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 ∼1.73 μmol/kg = ∼795 μg/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 ∼1.73 μmol/kg, or ∼795 μg/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 °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 μg/kg (∼0.020 μmol/kg) and 30 μg/kg (∼0.060 μmol/kg), respectively (<10× 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 comprise a stable matrix for 5-mthf fortification.