Determination of methotrexate in human plasma by HPLC and qualitative analysis of its metabolite by mass spectrometry / 药学实践杂志

ABSTRACT

Objective To develop an SPE‐HPLC method for the determination of methotrexate (M TX) in human plasma to monitor the clinical drug use of M TX ,and to identify the human metabolite of M TX by mass spectrometry .Methods M TX was extracted from human plasma using C18 SPE column and analyzed directly after elution .The separation of MTX was per‐formed on Diamonsil C18 column (150 mm × 4 .6 mm ,5 m) with a gradient mobile phase of methanol and 0 .5% acetic acid solu‐tion (containing 0 .3% triethylamine) at a flow rate of 1 .0 ml/min .The detection wavelength was 306 nm .QLMS and MRM‐IDA‐EPI scan modes were used to obtain the mass spectrum of MTX metabolite .Results The calibration curve of MTX in plasma was linear over the range of 0 .05‐100 μmol/L(r=0 .999 9) .The extraction recovery was above 95% and accuracy was between 97% and 105% .Both intra‐and inter‐day precision were less than 5% .7‐OH MTX was identified to be the metabolite through the analysis of the mass spectrum .Conclusion The method is sensitive ,reproducible ,easy to operate and suits for monitoring the concentration of M TX in clinical practice .