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.
Validated Method for the Determination of Deflazacort by a Flow‐Injection Analysis with UV Detection: Application to Pharmaceutical Formulations
Abstract A simple, precise, accurate, and fast flow‐injection analysis (FIA) method employing UV detection is described for the determination of deflazacort (DEF) in pharmaceutical tablets. The best carrier solvent system consisted of EtOH: sodium dihydrogen phosphate (0.2 M) (20:80, v/v) (pH 6.0). Related parameters were elucidated and a flow‐rate of 1.3 mL min−1 was used and the analyte was monitored at 247 nm. In the optimum condition, the repeatability was in the range of 0.27–0.41 as intra‐day precision. The linearization was tested considering intra‐ and inter‐day experiments in the range of 1.0 × 10−5–5.0 × 10−5 M and good correlation coefficients were obtained. The limit of detection (LOD) and the limit of quantification (LOQ) were calculated to be 2.35 × 10−7 and 7.04 × 10−7 M, respectively, as inter‐day results. The proposed method was applied to the determination of DEF in pharmaceutical preparations. Conventional UV‐spectrophotometry was used as a comparison method and their results were also compared to those of the FIA technique. Insignificant differences between FIA and UV‐spectrophotometric results were observed (p < 0.05). In conclusion, the tablets provide the general official requirements, 101.3% and 102.3%, respectively. Therefore, the proposed method is suggested to the routine laboratories for the determination of DEF in tablets.
Metabolism of deflazacort in the rat, dog and man.
The metabolism of [2'-14C]deflazacort, (11 beta, 16 beta)-21-(acetoxyl)-11-hydroxy-2'-methyl-5'H-pregna-1, 4-dieno[17,16-d]oxazole-3,20-dione, orally given to rats, dogs, and humans, has been studied. From the urine of the three species and from rat bile and liver preparations, five main metabolites I-V have been isolated and their structures investigated by physicochemical analysis: 1,(5 beta,11 beta,16 beta)-11,21-dihydroxy-2'-methyl-5'H-pregn-1-eno[17,16-d]oxazole-3,20-dione; II, (11 beta,16 beta)-11,21-dihydroxy-2'-methyl-5'H-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione; III, (6 beta,11 beta,16 beta)-6,11,21-trihydroxy-2'-methyl-5'H-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione; IV, (3 epsilon,11 beta,16 beta)-3,11,21-trihydroxy-2'-methyl-5'H-pregn-5-eno[17,16-d]oxazol-20-one. Metabolites II and III are quantitatively the most important in the urine of the rat, dog, and man; metabolite V, whose structure is uncertain, has been found in human and rat urine. In the formation of metabolites I-V the fused 2-methyloxazoline ring is unmetabolized, whereas the steroid moiety follows the general metabolic pathways reported for other related corticosteroids.