Simultaneous determination of low levels of methotrexate and cyclophosphamide in human urine by micro liquid chromatography/electrospray ionization tandem mass spectrometry

The aim of this study was to develop and validate a novel solid-phase extraction (SPE) liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of two antineoplastic drugs, cyclophosphamide (CP) and methotrexate (MTX), in human urine using trophosphamide as internal standard. The method showed good precision and accuracy (mean RSD 2.8% and 0.9%; bias 2.7% and 2.4% for MTX and CP, respectively). The lower limits of detection obtained, 0.2 microg/L(urine) for MTX and 0.04 microg/L(urine) for CP, were lower than the best previously reported values. The use of a 96-well SPE plate for matrix purification ensures a high throughput (50 samples/day), allowing the routine biological monitoring of CP and MTX as measures of occupational exposure at very low levels.