Characterization of grape seed procyanidins by comprehensive two-dimensional hydrophilic interaction × reversed phase liquid chromatography coupled to diode array detection and tandem mass spectrometry

Abstract

In this work, the development and optimization of a new methodology to analyze grape seed procyanidins based on the application of two-dimensional comprehensive LC is presented. This two-dimensional method involves the use of a microbore column containing a diol stationary phase in the first dimension coupled to either a C18 partially porous short column or a C18 monolithic column in the second dimension. The orthogonal hydrophilic interaction × reversed phase liquid chromatography (HILIC×RP-LC) system is interfaced through a ten-port two-position switching valve. The optimized HILIC×RP-LC separation followed by diode array and tandem mass spectrometry detection (HILIC×RP-LC-DAD-MS/MS) made possible the direct analysis of a complex grape seed extract and allowed the tentative identification of 43 flavan-3-ols, including monomers and procyanidin oligomers till a polymerization degree of 7 units with different galloylation degrees. To the best of our knowledge, this is the first time that this powerful analytical technique is employed to characterize complex procyanidin samples. This work successfully demonstrates the great capabilities of the HILIC×RP-LC-DAD-MS/MS coupling for the direct analysis of very complex natural samples like grape seeds.

Theory and Practice of UHPLC and UHPLC–MS

“…The unique performance of these particle columns, offering the advantages of higher efficiencies and rugged design, capable of high-pressure operation, was demonstrated in some studies Marchetti et al, 2007;Cavazzini et al, 2007;Cunliffe and Maloney, 2007;Donato et al, 2009). Because of these attributes, these columns were chosen as 2 D of LC Â LC systems, and several research groups (Mondello, Dugo, Jandera and Cifuentes) used them in natural products applications (Hajek et al, 2008;Dugo et al, 2008cDugo et al, , 2009aRusso et al, 2011;Mondello et al, 2011;Cacciola et al, 2012;Leme et al, 2014;Jandera, 2012;Cesla et al, 2009;Hájek and Jandera, 2012;Montero et al 2013aMontero et al ,b, 2014Montero et al , 2016aDonato et al, 2016). In two of the abovementioned references (Dugo et al, 2008c;Hajek et al, 2008) the performance of a partially porous short column was compared with that of a monolithic column.…”

“…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;…”

“…The combination of HILIC and RP-LC promises higher orthogonality with respect to RP-LC Â RP-LC, although hyphenation of the former modes is more complicated because of the relative elution strengths of the mobile phase used in each dimension. For such a reason, the employment of microflow rates in the 1 D is highly beneficial to minimize dilution and provide flow rates compatible with 2 D injection volumes (Kalili and de Villiers, 2009, 2013aBeelders et al, 2015;Kalili et al, 2013Kalili et al, , 2014Montero et al, 2013aMontero et al, ,b, 2014Montero et al, , 2016a. As an example in Fig.…”

Large-scale qualitative and quantitative characterization of components in Shenfu injection by integrating hydrophilic interaction chromatography, reversed phase liquid chromatography, and tandem mass spectrometry

“…Column-switching chromatographic separation shows significant improvement in separation potency and peak capacity in comparison with single-column LC analysis attributing to the integration of different separation modes [12,14,15]. Hydrophilic interaction liquid chromatography (HILIC) provides an alternative approach to effectively separate small polar compounds on polar stationary phases as normal phase LC, but the mobile phase used for HILIC is similar to those employed in the reversed phase LC (RPLC) mode [16]; hence, it has drawn increasing interests that the combination of HILIC and RPLC offers great potential for "entire-components" separation of complex matrix attributing to separation orthogonality and mobile phase compatibility between these two modes [15][16][17][18][19][20][21][22][23][24]. However, it is still problematic in practice to achieve online hyphenation of RP and HILIC columns because the stronger elution solvent in HILIC will be the weaker one in RPLC, and vice versa [24].…”

“…Hydrophilic interaction liquid chromatography (HILIC) provides an alternative approach to effectively separate small polar compounds on polar stationary phases as normal phase LC, but the mobile phase used for HILIC is similar to those employed in the reversed phase LC (RPLC) mode [16]; hence, it has drawn increasing interests that the combination of HILIC and RPLC offers great potential for "entire-components" separation of complex matrix attributing to separation orthogonality and mobile phase compatibility between these two modes [15][16][17][18][19][20][21][22][23][24]. However, it is still problematic in practice to achieve online hyphenation of RP and HILIC columns because the stronger elution solvent in HILIC will be the weaker one in RPLC, and vice versa [24]. In order to circumvent the bottleneck, off-line connection that the effluents of the first RP-C 18 or HILIC column are severally collected, concentrated, reconstituted, and re-injected into the second HILIC or RP-C 18 column, has been extensively constructed [14,15,25], however, indicating labor-and time-consuming procedures.…”