Quantitative determination of 21-hydroxy-deflazacort in human plasma using gradient semi-microbore liquid chromatography.

A sensitive and selective liquid chromatographic procedure to quantitate the deflazacort metabolite 21-hydroxy-deflazacort (DF-21OH) in human plasma was developed and validated. DF-21OH and fludrocortisone acetate (internal standard, IS) were isolated from human plasma (2 mL) by solid-phase extraction onto C-18 cartridges. Potential interferences were selectively removed and analytes were eluted with ethyl acetate. Following evaporation, the residue was reconstituted for HPLC analysis. Separation was achieved by gradient elution using a 5 microns YMC Basic column (2.0 x 100 mm) with mobile phases consisting of 20% methanol and 50% acetonitrile in 50 mM phosphate buffer (pH 3) at a temperature of 50 degrees C. Flow rate was maintained at 0.3 mL/min., and analytes were quantified spectrophotometrically at 246 nm. The assay was validated over the range 1.0 to 500 ng/mL DF-21OH. Calibration curves were prepared using a weighted (1/concentration) nonlinear quadratic regression algorithm. Peak-height ratios were proportional to the amount of DF-21OH added to plasma. Assay precision (%RSD) ranged from 4.2 to 11%, with a corresponding assay accuracy (% relative error) of +/- 2.8%. Absolute recovery of DF-21OH from plasma was 78-86% over the concentration range. The minimum quantitation limit was 1.0 ng/mL.