Development and Validation of RP-HPLC Method for the Simultaneous Quantification of Seven Flavonoids in Pericarpium Citri reticulatae

In this study, a reversed phase high performance liquid chromatography (RP-HPLC) method was developed and validated for simultaneous quantification of seven flavonoids including nobiletin, tangeretin, naringin, naringenin, hesperidin, neohesperidin, and hesperetin in Pericarpium Citri reticulatae (PCR). Ultrasonic-assisted extraction (UAE) conditions were optimized using response surface methodology (RSM) to obtain maximum extractive contents of the seven flavonoids. A Box–Behnken design (BBD) was employed to study the main effects and interactions of independent variables. Following UAE, chromatographic separation was accomplished on a C18 column with a linear gradient elution. Full validation of the assay was implemented. It was found that this method had good linearity and specificity. Limit of detection (LOD) and limit of quantification (LOQ) for the tested flavonoids were 0.17–0.49and 0.75–1.75 μg/mL, respectively. Precisions (relative standard deviation, RSD) for intra- and interday were better than 4.54 %. Reproducibility was less than 4.91 %. Analyzed samples were stable for at least 48 h. Analysis had a good robustness (RSD < 8.00 %). Recoveries for the flavonoids were 98.00–101.32 %. The established method was successfully applied to determine the seven components in real samples from different locations and under different stress conditions. Results demonstrated that this analytical method was rapid, comprehensive, and cost effective for quality control of Pericarpium C. reticulatae.