Polyphenol analysis in human milk by a rapid, cost-effective, and accurate micromethod: translational development.

Abstract

To develop a rapid method for analysing polyphenols, which are potentially active antioxidants against neonatal oxidative stress, from small human milk (HM) volumes. Acid and alkaline extractions were compared using two dyes: Folin-Ciocalteu and Fast Blue BB. Linearity, sensitivity, recovery percentage, polyphenol content, precision, and stability were assessed in 14 HM samples and compared using the Kruskal-Wallis H test (p<0.05). The best technique was applied to 284 HM samples to determine their polyphenolic content and its association with maternal diet by multifactorial linear regression. Acidic extraction successfully recovered the gallic acid reference standard, whereas alkaline extraction overestimated it. Calibration curves for all methods were linear (R2>0.96) up to 500 mg/L. All bicarbonate-based Folin-Ciocalteu methods assayed were stable and repeatable, whereas Fast Blue BB-based variants were not. HM polyphenols (mean=94.68 mg/L) positively correlated to the dietary intake of hydroxycinnamic acids, the most consumed polyphenolic family in this population. A bicarbonate-based Folin-Ciocalteu micromethod allowed the accurate determination of polyphenols in HM, which might be useful for translational research settings and HM banks.