Development And Validation Of Uhplc-Ms/Ms Assay For Therapeutic Drug Monitoring Of High-Dose Methotrexate In Children With Acute Lymphoblastic Leukemia

Purpose: Precise and timely detection of methotrexate (MTX) concentration played a key role in high-dose MTX individualization therapy in acute lymphoblastic leukemia (ALL) children to avoid serious adverse effects or nonresponse. This report described a sensibility and validation of ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for therapeutic drug monitoring (TDM) of methotrexate concentration in children's plasma.Methods: One-step protein precipitation of samples was accomplished by adding 200 mu L of acetonitrile to 100 mu L of plasma sample. The separation of plasma samples was carried out on a ZORBAX Eclipse Plus C18 Rapid Resolution HD column with gradient elution using a mobile phase constituted of acetonitrile and 1% formic acid. The detection was executed by electrospray ionization (ESI) of triple quadrupole tandem mass spectrometer (TQMS) in the multiple reaction monitoring (MRM) mode with the transitions m/z 455.2 -> 307.9 for methotrexate and rn/z 458.2 -> 311.2 for IS, separately. Linear concentration range of the calibration curve was 44-11,000 nmol/L and 44 nmol/L was the lower limit of quantification.Results: The methotrexate elution time was at 1.577 min, and the overall running time was only 3.3 min. The intra- and interday precision for all the analysis results was within 11.24%, and mean recoveries rate of methotrexate exceeded 87.98%.Conclusion: The described and fully validated UHPLC-MS/MS method was successfully applied in clinical TDM after infusion of high-dose methotrexate 1-5 g/m(2) to 41 childpatients.