title: UPLC-MS/MS assay of 21-aminosteroid (lazaroid U74389G) for application in pharmacokinetic study.
abstract: Lazaroids are potent inhibitors of lipid peroxidation, both in vitro and in vivo. Additionally, a member of the lazaroid family, U-74389G (LAZ) has been shown to have specific radio-protective and anti-proliferative effects. However, there is no quantitative analytical method developed for measuring the therapeutic levels of LAZ for the aforementioned effects. This article highlights the development and validation of a sensitive UPLC-MS/MS method for the quantification of LAZ, and its subsequent application in pharmacokinetic studies in rats with the lower limit of quantification (LLOQ) of 1.95 ng/mL. LAZ and internal standard diadzein (IS) were separated using ACQUITY UPLC(®) BEH C18 column. Gradient elution was used at a flow rate of 0.45 mL/min with mobile phases consisting of 0.1% formic acid in water and 0.1% formic in acetonitrile. LAZ (m/z 612→260) and IS (m/z 255→199) were detected by electrospray ionization (ESI) using multiple reaction monitoring (MRM) in a positive mode on QTRAP(®) 5500 System. The UPLC-MS/MS method was validated as per the US FDA Guidelines for Bio-analytical Validation. LAZ was extracted from rat plasma (100 μL) using protein precipitation by acetonitrile with mean recovery and matrix factor in range of 47.7-56.1%, and 85.6-89.4%, respectively. The calibration curve for LAZ was linear in the range of 1.95-250 ng/mL. The inter-day and intra-day accuracy and precision values for LLOQ, low, medium, high and very high concentration QC samples were within ±15%. LAZ was tested under different storage conditions, for short-term bench-top stability (1h and 3h at 25°C), long-term stability (1 month at -80°C), freeze-thaw cycle stability (1 cycle and 3 cycles) and stability of processed samples in auto-sampler (24h at 10°C) with stability values within ±15% range of nominal concentrations. The validated UPLC-MS/MS method was further applied to a pharmacokinetic study in rats after a single intravenous dose of LAZ at 5 mg/kg.
authors: Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX 77030, USA. Electronic address: psgadgil@uh.edu.
authors: P
authors: P
authors: Gadgil
authors: Pfizer, Groton, CT 06340, USA.
authors: F
authors: F
authors: Ibrahim
authors: Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX 77030, USA. Electronic address: Dchow@uh.edu.
authors: D S-L
authors: DS
authors: Chow
mesh_terms: D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D057230:Limit of Detection; D008297:Male; D011281:Pregnatrienes / Q000737:chemistry* / Q000493:pharmacokinetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D011281:Pregnatrienes; C074965:U 74389F
keywords: 21-Aminosteroid; LC–MS; Stability; U-74389G