Drug Design, Development and Therapy (Nov 2021)

A Validated HPLC-MS/MS Method for Quantification of Methotrexate and Application for Therapeutic Drug Monitoring in Children and Adults with Non-Hodgkin Lymphoma

  • Feng Z,
  • Gao J,
  • Gao X,
  • Hua L,
  • Nie X,
  • Sun Y,
  • Wang M

Journal volume & issue
Vol. Volume 15
pp. 4575 – 4583


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Zhangying Feng, Jinglin Gao, Xiaonan Gao, Li Hua, Xuyang Nie, Yaqi Sun, Mingxia Wang Department of Clinical Pharmacology, The Fourth Hospital of Hebei Medical University, Shijiazhuang, People’s Republic of ChinaCorrespondence: Mingxia WangDepartment of Clinical Pharmacology, The Fourth Hospital of Hebei Medical University, 12 Jiankang Road, Shijiazhuang, 050011, People’s Republic of ChinaTel +86311-66696233Email [email protected]: A simple, rapid and reliable method to quantify methotrexate (MTX) in human plasma by high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) was established and validated in two laboratories.Patients and Methods: Sample separation was achieved on a Synergi Hydro-RP column (50 mm× 2.0 mm, 2.5 μm) with a gradient elution program in 3.5 min after a simple protein precipitation with methanol (MeOH) and acetonitrile (ACN) (1:1). About 5 mM ammonium formate aqueous solution with 0.2% formic acid and ACN were used as mobile phase with a flow rate of 0.5 mL/min at 40 °C. Mass spectrometry detection using AB Sciex Triple Quad 4500 mass spectrometer (4500 QQQ) and Qtrap 5500 mass spectrometer (5500 Q-trap) were both characterized by electrospray ionization (ESI) for positive ions in multiple reaction-monitoring (MRM) mode. Quantitative ion pairs were m/z 455.1→m/z 308.0 for MTX and m/z 248.1→m/z 121.0 for tinidazole (TNZ) used as internal standard (IS).Results: Linear calibration curves were generated over the range of 5– 1000 ng/mL (r2> 0.99) on both the 4500 QQQ and 5500 Q-trap, both of the intra- and inter-batch precision were less than 7.67% and accuracy ranged from 96.33% to 108.94%. The recovery and matrix effect were 82.20– 93.98% and 102.69– 105.28%, respectively.Conclusion: An analytical method transfer was achieved by re-verification in two laboratories to ensure stability and reproducibility and this method has been applied for therapeutic drug monitoring (TDM) successfully in children and adults with NHL, and during routine TDM, two delayed elimination of MTX cases were observed and analyzed.Keywords: methotrexate, therapeutic drug monitoring, non-Hodgkin lymphoma, children, adult