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An analysis method was developed for the separation and quantification of hesperidin, neohesperidin, neohesperidin dihydrochalcone and hesperetin by using HPLC-UV. Single factor experiments and Box-Behnken Designs were used to optimize separation of four flavonoids, in which a gradient elution method was adopted with 99% acetonitrile and 0.1% formic acid as mobile phases at a flow rate of 0.9 mL/min. A C 18  column was used with a column temperature of 35 °C. LODs and LOQs were below 0.84 µg/mL and 2.84 µg/mL, respectively. Linearity with good correlation coefficients ( r  &gt; 0.99,  n  = 5) was attained, recovery rate of four flavonoids ranged from 88% to 130%, the RSD indicating results precision for analyzing hesperidin, neohesperidin, neohesperidin dihydrochalcone and hesperetin ranged from 1.2% to 4.6%. Finally, the present method could be successfully applied to identify and quantify hesperidin, neohesperidin and hesperetin in  Fructus Aurantii Immaturus  and  Pericarpium Citri Reticulatae .

Development of a HPLC-UV Method for the Separation and Quantification of Hesperidin, Neohesperidin, Neohesperidin Dihydrochalcone and Hesperetin