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.
US Clinical Bioanalytics Department, Marion Merrell Dow Inc., Kansas City, MO 64134.
D L
DL
Reynolds
S D
SD
Burmaster
L S
LS
Eichmeier
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005438:Fludrocortisone / Q000097:blood; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood*; D011282:Pregnenediones / Q000097:blood*; D012015:Reference Standards; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet
D007166:Immunosuppressive Agents; D011282:Pregnenediones; C048465:21-deacetyldeflazacort; D005438:Fludrocortisone