Food Chemistry 204 (2016) 400–408

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Food Chemistry

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Determination of benzoxazinoids in wheat and rye by HPLC-DAD and UPLC-QTOF MS ⇑ Juha-Matti Pihlava , Tuula Kurtelius

Natural Resources Institute Finland, 31600 Jokioinen, Finland article info abstract

Article history: Phenolic compounds in have received considerable interest. Besides the more typical phenolic acids Received 14 October 2015 and flavonoids, beer contains also lesser-known compounds, such as hordatines, their agmatine precur- Received in revised form 1 February 2016 sors and other phenolamines. Current work shows that beers brewed from wheat or rye , in addition Accepted 25 February 2016 to malts, contain benzoxazinoids, a group of nitrogen containing secondary metabolites typical to wheat and rye. In this work, HPLC-DAD was used for the quantification of major benzoxazinoids in 32 wheat and four rye beers. Of the wheat beers 22 samples and all of the rye beers contained benzoxazi- Keywords: noids, or their breakdown products. Concentrations of DIBOA (2,4-dihydroxy-1,4-benzoxazin-3-one) Beer (as aglycon) varied from 1.7 to 21.9 mg/l in wheat beers and from 5.6 to 31.6 mg/l in rye beers. Wheat Rye Breakdown products BOA (benzoxazolin-2-one), found in 15 beers, and MBOA (6-methoxy- Benzoxazinoids benzoxazolin-2-one), found in two beers, were measured at concentrations ranging from 2.4 to DIBOA 10.7 mg/l and 8.4 to 10.5 mg/l, respectively. BOA Identification of benzoxazinoids by UPLC-QTOF MS was done on selected beers. Benzoxazinoid profiles MBOA varied greatly between different wheat beers, and compared to rye beers the chemical diversity of ben- zoxazinoids was higher. As far as the authors know, this is the first time that other benzoxazinoids, rather than just the decomposition products BOA or MBOA, have been reported in beer. The results also show that benzoxazinoids can be present in beer glycosylated with three or four hexose units. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction (Quifer-Rada et al., 2015), as well as various forms of hordatines, their hydroxycinnamoyl agmatine precursors and other pheno- Beer can be considered as a relatively good source of phenolic lamides (Pihlava, 2014), have been reported in the literature. compounds in the human diet (Callemien & Collin 2010; Nardini, Polyphenols in beer can either originate from the , or as in Natella, Scaccini, & Ghiselli 2006; Zamora-Ros et al. 2013). Various the case of hordatines, from barley . By using wheat or rye phenolic compounds have been identified in beer, including phe- malt in addition to barley malt in , a special group of com- nolic acids and their conjugates, flavonoids, phenolamides and hor- pounds, namely benzoxazinoids, can be introduced into beers. datines (Ceslová, Holcapek, Fidler, Dršticková, & Lísa, 2009, Nardini Benzoxazinoids are nitrogen-containing secondary metabolites & Ghiselli, 2004; Pihlava, 2014; Quifer-Rada et al., 2015). Of these, found in poaceaous plants, such as rye, wheat, triticale and maize, phenolic acids have been a particular subject of interest, in a num- where they act as defense compounds against pathogens and ber of studies, due to their antioxidant properties and because fer- insects (Niemeyer, 2009). The chemical structures of benzoxazi- ulic acid can be a potential flavor precursor in beers (Callemien & noids, mentioned in this article, are presented in Fig. 1. Dietary Collin, 2010; Inns, Buggey, Booer, Nursten, & Ames, 2011; benzoxazinoids are absorbed and metabolized in humans Piazzon, Forte, & Nardini, 2010; Szwajgier, 2009; Vanbeneden, (Adhikari et al., 2013; Beckmann et al., 2013; Hanhineva et al., Gils, Delvaux, & Delvaux, 2007; Vanbeneden, Van Roey, Willems, 2014) and they are of special interest because of their possible pos- Delvaux, & Delvaux, 2008). Despite that number of phenolic com- itive health effects as reviewed by Adhikari et al. (2015). pounds have been characterized in beers over the years, just Benzoxazinoids are stored in plant cells as glucosides, but once recently, compounds, such as feruloylquinic acid, hexosides of caf- the cell structure is ruptured by herbivores or plant pathogens, the feic, coumaric and sinapic acids and certain flavonoid glycosides active aglycons, DIBOA (2,4-dihydroxy-1,4-benzoxazin-3-one) and/or DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one),

⇑ Corresponding author. are released enzymatically. Cyclic hydroxamic acids DIBOA and E-mail address: juha-matti.pihlava@luke.fi (J.-M. Pihlava). DIMBOA are then spontaneously decomposed to benzoxazolinones http://dx.doi.org/10.1016/j.foodchem.2016.02.148 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved. J.-M. Pihlava, T. Kurtelius / Food Chemistry 204 (2016) 400–408 401

Fig. 1. Structures of benzoxazinoids and their breakdown products mentioned in this article. DIBOA (2,4-dihydroxy-1,4-benzoxazin-3-one) and DIMBOA (2,4-dihydroxy- 7-methoxy-1,4-benzoxazin-3-one) are hydroxamic acids, HBOA (2-hydroxy-1,4-benzoxazin-3-one), HMBOA (2-hydroxy-7-methoxy-1,4-benzoxazin-3-one), DHBOA (2,7-dihydroxy-1,4-benzoxazin-3-one) and DHMBOA (8-OH-HMBOA) (2,8-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) are lactams, BOA (benzoxazolin-2-one) and MBOA (6-methoxy-benzoxazolin-2-one) are benzoxazolinones and HHPAA (2-hydroxy-N-(2-hydroxyphenyl)-acetamide) and HPAA (N-(2-hydroxyphenyl)-acetamide) are acetamides. The structure of 8-OH-HMBOA is hypothetical, not reported in the literature earlier.

