Determination of 21‐hydroxydeflazacort in human plasma by high‐performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry. Application to bioequivalence study

A liquid chromatographic atmospheric pressure chemical ionization tandem mass spectrometric method is described for the determination of 21-hydroxydeflazacort in human plasma using dexamethasone 21-acetate as an internal standard. The procedure requires a single diethyl ether extraction. After evaporation of the solvent under a nitrogen flow, the analytes are reconstituted in the mobile phase, chromatographed on a C18 reversed-phase column and analyzed by mass spectrometry via a heated nebulizer interface where they are detected by multiple reaction monitoring. The method has a chromatographic run time of less than 5 min and a linear calibration curve with a range of 1–400 ng ml−1 (r > 0.999). The between-run precision, based on the relative standard deviation for replicate quality controls, was ≤5.5% (10 ng ml−1), 1.0% (50 ng ml−1) and 2.7% (200 ng ml−1). The between-run accuracy was ±7.1, 3.8 and 4.8% for the above concentrations, respectively. This method was employed in a bioequivalence study of two DFZ tablet formulations (Denacen from Marjan Industria e Comercio, Brazil, as a test formulation, and Calcort from Merrell Lepetit, Brazil, as a reference formulation) in 24 healthy volunteers of both sexes who received a single 30 mg dose of each formulation. The study was conducted using an open, randomized, two-period crossover design with a 7-day washout interval. The 90% confidence interval (CI) of the individual geometric mean ratio for Denacen/Calcort was 89.8–109.5% for area under the curve AUC(0–24 h) and 80.7–98.5% for Cmax. Since both the 90% CI for AUC(0–24 h) and Cmax were included in the 80–125% interval proposed by the US Food and Drug Administration, Denacen was considered bioequivalent to Calcort according to both the rate and extent of absorption. Copyright © 2000 John Wiley & Sons, Ltd.