CC BY-NC-ND 4.0 · J Lab Physicians 2023; 15(03): 344-353
DOI: 10.1055/s-0042-1760668
Original Article

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

Naresh Kumar Tripathy
1   Department of Haematology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
Shravan Kumar Mishra
2   Department of Pathology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
1   Department of Haematology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
Shreya Srivastava
2   Department of Pathology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
Anshul Gupta
1   Department of Haematology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
Raghavendra Lingaiah
2   Department of Pathology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
› Institutsangaben


Objectives 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.

Materials and Methods 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.

Results 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.

Conclusion 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.


Artikel online veröffentlicht:
18. Januar 2023

© 2023. The Indian Association of Laboratory Physicians. This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (

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