Self-assembly pipette tip-based cigarette filters for micro-solid phase extraction of ketoconazole cis-enantiomers in urine samples followed by high-performance liquid chromatography/diode array detection

Abstract

A simple stereoselective HPLC/DAD method was developed for the determination of ketoconazole (KTZ) cis-enantiomers in human urine. In addition, the potential use of pipette tip-based cigarette filters for micro-solid phase extraction (PT-CFs-μ-SPE) of KTZ cis-enantiomers in human urine samples prior to HPLC/DAD was studied in this paper. In order to find a suitable procedure for extraction of KTZ cis-enantiomers in human urine, various parameters that probably affect the extraction efficiency including the washing solvent, eluent kind and volume, pH, quantity of the material, sample volume and ionic strength were systematically optimized. Optimum chromatographic separations among the KTZ cis-enantiomers have been achieved within 10 minutes using a Chiralpak® IA column (100 × 4.6 mm, 3 μm) as the stationary phase with a mobile phase consisting of 100% ethanol at a flow rate of 1 mL min⁻¹. Detection was performed at 250 nm. The performance criteria for linearity, sensitivity, precision, accuracy, recovery, robustness and stability have been assessed and were within the recommended guidelines. The mean extraction recoveries/relative standard deviation for (+)-(2R,4S)-KTZ and (−)-(2S,4R)-KTZ were 100.74 ± 9.52% and 106.90 ± 9.26%, respectively. The method showed to be linear over the concentration range of 12.5 to 400 ng mL⁻¹ for each enantiomer with the correlation coefficients of 0.9946 and 0.9910, for (+)-(2R,4S)-KTZ and (−)-(2S,4R)-KTZ, respectively. Within-day and between-day precision and accuracy assays for these analytes were studied at three concentration levels and were lower than 15%. The method was successfully employed in a preliminary cumulative urinary excretion study after administration of racemic cis-KTZ to a healthy volunteer.