Simultaneous determination of cinobufagin and its five metabolites in rat plasma by UPLC-MS/MS for characterization of metabolic profiles and pharmacokinetic study
Cinobufagin is one of main ingredients of Venenum bufonis and has many pharmacological activities and toxicities. It is very important but difficult to simultaneously determine the cinobufagin and its metabolites because of the strong physiological activity and the low concentration in blood. In this study, a convenient and efficient method was developed to simultaneously determine cinobufagin and its five metabolites in rat plasma (semi-quantification). BF211, the derivative of bufalin, was added into plasma sample as internal standard (IS). An ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 um) was used in the separation at 40 ℃ and the mobile phase containing the acetonitrile and water added 0.05 % formic acid was running under gradient elution at 0.4 mL/min. The UPLC-MS/MS with an electrospray ionization source in positive ion mode was used in the quantification of cinobufagin and its five metabolites in the mode of multiple reaction monitoring (MRM). The monitored ion pairs were m/z 443.5 → 365.3 for the transition of cinobufagin, m/z 513.7 → 145.3 for IS, m/z 443.2 → 365.2, 401.2 → 265.2, 417.2 → 363.2 for 3-epi-cinobufagin, desacetylcinobufagin and hydroxyl-desacetylcinobufagin respectively. The calibration curve of cinobufagin showed good linearity over the range of 1.0 – 200 ng/mL (y = 0.201x+0.105, R2 > 0.990) and the limit of quantitation (LOQ) was 1.0 ng/mL. According to the results, the specificity, accuracy, precision, extraction recovery, matrix effect and stability of the method were satisfactory. The results of pharmacokinetic study showed that the cinobufagin was absorbed quickly (Tmax = 0.42 ± 0.29 h) and the main metabolite was desacetylcinobufagin (Cmax = 897.95 ± 237.35 ng/mL). This pharmacokinetic study could be used as essential data to interpret the pharmacokinetic-pharmacodynamic relationships of cinobufagin.