基于响应面法优化高效液相色谱法检测凉茶颗粒中7种绿原酸及其类似物的微波辅助萃取条件

Condition Optimization on Microwave-Assisted Extraction of Determination of 7 Chlorogenic Acids and Their Analogues in Herbal Tea Granules by High Performance Liquid Chromatography Based on Response Surface Methodology

在采用高效液相色谱法测定时,以凉茶颗粒样品中7种绿原酸及其类似物总含量为响应值,通过单因素法和响应面法,优化了萃取溶剂甲醇溶液的体积分数(因素A)、萃取时间(因素B)、萃取温度(因素C)和料液比[样品用量和提取溶剂体积的比值,单位g:mL,因素D]等微波辅助萃取条件。取1.00 g凉茶颗粒样品,加入20 mL 70%(体积分数,下同)甲醇溶液,涡旋溶解1 min,置于微波萃取仪中,于80℃萃取2.0 min。萃取液过0.22μm滤膜,滤液按照色谱条件测定。在色谱分析中,以ZORBAX Eclipse Plus C_(18)色谱柱为固定相,不同体积比的0.1%(体积分数)磷酸溶液和甲醇的混合溶液为流动相进行梯度洗脱分离,采用二极管阵列检测器在325 nm波长下检测。结果显示:采用Box-Behnken中心组合设计,通过四因素三水平试验建立了二次多项回归模型,其中A2,B2对7种目标物总含量的影响极显著,AD,D2影响显著,且交互因素的影响和等高线、响应面所得的一致;在优化的微波辅助萃取条件下,7种目标物的总测定值为32.98 mg·kg^(-1),是预测值(41.10 mg·kg^(-1))的80.3%,说明回归模型的可靠性较高;7种目标物的质量浓度均在0.01~4.0 mg·L^(-1)内和峰面积呈线性关系,检出限(3S/N)为0.12~0.60 mg·kg^(-1);按标准加入法进行回收试验,7种目标物的回收率为97.5%~105%,测定值的相对标准偏差(n=6)为1.4%~6.9%。

When using high performance liquid chromatography for determination,the total content of 7 chlorogenic acids and their analogues in the herbal tea granule sample was used as the response values,and microwave-assisted extraction conditions,including the volume fraction of the methanol solution(extraction solvent,factor A),extraction time(factor B),extraction temperature(factor C),and solid-liquid ratio(ratio of sample amount to extraction solvent volume,g:mL,factor D)were optimized through single factor method and response surface methodology.The 20 mL of 70%(volume fraction)methanol solution was added into 1.00 g of herbal tea granule sample for dissolution for 1 min by vortex.The mixture was placed into a microwave extractor,and extraction process was made at 80℃for 2.0 min.The extract was passed through a 0.22μm filter membrane,and the filtrate was determined according to chromatographic conditions.In chromatographic analysis,ZORBAX Eclipse Plus C_(18)chromatographic column was used as the stationary phase,and mixed solutions composed of 0.1%(volume fraction)phosphoric acid solution and methanol at different volume ratios were used as the mobile phase for gradient elution separation.A diode array detector was used for detection at the wavelength of 325 nm.It was shown that the quadratic polynomial regression model was established through four factors and three levels experiment with the Box-Behnken central combination design.A~2 and B~2 had the significant effect on the total content of 7 targets,while AD and D~2 had the significant effect.The effect of interaction factors was consistent with those obtained from contour lines and response surfaces.Under optimized microwave assisted extraction conditions,the total determined value of the 7 targets were 32.98 mg·kg^(-1),which was 80.3%of the predicted value(41.10 mg·kg^(-1)),indicating that the regression model had high reliability.Linear relationships between values of the mass concentration and the peak area of 7 targets were kept in the range of 0.01-4.0 mg·L^(-1),with detection limits(3S/N)in the range of 0.12-0.60 mg·kg^(-1).Test for recovery was made according to the standard addition method,giving recoveries of 7 targets in the range of 97.5%~105%,and RSDs(n=6)of the determined values ranged from 1.4%to 6.9%.