Abstract:

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.

Key words: Box-Behnken design, fluorescence detection (FLD), high performance liquid chromatography(HPLC), Plackett-Burman design, plasma, quality by design, response surface methodology, rhubarb anthraquinones