超高效液相色谱法同时测定肿节风中6个成分的含量

Quantitative Analysis of 6 Components in Hebra Sarcandrae by Ultra Performance Liquid Chromatography

建立了同时测定肿节风中新绿原酸、绿原酸、隐绿原酸、咖啡酸、迷迭香酸、异嗪皮啶6个成分含量的超高效液相色谱方法。采用Waters Acquity UPLC BEH C_(18)柱(2.1 mm×50 mm,1.7μm)分离,以乙腈-水(各含0.1%甲酸)为流动相进行梯度洗脱,流速为0.5 mL·min^(-1),检测波长330 nm,柱温35℃;以异嗪皮啶为参照物,计算其与其余5种目标物的相对校正因子,并考察了不同色谱仪、色谱柱、流动相、流速、柱温对相对校正因子重现性的影响;通过相对校正因子计算各成分的含量,实现一测多评,同时采用外标法测定肿节风中各成分的量,并对一测多评计算值与外标法实测值进行配对t检验。肿节风中新绿原酸、绿原酸、隐绿原酸、咖啡酸、迷迭香酸、异嗪皮啶分别在0.522 0~26.09,1.482~74.13,0.591 0~29.49,0.632 5~31.29,2.612~130.5,0.970 4~48.73μg·mL^(-1)范围内线性关系良好(r≥0.999 7),仪器精密度、方法重复性、样品稳定性的相对标准偏差(RSD)均不大于1.9%,平均加标回收率为98.6%~101.5%,RSD(n=6)不大于1.9%。所建立的相对校正因子重现性良好,外标法实测值与一测多评法计算值无显著性差异(P>0.05),均可用于肿节风中5个咖啡酰类和1个香豆素类成分的同时定量测定。

An ultra performance liquid chromatographic（ UPLC） method was developed to quantitatively analyze six components, i. e., neochlorogenic, chlorogenic acid,4-dicaffeoylquinic acid,caffeic acid,rosmarinic acid and isofraxidin in Hebra Sarcandrae. The UPLC analysis was carried out on a Waters Acquity BEH C_（18）column（ 2. 1 mm × 50 mm,1. 7 μm） by gradient elution with acetonitrile and water（ both containing 0. 1% formic acid） as mobile phase at a flow rate of 0. 5 mL·min^（-1）.The column temperature was maintained at 35 ℃ and the detection wavelength was set at 330 nm.Isofraxidin was used as an internal reference substance to get the average relative correction factors（ RCFs） of the other 5 components（ neochlorogenic,chlorogenic acid,4-dicaffeoylquinic acid,caffeic acid,rosmarinic acid）,and chromatographic column,mobile phase,flow rate,column temperature were investigated to validate the durability of the achieved RCFs. The contents of 6 components were determined by both quantitative analysis of multicomponents by single marker（ QAMS） and external standard method（ ESM）,and the quantitative results were compared between the two methods by using paired t-test. The results showed that the linear ranges for neochlorogenic,chlorogenic acid,4-dicaffeoylquinic acid, caffeic acid and, rosmarinic acid and isofraxidin were 0. 522 0-26. 09,1. 482- 74. 13,0. 591 0- 29. 49,0. 632 5- 31. 29,2. 612- 130. 5,0. 970 4- 48. 73μg·mL^（-1）（ r≥0. 999 7）,respectively. The RSD values（ n = 6） of precision,reproducibility,and sample stability were not more than 1. 9%. The average recoveries of 6 components were between98. 6% and 101. 5% with RSDs not more than 1. 9%. The achieved RCFs are good（ RSD 5. 0%）.There is no significant difference in the contents obtained by the two methods with P values greater than 0. 05,so both of the methods could be applied in the simultaneous determination of five caffeoyl derivatives and one coumarin in Hebra Sarcandrae.