HRMSn Profiling Method To Identify and Quantify

Jul 17, 2014 - A computational tool for accelerated analysis of oligomeric proanthocyanidins in plants. Mengliang Zhang , Jianghao Sun , Pei Chen. Jou...
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UHPLC-PDA-ESI/HRMSn Profiling Method To Identify and Quantify Oligomeric Proanthocyanidins in Plant Products Long-Ze Lin,*,† Jianghao Sun,† Pei Chen,† Maria J. Monagas,§ and James M. Harnly† †

Food Composition and Methods Development Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Service, U.S. Department of Agriculture, Building-161, BARC-East, 10300 Baltimore Avenue, Beltsville, Maryland 20705, United States § United States Pharmacopeia, 12601 Twinbrook Parkway, Rockville, Maryland 20852, United States ABSTRACT: Oligomeric proanthocyanidins were successfully identified by UHPLC-PDA-HRMSn in a selection of plantderived materials (jujube fruit, Fuji apple, fruit pericarps of litchi and mangosteen, dark chocolate, and grape seed and cranberry extracts). The identities of 247 proanthocyanidins were theoretically predicted by computing high-accuracy masses based on the degree of polymerization, flavan-3-ol components, and the number of A type linkages and galloyls. MSn fragments allowed characterization on flavan-3-ol based on the monomer, connectivity, and location of A-type bonds. Identification of doubly or triply charged ions of 50 PAs was made on the basis of theoretical calculations. A single catechin standard and molar relative response factors (MRRFs) were used to quantify the well-separated PAs. The ratios of the SIM peak counts were used to quantify each of the unseparated isomers. This is the first report of direct determination of each of the proanthocyanidins in plant-derived foods and proanthocyanidins containing an epifisetinidol unit in grape seeds. KEYWORDS: oligomeric proanthocyanidins, identification, quantification, plant products, UHPLC-PDA-ESI/HRMSn profiling method

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INTRODUCTION Proanthocyanidins (PAs) are various length polymers of flavanols (catechins and their enantiomers) linked through a single C4−C8 or C4−C6 bond (B-type PAs) or with an additional C2−O−C7 or C2−O−C5 bond (A-type PAs) as shown in Figure 1. There are a variety of different classes of PAs, depending on the substitution pattern of the monomeric flavan-3-ols (mainly epicatechins, epigallocatechins, and epiafzelechins to form procyanidins, propelargonidins, and prodelphinidins), acyls (usually galloyl), glycosyls, and other substituents.1−4 The highly polymerized PAs are reported to have molecular weights up to 30000 Da. However, these PAs may not be efficiently extracted from plant materials.1−3 PAs are the main polyphenolic components in many different plant-derived foods, such as grains, berries, fruits, nuts, and teas, and are reported to have a variety of health-promoting benefits.1−7 As the degree of polymerization increases, the compounds become less soluble in aqueous solution and less bioavailable in the intestine. Fermentation in the colon, however, leads to absorption of many of the metabolic products. The most absorbed PAs in the intestine have a degree of polymerization (DP) less than or equal to 4 (DP ≤ 4).1−7 Accurate analytical methods for the separation, identification, and quantification of individual oligomeric PAs in foods are necessary to establish the relationship between dietary intake of polyphenols and health outcomes from biological, epidemiological, and clinical studies. PAs have a high structural diversity with many regioisomeric (order of linkage for the flavan-3-ols) and stereoisomeric (physical structure of individual flavan-3-ols) forms, which makes identification and quantification difficult tasks. In general, analytical methods have focused on each oligomer as © 2014 American Chemical Society

