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

Abstract

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 C₁₈ 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 > 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.

Graphical Abstract

Keywords:

• HPLC-UV
• Flavonoids
• Simultaneous quantification
• Box–Behnken design
• Response surface methodology

Disclosure statement

The authors declare that they have no competing interest in this paper.

Additional information

Funding

The authors would like to thank the Science and Technology Planning Project of Guangzhou (201903010066), the Science and Technology Planning Project of the Xinjiang Production and Construction Corps, China (2018AB009), and the Natural Science Foundation of Xinjiang Uygur Autonomous Region, China (2018D01A03).