Quantification of flavan-3-ols and phenolic acids in milk-based food products by reversed-phase liquid chromatography–tandem mass spectrometry

This article reports the development and validation of a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the comprehensive quantification of flavan-3-ol and phenolic acid constituents of milk-based food products. Isotope dilution-based sample preparation consisted of protein precipitation using acidic methanol enriched with the stable isotope labelled internal standards and ultrafiltration to preserve overall polyphenol composition, but to eliminate milk proteins in order to comply with LC. Reversed-phase liquid chromatography was optimized to achieve separation of 22 analytes in 8 min in order to reduce suppression effects, achieve a wide dynamic range and, most importantly, to resolve isomeric compounds. Negative-ion electrospray mass spectrometric detection and fragmentation of analytes was optimized, final transitions were selected for maximized selectivity, reliable quantification and reduction of false positives. The quantitative performance of the method was validated, the main features include: (1) range of lower limits of detection 5–15 ng/mL for flavan-3-ols, 60–100 ng/mL for procyanidins, 1–60 ng/mL for other phenolic acids, (2) lower limits of quantification 15–45 ng/mL for flavan-3-ols, 200–300 ng/mL for procyanidins, 3–200 ng/mL for other phenolic acids, (3) averaged intra-day precision 9.5%, (4) calibrated range 60–300,000 ng/mL for flavan-3-ols, 900–900,000 ng/mL for procyanidins, 9–225,000 ng/mL for other phenolic acids, (5) averaged accuracy 99.5%. Applications for yoghurt and ice-cream products are given. The presented data suggest that this method will help to better characterize the polyphenol composition of milk-based food products for quality control, assessment of dietary intake and for polyphenol bioavailability/bioefficacy studies.