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 ml) spiked with known concentrations of ketorolac tromethamine and 10 μg of ketoprofen as the internal standard (IS) were acidified with 200 μl of 1N HCl and extracted with 7 ml of n-hexane-ether (7:3 v/v). Extracts were centrifuged and organic layer was back-extracted with 400 μl of 0.1% tromethamine solution. Twenty μl 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 r²=0.9944; in range 50-2000 ng/ml, r²=0.9998). The mean intra-day accuracy and precision for this HPLC method were found to be 3.6 and 3.7%, respectively. The mean inter-day accuracy and precision were found to be 4.0 and 3.7%, respectively, 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.