高效液相色谱法同时测定水果蔬菜中L-抗坏血酸、D-异抗坏血酸、脱氢抗坏血酸及总维生素C的含量

Determination of L-Ascorbic Acid,D-Isoascorbic Acid,Dehydroascorbic Acid and Total Vitamin C in Fruit and Vegetable by HPLC

建立了同时测定水果或蔬菜中L-抗坏血酸、D-异抗坏血酸、脱氢抗坏血酸及总维生素C含量的高效液相色谱分析方法。用偏磷酸提取水果或蔬菜样品中L-抗坏血酸、D-异抗坏血酸和脱氢抗坏血酸,提取液中的L-抗坏血酸、D-异抗坏血酸可直接进行检测,脱氢抗坏血酸在磷酸钠溶液中（pH 7.0-7.2）用L-半胱氨酸还原成L-抗坏血酸,之后测定以L-抗坏血酸表达总维生素C含量,脱氢抗坏血酸含量由总维生素C含量减去L-抗坏血酸含量获得。采用C18色谱柱分离,以甲醇-磷酸盐缓冲溶液（pH 3.5）为流动相,在245 nm下检测,外标法定量。结果表明,在0.5-50 mg/L的浓度范围内L-抗坏血酸和D-异抗坏血酸的线性关系良好,相关系数大于0.999,检出限分别为42.0,19.4μg/kg,脱氢抗坏血酸的检出限为262μg/kg。低、中、高3个浓度的加标水平下,3种物质的加标回收率为82.8%-111.3%,相对标准偏差（RSD）小于15%。该方法操作简单,灵敏度高,准确性好,适用于水果和蔬菜中维生素C的测定。

A reliable and simple method was developed for the determination of L-ascorbic acid, D- isoascorbic acid, dehydroascorbic acid and total vitamin C in fruit and vegetable by high performance liquid chromatography （HPLC）. The fruit or vegetable sample were extracted with metaphosphoric acid. L-Ascorbic acid and D-isoascorbie acid in extract were directly determined by HPLC with ultraviolet spectrophotometric detection. Dehydroascorbie acid in extract was reduced to L-ascorbie acid by L-cysteine in trisodium phosphate （pH 7.0 -7.2） solution, and then total vitamin C was determined. The dehydroaseorbic acid was calculated by substracting L - Ve from total vitamin C. The analytes were separated on a C18 column using methanol -0. 01 mol/L phosphate buffer （pH 3.5 ） as mobile phase, and analyzed quantitatively at UV wavelength of 245 nm by the external standard method. The calibration curves for L-ascorbic acid and D-isoascorbic acid were linear in the range of 0. 5 - 50 mg/L with correlation coefficients more than 0. 999. The limits of detection were 42. 0 μg/kg for L-ascorbic acid, 19. 4 μg/kg for D-isoascorbic acid and 262 μg/kg for dehydroaseorbic acid. The average recoveries of three substances in four matrices ranged from 82. 8% to 111.3% with relative standard deviations（RSD） less than 15% at three spiked and accurate, and was suitable for the determination of levels. The method was simple, sensitive L-ascorbic acid, D-isoascorbic acid, dehydroascorbic acid and total vitamin C in fruit and vegetable samples.