Sample preparation via solid phase extraction in the 96-well format for HPLC/UV detection-based biofluid assays. Application to the determination of a novel cyclooxygenase II inhibitor in human plasma and urine

Methods using solid phase extraction in the 96-well format, together with HPLC and UV detection, are described for the determination of low ng/mL concentrations of 2-(3,5-difluorophenyl)-3-(4-methanesulfonylphenyl)-cyclo-pent-2-enone, a cyclooxygenase II inhibitor, in human plasma and urine. The plasma assay utilizes a packed-bed SPE plate, while the urine assay employs a 96 well plate containing membrane-based extraction disks. Both matrices are diluted with 17% acetonitrile in water prior to application to the extraction plates. Following sample application, the plates are washed with acetonitrile/water mixtures to remove endogenous components prior to elution of the analytes with 100% acetonitrile. The urine extracts are diluted with water and injected directly into the HPLC system. The plasma extracts, however, require evaporation and reconstitution prior to injection. An HPLC/column switching system is employed to separate the analyte and an internal standard from co-extracted endogenous species. The compounds are detected based on their UV absorbance at 288 mn. The 96-well SPE assays were validated over concentration ranges of 5 to 500 ng/mL for plasma (1 mL sample) and 10 to 1000 ng/mL for urine (0.5 mL sample). Replicate analyses (n=5) of spiked plasma and urine standards yielded linear responses with coefficients of variation below 10% and accuracy within 6% of nominal concentrations.