a class and have been unable to identify the PAs within each class. Matrix-assisted laser desorption ionization−time-of-flightmass spectrometry (MALDI-TOF-MS) has been used to detect PA metal adducts and to determine the types and DP values of the compounds.1,8−12 ESI-MSn has also been used to identify PA molecular ions and their fragments.8,13−23 However, neither of these methods can identify the PA isomers.1,7−16 Normal and reverse phase HPLC methods have been used to separate PA oligomers and tandem MS has been used to characterize the PAs for DP ≤ 6 (typically m/z 50− 2000).1,12,19−23 Doubly and triply charged negative molecular ions of some higher oligomers (DP > 6) have been detected using negative ionization.1,4,14−19 Reverse phase HPLC-PDSMS analysis of thiolytically degraded products of PAs has been used to identify the PA terminal (with the C8 connection) and extension units (with the C4 connection) and to determine the mean DP value (mDP).1−4,11−17 Both 1H and 13C NMR analyses have been used to identify PA flavan-3-ols and the cis or trans stereochemistries of PAs.10,11 Until now, however, there has been only limited application of UHPLC-HRMSn to the study of oligomeric PAs.20−23 Total PA concentration has been estimated using colorimetric methods. In addition, total concentrations for each oligomeric class (DP = 2−10) have been estimated using fluorescence detection and relative response factors (based on mass) following separation by normal phase chromatography.2−4,6 HPLC-PDA-MS analysis of PA thiolytic degraded Received: Revised: Accepted: Published: 9387

February 27, 2014 July 17, 2014 July 17, 2014 July 17, 2014 dx.doi.org/10.1021/jf501011y | J. Agric. Food Chem. 2014, 62, 9387−9400

Journal of Agricultural and Food Chemistry

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Plant Materials and Extraction. Fresh fruits of jujube (Ziziphus jujuba Mill), Fuji apple (Malus domestica Borkh cv. Fuji), litchi (Litchi chinensis Sonn.), and mangosteen (Garcinia mangostana Linn.) were purchased from local food stores. Dark chocolate was purchased from a local Trader Joes store in Maryland, USA. The extracts of grape seed and cranberry were kindly supplied by Triarco Industries, Inc. (Paterson, NJ, USA). The fruit pericarps of litchi and mangosteen and the skins of fresh jujubes and apples were lyophilized, and the dried materials were powdered.24−26 Each of the powdered fruit samples (250 mg) was extracted with 5.000 mL of a methanol/water (60:40, v/v) solvent using sonication for 60 min at room temperature. The slurry mixture was centrifuged at 2500 rpm for 15 min. The supernatant (4.000 mL) was taken from the tube and filtered through a 17 mm (0.45 μm) PVDF syringe filter (VWR Scientific, Seattle, WA, USA) for injections.24−26 A second extraction using acetone/methanol/water (2:2:1, v/v/v, 4.000 mL) was treated in the same way to check the extraction efficiency of the general extraction method. The result showed that >95% of the mass for each main compound was extracted from the plant material by the first extraction. Powdered chocolate samples (2000 mg) were extracted with 40 mL of the same aqueous methanol and treated as described above, and the supernatant was taken to dryness under vacuum at 40 °C. The approximately 30 mg of the residue was dissolved in water (1 mL) and passed through Sep-PakVac RC (500 mg) C18 cartridge (Waters Corp., Milford, MA, USA). After washing with water (5 mL), the PAs were eluted with methanol (5 mL) and again taken to dryness under vacuum. The residue was dissolved in 1.000 mL of the methanol/water solvent and filtered for injection. The grape seed (10.80 mg) and cranberry (10.20 mg) extracts were dissolved in the same aqueous methanol (1.0 mL) and filtered. Triplicate injections (1 μL) of each solution were used to determine the average concentration and the relative standard deviation for each of the PAs in the extract. Dried catechin was used as the external calibration standard; 4 mg was placed in a 10 mL volumetric flask, dissolved in the methanol/water (60:40, v/v) solvent, and brought to volume. This stock solution was diluted 1:4 and 1:16. The stock and each dilution were injected onto the column three times and used to construct a calibration curve. UHPLC-PDA-ESI/HRMSn Conditions. The UHPLC-HRMS system used consisted of an LTQ Orbitrap XL mass spectrometer with an Accela 1250 binary pump, a PAL HTC Accela TMO autosampler, a PDA detector (ThermoScientific, San Jose, CA, USA), and a G1316A column compartment (Agilent, Palo Alto, CA, USA). The separation was carried out on a U-HPLC column (200 mm × 2.1 mm i.d., 1.9 μm, Hypersil Gold AQ RP-C18) (Thermo- Scientific) with an HPLC/ UHPLC precolumn filter (UltraShield Analytical Scientific Instruments, Richmond, CA, USA) at a flow rate of 0.3 mL/min. The mobile phase consisted of a combination of A (0.1% formic acid in water, v/v) and B (0.1% formic acid in acetonitrile, v/v). The linear gradient was from 4 to 20% B (v/v) at 40 min, to 35% B at 60 min, and to 100% B at 61 min and held at 100% B to 65 min. The PDA recorded spectra from 200 to 700 nm and provided real-time monitoring at 280 and 330 nm.26 The HRMS was operated in the negative ionization mode using the following conditions: sheath gas at 70 (arbitrary units), aux and sweep gas at 15 (arbitrary units), spray voltage at 4.8 kV, capillary temperature at 300 °C, capillary voltage at 15 V, and tube lens at 70 V. The mass range was from m/z 50 to 2000 with a resolution of 15000, FTMS AGC target at 2e5, FT-MS/MS AGC target at 1e5, isolation width of 1.5 amu, and maximum ion injection time of 500 ms. The most intense ion was selected for the data-dependent scan to provide MS2 to MS5 product ions with a normalized collision energy at 35%.26 The selective ion monitoring (SIM) mode was used to select the molecular ions of the isomers from each of the PA groups in grape seed extract for their quantification.

