ArticleJournal of AOAC International2026
An HPLC-MS-Based Method for Determination of the D- and L- 5-Methyltetrahydrofolate Isomer Ratio in Dietary Supplements.
Article in Journal of AOAC International, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
background5-Methyltetrahydrofolate (5-MTHF), a reduced form of folate (vitamin B9) marketed as a potentially more bioavailable alternative to folic acid, is a key ingredient in dietary supplement (DS), particularly prenatal multivitamin/minerals. Its synthesis may produce racemic mixtures of bioactive L-5-MTHF and inactive D-5-MTHF, raising concerns about labeling accuracy, integrity, and health impacts. DS manufacturers consider D-5-MTHF an impurity and do not use racemic mixtures of 5-MTHF in their products.
objectiveThis study aimed to develop and validate an HPLC-MS-based method to separate and quantify the D and L-5-MTHF diastereomers in various DS matrixes and dosage forms.
methodsA single-laboratory validated method was developed for determination of 5-MTHF isomers in different dosage forms of DS using targeted selected ion monitoring (targeted SIM) with a ChiralPak HSA column. L-cysteine was selected as an extraction stabilizer, and the mobile phase was composed of 100 mM ammonium acetate and 1% (v/v) acetic acid in acetonitrile (ACN) at a 97:3 ratio.
resultsThe HPLC/MS method was able to separate D- and L-5-MTHF diastereomers by isocratic elution within 20 min. It showed great linearity with the concentration range of 1 to 160 µg/mL of 5-MTHF with R2 >0.99. The method was validated for the limit of detection (LOD), limit of quantitation (LOQ), linear range, specificity, and recovery according to the guidelines described in Q2(R1) Validation of Analytical Procedures: Text and Methodology Guidance for Industry. The method was then applied for the analysis of 101 samples, and two samples were found to exceed the USP limit of ≤1% D-5-MTHF relative to L-5-MTHF (1.39 and 50.28%).
conclusionsThis performance of HPLC-MS method established effectively determines D- and L-5-MTHF ratios in complex DS, serving as a reliable tool to detect adulteration with racemic mixtures in prenatal and other 5-MTHF-labeled containing DS. It is significantly faster than previously reported methods and possesses great selectivity in separation of the diastereomers in different forms of DS. HIGHLIGHTS: Successfully validated an HPLC-MS method for quantifying D- and L-5-MTHF diastereomers in diverse DS dosage forms, aiding enantiomeric purity assessment.
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