Development and Validation of UHPLC-MS/MS Assay for Therapeutic Drug Monitoring of High-dose Methotrexate in Children with Acute Lymphoblastic Leukemia

Le-jing Lian,1 Bin Lin,2 Xiao Cui,1 Jie He,1 Zhe Wang,1 Xiao-dong Lin,1 Wei-jian Ye,1 Rui-jie Chen,1 Wei Sun1 1Department of Pharmacy, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, People’s Republic of China; 2Department of Clinical Pharmacy, Changxing People’s Hospital, Changxing 313100, People’s Republic of ChinaCorrespondence: Wei Sun; Rui-jie Chen Tel +8613968803162; +8613806890233Email 33sunwei@163.com; crj@wzhealth.comPurpose: 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 μL of acetonitrile to 100 μL 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 m/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/m2 to 41 childpatients.Keywords: high-dose methotrexate, UHPLC-MS/MS, therapeutic drug monitoring, children, acute lymphoblastic leukemia