Quantitative Analysis Of An Antitumor Candidate Compound AB2 By LC-MS/MS And Its Metabolites Identification In Vitro

The candidate compound AB2 is a cardiac glycosides with extensive pharmacological activities,which has been widely used in the clinical treatment of heart failure,arrhythmia,etc.Research shows that it also has significant effect in anti-tumor.As an innovative drug,AB2 is currently in the non-clinical stage and is expected to become a new anti-tumor drug for clinical use.A sensitive LC-MS/MS assay has been developed and validated for the determination of AB2 concentration in rat plasma in this study.Validation results showed that the method had high specificity and sensitivity,short analysis time,good accuracy and repeatability.In addition,the method required very small volume of plasma.It is suitable for pharmacokinetic study of AB2 in rat.To characterize the metabolite profile,metabolic stability of AB2 was evaluated in different species in vitro by a liver microsome incubation experiment.We compared metabolic differences between human,cynomolgus monkeys,beagle dogs,mice and rats.Furthermore,the high resolution mass spectrometry TripleTOF 5600 was used to identify the phase I metabolites.The analysis of AB2 was on a SB-C₁₈ reversed-phase column.For mass spectrometric detection,a Sciex API 4000 mass spectrometer with electrospray ionization?ESI?was used to monitor analytes in the positive ion-multiple reaction monitoring?MRM?mode.The characteristic ion pairs were m/z 552.4?375.4 for AB2 and m/z 566.2?371.1 for Impurity-A,the internal standard?IS?.The sample was extracted by liquid-liquid extraction with a short analytical time for 5 min.The standard curve was linear?correlation coefficient r>0.9950?by weighted?1/X²?regession over the range of 0.8²00 ng/mL in rat plasma.The intra-day and inter-day accuracy were 4.79%⁸.77%and-3.53%⁴.23%,respectively.The intra-day and inter-day precision were 2.82%⁶.15%and 1.15%⁵.24%,respectively.The recoveries of AB2 and internal standard in rat plasma were 83.0%⁹1.8%and 82.1%,respectively.The matrix effect was 100%¹13%and corrected with internal standard.No carryover effect occured in this study.The established LC-MS/MS method in this study has been successfully applied to the pharmacokinetic study of AB2 in rat.After incubation of AB2 in the liver microsomes for 90 min,the AB2 residual ratio in human,cynomolgus monkey,beagle dog,mice and rat liver microsomes were29.8±11.0%,14.1±5.43%,92.4±2.02%,75.2±0.00%and 100±26.7%,respectively.The metabolism level in vitro of different species from high to low follows as:cynomolgus monkey>human>SD rat?beagle dog?CD1 mice.Cynomolgus monkey behaved more closely to human.The in vitro half-life of monkey and human were 33.0 min and 57.8 min,the intrinsic clearance of monkey and human were 42.0,24.0?L·min^-1·mg^-1,respectively.Howerer,there was almost no reduce of AB2 in rat,mice and beagle dog,suggesting that AB2 may not been metabolized in these species.High resolution mass spectrometry?HRMS?of TripleTOF 5600 was used to identify the metabolites.We identified M1 as one of the main metabolites,which was generated through dealkylation reaction catalyzed by cytochrome P450.The metabolic site of M1 located on the glycosyl of AB2,namely the methoxy group at the 3-C position of the sugar molecule structure was demethylated.However,the exact structure of M1 still needs to be further verified by comparison with the authentic compound.