A novel extractionless hplc fluorescence method for the determination of glyburide in the human plasma: application...

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

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.

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.

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