A Rapid Method for Determination of Serum Methotrexate Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry and Its Application in Therapeutic Drug Monitoring.

 Methotrexate (MTX) has anticancer therapeutic potential with multiple doses-related adverse effects and toxicities. Immunoassays for therapeutic monitoring of serum MTX have their own limitations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered as the reference method; however, commercially availability of them is limited. We aimed to adapt/develop an in-house LC-MS/MS method for therapeutic monitoring of serum MTX.  Serum protein precipitation was performed using acetonitrile-water containing 250 μM solution of aminoacetophenone as internal standard (IS). Chromatographic separation was achieved on a C18 column with mobile phase of 0.1% solution of formic acid (solvent A) and acetonitrile (solvent B) at a flow rate of 0.4 mL/min. MS was performed under positive ion mode with mass transition for MTX and IS as m/z 455.1→308.1 and 136.2→94.1, respectively. The method was validated by following Bioanalytical Method Validation Guidance for Industry, 2018 and applied on leukemia patients' samples on MTX therapy.  The correlation coefficient of eight serially diluted calibration standards of 0.09 to 12.5 μM was >0.99 and had linearity with > 95% precision and accuracy at analytical quality control levels. The lower limit of MTX quantification achieved was 0.09 μM with good intensity and sharp peak as compared with blank sample. The total run time of the assay was 5 minutes. The serum MTX levels obtained by this method in leukemia patients exhibited clinical correlation and an excellent agreement with commercial immunoassay used in parallel.  We were able to develop a rapid, sensitive, and cost-effective LC-MS/MS method suitable for therapeutic drug monitoring of MTX in routine clinical diagnostic laboratories.