Full Length Research Paper

A rapid high-performance liquid chromatography (HPLC) method for the extraction and quantification of folates in dairy products and cultures of Propionibacterium freudenreichii

Jessy Van Wyk¹* and Trevor J. Britz²

¹Department of Food Technology, Cape Peninsula University of Technology, Bellville 7535, South Africa.

²Department of Food Science, University of Stellenbosch, Stellenbosch 7600, South Africa.

*Corresponding author. E-mail: vanwykj@cput.ac.za. Tel: +27 21 959 6176 or +27 0822006876. Fax: +27 21 959 6095.

Abbreviations: AOAC, Association of Official Analytical Chemists; HPLC, high-performance liquid chromatography; MA, microbiological assay; NTDs, neural tube defects; PAB, propionibacteria; PGA, pteroylglutamic acid; THF, tetrahydrofolate; RSD, relative standard deviation.

Accepted 31 October, 2011

   Abstract

Nutritional folate deficiencies in Southern African communities necessitated mandatory fortification. Current microbiological assays (MA) used to measure food folates, essential for quality control and regulatory purposes, are time-consuming. This study describes an alternative extraction and detection method for folates in dairy products and Propionibacterium freudenreichii cultures. Folates were extracted by heating with a phosphate buffer (pH 6.0). Polyglutamates were deconjugated with chicken pancreas and hog kidney deconjugases. Samples were purified using strong anion exchange solid phase extraction. Reversed-phase high-performance liquid chromatography (HPLC) using an acetonitrile-phosphate buffer (pH 2.2) gradient effectively separated four vitamers. Fluorescence (tetrahydrofolate (THF), 5-CH₃-THF and 5-CHO-THF), and UV detection (folic acid) were used, calibration curves were linear (R²>0.0997), and detection and quantification limits were 0.0006 to 0.015 and 0.002 to 0.05 mg/ml, respectively. Accuracy was 80 to 108% and intra- and inter-day precision [%relative standard deviation (%RSD)] were lower than 4%. The method, validated against the standard MA, is a selective, sensitive, reliable and rapid alternative.

Key words: Propionibacterium freudenreichii, microbiological folate assay, high-performance liquid chromatography (HPLC), folate.