An HPLC-MS-based method for determination of the D- and L- 5-methytetrahydrofolate isomer ratio in dietary supplements

Authors: WANG, XIAOJUAN - University Of Maryland; PANDEY, DEEPESH - University Of Maryland; VAVILALA, SUMA - University Of Maryland; OH, LAURA - University Of Maryland; GUSEV, PAVEL - University Of Maryland; ANDREWS, KAREN - University Of Maryland; Pehrsson, Pamela; Harnly, James; Sun, Jianghao

Submitted to: Journal of AOAC International
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/4/2025
Publication Date: 9/16/2025
Citation: Wang, X., Pandey, D., Vavilala, S., Oh, L., Gusev, P., Andrews, K.W., Pehrsson, P.R., Harnly, J.M., Sun, J. 2025. An HPLC-MS-based method for determination of the D- and L- 5-methytetrahydrofolate isomer ratio in dietary supplements. Journal of AOAC International. 109(2):154-162. https://doi.org/10.1093/jaoacint/qsaf089.
DOI: https://doi.org/10.1093/jaoacint/qsaf089

Interpretive Summary: L-5-Methytetrahydrofolate (L-5-MTHF) is the form of folate that is found in food, and it is the form that the body uses to carry out its functions. D-5-Methytetrahydrofolate (D-5-MTHF) has the same chemical formula as L-5-MTHF but different in molecular arrangement. The D-form is considered as an impurity. D-5-MTHF is not biologically active and cannot be used by the human body in the same way as 5-L-MTHF. The synthesis of 5-L-MTHF may produce a racemic mixture (50:50 ratio of a mixture of L (active) and D (inactive) forms) of diastereomers. Some dietary supplements (DS) may use racemic mixtures of 5-MTHF in their products. However, analytical method is lacking for determination of D- and L-5-MTHF in dietary supplements. This manuscript described a high performance liquid chromatography mass spectrometry based method for separation the D- and L-5-MTHF. The method was fully validated and applied to four different dosage forms including tablets, capsules, soft gels, and gummies.

Technical Abstract: Background 5-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. 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 matrixes 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 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%). Conclusions 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 methods and possesses great selectivity in separation of the diastereomers in different forms of DS.