Figure 1. Structures of flavan-3-ol units and common fragmentation patterns for proanthocyanidins.

mixtures has also been used for quantification of PAs.1−4,6,10−12 However, direct quantification of the different PAs comprising each oligomeric class is still problematic due to the difficulty of separation and the lack of standards.1−4 As a part of a project to systematically identify and quantify food phenolic compounds, a standardized HPLC-PDA-ESI/MS method was developed for the identification and quantification of food polyphenols, including some PAs.24 Quantification was based on UV absorbance and molar relative response factors (MRRFs).25 This method has been upgraded and now uses ultrahigh-performance liquid chromatography−photodiode array detection−high-resolution mass spectrometry operated in the tandem mode (UHPLC-PDA-ESI/HRMSn).26 In the current study, this method was employed to identify nearly 300 oligomeric PAs in selected plants (fruit pericarps of litchi and mangosteen), extracts (from grape seed and cranberry), and food samples (jujube, Fuji apple, and chocolate) and to quantify PAs in grape seed extract. The main PAs in each of the oligomeric classes were quantified.

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MATERIALS AND METHODS

Chemicals. Formic acid, HPLC grade methanol, and acetonitrile were purchased from VWR International, Inc. (Clarksburg, MD, USA). HPLC grade water was prepared from distilled water using a Milli-Q system (Millipore Laboratory, Bedford, MA, USA). Standards. (+)-Catechin, (−)-epicatechin, (−)-gallocatechin-3-Ogallate, (−)-epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, procyanidin C1, and procyanidin A2 were obtained from Chromadex, Inc. (Irvine, CA, USA). The standards were vacuumdried using a vacuum drying box (National Appliance Co., Portland, OR, USA) at 110 °C until a constant weight was reached (about 24 h). These dried standards were used to determine the MRRF that were used for calibration.25 9388

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Table 1. Computed High-Resolution Mass, Molecular Weight, Molecular Ions, and Composition of Common Oligomeric PAsa DP dimers

trimers

HRMW (Da)

HR [M − H]− (m/z)

C

H

O

EA/EF

B-type propelargonidin B-type proanthocyanidin B-type proanthocyanidin B-type procyanidin A-type procyanidin B-type procyanidin galloylated procyanidin galloylated procyanidin B-type proanthocyanidin B-type proanthocyanidin B-type prodelphinidin galloylated proanthocyanidin galloylated prodelphinidin

546.1518 560.1311 562.1467 578.1416 576.1260 578.1416 730.1524 882.1632 592.1209 594.1365 610.1314 746.1473

545.1440 559.1233 561.1389 577.1338 575.1182 577.1338 729.1446 881.1554 591.1131 593.1287 609.1236 745.1395

