A unified UPLC-MS/MS method for simultaneous quantification of intact anthocyanins and their metabolites in rat plasma

A rapid and sensitive UPLC-MS/MS method was established and validated for the simultaneous quantification of mulberry anthocyanins and their major metabolites in rat plasma. The method successfully achieved, for the first time, the simultaneous quantification of anthocyanin prototype compounds, including cyanidin-3-O-glucoside (C3G) and cyanidin-3-O-rutinoside (C3R) and their major metabolites, namely protocatechuic acid (PCA), 4-hydroxybenzoic acid (4-HBA), p-coumaric acid (p-CA), and vanillic acid (VA) in the same analysis. Plasma samples were prepared using a straightforward protein precipitation procedure with methanol, followed by chromatographic separation on a Waters ACQUITY Premier BEH C18 column under gradient elution conditions. Quantitative analysis was performed in multiple reaction monitoring (MRM) mode using electrospray ionization in both positive and negative ion modes, with an internal standard for calibration. Method validation covered specificity, linearity, limits of detection and quantification, precision, recovery, and stability. All analytes exhibited satisfactory linearity with correlation coefficients (R2) exceeding 0.990. Limits of quantitation of the method were 0.25-10.00 ng mL-1. Within- and between-day precisions ranged from 2.40% to 15.73% and 4.95% to 16.92%, respectively, and extraction recoveries were in the range of 81.95-99.74%. Under various conditions tested, all compounds were stable for at least 6 h. The method was demonstrated to be simple, sensitive, selective, and robust, and was successfully applied for the determination of anthocyanins and their metabolites in rat plasma after oral administration. These results would be beneficial for the pharmacokinetic and metabolic research of anthocyanins in complex biological matrices.