白芷中香豆素类化合物的超临界流体色谱分离方法研究

Study on separation of coumarins in Angelica dahurica by supercritical fluid chromatography

目的:建立超临界流体色谱法(SFC法)同时测定白芷中7个香豆素类化合物(水合氧化前胡素、佛手柑内酯、氧化前胡素、欧前胡素、蛇床子素、异欧前胡素、软木花椒素)的含量,并与高效液相色谱法(HPLC法)进行比较。方法:采用Agilent ZORBAX RX-SIL(150 mm×4.6 mm, 5μm)色谱柱,以二氧化碳(A)-乙醇(B)为流动相,梯度洗脱,流速1.5 mL·min^(-1),柱温45℃,背压12.75 MPa,进样体积为2μL,检测波长280 nm。结果:7个香豆素类化合物在一定质量浓度范围内均具有良好的线性关系(r>0.999 0),精密度的RSD为0.79%~2.9%。7个香豆素类化合物在24 h内具有良好的稳定性,重复性的RSD为1.8%~3.0%,回收率为93.8%~102%。27批白芷饮片中,7个香豆素类化合物的含量由高到低的顺序为欧前胡素、水合氧化前胡素、异欧前胡素、氧化前胡素、佛手柑内酯、软木花椒素、蛇床子素,含量范围分别为1.00~5.70、0.24~3.10、0.31~1.50、0.01~2.50、0.03~0.39、0.01~1.20、0.01~0.54 mg·g^(-1)。在一定程度上,SFC法HPLC法具有相反的色谱行为。结论:建立的SFC方法能够快速分离含2对同分异构体的7个香豆素类化合物。SFC法的保留及选择性受色谱柱、改性剂的影响较大。与HPLC法相比,SFC法更迅速、经济环保。

Objective:To establish a supercritical fluid chromatography method for the simultaneous determination of 7 coumarins(oxypeucedanin hydrat,bergapten,oxypeucedanin,imperatorin,osthole,isoimperatorin,suberosin)in Angelica dahurica,and to compare with high performance liquid chromatography(HPLC)method.Methods:The Agilent ZORBAX RX-SIL(150 mm×4.6 mm,5μm)column was used,the mobile phase was CO,(A)-ethanol(B)with gradient elution,flow rate was 1.5 mL·min^(-1),the column temperature was 45℃,back pressure was 12.75 MPa,the injection volume was 2μL,and the detection wavelength was 280 nm.Results:The seven coumarins all had good linear relationship(r>0.9990),the RSDs of precision were 0.79%-2.9%,seven coumarins remained stable within 24 h,the RSDs of repeatability were 1.8%-3.0%,and the recoveries were 93.8%-102%.In 27 batches of Angelica dahurica decoction piece,seven of coumarin compounds content from high to low were imperatorin,oxypeucedanin hydrat,isoimperatorin,oxypeucedanin,bergapten,suberosin,and osthole,the contents ranged from 1.00 to 5.70,0.24 to 3.10,0.31 to 1.50,0.01 to 2.50,0.03 to 0.39,0.01 to 1.20,0.01 to 0.54 mg:g^(-1),respectively.To some extent,the SFC method and the HPLC method showed the opposite chromatographic behavior.Conclusion:The established SFC method can rapidly separate 7 coumarins including 2 pairs of isomers.The retention and selectivity of SFC method are greatly affected by column and modifier.Compared with HPLC method,the SFC method is more rapid,economic and environmental friendly.