30 30 30 30 30 30 37 44 30 30 30 37

26 24 26 26 24 26 30 34 24 26 26 30

10 11 11 12 12 12 16 20 13 13 14 17

2 1 1 1 0 0 0 0 0 0 0 0

0 0 0 1 0 0 0 0 1 1 2 1

0 0 0 0 0 0 1 2 0 0 0 1

0 1 0 0 1 0 0 0 1 0 0 0

914.1530

913.1452

44

34

22

0

2

2

0

B-type propelargonidin A-type proanthocyanidin B-type proanthocyanidin A-type proanthocyanidin B-type proanthocyanidin galloylated proanthocyanidin A-type procyanidin A-type procyanidin B-type procyanidin galloylated procyanidin galloylated procyanidin B-type proanthocyanidin B-type proanthocyanidin B-type prodelphinidin galloylated prodelphinidin

818.2199 832.1992 834.2148 848.1941 850.2097 986.2256

817.2121 831.1914 833.2070 847.1863 849.2019 985.2178

45 45 45 45 45 52

38 36 38 36 38 42

15 16 16 17 17 20

3 2 2 1 1 2

0 0 0 0 0 0

0 0 0 0 0 1

0 1 0 1 0 0

862.1734 864.1890 866.2046 1018.2154 1170.2262 882.1995 898.1944 914.1893 1034.2103

861.1656 863.1812 865.1968 1017.2076 1169.2184 881.1917 897.1866 913.1815 1033.2025

45 45 45 52 59 45 45 45 52

34 36 38 42 46 38 38 38 42

18 18 18 22 26 19 20 21 23

0 0 0 0 0 0 0 0 0

0 0 0 0 0 1 2 3 1

0 0 0 1 2 0 0 0 1

2 1 0 0 0 0 0 0 0

60 60 60 60 60 60 60 60 67 74 60 60

48 50 48 50 44 46 48 50 54 58 48 50

22 22 23 23 24 24 24 24 28 32 25 25

2 2 1 1 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 1 1

0 0 0 0 0 0 0 0 1 2 0 0

1 0 1 0 3 2 1 0 0 0 1 0

proanthocyanidin

HR HR [M − 2H]2− [M − 3H]3− (m/z) (m/z)

EG galloyl

A-bond

tetramers

A-type proanthocyanidin B-type proanthocyanidin A-type proanthocyanidin B-type proanthocyanidin A-type procyanidin A-type procyanidin A-type procyanidin B-type procyanidin galloylated procyanidin galloylated procyanidin A-type proanthocyanidin B-type proanthocyanidin

1120.2622 1122.2778 1136.2571 1138.2727 1148.2208 1150.2364 1152.2520 1154.2676 1306.2784 1458.2892 1168.2469 1170.2625

1119.2544 1121.2700 1135.2493 1137.2649 1147.2130 1149.2286 1151.2442 1153.2598 1305.2706 1457.2814 1167.2391 1169.2547

pentamers

A-type proanthocyanidin B-type proanthocyanidin A-type proanthocyanidin B-type proanthocyanidin A-type procyanidin A-type procyanidin A-type procyanidin B-type procyanidin galloylated procyanidin B-type proanthocyanidin

1392.3303 1410.3408 1424.3201 1426.3357 1436.2838 1438.2994 1440.3150 1442.3306 1594.3414 1458.3255

1391.3225 1409.3330 1423.3123 1425.3279 1435.2760 1437.2916 1439.3072 1441.3228 1593.3336 1457.3177

695.1574 704.1626 711.1523 712.1601 717.1341 718.1419 719.1497 720.1575 796.1629 728.1550

75 75 75 75 75 75 75 75 82 75

60 62 60 62 56 58 60 62 66 62

27 28 29 29 30 30 30 30 34 31

3 2 l 1 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 1

0 0 0 0 0 0 0 0 1 0

1 0 1 0 3 2 1 0 0 0

hexamers

B-type proanthocyanidin A-type proanthocyanidin

1682.4089 1696.3882

1681.4011 1695.3804

840.1967 847.1863

90 90

74 72

33 34

3 2

0 0

0 0

0 1

9389

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Table 1. continued proanthocyanidin

HRMW (Da)

HR [M − H]− (m/z)

B-type proanthocyanidin A-type proanthocyanidin A-type proanthocyanidin B-type proanthocyanidin A-type procyanidin A-type procyanidin A-type procyanidin B-type procyanidin galloylated procyanidin B-type proanthocyanidin galloylated proanthocyanidin

