High-performance liquid chromatographic method for the determination of indomethacin and its two primary metabolites in urine.

Quantitation of total amounts (i.e., free compound plus glucuronide conjugate) of indomethacin (INDO) and its deschlorobenzoyl (DBI) and desmethyl (DMI) metabolites in human urine is described. An aliquot (0.4 ml) of urine is incubated with glucuronidase (1000 U, 2 h, 37 degrees C) and extracted with 5 ml of dichloromethane containing the internal standards: the fluoro analogue of INDO, F-INDO, and indole-3-propionic acid (IPA). The organic phase is concentrated, dissolved in mobile phase and aliquots are injected onto the high-performance liquid chromatograph. INDO and DMI are measured with UV detection at 254 nm over a linear range of 0.25-125 micrograms/ml. Retention times for DMI, F-INDO and INDO are 4.0, 6.8 and 12.1 min, respectively, using a C8 reversed-phase column with an acetonitrile-0.1 M acetate, pH 5.0 (30:70) mobile phase at a 2.5 ml/min flow-rate. DBI is measured using fluorescence detection (excitation = 305 nm, emission = 370 nm) over a linear range of 0.25-12.5 micrograms/ml. Retention times for DBI and IPA are 4.5 and 7.8 min, respectively, on the same C8 column with an acetonitrile-0.025 M acetate, pH 4.0 (22:78) mobile phase at a 2.0 ml/min flow-rate. Inter- and intra-day precision were smaller than 10% for INDO, DMI and DBI over the concentration ranges indicated.