Multiseparation of anthocyanins and anthocyanidins by high performance liquid chromatography combined with response surface methodology
Vu Thi Trang1,2, Le Hoang Duc2, Nguyen Hoai Thu2, Le Thi Hong Hao1,2, Nguyen Xuan Trung2
1National Institute for Food Control, 65 Pham ThanDuat, Hanoi, Vietnam
2VNU University of Science, Vietnam National University-Hanoi, 19 Le Thanh Tong, Hanoi, Vietnam
A method has been developed for the separation of anthocyanins and anthocyanidins with high performance liquid chromatography (HPLC). Experimental designs were applied for multivariate optimization of the HPLC experimental conditions with using response surface methodology (RSM). Three independent factors, namely formic acid concentration, initial ratio of acetonitrile in gradient program and flow rate, were used to design mathematical models. The responses were resolutions of four peak couples which were the most difficult to separate including: pelargonidin-3-glucoside and delphinidin, delphinidin and peonidin-3-glucoside, peonidin-3-glucoside and malvidin-3-glucoside, peonidin and malvidin. Variance analysis proved a chosen model was highly fit and the RSM method yielded good results in improving separation of anthocyanins and anthocyanidins. The optimized HPLC parameters were as follows: C18 column (250 mm × 4.6 mm × 5 µm), mobile phase is FA 10 % and ACN used as a gradient; flow rate 0.8 mL.min-1. Using these optimum conditions, separation of compounds with good resolutions and a run time of less than 30 min were archived. The results for method validation satisfied the requirement of AOAC, linearity range from 0.2–10 ppm with R2 ≥ 0.9955, LOD from 0.05–0.1 mg/kg, RSD from 4.79–6.45 % and the recovery is from 85.4 – 109.6 %. The method was applied to determone anthocyanins and anthocyanidins in some fruits and vegetables samples with the content of anthocyanidins being from 5.74 – 218.27 mg/100g. Anthocyanins primarily concentrate in peel of fruits and vegetables, black bean peel contains most anthocyanins.
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