UPLC–MS/MS assay of 21-aminosteroid (lazaroid U74389G) for application in pharmacokinetic study

• Development of UPLC–MS/MS assay of lazaroid U-74389G (LAZ) in rat plasma.
• Assay validation as per US-FDA Guidance for Industry: Bioanalytical Method Validation.
• Application of the UPLC–MS/MS assay to a pre-clinical PK study of LAZ in rats.

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 (1 h and 3 h 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 (24 h 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.

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