The stereospecific HPLC assays reported for ketoprofen (KT) mainly utilize indirect approaches. These assays involve the formation of amide diastereomeric derivatives, which are then separated by chromatography. The advantages of indirect methods include versatility, good sensitivity and cost effectiveness; however, lengthy preparation time is often required. Therefore, we have developed a new direct stereospecific HPLC assay for KT enantiomers to improve preparation time and sensitivity. The KT enantiomers and indomethacin, internal standard (I.S.), were resolved using a Chiralpac AD column attached to 5 cm Supelcosil LC-SI at constant temperature (30 degrees C). The mobile phase consisted of hexane-isopropanol-trifluoroacetic acid (90:10:0.1). Under chromatographic conditions employed R-KT, S-KT and I.S. were eluted at 12, 14 and 16 min, respectively. A linear concentration response relationship was found (0.05-5.0 micrograms/ml of enantiomers) which covered normally observed concentrations in plasma after conventional doses of KT. The minimum quantifiable concentration of the assay was found to be 0.025 or 0.25 microgram/ml based on 1 ml of human or 0.1 ml of rat plasma samples, respectively. This direct HPLC method is suitable for pharmacokinetic studies of KT enantiomers and offers the advantages of shorter sample preparation and run time. This method is at least as sensitive as assays currently in use.
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