HPLC-qTOF-MS/MS-Based Profiling of Flavan-3-ols and Dimeric Proanthocyanidins in Berries of Two Muscadine Grape Hybrids FLH 13-11 and FLH 17-66

Abstract

FLH 13-11 FL and FLH 17-66 FL are two interspecific hybrid varieties of muscadine grape resulting from the cross of Vitis munsoniana (Simpson) ex Munson and V. rotundifolia. However, profiles of flavan-3-ols and proanthocyanidins in these two hybrids have not been characterized. Herein, we report the use of high-performance liquid chromatography-quadrupole, time-of-flight, tandem mass spectrometry (HPLC-qTOF-MS/MS) to characterize these two groups of metabolites in berries. Ripe berries collected from two consecutive cropping years were used to extract metabolites. Metabolites were ionized using the negative mode. Collision-induced dissociation was performed to fragmentize ions to obtain feature fragment profiles. Based on standards, MS features, and fragments resulted from MS/MS, four flavan-3-ol aglycones, 18 gallated or glycosylated conjugates, and eight dimeric procyanidins, were annotated from berry extracts. Of these 30 metabolites, six are new methylated flavan-3-ol gallates. Furthermore, comparative profiling analysis showed obvious effects of each cultivar on the composition these 30 metabolites, indicating that genotypes control biosynthesis. In addition, cropping seasons altered profiles of these metabolites, showing effects of growing years on metabolic composition. These data are informative to enhance the application of the two cultivars in grape and wine industries in the future.

Keywords: muscadine; proanthocyanidins; tandem mass spectrometry.

Figure 1

Schemes of flavan-3-ols (monomers) and dimeric proanthocyanidins: (a) four types of stereo configurations of different monomeric flavan-3-ols, such as (+)-catechin, (−)-epicatechin, (−)-epigallocatechin, and (−)-epicatechin; (b) two examples of dimeric proanthocyanidins, procyanidin A2 (A-type) and procyanidin B1 (B-type).

Figure 2

Comparison of metabolite peak profiles between berry extracts of FLH 13-11 and FLH 17-66 in two years and nine standards. Metabolite peaks detected by HPLC were recorded at 280 nm. (a) Two chromatograms show peaks annotated to be flavan-3-ols and dimeric PAs in berries of FLH 13-11 from the 2011 and 2012 cropping years. (b) Two chromatograms show peaks annotated to be flavan-3-ols and dimeric PAs in berries of FLH 17-66 from the 2011 and 2012 cropping years. (c) A chromatogram shows retention times of nine standards including five flavan-3-ol aglycones, two conjugates, and two dimeric PA standards (procyanidin B1 and B2). Compound peaks detected in one variety only and two varieties are highlighted with red and black color, respectively. Standards and abbreviations, (+)-catechin (Cat), (−)-epicatechin (EpiCat), (−)-gallocatechin (gCat), (−)-epigallocatechin (EpigCat), (−)-catechin gallate (CatG), (−)-epigallocatechin gallate (EpigCatG), (−)-gallocatechin gallate (gCatG), procyanidin B1 (Proc B1), and procyanidin B2 (Proc B2). Abbreviation for compounds annotated by HPLC-qTOF-MS/MS analysis described below, (+)-Cat3Glu: (+)-catechin 3-glucoside, (−)-Cat3Glu: (−)-catechin 3-glucoside, (+)-EpiCat: (+)-epicatechin, (+)-gCat3Glu: (+)-gallocatechin 3-glucoside, (−)-gCat3Glu: (−)-gallocatechin 3-glucoside, (+)-EpigCat3Glu: (+)-epigallocatechin 3-glucuside, (−)-EpigCat3Glu: (−)-epigallocatechin 3-glucoside, OMe(+)-CatG: Methyl-O-(+)-catechin-3-gallate, OMe(−)-CatG: Methyl-O-(−)-catechin-3-gallate, OMe(−)-gCatG: Methyl-O-(−)-gallocatechin-3-gallate, OMe(+)-EpigCatG: Methyl-O-(−)-epigallocatechin-3-gallate, OMe(−)-EpigCatG: Methyl-O-(−)-epigallocatechin-3-gallate, and OMe(+/−)-EpiCatG: Methyl-O-(+/−)-epicatechin-3-gallate. Proc B4, B5, B6, B7, B8, and A2: procyanidin B4, B5, B6, B7, B8, and A2.

Figure 3

Extracted ion chromatogram (EIC) of primary mass spectrum (MS1) and m/z features of secondary ion fragments (MS2) derived from HPLC-qTOF-MS/MS annotate this peak to be (+)-catechin, (MW, 290.26). (a) Chromatogram of FLH 13-11 extract (2012) recorded at 280 nm, (b) total ion chromatogram of FLH 13-11 extract (2012), (c) EIC of primary ion 289.1574 [M − H]⁻, (d) enhanced charge capacity (ECC) ion product for m/z 289.1574, (e) a MS profile showing an extracted m/z 289.1569, and (f) fragments from collision-induced dissociation (CID) of m/z 289.1569 showing m/z 109.0815, 121.0136, 123.1001–125.0089, 137.0078, 145.0888–151.0230, 159.0238–164.0868, 173.0911, 191.1035, 203.1362, 219.1033, 229.092, and 247.1028 (Table 1).

