Toward Unraveling Grape Tannin Composition: Application of Online Hydrophilic Interaction Chromatography × Reversed-Phase Liquid Chromatography–Time-of-Flight Mass Spectrometry for Grape Seed Analysis

Abstract

Despite the significant importance of tannins in viticulture and enology, relatively little is known about the detailed chemical composition of these molecules. This is due to challenges associated with the accurate analytical determination of the highly structurally diverse proanthocyanidins which comprise tannins. In this contribution, we address this limitation by demonstrating how online comprehensive two-dimensional liquid chromatography (LC × LC) coupled to high resolution mass spectrometry (HR-MS) can be exploited as a powerful analytical approach for the detailed characterization of grape seed tannins. Hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RP-LC) were employed in the two dimensions to provide complementary information in terms of separation according to hydrophilicity and hydrophobicity, respectively. Online coupling of HILIC × RP-LC with fluorescence detection and electrospray ionization MS delivered high resolution analysis in a practical analysis time, while allowing selective detection and facilitating compound identification. Time-of-flight (TOF) MS provided high acquisition rates and sensitivity coupled to accurate mass information, which allowed detection of procyanidins up to a degree of polymerization (DP) of 16 and a degree of galloylation up to 8 in a red grape seed extract. This analytical methodology promises to shed new light on these important grape constituents and potentially on their evolution during wine production.

Theory and Practice of UHPLC and UHPLC–MS

“…Later, the performance of off-line, online stop-flow, and comprehensive HILIC chromatography combined with RPLC was compared in terms of peak capacities, analysis times, and peak production rates (Kalili and de Villiers, 2013). An online comprehensive LC Â LC system is best for samples requiring peak capacities up to 600.…”

“…The compatibility of the mobile phases used in 2D HILIC-RP systems can be improved in several ways (Kalili and de Villiers, 2013):…”

“…In this regard, remarkable efforts have been made to extend the pressure capability of the pumping systems to 1300 bar. In addition, compared to old generation HPLC instruments, the contribution of the system to band broadening was dramatically reduced by using short and narrow connecting tubes, low injected volumes, small UV-cell volumes, and fast acquisition rates Jandera, 2012, 2016;Krol-Kogus et al, 2014;de Villiers, 2009, 2010;Beelders et al, 2015;Kalili et al, 2013;Montero et al, 2014;Willemse et al, 2014Scoparo et al, 2012).…”

“…On the one hand, for NP  RP separation of carotenoids, either silica or cyanopropyl (CN) stationary phases have been investigated (Dugo et al , 2008d(Dugo et al ,e, 2009cCacciola et al, 2012Cacciola et al, , 2016a, whereas for triacylglycerol (TAG) separation, an Ag  RP combination turned out to be useful for this task Dugo et al, 2006b,c;Van der Klift et al, 2008;Hu et al, 2013;Wei et al, , 2015Costa et al, 2015;. On the other hand, for RP  RP separation of phenolic antioxidants, the effect of several stationary phase chemistries (C8, C18, CN, Diol, F 5 , NH 2 , PEG, Phenyl, RPAmide, and other laboratory-made stationary phases) on the separation selectivity and resolution has been investigated (Blahovà et al, 2006;Cacciola et al, 2006Cacciola et al, , 2007aCacciola et al, ,b,c, 2011Hyilompolo, 2007, 2008;Pol et al, 2007, Pol andDugo et al, 2008cDugo et al, , 2009aJandera et al, 2008aJandera et al, ,b, 2012Hájek et al, 2008Cesla et al, 2009;Kalili and de Villiers, 2009, 2013aZhou et al, 2009;Russo et al, 2011;Fu et al, 2012;Hájek and Jandera, 2012;Kalili et al 2013Kalili et al , 2014Montero et al, 2013aMontero et al, ,b, 2014Montero et al, , 2016aKrol-Kogus et al, 2014;Krauze-Baranowska et al, 2014;Leme et al, 2014;Tanaka et al, 2014;…”

“…For example, 2D HILIC Â RP separation of phenolic compounds in green tea was reported using a diol HILIC column in the first dimension and a gradient of water and methanol in acetonitrile with acetic acid additive. 50 mL fractions were collected with 1 min frequency and evaporated under nitrogen to 2 mL before introduction onto a C18 column in the second dimension (Kalili and de Villiers, 2013).…”