Analysis of 21-hydroxy deflazacort in human plasma by UPLC–MS/MS: Application to a bioequivalence study in healthy volunteers

• Published in: Journal of pharmaceutical and biomedical analysis 2013-11, Vol.85, p.213-217
• Main Authors: Patel, Daxesh P., Sharma, Primal, Patel, Bhargav M., Sanyal, Mallika, Singhal, Puran, Shrivastav, Pranav S.
• Format: Article
• Language: English
• Subjects: 21-Hydroxy deflazacort; Bioequivalence study; Chromatography, High Pressure Liquid - methods; Humans; Incurred sample reanalysis; Pregnenediones - blood; Pregnenediones - pharmacokinetics; Reproducibility of Results; Sensitive; Tandem Mass Spectrometry - methods; Therapeutic Equivalency; UPLC–MS/MS
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2013.07.035

•Sensitive and rapid UPLC–MS/MS method for 21-hydroxy deflazacort in human plasma.•The method employs only 100μL plasma for efficient processing by SPE.•First report on bioequivalence study in healthy Indian volunteers.•Method reproducibility was ascertained by incurred sample reanalysis. A sensitive and rapid ultra performance liquid chromatography–tandem mass spectrometric (UPLC–MS/MS) method has been developed for the determination of 21-hydroxy deflazacort in human plasma using betamethasone as the internal standard (IS). After solid-phase extraction from 100μL human plasma, the analyte and IS were analyzed on Waters Acquity UPLC BEH C18 (50mm×2.1mm, 1.7μm) column using acetonitrile-4.0mM ammonium formate, pH 3.5 (90:10, v/v) as the mobile phase. The protonated analyte was quantified by selected reaction monitoring in the positive ionization mode by triple quadrupole mass spectrometer. The calibration plots were linear over the concentration range 0.50–500ng/mL. Intra-batch and inter-batch precision (% CV) and accuracy (%) for five quality control samples ranged within 1.40–4.82% and 98.0–102.0% respectively. The overall mean extraction recovery of 21-hydroxy deflazacort from plasma ranged from 95.3 to 97.3%. Matrix effect was assessed by post-column analyte infusion and the extraction recovery was >95.0% across four quality control levels for the analyte and IS. Stability was evaluated under different conditions like bench top, autosampler, processed sample (at room temperature and in cooling chamber), freeze-thaw and long term stability. The method was applied to support a bioequivalence study of 30mg deflazacort tablet formulation in 28 healthy subjects. Assay reproducibility was demonstrated by reanalysis of 115 incurred samples.