High-performance liquid chromatographic determination of tolmetin, tolmetin glucuronide and its isomeric conjugates in plasma and urine

A rapid and sensitive analytical procedure is described for the simultaneous measurement of tolmetin (T), tolmetin glucuronide (1 beta-TG) and the isomers of tolmetin glucuronide in plasma and urine. A reversed-phase liquid chromatographic system is used with an ion-pairing mobile phase of methanol-tetrabutylammonium hydrogensulfate buffered to pH 4.5 and kept at a constant temperature of 50 degrees C. Detection is by UV at 313 nm. Plasma (0.5 ml) and urine (0.1 ml) are collected in pre-cooled containers and immediately adjusted to pH 3.0 to minimize TG isomerization and hydrolysis. Samples are then deproteinated with acetonitrile, the supernatant is evaporated to dryness and reconstituted in an acetate buffer (pH 4.5), and 50 microliters are injected onto the system. Using zomepirac as the internal standard, the measurable, linear concentration ranges are 0.05-50 micrograms/ml for T in plasma and 0.025-50 micrograms/ml for T in urine. Chromatographic peaks representing T,1 beta-TG and three isomers of TG were identified, all with retention times less than 10 min. The need for special handling of biological samples is discussed.