Determination of ketorolac in human serum by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) assay has been developed for the determination of ketorolac in human serum using a new extraction method with a good recovery. Human serum samples (1.0 mi) spiked with known concentrations of ketorolac tromethamine and 10 mu g of ketoprofen as the internal standard (IS) were acidified with 200 mu l of 1N HCl and extracted with 7 mi of n-hexane-ether (7:3 v/v). Extracts were centrifuged and organic layer was back-extracted with 400 mu l of 0.1% tromethamine solution. Twenty mu l of centrifuged aqueous layer was injected onto a reversed-phase octyl column and eluted with a mixture of acetonitrile, water, methanol, and triethylamine [35.55:10:0.1 (v/v), pH 3.0] at a flow rate of 1.0 ml/min. Ultraviolet detection of ketorolac and IS was carried out at 300 nm. The calibration curve obtained using peak area ratios showed a good linearity (in concentration range 10-150 ng/ml r(2)=0.9944; in range 50-2000 ng/ml, r(2)=0.9998). The mean intra-day accuracy and precision for this HPLC method were found to be 3.6 and 3.7%, respec tively. The mean inter-day accuracy and precision were found to be 4.0 and 3.7%, respec tively, in the concentration range 50-2000 ng/ml. The recovery of ketorolac from serum was 92.0 (+/-5.7)% at the concentration of 100 ng/ml. This method proved to be readily applicable to the assay of ketorolac in human serum.