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

Abstract

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 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

Acknowledgments

We thank Dr Jiuxiang Wang and Dr Yuan Li (the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University) for their professional answers and insightful suggestions. The study was supported by the National Natural Science Foundation of China (Grant Nos. 81600216 and 81900331), by Science and Technology Research Project of Wenzhou City (Grant Nos. Y20160534 and 2020Y0390) and by the Special Project for Significant New Drug Research and Development in the Major National Science and Technology Projects of China (2020ZX09201002).

Ethics Statement

The studies involving human participants were reviewed and approved by the Ethics Committee of The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University (Wenzhou 325027, China). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

Disclosure

The authors report no conflicts of interest in this work.