甲基吡喃花色苷的制备优化及HPLC-MS/MS分析

Optimized method for preparation of methyl pyranoanthocyanins and analyzed by HPLC-MS/MS

以葡萄皮红色素提取物为原料,正交实验法研究花色苷在丙酮介质中反应合成甲基吡喃花色苷的最佳条件,探讨了反应物比例、底物浓度、温度、pH等因素对甲基吡喃花色苷生成量的影响,高效液相色谱(HPLC)监测反应过程,并利用高效液相色谱-二极管阵列检测-串联离子阱多级质谱(HPLC-DAD-EI-MS/MS)法对反应产物进行定性和定量分析。结果表明最佳反应条件是:反应物比例(葡萄皮红色素提取物mg∶丙酮mL)15∶1,葡萄皮红色素提取物浓度2.5mg/mL,反应pH3.0、温度45℃。高效液相色谱串联质谱结合多级质谱裂解分析表明,反应主要产物为甲基吡喃锦葵花色苷,反应第9d其得率为49.4%。经分离纯化后的甲基吡喃花色苷特征吸收峰在478nm,与原花色苷比较明显发生蓝移。实验结果为甲基吡喃花色苷的高效制备、功能与理化性质研究提供了一种经济有效的研究方法和物质基础。

Based on the raw material of red pigments extracts of grape skins,the optimized conditions for the synthesis of methyl pyranoanthocyanin by reactions of anthocyanins in acetone solutions were studied by orthogonal experiment design. The factors which had effects on reaction yield of methyl pyranoanthocyanin were investigated in terms of ratio of reactants（grape anthocyanins to acetone）,concentration of extracts of red grape skins,reaction temperature and pH. HPLC was used to monitor the reaction process and the resulting product pigment was analyzed by HPLC-DAD-EI-MS/MS qualitatively and quantitatively. The optimal synthesis conditions were as follows：the ratio of extracts of red grape skins（mg） to acetone（mL） was 15：1, concentration of grape skin pigments was 2.5mg/mL,reaction temperature was set at 45℃ and pH was 3.0. The main reaction product was confirmed by HPLC-MS/MS to be methyl pyranoMv with the yield of 49.4% after nine days. The characteristic spectral of the purified methyl pyranoMv pigment was at 478nm,which was distinctly blue-shifted when compared to original anthocyanins. The experimental results provided an effective and economic approach to the high efficient preparation of methyl pyranoMv pigment for further studies of functional and physical-chemical properties.