A Validated Liquid Chromatographic Method for Determining Folates in Vegetables, Milk Powder, Liver, and Flour

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Abstract
A liquid chronoat (LG) 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 LG with fluorescence and diede 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 tetrahy-drofolic acid, 5-methyltetrahydrofolic acid, and 5-formyltetrahydrofolic acid. 10-Formylffolic 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 arnount 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 folic acid were 1ng/ml. Repetability 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 μg/100 g. Recoveries ranged from 73 to 109%. Use of amylase and protease was advantageous. Use of a comercially available folate-binding protein for cleanup saved time and money and was effective. Results for 5-methyltetrahydrofolioc acid were in good agreement with results obtained with other LC methods. Results for total folates were lower than results obtained with microbiological methods.

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