Optimization and Validation of an HPLC–PDA Method for the Determination of (6S)-5-Methyltetrahydrofolate in Diverse Dietary Supplement Formulations

Analytical methods for (6S)-5-methyltetrahydrofolate (5-MTHF) have mainly been reported for specific formulation types, such as tablets, chewable tablets, powders, and liquid formulations, despite its increasing use in dietary supplement and health functional food formulations with diverse matrix compositions. In this study, high-performance liquid chromatography with photodiode array detection analytical conditions and sample preparation approaches were systematically compared and optimized to improve applicability across different formulation matrices. Chromatographic performance was evaluated under standard solution conditions, followed by a comparative assessment of sample preparation conditions in tablet, chewable tablet, powder, and liquid formulations. An ultrasonic pretreatment approach without thermal treatment provided consistent recovery and repeatability across all the tested formulations. The optimized analytical conditions showed linear detector response (R2 = 0.9992), precision with relative standard deviation values of 0.52–4.06%, recoveries of 81.84–102.56%, and an estimated expanded measurement uncertainty of approximately 10%. These results indicate that the optimized HPLC–PDA analytical conditions are applicable to the determination of 5-MTHF across diverse dietary supplement and health functional food formulations.