1698.4038 1710.3675 1712.3831 1714.3987 1724.3468 1726.3624 1728.3780 1730.3936 1882.4044 1746.3885 1898.3993

1697.3960 1709.3597 1711.3753 1713.3909 1723.3390 1725.3546 1727.3702 1729.3858 1881.3966 1745.3807 1897.3915

848.1941 854.1760 855.1838 856.1916 861.1656 862.1734 863.1812 864.1890 940.1944 872.1865 948.1919

heptamers

A-type proanthocyanidin A-type proanthocyanidin B-type proanthocyanidin A-type procyanidin A-type procyanidin A-type procyanidin B-type procyanidin galloylared procyanidin A-type proanthocyanidin B-type proanthocyanidin

1980.4200 1996.4149 2002.4617 2012.4098 2014.4254 2016.4410 2018.4566 2170.4674 2032.4359 2034.4515

1979.4122 1995.4071 2001.4539 2011.4020 2013.4176 2015.4332 2017.4488 2169.4596 2031.4281 2033.4437

octamers

B-type proanthocyanidin B-type proanthocyanidin A-type procynidin A-type procynidin B-type procynidin galloylated procyanidin B-type proanthocyanidin

2274.5298 2290.5247 2302.4884 2304.5040 2306.5196 2454.4992 2322.5145

nonamers

B-type proanthocyanidin B-type proanthocyanidin A-type procyanidin B-type procyanidin galloylated procyanidin B-type proanthocyanidin

decamers

B-type proanthocyanidin B-type proanthocyanidin A-type procyanidin B-type procyanidin galloylated procyanidin B-type proanthocyanidin galloylated proanthocyanidin

DP

HR HR [M − 2H]2− [M − 3H]3− (m/z) (m/z)

C

H

O

EA/EF

570.4584 573.7745 574.4463 575.1182 575.7901 626.4603 581.1217 631.7920

90 90 90 90 90 90 90 90 97 90 97

74 70 72 74 68 70 72 74 78 74 78

34 35 35 35 36 36 36 36 40 37 41

2 1 1 1 0 0 0 0 0 0 0

EG galloyl 0 0 0 0 0 0 0 0 0 1 1

0 0 0 0 0 0 0 0 1 0 1

A-bond 0 2 1 0 3 2 1 0 0 0 0

989.2022 997.1997 1000.2231 1005.1971 1006.2049 1007.2127 1008.2205 1084.2259 1015.2102 1016.2180

659.1322 664.4638 666.4794 669.7955 670.4673 671.1392 671.8111 722.4813 676.4708 677.1427

