Quantitation of Key Antioxidants and Their Contribution to the Oxidative Stability of Beer

Abstract

A sensitive high-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-MS/MS_MRM) method, leveraging both technique and internal calibration, was developed for the simultaneous and comprehensive quantitative analysis of 46 antioxidants and antioxidant precursors in different beer types without any cleanup procedure. Combined with their in vitro antioxidant activity, a dose-activity estimation exposed a group of 10 key antioxidants, namely, tryptophan, tyrosine, hordatine A, hordatine B, procyanidin B₃, prodelphinidin B₃, tachioside (3-methoxy-4-hydroxyphenyl-β-d-glucopyranoside), (+)-catechin, tyrosol, and ferulic acid. To study the effect of antioxidants in spiking and aging studies, another liquid chromatography-MS (LC-MS)-based method was developed, monitoring markers for oxidation in beer. A positive effect of the antioxidants on the flavor stability at naturally relevant concentrations was shown by a slowing of oxygen-dependent aging reactions highlighted in beer storage trials under oxygen atmosphere. Thereby, a doubling of the natural concentration of all investigated antioxidants in beer revealed a limit inhibition of 67% on the degradation of cis-isocohumulone to hydroxy-cis-alloisocohumulone.

Keywords: LC-MS/MS; antioxidants; beer; flavor stability; hordatines; phenols; tachioside.

Figure 1

Chemical structures of antioxidants and antioxidant precursors investigated in beer: p-hydroxybenzoic acid (1), vanillic acid (2), syringic acid (3), p-coumaric acid (4), caffeic acid (5), ferulic acid (6), sinapic acid (7), tyrosol (8), p-hydroxyphenyllactic acid (9), cyclo (Pro-Tyr) (10), N-feruloyltyramine (11), pinoresinol (12), syringaresinol (13), 5-feruloylquinic acid (14), 4-feruloylquinic acid (15), 3-feruloylquinic acid (16), arbutin (17), tachioside (18), co-multifidol glucoside (19a), n-multifidol glucoside (19b), ad-multifidol glucoside (19c), (+)-catechin (20), (−)-epicatechin (21), procyanidin B₃ (22), prodelphinidin B₃ (23), kaempferol glucoside (24), quercetin glucoside (25), isorhamnetin glucoside (26), kaempferol malonylglucoside (27), quercetin malonylglucoside (28), isoxanthohumol (29), xanthohumol (30), saponarin (31), tryptophan (32), tyrosine (33), hordatine A (34), hordatine B (35), hordatine C (36), 4-(2-formylpyrrol-1-yl)butyric acid (37), 4-[2-formyl-5-(hydroxymethyl)pyrrol-1-yl]butyric acid (38), 4-hydroxyphenyl-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside (arbutintrioside, 39), 4-hydroxy-3-methoxyphenyl-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside (tachiotrioside, 40), 4-hydroxy-3-methoxyphenyl-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside (tachiodioside, 41), hordatine A glucoside (42), hordatine B glucoside (43), and hordatine C glucoside (44).

Figure 2

Chemical structures of compounds applied as internal standards: 4-methoxyphenyl glucoside (45), 3,4,5-trimethoxybenzoic acid (46), o-coumaric acid (47), dihydrorobinetin (48), apigenin (49), tryptophan-d₅ (32a), and tyrosine-d₄ (33a).

Figure 3

HPLC-MS/MS analysis of a beer sample showing the mass transition traces of the phenolic compounds and internal standards analyzed in ESI^– mode. Signal intensity of each mass transition is normalized and numbering of compounds refers to chemical structures given in Figures 1 and 2.

Figure 4

HPLC-MS/MS analysis of a beer sample showing the mass transition traces of the antioxidants and internal standards analyzed in ESI⁺ mode. Signal intensity of each mass transition is normalized and numbering of compounds refers to chemical structures given in Figures 1 and 2, as ECHO standards are marked with an “S”.

Figure 5

Hierarchical cluster analysis of the concentrations of antioxidants and antioxidant precursors in commercial beer samples scaled logarithmically. Numbering of compounds refers to chemical structures given in Figure 1.

Figure 6

(A) Score plot after PCA of beer samples stored under oxygen atmosphere and investigated by UPLC-TOF-MS, plotting the first two principal components. (B) S-plot calculated after OPLS-DA using the UPLC-TOF-MS data, with the p1-value of the loadings on the x-axis against the correlation between peak area and classification (fresh beer as −1 and beer aged for 4 weeks as +1) on the y-axis, revealing the marker compounds trans-isohumulones (50a–c), cis-isohumulones (51a–c), hydroxyl-trans-alloisohumulones (52a–c), and hydroxyl-cis-alloisohumulones (53a–c).

Figure 7

Concentrations of 51b (A) and 53b (B) in beer samples spiked with antioxidants () and then stored at 40 °C for 7 days () and 21 days (). Numbering and constituents of batches are given in Table 3.