HPLC法拆分并测定草豆蔻中(±)-山姜素和(±)-乔松素对映体含量

Study on enantioseparation and content determination of (±)-alpinetin and (±)-pinocembrin in Alpiniae Katsumadai Semen using HPLC

关键词:  山姜素对映体  乔松素对映体  高效液相色谱法  草豆蔻  手性分离分析

Key words:  (±)-alpinetin  (±)-pinocembrin  HPLC  Alpiniae Katsumadai Semen  chiral separation

摘要:

目的 建立草豆蔻药材中(±)-山姜素和(±)-乔松素同时手性和非手性分离分析方法,并将所建立的方法应用于草豆蔻药材和不同炒制时间炮制品中两个成分的对映体的含量测定。方法 采用手性柱-高效液相色谱法对山姜素和乔松素进行对映体拆分研究,优化了手性柱种类、流动相比例、流速和柱温的色谱条件,并在最佳分离条件下测定了不同批次草豆蔻药材和不同炒制时间草豆蔻药材中两对映体的含量。结果 确定了山姜素和乔松素对映体的手性和非手性分离条件为色谱柱：PHENOMENEX Lux i-Amylose-3 (4.6 mm×250 mm,5 μm),流动相为乙腈-水(62:38,v/v),流速为0.4 mL.min_-1^,柱温为25 ℃,检测波长为290 nm。在最佳分离条件下,山姜素两个对映体和乔松素两个对映体的分离度能达到1.5,并且草豆蔻中其他成分并不干扰两个成分对映体的含量测定。将所建立的对映体分离分析方法应用于不同批次草豆蔻药材和不同炒制时间草豆蔻药材中两对映体的含量测定,所测定的三个批次的草豆蔻药材中(+)-乔松素的含量为1.66-3.06 mg.g_-1^{,而(-)-乔松素的含量为}2.27-3.66 mg.g_-1^{,两个对映体的}EF值为0.41-0.46；山姜素两个对映体的含量分别为3.00-5.46和3.29-5.63 mg.g_-1 ^{,两个对映体的}EF值 为0.48-0.49。而随着炒制时间的增加,山姜素两个对映体和乔松素两个对映体的含量均呈现先升高再下降的趋势,两者均是在炒制10分钟时呈现含量最大值,但是炒制对于两个成分的对映体的EF影响不大。 结论 所建立的草豆蔻药材中(±)-山姜素和(±)-乔松素同时手性和非手性分离分析方法准确高、重复性好,可用于基于对映体层面的草豆蔻药材的质量控制和质量评价。

Abstract:

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, v/v) 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 Rs of the alpinetin isomers and pinocembrin isomers can reach 1.5, and other components of Alpiniae Katsumadai Seme do not interfere with the content determination of the two aanalytes. 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 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 minutes. 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 contro of Alpiniae Katsumadai Seme based on the enantiomeric layer.