HPLC法同时测定四物汤中8个成分及其在水提醇沉过程中的传递

目的：建立HPLC波长切换法同时测定四物汤中5-羟甲基糠醛、绿原酸、咖啡酸、芍药苷、阿魏酸、毛蕊花糖苷、洋川芎内酯A、藁本内酯8个成分的含量，并分析其在水提醇沉过程中的传递情况。方法：按照四物合剂制法制备水煎液、醇沉上清液、醇沉沉淀液样品进行测定；采用Agilent HC-C₁₈色谱柱（250 mm×4.6 mm，5 μm），流动相为乙腈（A）-0.5%磷酸水溶液（B），梯度洗脱，流速1.0 mL·min^-1，柱温23℃；进样量10 μL；检测波长为280 nm（0~10 min检测5-羟甲基糠醛，45~46 min，检测洋川芎内酯A）、327 nm（10~20 min检测绿原酸、咖啡酸）、230 nm（20~25 min检测芍药苷）、316 nm（25~29 min检测阿魏酸）、334 nm（29~45 min检测毛蕊花糖苷）、324 nm（46~50 min检测藁本内酯）。结果：5-羟甲基糠醛、绿原酸、咖啡酸、芍药苷、阿魏酸、毛蕊花糖苷、洋川芎内酯A、藁本内酯的质量浓度分别在0.61~19.50 μg·mL^-1（r=1.000）、0.99~31.80 μg·mL^-1（r=0.999 5）、0.18~5.88 μg·mL^-1（r=0.999 7）、10.38~332.00 μg·mL^-1（r=1.000）、0.91~29.10 μg·mL^-1（r=0.999 7）、0.53~8.46 μg·mL^-1（r=0.999 1）、1.49~47.80 μg·mL^-1（r=0.999 4）、0.71~22.60 μg·mL^-1（r=0.999 1）范围内与峰面积呈良好线性关系；平均回收率分别为99.6%、100.1%、98.7%、98.1%、100.9%、99.2%、98.7%、97.0%，RSD分别为1.1%、1.1%、1.2%、0.90%、1.7%、1.9%、0.80%、1.5%。与水煎液相比，醇沉上清液样品中8个成分的损失比例为15%~20%，醇沉沉淀中残留了少量的有效成分。结论：所建含量测定方法合理可行，重复性好，可用于四物汤的质量控制；四物汤水提醇沉过程中伴随着有效成分“量”的变化，在工艺过程中应该注意对工艺参数的优选，以保留有效成分。

Objective:To establish an HPLC wavelength switching method for simultaneously determining the contents of 5-hydroxymethylfurfural,chlorogenic acid,caffeic acid,paeoniflorin,ferulic acid,acteoside,senkyunolide A and ligustilide in Siwu decoction. And to analyze transmitting of the components in water extraction and ethanol precipitation process. Methods:Siwu decoction sample,Siwu ethanol supernatant sample and Siwu ethanol sediment sample were prepared according to the preparation method of Siwu mixture,and the samples were determined by HPLC. Separation was performed on an Agilent HC-C₁₈ column(250 mm×4.6 mm,5 μm) by a gradient elution of acetonitrile(A)-0.5% phosphoric acid solution(B). The flow rate was 1.0 mL·min^-1 and the column temperature was 23℃. The injection volume was 10 μL. The detection wavelengths were set at 280 nm (0-10 min for 5-hydroxymethylfurfural and,45-46 min for senkyunolide A),327 nm (10-20 min for chlorogenic acid and caffeic acid),230 nm (20-25 min for paeoniflorin),316 nm (25-29 min for ferulic acid),334 nm (29-45 min for acteoside),and 324 nm (46-50 min for ligustilide). Results:The 5-hydroxymethylfurfural,chlorogenic acid,caffeic acid,paeoniflorin,ferulic acid,acteoside,senkyunolide A and ligustilide showed good linearity in the ranges of 0.61-19.50 μg·mL^-1(r=1.000),0.99-31.80 μg·mL^-1(r=0.999 5),0.18-5.88 μg·mL^-1(r=0.999 7),10.38-332.00 μg·mL^-1(r=1.000),0.91-29.10 μg·mL^-1(r=0.999 7),0.53-8.46 μg·mL^-1(r=0.999 1),1.49-47.80 μg·mL^-1(r=0.999 4) and 0.71-22.60 μg·mL^-1(r=0.999 1),respectively. The average recoveries were 99.6% (RSD=1.1%),100.1% (RSD=1.1%),98.7% (RSD=1.2%),98.1% (RSD=0.90%),100.9% (RSD=1.7%),99.2% (RSD=1.9%),98.7% (RSD=0.80%) and 97.0% (RSD=1.5%),respectively. Compared with the Siwu decoction sample,the loss rate of eight active components in Siwu ethanol supernatant sample was 15%-20%. There were a few active components in the Siwu ethanol sediment sample. Conclusion:The HPLC wavelength switching method is reasonable,feasible and repeatable,which can be used for the quality control of Siwu decoction. In water extraction and ethanol precipitation process of Siwu decoction,the active components' content changes. So,we should pay attention to optimize the process parameters to retain the active components,in water extraction and ethanol precipitation process.

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