A validated LC-MS/MS assay for simultaneous quantification of methotrexate and tofacitinib in rat plasma

application to a pharmacokinetic study

Kuldeep Sharma, Kalpeshkumar Giri, Vinay Dhiman, Abhishek Dixit, Mohd Zainuddin, Ramesh Mullangi*

*Corresponding author for this work

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

A highly sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for simultaneous quantification of methotrexate (MTX) and tofacitinib (TFB) in rat plasma (50 μL) using phenacetin as an internal standard (IS), as per the US Food and Drug Administration guidelines. After a solid-phase extraction procedure, the separation of the analytes and IS was performed on a Chromolith RP18e column using an isocratic mobile phase of 5 m m ammonium acetate (pH 5.0) and acetonitrile at a ratio of 25:75 (v/v) using flow-gradient with a total run time of 3.5 min. The detection was performed in multiple reaction monitoring mode, using the transitions of m/z 455.2 → 308.3, m/z 313.2 → 149.2 and m/z 180.3 → 110.2 for MTX, TFB and IS, respectively. The calibration curves were linear over the range of 0.49-91.0 and 0.40-74.4 ng/mL for MTX and TFB, respectively. The intra- and interday accuracy and precision values for MTX and TFB were <15% at low quality control (QC), medium QC and high QC and <20% at lower limit of quantification. The validated assay was applied to derive the pharmacokinetic parameters for MTX and TFB post-dosing of MTX and TFB orally and intravenously to rats.

Original languageEnglish
Pages (from-to)722-732
Number of pages11
JournalBiomedical Chromatography
Volume29
Issue number5
DOIs
Publication statusPublished - May 2015
Externally publishedYes

Keywords

  • LC-MS/MS
  • Method validation
  • Methotrexate
  • Pharmacokinetics
  • Rat plasma
  • Tofacitinib

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