105 105 105 105 105 105 105 112 105 105

80 80 86 80 82 84 86 90 84 86

40 41 41 42 42 42 42 46 43 43

2 1 1 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 1 1

0 0 0 0 0 0 0 1 0 0

3 3 0 3 2 1 0 0 1 0

2273.5220 2289.5169 2301.4806 2303.4962 2305.5118 2453.4914 2321.5067

1136.2571 1144.2546 1150.2364 1151.2442 1152.2520 1226.2418 1160.2495

757.1688 762.5004 766.4883 767.1602 767.8321 817.1586 773.1637

120 120 120 120 120 127 120

98 98 94 96 98 98 98

46 47 48 48 48 52 49

2 1 0 0 0 0 0

0 0 0 0 0 0 1

0 0 0 0 0 1 0

0 0 2 1 0 2 0

2562.5928 2578.5877 2592.5670 2594.5826 2742.5622 2610.5775

2561.5850 2577.5799 2591.5592 2593.5748 2741.5544 2609.5697

1280.2886 1288.2861 1295.2757 1296.2835 1370.2733 1304.2810

853.1898 858.5214 863.1812 863.8531 913.1796 869.1847

135 135 135 135 142 135

110 110 108 110 110 110

52 53 54 54 58 55

2 1 0 0 0 0

0 0 0 0 0 1

0 0 0 0 1 0

0 0 1 0 2 0

2850.6558 2866.6507 2878.6144 2882.6456 3030.6252 2898.6405 3050.6513

2849.6480 2865.6429 2877.6066 2881.6378 3029.6174 2897.6327 3049.6435

1424.3201 1432.3176 1438.2994 1440.3150 1514.3048 1448.3125 1524.3179

949.2108 954.5424 958.5303 959.8741 1009.2006 965.2057 1015.8760

150 150 150 150 157 150 157

122 122 118 122 122 122 126

58 59 60 60 64 61 65

2 1 0 0 0 0 0

0 0 0 0 0 1 1

0 0 0 0 1 0 1

0 0 2 0 2 0 0

Composition is used for the numbers of the atoms of carbon, hydrogen, and oxygen of the molecular formula and the numbers of the flavan-3-ol units, A-type bonds, and galloyls. Abbreviations: DP, degree of polymerization; G, galloyl; EC, EA, EG, epicatechin, epiafzelechin, and epigallocatechin, respectively; C, H, O, carbon, hydrogen, and oxygen.

a

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RESULTS AND DISCUSSION

Similarly, the abbreviations for epiafzelechin (EA), epigallocatechin (EG), epifisetinidol (EF), and robinetinidol (ER) will be used to represent their isomers in PAs. In this paper, the PAs formed with only EA, EC, or EG units are called propelargonidin, procyanidin, or prodelpeinidin, whereas those formed from two different units are called proanthocyanidins. The B-type PA dimers have two flavan-3-ol units (i.e., four chiral centers) and an additional asymmetric center at C4.

Exact Masses and Molecular Formula for Proanthocyanidins. Chemically, each flavan-3-ol unit of a PA has two stereogenic (or chiral) centers (Figure 1), which can result in four (or 22) stereoisomers, that is, (2R,3S)-catechin or (+)-C, (2R,3R)-epicatechin or (+)-EC, (2S,3R)-catechin, or (−)-C, and (2S,3S)-epicatechin or (−)-EC. In this paper, EC will be used to represent all four isomers in the text, tables, and figures. 9390

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Table 2. Proanthocyanidins Found in Seven Samples HR [ M − H]− (m/z)

mol formula

major MS2 ions (m/z)(%)

DP

proanthocyanidin

monomer

epiazfelechin catechin epicatechin epigallocatechin epicatechin-3-gallate catechin-3-gallate gallocatechin-3gallate epigallocatechin-3gallate

L, C ALL ALL standard G G, M standard

273.0761 289.0710 289.0714 305.0665 441.0827 441.0827 457.0775

C15H13O5 C15H13O6 C15H13O6 C13H13O7 C22H17O10 C22H17O10 C22H17O11

167(100) 245(100), 205(35), 179(12) 245(100), 205(33), 179(11) 305(100), 221(19), 219(29), 179(20) 331(16), 289(100), 271(9), 169(20) 331(19), 289(100), 271(10), 193(6), 169(21) 331(67), 305(36), 287(10), 193(10), 169(100)

M

457.0779

C22H17O10

331(53), 305(38), 287(9), 269(7), 193(9), 169(100)

EA→EC(1) EC→EA(1) EF→EC(1) EF→EC(2)

M(2) M(1) G(2) G(3)

561.1393 561.1380 561.1382 561.1383

C30H25O11 C30H25O11 C30H25O11 C30H25O11

EF→EC(3) EC→A→EC(1) EC→A→EC(2)

561.1389 575.1190 575.1181

C30H25O11 C30H23O12 C30H23O12

575.1179 575.1202 577.1345

C30H23O12 C30H23O12 C30H25O12

EC→EC(2) EC→EC(3)

G(5) A(2) C(11), D(1), L(8), M(5) C(1) C(1) B(1), B(2), G(10), M(3), A(3) G(3) D(1), M(1)

543(34), 435(50), 425(19), 407(19), 289(100), 271(13), 245(7) 543(9)435(100), 409(64), 391(7), 299(44), 287(50), 273(57), 161(8) 451(40), 435(89), 423(100), 409(49), 325(17), 289(13), 271(26) 451(100), 435(78), 423(91), 409(56), 397(17), 299(25), 289(15), 271(48) 451(42), 435(100), 423(100), 409(39), 325(13), 289(15), 271(21) 539(23), 449(82), 423(100), 411(13), 407(19), 289(26), 285(18) 557(15), 539(30), 453(20), 452(16), 449(100), 447(20), 423(30), 407(20), 289(26), 287(16), 285(27) 449(27), 413(13), 395(88), 377(100), 333(21) 535(22), 509(47), 391(29), 347(100), 329(84), 285(22) 559(17), 451(37), 425(100), 407(53), 299(8), 289(26), 287(8)

