CHAPTER 25: Quantitation of Folates by Stable Isotope Dilution Assays

Folates are a group of water-soluble vitamins involved in the metabolism of one-carbon groups. The most common analytical method for folates is a microbiological assay, which is not able to differentiate between single folate vitamers. Stable isotope dilution assays (SIDAs) for folates are based on the use of stable isotopologues as internal standards that are detected by liquid chromatography coupled to mass spectrometry. SIDAs are becoming increasingly used as they enable differentiation between vitamers and correction for losses during sample preparation. [¹³C]-labelled and [²H₄]-labelled folates are mainly used as internal standards in folate SIDAs. Applications of folate SIDAs are reported for foods with or without fortification by folic acid and for clinical samples such as blood serum, plasma, red blood cells, urine and ileostomy effluents. In method comparisons of SIDAs with microbiological assays, good agreement is obtained if 5-methyltetrahydrofolate is used as the calibrator for the microbiological assay. Method comparisons of SIDA with microbiological assay for food folates revealed good agreement for fortified foods. For endogenous food folates, the vitamer distribution has to be considered for microbiological assays to obtain accurate results.