Simultaneous determination of AZD1152 (prodrug) and AZD1152-hydroxyquinazoline pyrazol anilide by reversed phase liquid chromatography

A simple, selective and sensitive reversed phase liquid chromatography method utilizing ultraviolet detection has been developed and validated for the simultaneous determination of the prodrug AZD1152 and its active product AZD1152-hydroxyquinazoline pyrazol anilide (hQPA) in human and mouse plasma and mouse tissues. Isocratic separation was achieved using an 5 μm UptiSphere HDO C-18 column (150 mm × 4.6 mm) with guard column in combination with a mobile phase comprised of phosphate buffered water (50 mM; pH 3.4) and acetonitrile (81.5: 18.5; v/v). UV detection at 318 nm was used. Sample preparation involved a single-step protein precipitation with ethanol. Ex vivo conversion of AZD1152 by endogenous phosphatases was prevented by immediate cooling of the samples in ice-water and addition of sodium fluoride and EDTA. The validation parameters included specificity, recovery, accuracy, precision, sensitivity and stability. The lower limit of quantification in human plasma for AZD1152 and hQPA was 25 ng/ml. The applicability of the method was demonstrated by successful determination of AZD1152 and hQPA in human plasma and in plasma, brain, liver, kidney and ileum samples from mice dosed with AZD1152.