577.1335 577.1340

C30H25O12 C30H25O12

EC→EC(4) EC→EG(1) (EC→EC)g(1) (EC→EC)g(2)

D(1), G(1), M(1) G(1) G(5) G(2)

577.1335 593.1279 729.1434 729.1435

C30H25O12 C30H25O13 C37H29O16 C37H29O16

(EC→EC)g(3)

G(1)

729.1437

C37H29O16

(EC→EC)2g

G(2)

881.1541

C44H33O20

559(57), 467(20), 451(100), 425(86), 407(59), 289(65) 559(75), 533(46), 451(29), 439(67), 425(75), 407(20), 393(100), 289(29), 269(35) 559(100), 533(31), 451(21), 439(34), 425(32), 407(18), 393(35) 575(13), 525(6), 467(24), 441(100), 427(6), 423(12), 305(16) 603(14), 577(100), 559(46), 451(13), 425(20), 407(50) 711(23), 603(45), 577(99), 559(88), 451(46), 441(42), 407(100), 289(19) 711(35), 619(29), 603(100), 577(80), 559(51), 451(31), 441(29), 433(18), 407(28), 289(15), 245(17) 729(100), 711(26), 559(20), 407(23)

EA→EA→EC(1) EA→A→EC→EC (1) EA→EC→EC(1)

M(1) M(1), L(1)

833.2083 847.1853

C45H37O16 C45H37O17

816(23), 707(81), 561(91), 543(100), 435(23), 289(35) 711(30), 693(12), 557(34), 435(37), 411(100), 289(13)

M(5)

849.2026

C45H37O17

EA→EC→EC(2) EA→EC→EC(3)

G (4) M (2)

849.2014 849.2017

C45H37O17 C45H37O17

EA→EC→EC(4)

M (2)

849.2204

C45H37O17

EA→EC→EC(5) EA→ECEC(6) EF→EC→EC(7)

G(2) G(2) G(2)

849.2010 849.2018 849.2010

C45H37O17 C45H37O17 C45H37O17

EC→EC→A→EC (1) EC→EC→A→EC (2) EC→A→EC→EC (1) EC-(4β-8)-EC-(4β8)-EC(2) EC-(4β-8)-EC-(4β8)-EC(2) EC→EC→EC(3)

L(3), M(6), C(1)

863.1814

C45H35O18

M(2)

863.1823

C45H35O18

C(2)

863.1804

C45H35O18

C(1), A(4)

865.1971

C45H37O18

723(31), 697(31), 577(100), 571(15), 559(51), 451(17), 425(28), 407(23),289(9), 287(15) 831(94), 723(69), 697(26), 679(79), 561(100) 831(45), 723(68), 697(16), 679(70), 561(38), 559(100), 433(36), 407(50), 289(19) 723(100), 697(37), 679(49), 577(51), 571(39), 561(39), 451(43), 425(24), 407(37), 289(32) 831(16), 723(30), 697(100), 679(71), 561(14), 545(12) 831(100), 723(61), 679(35), 561(41) 739(21), 697(100), 679(59), 559(67), 545(26), 527(16), 451(12), 407(11), 397(17), 289(13) 737(72), 711(62), 693(42), 591(69), 575(100), 573(58), 449(34), 439(32), 289(89), 287(67) 845(13), 737(19), 711(100), 693(41), 575(94), 573(15), 451(23), 411(17) 737(8), 711(100), 693(8), 575(9), 573(41), 559(7), 531(10), 451(47), 411(43), 299(6), 289(19), 285(7) 847(18), 749(48), 695(100), 577(68), 575(31), 425(27), 407(30)

M(12), G(10), J(7), D(3), L(2) M(4), G(1), A(2), D (2) M(1) J(2), D(2)

865.1971

C45H37O18

847(18), 749(48), 695(100), 577(68), 575(31), 425(27), 407(30)

