HPLC determination of ketoprofen enantiomers in human serum using a nonporous octadecylsilane 1.5 microns column with hydroxypropyl beta-cyclodextrin as mobile phase additive.

A sensitive and stereospecific high-performance liquid chromatography (HPLC) method for the quantitation of ketoprofen enantiomers in human serum was developed. The assay involves the use of an octadecylsilane solid-phase extraction for serum sample clean-up prior to HPLC analysis. Chromatographic resolution of the ketoprofen enantiomers was performed on a nonporous octyldecylsilane column with hydroxypropyl beta-cyclodextrin as the mobile phase additive. The composition of the mobile phase was 98:2 v/v aqueous 0.1% trifluoroacetic acid (TFA), pH 4.00 (adjusted with triethylamine (TEA))/acetonitrile containing 10 mM hydroxypropyl beta-cyclodextrin (beta-CD) at a flow rate of 0.8 ml min-1. Recoveries of R(-)-ketoprofen was 95.4 +/- 2.16% and for S(+)-ketoprofen 96.2 +/- 1.31%. Linear calibration curves were obtained in the range 0.025-15 micrograms ml-1 range for each enantiomer in serum. The detection limit based on a S/N = 3 ratio was 10 ng ml-1 for each enantiomer in serum with ultraviolet detection at 220 nm. The limit of quantitation for each enantiomer was 25 ng ml-1. Precision calculated as % relative standard deviation (%R.S.D.) and accuracy calculated as % error were in the range 0.2-5.2% and 0.3-2.2%, respectively, for the R enantiomer and 0.3-6.2 and 0.2-3.2%, respectively, for the S enantiomer.