The retention of ketorolac enantiomers on a human serum albumin (HSA)-based HPLC chiral stationary phase (CSP) was investigated to assess the utility of immobilized protein for probing the binding of (R)- and (S)-ketorolac to native HSA. Results from the chromatographic study were compared with enantiomorph binding data obtained from HSA ultrafiltration experiments conducted both in the presence and absence of the medium chain-length fatty acid octanoic acid. Without octanoic acid in the mobile phase containing 10% propan-2-ol in 20 mM phosphate buffer at 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 the column eluent reduced the capacity factors of both isomers consistent with displacement of drug from HSA binding sites. A reduction in the capacity factor ratio [(R):(S)] was observed as the octanoate concentration increased from 0.5 to 4.0 mM. The percentage unbound of (R)- and (S)-ketorolac present separately (2.0 μg/ml) in 40.0 mg/ml HSA solution (22°C and pH 7.4) was 0.245% and 0.643%, respectively, and both values increased as a function of increasing octanoate concentration in the HSA solution. A biphasic effect of octanoate on the percentage unbound ratio of (S):(R) was observed. In light of these findings, it would appear that silica-immobilized HSA is capable of qualitatively probing the enantioselective binding of ketorolac to HSA and moreover, more than one specific ketorolac binding site may exist on the HSA molecule.
|Number of pages||6|
|Journal||Journal of Chromatography B: Biomedical Sciences and Applications|
|Publication status||Published - 2 Dec 1994|