A novel extractionless HPLC fluorescence method for the determination of glyburide in the human plasma: Application to a bioequivalence study

Purpose. To develop a simple, sensitive and rapid HPLC fluorescence method with single step sample preparation for the determination of glyburide in t he human plasma. Methods. Glyburide and ketoconazole (internal standard) we re extracted from the 0.5 mL plasma by addition of 0.5 mL acetonitrile and 50 L CuSO4 solution (5% w/v in water). The separation was achieved on the K ingsorb 3 mum, C8 reverse phase column at ambient temperature with a mobile phase consisted of 45% buffer solution (0.05 M NH4H2PO4), 40% acetonitrile and 15% methanol adjusted to pH 5.7 by diluted ammonia solution. A fluores cence detector was set at 235 nm excitation wavelength and 354 nm emission wavelengths to monitor eluted components. Results. The internal standard an d glyburide eluted at about 6.7 and 9.6 min, respectively at the flow rate of 1 mL/min. The regression equation was established for every calibration curves (5 ng/mL to 400 ng/mL), which resulted in the correlation coefficien t of 0.99 or greater. The absolute recovery ranged from 94.32 to 98.12% and the relative recovery ranged from 91.12 to 97.15%. The intraday coefficien t of variation (CV) ranged from of 6.52 to 12.35% and interday varied from 6.21 to 16.07%. The limit of quantitation (LOQ) of glyburide was set to fiv e ng/mL. Conclusion. This simple, rapid and sensitive method is suitable fo r pharmacokinetic, bioavailability and biequivalence studies.