DETERMINATION OF KETOROLAC ENANTIOMERS IN PLASMA USING ENANTIOSELECTIVE LIQUID-CHROMATOGRAPHY ON AN ALPHA(1)-ACID GLYCOPROTEIN CHIRAL STATIONARY-PHASE AND ULTRAVIOLET DETECTION

A chirally selective high-performance liquid chromatographic assay was developed to measure the R(+) and S(-) enantiomers of ketorolac in plasma for pharmacokinetic studies. Naproxen sodium [S(+) enantiomer] (10 mu g) was used as an internal standard. Plasma samples (0.5 ml) were acidified (50 mu l of 4 M H3PO4 to pH 1.5), extracted into 0.4 ml of 10% pentan-2-ol in hexane and back-extracted into 0.15 ml of base (20 mM NaOH pH to 7-8), of which samples (5 mu l) were chromatographed on a 100 x 4 mm I.D. column packed with an HPLC chiral stationary phase based upon immobilized alpha(1)-acid glycoprotein (Chiral AGP-CSP) with 4% propan-2-ol in 0.1 M NaH2PO4 pH 5.5, at 0.9 ml/min. Detection was at 325 nm and run time was 10 min. Retention times of R- and S-ketorolac and of S(+)-naproxen were 3.3, 4.8 and 6.4 min, respectively. The metabolite p-hydroxyketorolac was not resolved enantiomerically and had a retention time of 2.2 min. The assay was linear over the range 0.5-10 mg/l, with precisions <5% C.V. Good separations (alpha > 1.35) and resolutions (R(s) > 3.23) between peaks were achieved. The sensitivity could be extended to 35 mu g/l with less precision by increasing the injection volume to 100 mu l.