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A simple, rapid and reliable liquid chromatography–mass spectrometry method for determination of methotrexate in human plasma and its application to therapeutic drug monitoring
Author(s) -
Wu Dan,
Wang Yixuan,
Sun Yan,
Ouyang Nian,
Qian Jun
Publication year - 2015
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.3408
Subject(s) - chemistry , chromatography , protein precipitation , mass spectrometry , calibration curve , therapeutic drug monitoring , detection limit , selected reaction monitoring , liquid chromatography–mass spectrometry , electrospray ionization , tandem mass spectrometry , acetonitrile , high performance liquid chromatography , analytical chemistry (journal) , pharmacokinetics , medicine
A simple, rapid and reliable liquid chromatography–electrospray ionization tandem mass spectrometry method was established and validated for the determination of methotrexate in human plasma. After a straightforward protein precipitation by acetonitrile–water (70:30, v/v), methotrexate (MTX) and p ‐aminoacetophenone (used as internal standard, IS) were separated on a Column C 18 column (50 × 2.1 mm, 3 µm; Column Technology, Fremont, CA, USA) using a gradient elution with mobile phase of acetonitrile and 0.03% acetic acid aqueous solution at a flow rate of 0.5 mL/min. The total chromatographic runtime was 5 min for each injection. Quantification detection was performed in a triple‐quadruple tandem mass spectrometer under positive mode monitoring the following mass transitions: m / z 455.3 → 308.3 for MTX and m / z 136.1 → 94.4 for IS. The calibration curve was linear over the range of 0.05–25.0 µmol/L with a lower limit of quantification of 0.05 µmol/L. The intra‐ and interday precisions were <5.2%, the accuracy varied from −4.1 to 4.5%. The recovery was >94%. The LC‐MS/MS method showed an excellent agreement with the existing HPLC‐UV method using Passing–Bablok regression and Bland–Altman difference plot analysis. The validated LC‐MS/MS can be successfully applied to the routine therapeutic drug monitoring of MTX in clinical laboratories. Copyright © 2015 John Wiley & Sons, Ltd.