Simultaneous determination of cortisone and cortisol in serum by HPLC-DAD and application for pharmacokinetics

Abstract

To develop a high performance liquid chromatography method for the simultaneous determination of cortisone and cortisol in rat serum and apply it for pharmacokinetics. After addition of pirfenidone as internal standard (IS), a liquid-liquid extraction with ethylacetate was employed for the sample preparation. Samples were separated on Zorbax SB-C18 column at 25 ºC using mobile phase consisting of acetonitrile-water-0.1 % trifluoroacetic acid with flow rate of 0.9 mL/min, utilizing DAD detection at 246 nm. Excellent liner relationships of the cortisone and cortisol concentrations were obtained from 50 to 6000 ng/mL, with r = 0.9997, 0.9999 respectively, and the lower limit of quantitation (LLOQ) were both 50 ng/mL. The developed method was successfully applied to pharmacokinetic studies of cortisone and cortisol in rats following single dose of 20 mg/kg via intraperitoneal injection.