Abstract：OBJECTIVE To establish an HPLC-MS/MS method for determining erythrocyte methotrexate polyglutamates (MTXPGs) in patients with rheumatoid arthritis (RA).METHODS The indirect method was applied to determine the concentrations of free methotrexate (MTX) and total MTX (MTX+MTXPGs) respectively by using isotope MTX-d3 as the internal standard, and then concentration of erythrocyte MTXPGs was calculated. To determine free MTX, whole blood (after freeze-thaw cycles to break blood cells) was precipitated with methanol, and then analyzed by LC-MS/MS. Multiple reactions monitoring (MRM) was performed with electrospray ionization (ESI) at a positive ion mode.To determine total MTX, whole blood (after freeze-thaw cycles) was incubated with 200 mmol·L-1 ascorbic acid at 37 ℃ for 2.5 h to enzymatically convert the MTXPGs to MTX,and the protein was precipitated for sample analysis.RESULTS The calibration curve of MTX was linear over the range of 1-300 ng·mL-1 (r=0.999 4) and the limit of detection (LOD) was 0.5 ng·mL-1. The accuracy (RE) was within ±5% and the intra-and inter-day precisions (RSD) were less than 7.96% and 10.88%, respectively.CONCLUSION This method is rapid, sensitive, highly specific and reproducible, which is successfully applied for the erythrocyte MTXPGs monitoring in patients with RA.
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