Verification of 5-Methyltetrahydrofolic Acid in Nutritional Products

A simple method for the verification of supplemental (6R,S)-5-methyl-5,6,7,8-tetrahydrofolic acid, "5-MTHF," in nutritional products is described. Nutritional product samples are prepared for the liquid chromatographic/fluorescence detection (LC/FLD) determination of 5-MTHF by buffer dilution, 10-min centrifugation, and syringe filtration. Method performance has been defined by assessments of 5-MTHF linearity (r (2) averaged 0.9999 +/- 0.0001, and all relative calibration errors averaged <= 0.6%, for ten consecutive six-point standard curves), intermediate precision (rsd = 1.1%, n = 9, for three products fortified at similar to 1.73 mu mol/kg = similar to 795 mu g/kg, tested on each of 3 days), accuracy (spike recovery average = 92.8 +/- 1.0%, n = 9, for nutritional products spiked with 5-MTHF at similar to 1.73 mu mol/kg, or similar to 795 mu g/kg), and selectivity (absence of interference from reagent blanks, and from four compounds structurally related to 5-MTHF). The 5-MTHF recovery, as % of unheated controls, from a simulated heat treatment (20 min at 120 A degrees C) averaged 99.1 +/- 0.6%, n = 4. The limits of 5-MTHF detection (S/N = 3) and quantification (S/N = 10) were experimentally determined to be 10 mu g/kg (similar to 0.020 mu mol/kg) and 30 mu g/kg (similar to 0.060 mu mol/kg), respectively (< 10x the expected fortification rate). The method provides a simple and inexpensive means for verifying proper fortification, and for assessing the stability to processing and storage conditions, of free 5-MTHF in nutritional products. A finding of interest is the indication that liquid nutritional products may comprise a stable matrix for 5-MTHF fortification.