反相高效液相色谱法测定葡萄中的有机酸

Determination of organic acids in grape berry with reversed-phase high performance liquid chromatography

本研究建立了反相高效液相色谱检测葡萄果实有机酸的方法,并评价该方法的准确性和灵敏度。结果显示,用酸性水溶液提取果实有机酸,当应用色谱柱为KromasilC18,流动相为3%CH3OH-0.01mol/LKH2PO4(pH2.8)缓冲液,流速为0.8mL/min,柱温为25℃,检测波长为210nm时,在8min内可将提取液中的酒石酸、苹果酸、柠檬酸和草酸有效分离。此方法的平均回收率>90%,定量相对标准偏差均在5%以内,最低检测限在0.067～1.12g/L,表明此法具有快速、灵敏、可靠的特点,能够满足果实样品4种有机酸同时检测的要求。运用该方法,分析了赤霞珠和霞多丽果实发育过程中有机酸含量的变化,发现红、白两品种间有机酸变化趋势相似,即草酸和酒石酸含量逐渐下降,而苹果酸在转色前逐渐积累,而后迅速下降;柠檬酸含量变化不明显。

A method was developed for simultaneous determination of organic acids in grape berries with the reversed-phase high performance liquid chromatography （RT-HPLC）, and its accuracy, repeatability and limit of detection （LOD） of this method was evaluated. The results showed that the artaric acid, malic acid, citric acid and oxalic acid contents in grape berries isolated with acid aqueous solution could be well separated within 8min under such conditions as a chromatographic column of Kromasil C 18 employed, a mobile phase containing 97% 10mmol/L KH：PO4 （pH2.8 adjusted with phosphoric acid） and 3% methanol kept, a flow rate of 0.8mL/min set, column temperature of 25℃ maintained and a detection wavelength at 210nm chosen. The extract recovery of the organic acids exceeded 90%, the relative standard deviation of the organic acid quantity was lower than 5% and LOD was in a range of 0.067- 1.12 g/L, indicating this method had quite a few advantages of high accuracy, good precision and feasibility for simultaneous determination of organic acids in grape berries. This method was further used to investigate the contents of organic acids in the developing berries of Cabernet Sauvignon （Vitis vinifera, cv.） and Chardonnay （Vitis vinifera, cv.）, and the results revealed that similar variation trends of organic acids were present in white and red grape cultivars. Both oxalic acid and tartaric acid contents gradually decreased with the berry development, malic acid gradually accumulated at pre-veraison and then rapidly decreased at post-veraison, and citric acid content kept relatively stable.