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-μL portion of buffer solution containing 300 or 550 μM human serum albumin and 100 or 300 μM racemic KP or a 300-μL portion of human plasma containing 12.5–100 μM 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. The advantages of the present method over the widely used ultrafiltration method are (1) a very low concentration of unbound drug, due to strong binding to protein, can be determined enantioselectively without sample pretreatment and (2) undesirable adsorption of drug and leakage of proteins, which often cause trouble in the ultrafiltration method, can be avoided. Consequently, the therapeutic concentrations of unbound KP in human plasma could be determined with good reproducibility by injecting a relatively small volume (300 μL) of untreated plasma. The limit of determination (signal-to-noise ratio of 10 with UV detection at 262 nm) was 1.0 nM for each KP enantiomer. The present system was used to determine the profile of the concentration of unbound KP enantiomers in rat plasma versus time.