Liquid−Liquid Extraction in the 96-Well Plate Format with SRM LC/MS Quantitative Determination of Methotrexate and Its Major Metabolite in Human Plasma

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Abstract
A method involving the semirobotic liquid−liquid extraction (LLE) in deep-well 96-well plates was developed for the quantitation of the anti-cancer/antiinflammatory drug methotrexate (MTX) and its major metabolite, 7-hydroxymethotrexate (7OH-MTX) in human plasma. The extraction time for the sample preparation was relatively short with four 96-well plates (384 samples) prepared in approximately 90 min by one person. The sample extracts were each analyzed within 1.2 min using a positive ion turbo-ionspray selected reaction monitoring liquid chromatography/mass spectrometric (SRM LC/MS) method in which 768 samples were easily analyzed within 22 h (maximum of 820 samples in 24 h). Deuterated internal standards, MTX-d3 and 7OH-MTX-d3, were used. The calibration curves for MTX and 7OH-MTX were linear (R2 > 0.997) and ranged from 0.5 to 250 and 0.75 to 100 ng/mL, respectively. The limit of quantitation (LOQ) for MTX and 7OH-MTX was 0.5 and 0.75 ng/mL, respectively; persistent carryover from the autosampler limited the LOQ achievable. The limit of detection (LOD) was 0.05 ng/mL for MTX and 0.1 ng/mL for 7OH-MTX. The intra- and inter-assay precision and accuracy did not exceed 15% for both MTX and 7OH-MTX. The recoveries were 61% for MTX and 47% for 7OH-MTX. The method was validated and demonstrated to be robust with high precision and accuracy.

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