Figure 4

Extracted ion chromatogram (EIC) of primary mass spectrum (MS1) and m/z features of secondary ion fragments (MS2) derived from LC-MC/MS annotate this peak to be (−)-epicatechin (MW, 290.26). (a) Chromatogram of FLH 17-66 extract (2012) recorded at 280 nm; (b) total ion chromatogram of FLH 17-66 extract (2012); (c) EIC of primary ion 289.0357 [M − H]¯; (d) enhanced charge capacity (ECC) ion product for m/z 289.0357; (e) a MS profile showing an extracted m/z value, 289.0359; and (f) fragments from collision-induced dissociation (CID) of m/z 289.0359 showing m/z 109.0068, 123.0213, 136.9995, 151.0132, 203.0421, and 246.9843 (Table 1).

Figure 5

Extracted ion chromatogram (EIC) of primary mass spectrum (MS1) and m/z features of secondary ion fragments (MS2) derived from LC-MC/MS annotate this peak to be (+)-catechin 3-O-glucoside (MW, 452.41). (a) Chromatogram of FLH 13-11 extracts (2012) recorded at 280 nm; (b) total ion chromatogram of FLH 13-11 extracts (2012); (c) EIC of primary ion 451.1255 [M − H]¯; (d) enhanced charge capacity (ECC) ion product for m/z 451.1255; (e) a MS profile showing an extracted m/z value, 451.1256 and (f) fragments from collision-induced dissociation (CID) of m/z 451.1256 showing m/z 109.0471, 110.9518, 125.0775, 149.0708, 169.0195, and 247.1017 (Table 1).

Figure 6

Extracted ion chromatogram (EIC) of primary mass spectrum (MS1) and m/z features of secondary ion fragments (MS2) derived from LC-MC/MS annotate this peak to be (−)-epicatechin 3-O-glucoside (MW, 452.41). (a) Chromatogram of FLH 17-66 extracts (2012) recorded at 280 nm; (b) total ion chromatogram of FLH 17-66 extract (2012); (c) EIC of primary ion 451.0918 [M − H]¯; (d) enhanced charge capacity (ECC) ion product for m/z 451.0918; (e) a MS profile showing an extracted m/z value, 451.0368; and (f) fragments from collision-induced dissociation (CID) of m/z 451.0368 showing m/z 124.9861, 172.9705, 244.9449, 272.9346, 286.9428, and 300.9102 (Table 1).

Figure 7

Extracted ion chromatogram (EIC) of primary mass spectrum (MS1) and m/z features of secondary ion fragments (MS2) derived from LC-MC/MS annotate this peak to be (+)-catechin gallate (MW, 442.37). (a) Chromatogram of FLH 17-66 extracts (2012) recorded at 280 nm absorbance; (b) total ion chromatogram of FLH 17-66 extract (2012); (c) EIC of primary ion 441.0366 [M − H]¯; (d) enhanced charge capacity (ECC) ion product for m/z 441.0366; (e) a MS profile showing an extracted m/z value, 441.0378; and (f) fragments from collision-induced dissociation (CID) of m/z 441.0378 showing m/z 109.0071, 125.0001, 136.9989, 151.0130, 179.0047, 245.0485, and 289.0349 (Table 1).

Figure 8

Extracted ion chromatogram (EIC) of primary mass spectrum (MS1) and m/z features of secondary ion fragments (MS2) derived from LC-MC/MS annotate this peak to be (−)-epicatechin gallate (MW, 442.37). (a) Chromatogram of FLH 17-66 extract (2012) recorded at 280 nm; (b) total ion chromatogram of FLH 17-66 extract (2012); (c) EIC of primary ion 441.0366 [M − H]¯; (d) enhanced charge capacity (ECC) ion product for m/z 441.0366; (e) a MS profile showing an extracted m/z value, 441.0362; and (f) fragments from collision-induced dissociation (CID) of m/z 441.0362 showing m/z 109.0065, 123.0209, 137.0003, 151.0112, 179.0064, 245.0452, 271.0298, 289.0319, 313.9907, 331.9910, and 358.9829 (Table 1).

Figure 9

Extracted ion chromatogram (EIC) of primary mass spectrum (MS1) and m/z features of secondary ion fragments (MS2) derived from LC-MC/MS annotate this peak to be procyanidin A2 (MW, 576.51). (a) Chromatogram of FLH 17-66 extract (2012) recorded at 280 nm; (b) total ion chromatogram of FLH 17-66 extract (2012); (c) EIC of primary ion 575.0652 [M − H]¯; (d) enhanced charge capacity (ECC) ion product for 575.0652 m/z; (e) A MS profile showing an extracted m/z value, 575.0652; and (f) fragments from collision-induced dissociation (CID) of m/z 575.0652 showing m/z 109.0070, 124.9985, 136.9988, 152.9935, 168.9837, 242.9965, 270.9877, 285.0011, 296.9985, 327.0119, and 425.0363 (Table 1).

Figure 10

Extracted ion chromatogram (EIC) of primary mass spectrum (MS1) and m/z features of secondary ion fragments (MS2) derived from LC-MC/MS annotate this peak to be procyanidin B2 (MW, 578.52). (a) Chromatogram of FLH 17-66 extract (2012) recorded at 280 nm; (b) total ion chromatogram of FLH 17-66 extract (2012); (c) EIC of primary ion 577.0828 [M − H]¯; (d) enhanced charge capacity (ECC) ion product for m/z 577.0828; (e) a MS profile showing an extracted m/z value, 577.0620; and (f) fragments from collision-induced dissociation (CID) of m/z 577.0620 showing m/z 109.0032, 124.9960, 136.9946, 151.0073, 160.9936, 245.0374, 289.0235, 407.0181, and 425.0330 (Table 1).

Figure 11

A Venn diagram showing effects of cultivars and cropping years on formation of 30 annotated metabolites. Arabic numerals are flavaan-3-ols and dimeric proanthocyanidins extracted from samples. Yellow and blue colors represent FLH13-11 and FLH 17-66, respectively.