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 (