An HPLC-MS-Based Method for Determination of the D- and L- 5-Methytetrahydrofolate Isomer Ratio in Dietary Supplements

Background 5-Methytetrahydrofolate (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. Objective This 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 matrices and dosage forms. Methods A 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. Results The 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 µg/mL to 160 µg/mL of 5-MTHF with R 2>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 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%). Conclusion This 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 method and possesses great selectivity in separation of the diastereomers in different form 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.