Enantioseparation and Content Determination of (±)-Alpinetin and (±)-Pinocembrin in Alpiniae Katsumadai Semen by HPLC

OBJECTIVE To establish the method for the simultaneous chiral and achiral separation of (±)-alpinetin and (±)-pinocembrin in Alpiniae Katsumadai Seme, which was applied to the determination of two enantiomers of each analytes in different batches of Alpiniae Katsumadai Seme and different processing time.

METHODS The enantiomeric separation method was studied by chiral columns-HPLC, as well as the chromatographic conditions including type of chiral column, mobile phase ratio, flow rate and column temperature were optimized. The contents of (±)-alpinetin and (±)-pinocembrin in different batches of Alpiniae Katsumadai Seme and different processing time were determined under the optimal separation conditions.

RESULTS The chiral and achiral separation conditions of the enantiomers of (±)-alpinetin and (±)-pinocembrin were determined as follows: PHENOMENEX Lux i-Amylose-3(4.6 mm×250 mm, 5 μm) as chiral column, mobile phase of acetonitrile-water(62∶38) at a flow rate of 0.4 mL·min⁻¹. The detection wavelength was set at 290 nm at a column temperature of 25 ℃. Under the optimal separation conditions, the resolution of two alpinetin isomers and pinocembrin isomers >1.5, and other components of Alpiniae Katsumadai Seme did not interfere with the content determination of the two analytes. The content of (+)-pinocembrin in three batches of Alpiniae Katsumadai Seme was 1.66–3.06 mg·g⁻¹, whileas the content of (–)-pinocembrin was 2.27–3.66 mg·g⁻¹, with the EF values of the two enantiomers were 0.41–0.46. The contents of the two enantiomers of alpinetin were 3.00–5.46 and 3.29–5.63 mg·g⁻¹, respectively, as well as the EF values of the two enantiomers were 0.48–0.49. With the increase of processing time, the contents of the two enantiomers of alpinetin and pinocembrin increased and reached a maximum value at 10 min. However, a decreasing trend of the contents of alpinetin isomers and pinocembrin isomers was observed, as the treatment time continued to increase. It's worth mentioning that processing of Alpiniae Katsumadai Seme had little effect on EF values of the (±)-alpinetin and (±)-pinocembrin.

CONCLUSION The established method for simultaneous chiral and achiral separation and analysis of (±)-alpinetin and (±)-pinocembrin in Alpiniae Katsumadai Seme is accurate and repeatable, which can be used for quality control and evaluation of Alpiniae Katsumadai Seme based on the enantiomeric layer.