Verification of 5-Methyltetrahydrofolic Acid in Nutritional Products

• Published in: Food analytical methods Vol. 10; no. 10; pp. 3255 - 3263
• Main Authors: Johns, Paul W., Baxter, Jeffrey H., Terp, Megan C.
• Format: Journal Article
• Language: English
• Published: New York Springer US 01-10-2017; Springer Nature B.V
• Subjects: Analytical Chemistry; Centrifugation; Chemistry; Chemistry and Materials Science; Chemistry/Food Science; Dilution; Fluorescence; Food Science; Heat treatment; Linearity; Microbiology; Recovery; Selectivity; Shelf life; Stability analysis; Storage conditions; Tetrahydrofolic acid; Validation; 5-Methyltetrahydrofolic acid; Stability; Metafolin; Folate
• ISSN: 1936-9751; 1936-976X
• DOI: 10.1007/s12161-017-0898-y

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 may comprise a stable matrix for 5-MTHF fortification.