应用质量源于设计的理念优化大鼠血浆中大黄蒽醌的分析方法

应用质量源于设计理念建立一种高效液相色谱-荧光检测法(HPLC-FLD)用于测定大鼠血浆中5种大黄蒽醌。用Plackett-Burman设计考察流动相中甲醇含量、pH值、流速、柱温和进样体积对色谱峰的分离度、理论塔板数、最末洗脱峰的保留时间和拖尾因子的影响,结果显示流动相中甲醇含量、流速和柱温对色谱系统的影响显著(p<0.05)。继而采用Box-Behnken设计结合响应面法考察上述三因素对分离度、保留时间和理论塔板数的影响。用Derringer渴求函数评价了响应值的综合作用。得出最优色谱条件为:以甲醇-0.1%(v/v)磷酸水溶液(81.4:18.6, v/v)为流动相等度洗脱,流速1.1 mL/min,柱温31℃,荧光检测激发波长为440 nm,发射波长为540 nm。建立的模型显示良好的预测性。结果表明:质量源于设计的理念可有效地应用于优化高效液相色谱分析方法。

Application of quality by design concept for the optimization of an analytical method for rhubarb anthraquinones in rat plasma

A robust high performance liquid chromatography with fluorescence detection method (HPLC-FLD) was developed for the determination of five rhubarb anthraquinones in rat plasma using a quality by design approach. A Plackett-Burman design was utilized to screen the effects of methanol content, the pH value of mobile phase, the flow rate, column temperature, and injection volume on peak resolution, number of the theoretical plate, retention time of the last eluted peak, and tailing factor. The results showed that the methanol content in mobile phase, flow rate, and column temperature were statistically significant (p < 0.05). A Box-Behnken experimental design with response surface methodology (RSM) was then utilized to evaluate the effects of these three factors on the selected responses. Derringer’s desirability function was used for the evaluation of the chromatographic goals. The optimum conditions for separation were as follows: an isocratic mobile phase consisting of methanol/0.1%H₃PO₄ (81.4/18.6, v/v), a flow rate of 1.1 mL/min, and a column temperature of 31℃. The excitation and emission wavelengths were 440 nm and 540 nm, respectively. The proposed method showed good prediction ability. The results clearly showed that quality by design concept could be effectively applied to optimize the HPLC method.