High-performance frontal analysis-high-performance liquid chromatographic system for stereoselective determination of unbound ketoprofen enantiomers in plasma after direct sample injection.

An on-line, high-performance frontal analysis (HPFA)-high-performance liquid chromatographic system was developed for the enantioselective determination of a low level of unbound ketoprofen (KP) that is in equilibrium with KP that is bound to protein. The system consists of three subsystems (HPFA system, preconcentration system, and chiral separation system) connected in series in the stated order via column-switching valves. When either a 300-microL portion of buffer solution containing 300 or 550 microM human serum albumin and 100 or 300 microM racemic KP or a 300-microL portion of human plasma containing 12.5-100 microM racemic KP was directly injected onto the HPFA column with the mobile phase at a low flow rate, KP was separated from proteins and eluted as a zonal peak with a plateau. The KP concentration in the eluant of the plateau region was the same as the unbound-KP concentration that was in equilibrium with protein-bound KP in the initial sample solution. A 1-mL portion of the eluant of the plateau region was switched to the preconcentration system, where KP was adsorbed and condensed on an octadecylsilyl silica (ODS) column. The adsorbed KP was eluted out of the ODS column and transferred to the chiral separation system, via another switching valve, where the enantiomers of unbound KP were separated and determined. The results agree well with those obtained by the conventional ultrafiltration method.(ABSTRACT TRUNCATED AT 250 WORDS)