Evaluating folate extraction from infant milk formulae and adult nutritionals: Enzymatic digestion versus enzyme-free heat treatment

•Addition of enzymes to extract folate from milk formulae was evaluated.•Enzyme-free heat treatment was more compatible with UPLC-MS/MS.•UPLC-MS/MS was used for quantitative analysis of folic acid and 5-methyltetrahydrofolate.

This study compares enzymatic treatments to release folic acid (FA) and endogenous 5-methyltetrahydrofolate (5-MTHF) from infant milk formulae with enzyme-free heat extraction. The limits of detection and quantitation of FA were 1.4 ng/mL and 3.1 ng/mL, respectively; 7.5 ng/mL and 16.2 ng/mL for 5-MTHF. Absolute mean recoveries were 85% (FA) and 95% (5-MTHF). The RSD of the within-run variability was 6% and the inter-day variability was 8%. Averaged measurements of FA and 5-MTHF in SRM-1849a were within the certified value range. Analysed folate levels in three brands were greater than label values, because of inherently high 5-MTHF occurring in samples. The results indicate that enzyme-free heat treatment prior to UPLC-MS/MS analysis gives better sensitivity and reduces chromatographic interferences for the determination of FA and 5-MTHF in milk formulae than enzymatic treatments. Enzyme-free heat treatment is more compatible with UPLC-MS/MS than folate extraction techniques involving the addition of enzymes to milk.