Abstract: In this study, the extraction of 5-methyl-tetrahydrofolate (5-MTHF) from maize was optimized, and the accuracy of high performance liquid chromatography (HPLC) for determination of 5-MTHF in maize was evaluated. The extraction parameters were optimized as follows: extraction buffer pH 6.5, α-amylase concentration 12 U/mL, protease concentration 0.028 U/mL , rat serum concentration 14 μL/mL, and amount of eluate collected from solid phase extraction (SPE) column chromatography 2.5 mL. The HPLC method showed a good linear relationship in the concentration range of 0.003 to 1.000 μg/mL with correlation coefficient of 0.999 9. The limit of detection (LOD) was 0.001 μg/mL, and the limit of quantification (LOQ) was 0.003 μg/mL. The precision expressed as relative standard deviation (RSD) was 1.90%, and the recovery of 5-MTHF from spiked sample was 104.57%. The concentrations of 5-MTHF and folate in folate-fortified maize (Jingke Nuo 928) were 181.18 and 253.65 μg/100 g dry mass, respectively. The optimized HPLC method is suitable for the determination of 5-MTHF and folate in maize, and can provide a reference for the determination of folate concentration in other fortified grains.

Key words: maize; 5-methyl-tetrahydrofolate; high performance liquid chromatography; total folate