Konings E J
Inspectorate for Health Protection, Commodities and Veterinary Public Health, Maastricht, The Netherlands.
J AOAC Int. 1999 Jan-Feb;82(1):119-27.
A liquid chromatographic (LC) method was elaborated for determining folates in foods. Folates were extracted by homogenizing in buffer and heat treatment. A portion was incubated with an enzyme preparation containing conjugase, amylase, and protease. After purification by affinity chromatography, folate monoglutamates were determined by reversed-phase LC with fluorescence and diode array detection. Gradient elution with phosphate buffer and acetonitrile was used to separate vitamers. The most abundant folate forms naturally present in foods were detected, including tetrahydrofolic acid, 5-methyltetrahydrofolic acid, and 5-formyltetrahydrofolic acid. 10-Formylfolic acid could be detected by applying a second fluorescence detector. Folic acid, used for fortification, might also be quantitated with this system. The difference between folate concentrations in sample extracts, with and without treatment of conjugase, is a measure of the quantity of polyglutamates in the food matrixes. An additional treatment with conjugase, amylase, and protease reflects the amount of matrix-bound folates. The LC system gave a linear response over the range 0-100 ng/mL. Detection limit for these compounds were 7 pg/mL for tetrahydrofolic acid and 5-methyltetrahydrofolic acid and 59 pg/mL for 10-formylfolic acid (signal-to-noise ratio > or = 3) when 100 microL was injected. Detection limits for 5-formyltetrahydrofolic acid and folic acid were 1 ng/mL. Repeatability relative standard deviation values for separate folates in 3 candidate Certified Reference Materials (CRMs)--mixed vegetables (CRM 485), pig liver (CRM 487), and whole-meal flour (CRM 121)--and a Certified Reference Material milk powder (CRM 421) varied from 3.3 to 21.0% for the concentration range 1.8-1440 micrograms/100 g. Recoveries ranged from 73 to 109%. Use of amylase and protease was advantageous. Use of a commercially available folate-binding protein for cleanup saved time and money and was effective. Results for 5-methyltetrahydrofolic acid were in good agreement with results obtained with other LC methods. Results for total folates were lower than results obtained with microbiological methods.
建立了一种液相色谱（LC）法用于测定食品中的叶酸。通过在缓冲液中匀浆和热处理提取叶酸。取一部分样品与含有结合酶、淀粉酶和蛋白酶的酶制剂一起孵育。经亲和色谱纯化后，通过反相LC结合荧光和二极管阵列检测来测定叶酸单谷氨酸酯。使用磷酸盐缓冲液和乙腈进行梯度洗脱以分离维生素变体。检测到了食品中天然存在的最丰富的叶酸形式，包括四氢叶酸、5-甲基四氢叶酸和5-甲酰基四氢叶酸。通过使用第二个荧光检测器可以检测到甲酰叶酸。用于强化的叶酸也可以用该系统进行定量。样品提取物中经结合酶处理和未经结合酶处理的叶酸浓度之差可衡量食品基质中多谷氨酸酯的含量。用结合酶、淀粉酶和蛋白酶进行额外处理可反映与基质结合的叶酸量。LC系统在0-100 ng/mL范围内呈线性响应。当进样100 μL时，这些化合物的检测限为：四氢叶酸和5-甲基四氢叶酸为7 pg/mL，10-甲酰基叶酸为59 pg/mL（信噪比≥3）。5-甲酰基四氢叶酸和叶酸的检测限为1 ng/mL。在3种候选有证标准物质（CRM）——混合蔬菜（CRM 485）、猪肝（CRM 487）和全麦粉（CRM 121）以及一种有证标准物质奶粉（CRM 421）中，单独叶酸的重复性相对标准偏差值在1.8-1440 μg/100 g浓度范围内为3.3%至21.0%。回收率在73%至109%之间。使用淀粉酶和蛋白酶是有利的。使用市售的叶酸结合蛋白进行净化既节省时间和金钱又有效。5-甲基四氢叶酸的结果与其他LC方法获得的结果高度一致。总叶酸的结果低于微生物法获得的结果。
