INFLUENCE OF OCTANOIC-ACID ON THE REVERSIBLE PROTEIN-BINDING OF KETOROLAC ENANTIOMERS TO HUMAN SERUM-ALBUMIN (HSA) - COMPARATIVE LIQUID-CHROMATOGRAPHIC STUDIES USING A HSA CHIRAL STATIONARY-PHASE

The retention of ketorolac enantiomers on a human serum albumin (HSA)- based HPLC chiral stationary phase (CSP) was investigated to assess th e utility of immobilized protein for probing the binding of (R)- and ( S)-ketorolac to native HSA. Results from the chromatographic study wer e compared with enantiomorph binding data obtained from HSA ultrafiltr ation experiments conducted both in the presence and absence of the me dium chain-length fatty acid octanoic acid. Without octanoic acid in t he mobile phase containing 10% propan-2-ol in 20 mM phosphate buffer a t pH 6.5, racemic ketorolac was stereochemically resolved with the HSA -CSP with large enantiomeric capacity factors [106.2 and 28.7 for (R)- and (S)-ketorolac, respectively]. The inclusion of octanoic acid in t he column eluent reduced the capacity factors of both isomers consiste nt with displacement of drug from HSA binding sites. A reduction in th e capacity factor ratio [(R):(S)] was observed as the octanoate concen tration increased from 0.5 to 4.0 mM. The percentage unbound of (R)- a nd (S)-ketorolac present separately (2.0 mu g/ml) in 40.0 mg/ml HSA so lution (22 degrees C and pH 7.4) was 0.245% and 0.643%, respectively, and both values increased as a function of increasing octanoate concen tration in the HSA solution. A biphasic effect of octanoate on the per centage unbound ratio of (S):(R) was observed. In light of these findi ngs, it would appear that silica-immobilized HSA is capable of qualita tively probing the enantioselective binding of ketorolac to HSA and mo reover, more than one specific ketorolac binding site may exist on the HSA molecule.