A 384-well solid-phase extraction for LC/MS/MS determination of methotrexate and its 7-hydroxy metabolite in human urine and plasma

A solid-phase extraction procedure, in a 384-well format, has been develope d for methotrexate and its primary metabolite, 7-hydroxymethotrexate, in hu man urine and plasma. This format has not been utilized previously for soli d-phase extraction of drugs from biological fluids, The 384-well plates con tained a C-18 stationary phase bonded to silica particles which are incorpo rated into a glass-fiber membrane. Methotrexate and 7-hydroxymethotrexate h ave been quantified across the curve range of 1 to 50 mug/mL and 50 to 1000 ng/mL, respectively, in urine and from 5 to 250 ng/mL and 5 to 100 ng/mL, respectively, in plasma. Both analytes are quantified by linear regression using 20-muL sample aliquots. Experiments to evaluate the influence of part icle size, elution volume, and injection volume on signal intensity were co nducted and are reported, along with the results of experiments examining c ross contamination between wells. Recovery was determined to be greater tha n or equal to 95% from urine. Results from a run of 384 samples analyzed ov er a 14-h period indicate that 384-well SPE can be successfully utilized to increase analytical run sizes and sample throughput for LC/MS/MS determina tion of small drug molecules in biological samples.