Indian Journal of Science • Analysis

A selective, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method has been developed and validated for the chromatographic separation and quantitation of Methotrexate in human plasma. Sample clean-up involved solid phase extraction (SPE) of the drug and their respective labelled internal standards from 200µL human plasma. Analyte was chromatographically separated on a ACE CN (150mm × 4.6mm, 5µm particle size) analytical column using 10mM ammonium formate, pH 7.0±0.1 and methanol (60:40, v/v) as the mobile phase. The parent → product ion transitions for both the drug (m/z 455.00 → 308.00) and metabolite (m/z 458.00 → 311.00) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The method was validated over the concentration range of 1.00–250ng/mL. Matrix factor was calculated by comparing the mean area of analyte or internal standard obtained from blank extracts spiked after extraction and from neat solutions. ISTD Normalized matrix factor for drug was found to 0.92. % over all recovery for drug was found 83.69%. The method was successfull