Development and validation of HPLC-MS/MS technique for the quantitative determination of a new derivative of sydnonymine and its metabolites in rabbit plasma

Introduction. BBP2023 is a new drug candidate of the sydnonymine group with cerebral vasodilating activity and procognitive properties. The pharmacological effects of BBP2023 are due to the presence of active metabolites. To conduct pharmacokinetic studies at the preclinical stage, it is necessary to develop a bioanalytical method for the determination of BBP2023 and its metabolites in rabbit blood plasma. Aim. Development and validation of a chromatography-mass spectrometric method for the quantitative determination of a new derivative of sydnonymine and its metabolites in rabbit blood plasma. Materials and methods. Quantitative determination of BBP2023 in rabbit blood plasma was performed by HPLC-MS/MS. The biomaterial sample preparation was based on the method of protein precipitation with methanol. Chromatographic separation was performed with a mixture of deionized water and acetonitrile in a gradient mode on a ZORBAX Eclipse XDB-C18 analytical column (4.6 × 50 mm, 5 μm) using an Agilent 1260 Infinity II chromatograph. Detection was performed in the MRM mode using an AB Sciex QTRAP 3200 MD mass spectrometer. N-(ethoxycarbonyl)-3-(4-methylpentan-2-yl)sydnonymine was used as the internal standard. Results and discussion. The method for sample preparation of rabbit blood plasma has been developed, optimal conditions for chromatography and detection of the studied compound and its metabolites have been selected. The bioanalytical method has been validated according to the following parameters: selectivity, matrix effect, recovery rate, carry-over effect, linearity of the calibration range, lower limit of quantitative determination, intraand inter-run accuracy and precision, as well as stability of analytes and the internal standard at the analysis stages. Conclusion. A method for the quantitative determination of a new derivative of sydnonymine BBP2023 and its metabolites in rabbit blood plasma using HPLC-MS/MS has been developed and validated. The confirmed analytical ranges for BBP2023, BBP2023 A and BBP2023 C were 5.0–500.0 ng/ml (5.0–1000.0 ng/ml for geranamine). The method was used in the analytical part of the preclinical pharmacokinetic study of BBP2023 in rabbits.