BOA (benzoxazolin-2-one) and MBOA (6-methoxy-benzoxazolin- graphic system connected to quadrupole time-of-flight mass 2-one) (Niemeyer, 2009). Further decomposition products are, for spectrometer (UPLC-QTOF MS). example, HPAA (N-(2-hydroxyphenyl)-acetamide) and HHPAA (2- hydroxy-N-(2-hydroxyphenyl)-acetamide), as well as numerous 2. Materials and methods other transformation products known to be formed by soil microbes (Fomsgaard, Mortensen, & Carlsen, 2004). 2.1. Chemicals and reagents Only recently has it been acknowledged that benzoxazinoids are also part of our diet via the consumption of rye- or wheat- Methanol was of LC–MS-quality (VWR International, Leuven, containing food products, such as (Beckmann et al., 2013; Belgium). Deionized water was purified in a Milli-Q water purifica- Hanhineva et al., 2014; Pedersen, Laursen, Mortensen, & tion system (Millipore, Bedford, MA, USA). Acetonitrile and water Fomsgaard, 2011; Tanwir, Fredholm, Gregersen, & Fomsgaard, containing 0.1% formic acid were of LC–MS ultra-quality (Sigma– 2013), although the concentrations of benzoxazinoids in wheat Aldrich, St. Louis, MO, USA). are much lower than in rye (Pedersen et al., 2011; Tanwir et al., BOA and MBOA were purchased from Sigma–Aldrich (St. Louis, 2013). The main form of benzoxazinoid in rye grains is the MO, USA). DIBOA used for quantification was isolated at Natural dihexoside of DIBOA (Hanhineva et al., 2011; Tanwir et al., 2013). Resources Institute Finland (formerly MTT Agrifood Research Fin- After fermentation and baking of commercial whole-grain land) in Jokioinen from germinated rye grains (Kurri 2007). The sourdough rye , the order of benzoxazinoids (as peak purity of DIBOA (>95%) was checked by LC–MS, HPLC-DAD, 1H intensity in arbitrary units) is reported to change as follows: NMR and 13C NMR (Kurri 2007) and confirmed by inter- DIBOA-hexoside > DIBOA-dihexoside > BOA > HBOA (2-hydroxy-1, laboratory comparison. 4-benzoxazin-3-one)-dihexoside > HBOA-hexoside > HPAA. In wheat bread enriched with rye bran, the benzoxazinoids are in the order 2.2. Samples of signal intensities: DIBOA-hexoside > DIBOA-dihexoside > BOA = HHPAA > HPAA > HBOA-dihexoside > HBOA-hexoside (Hanhineva Wheat (n = 32) and rye beers (n = 4) were purchased from local et al., 2014). grocery stores or in case the alcohol content was above 4.7%, from While it is currently widely known that rye and wheat breads the national alcoholic beverage retailing monopoly in Finland (Alko contain benzoxazinoids, the only references, so far, relating to ben- Inc). One of the rye beers was obtained from a brew pub (Tampere, zoxazinoids in beer have been made by Pothou, Melliou, Finland). All beers also contained barley malt as an ingredient, Skaltsounis, Liouni, and Magiatis (2013) and Manoukian, Melliou, except one low-alcohol sample (-beer ID 124) in which only Liouni, and Magiatis (2016). The decomposition products BOA rye malt was used. All the beers in the analysis were top- and MBOA were found as minor volatile compounds in wheat fermented, -type beers, except for one wheat (ID 3) and one beers, especially in those including malted wheat. Because the con- rye beer (ID 164) that were bottom-fermented -type beers. tent of benzoxazinoids increases during germination of rye and Information about the alcohol by volume (ABV), country of origin wheat grains (Katina et al., 2007; Pedersen et al., 2011), it is not and cereal ingredients of the beer samples is listed in Table 1. very surprising that these compounds can also be found in rye and wheat beers. 2.3. Sample preparation The aim of this study was to provide quantitative data on the major benzoxazinoids in 32 wheat and four rye beers by high per- An aliquot of sample was taken from a bottled beer and trans- formance liquid chromatography with diode array detector (HPLC- ferred to a plastic centrifuge tube and stored in a freezer. Prior to DAD) and to tentatively identify the various forms of benzoxazi- analysis the samples were thawed at room temperature. For noids in selected beers using ultra-performance liquid chromato- HPLC-DAD analysis, samples were filtered through a 0.45 lm PTFE Download English Version: https://daneshyari.com/en/article/7589111

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