal in treating immune-mediated inflammatory diseases. Intracellularly, methotrexate is metabolized to methotrexate-polyglutamates (MTX-PG1-7), comprising up to six additional glutamate moieties, crucial for cellular retention and therapeutic efficacy. Hitherto, quantification of MTX-PG1-6 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-PG1-6 in PBMCs. Stable-isotope labelled internal standard mixture of MTX-PG1-6 was added to 5 million PBMCs, followed by deproteinization with perchloric acid, and additional sample clean-up using solid phase extraction columns. MTX-PG1-6 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-PG1-6 in PBMCs derived from five methotrexate-treated rheumatoid arthritis patients. For all MTX-PG1-6, LLOQs were < 1 fmol-MTX-PG1-6/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-PG1-6 in PBMCs, thus facilitating PBMC-based therapeutic drug monitoring studies and understand associations between MTX-PG1-6 concentration and therapy efficacy or adherence.
U2 - 10.1016/j.jpba.2025.116882
DO - 10.1016/j.jpba.2025.116882
M3 - Article
C2 - 40222304
SN - 0731-7085
VL - 262
JO - Journal of Pharmaceutical and Biomedical Analysis
JF - Journal of Pharmaceutical and Biomedical Analysis
M1 - 116882
ER -