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

Abstract

Purpose: To develop a simple, sensitive and rapid HPLC fluorescence method with single step sample preparation for the determination of glyburide in the human plasma.

Methods: Glyburide and ketoconazole (internal standard) were extracted from the 0.5 mL plasma by addition of 0.5 mL acetonitrile and 50 microL CuSO(4) solution (5% w/v in water). The separation was achieved on the Kingsorb 3 microm, C8 reverse phase column at ambient temperature with a mobile phase consisted of 45% buffer solution (0.05 M NH(4)H(2)P(4)), 40% acetonitrile and 15% methanol adjusted to pH 5.7 by diluted ammonia solution. A fluorescence detector was set at 235 nm excitation wavelength and 354 nm emission wavelengths to monitor eluted components.

Results: The internal standard and 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 coefficient 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 coefficient 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 five ng/mL.

Conclusion: This simple, rapid and sensitive method is suitable for pharmacokinetic, bioavailability and biequivalence studies.