玉米中5-甲基四氢叶酸提取方法的优化及高效液相色谱法定量分析

Optimization of Extraction Process for 5-Methyltetrahydrofolate from Maize and Determination of Its Content by HPLC

优化玉米中5-甲基四氢叶酸(5-methyl-tetrahydrofolate,5-MTHF)提取方法,评价高效液相色谱(high performance liquid chromatography,HPLC)测定玉米5-MTHF含量的准确性。结果表明:优化最佳提取参数为叶酸提取液pH值为6.5,α-淀粉酶、蛋白酶、大鼠血清的添加量分别为12 U/mL、0.028 U/mL、14μL/mL,固相萃取净化柱收集洗脱液2.5 mL;HPLC法测定玉米中5-MTHF表明,在0.003~1.000μg/mL有良好的线性关系,相关系数为0.9999,检出限为0.001μg/mL,定量限为0.003μg/mL,精密度为1.90%,样品加标平均回收率为104.57%;测定叶酸强化玉米(京科糯928)中5-MTHF与总叶酸含量分别为181.18μg/100 g与253.65μg/100 g(干质量),提取优化后的HPLC检测方法适用于玉米中5-MTHF与总叶酸的测定,可为其他强化谷物中叶酸含量的测定提供参考。

In this study,the extraction of 5-methyl-tetrahydrofolate(5-MTHF)from maize was optimized,and the accuracy of high performance liquid chromatography(HPLC)for determination of 5-MTHF in maize was evaluated.The extraction parameters were optimized as follows:extraction buffer pH 6.5,α-amylase concentration 12 U/mL,protease concentration 0.028 U/mL,rat serum concentration 14μL/mL,and amount of eluate collected from solid phase extraction(SPE)column chromatography 2.5 mL.The HPLC method showed a good linear relationship in the concentration range of 0.003 to 1.000μg/mL with correlation coefficient of 0.9999.The limit of detection(LOD)was 0.001μg/mL,and the limit of quantification(LOQ)was 0.003μg/mL.The precision expressed as relative standard deviation(RSD)was 1.90%,and the recovery of 5-MTHF from spiked sample was 104.57%.The concentrations of 5-MTHF and folate in folate-fortified maize(Jingke Nuo 928)were 181.18 and 253.65μg/100 g dry mass,respectively.The optimized HPLC method is suitable for the determination of 5-MTHF and folate in maize,and can provide a reference for the determination of folate concentration in other fortified grains.