865.1961

C45H37O18

865.1939 865.1955

C45H37O18 C45H37O18

847(40), 779(51), 739(56), 713(57), 695(68), 577(89), 575(100), 449(22), 407(35), 289(27), 287(24) 801(41), 789(49), 779(100), 720(70), 695(51), 577(74), 575(55) 847(38), 739(100), 713(58), 695(87), 577(64), 575(35), 451(37), 449(26), 407(30), 287(29)

dimer

EC→A→EC(3) EC→A→EC(4) EC→EC(1)

trimer

EC→EC→EC(4) EC→EC→EC(5)

plant source

a

9391

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Table 2. continued DP

tetramer

pentamer

proanthocyanidin

plant source

a

HR [ M − H]− (m/z)

mol formula

(EC→EC→EC)g(1)

G(4)

1017.2069

C52H41O22

(EC→EC→EC)g(2) (EC→EC→EC)g(3) (EC→EC→EC)g(4) (EC→EC→EC)→2g (1)

G(1) G(2) G(3) G(1)

1017.2054 1017.2054 1017.2056 1169.2184

C52H41O22 C52H41O22 C52H41O22 C59H45O26

EC→EA→A→EC →EC(1) EA→EC→EC→EC (1) EA→EC→EC→EC (2) HA→EC→HC→EC (3) EC→A→EC→EC→A→EC(1) EC→A→EC→EC→A→EC(2) EC→EC→A→EC→EC(1) EC→A→EC→EC→EC(2) EC→A→EC→EC→EC(3) EC→A→EC→EC→EC(3) EC→EC→EC→A→EC(4) EC→EC→EC→. A→EC(5) EC→EC→EC→EC (1) EC→EC→EC→EC (2) EC→EC→EC→EC (3) EC→EC→EC→EC (4) EC→EC→EC→EC (5) EC→EC→EC→EC (6) EC→EC→EC→EC (7)

L(1)

1135.2472

C60H47O23

M(3)

1137.2649

C60H49O23

M(3)

1137.2666

C60H49O23

M(1)

1137.2651

C60H49O23

L(2), C(1)

1149.2268

C60H45O24

L(1)

1149.2285

C60H45O24

L(3)

1151.2423

C60H47O24

L(2), C(1)

1151.2419

C60H47O24

L(1)

1151.2419

C60H47O24

L(1)

1151.2421

C60H47O24

L(1)

1151.2416

C60H49O24

L(1)

1151.2419

C60H47O24

J(12), G(3), M(3), A (1), D(2) D(2), J(2), M(2), G (2) J(2)

1153.2571

C60H49O24

1153.2565

C60H49O24

1153.2582

C60H49O24

J(2)

1153.2577

C60H49O24

J(1)

1153.2577

C60H49O24

J(2), A(1), D(1)

1153.2590

C60H49O24

A(1)

1153.2590

C60H49O24

L(2)

1439.3058

C75H59O30

L(1)

1439.3058

C75H59O30

L(1)

1439.3058

C75H59O30

L(1)

1439.3058

C75H59O30

L(1)

1439.3058

C75H59O30

L(2)

1439.3058

C75H59O30

L(1)

1439.3058

C75H59O30

L(1)

1439.3058

C75H59O30

M(1)

1441.3229

C75H61O30

EC→EC→EC→EC→A→EC(1) EC→EC→EC→EC→EC(2) EC→EC→EC→EC→EC(3) EC→EC→EC→EC→EC(4) EC→EC→EC→EC→A→EC(5) EC→EC→EC→EC→A→EC(6) EC→EC→EC→EC→EC(7) EC→EC→A→EC→EC→EC(S) EC→EC→EC→EC→EC(1)

major MS2 ions (m/z)(%) 999(31), 891(47), 865(40), 847(57), 739(19), 729(100), 727(23), 695(28), 677(32), 575(20) 999(100), 891(48), 865(50), 847(62), 729(40), 695(39), 677(25) 999(19), 891(54), 865(33), 847(100), 729(83) 999(20), 891(24), 865(100), 847(53), 727(24), 695(24) not recorded

983(36), 965(22), 847(100), 845(30), 829(11), 693(26), 557(22), 411(15) 1119(42), 1011(59), 865(100), 847(51), 739(26), 577(46), 559(33), 407(26) 1119(42), 1011(67), 985(35), 967(85), 849(87), 847(100), 723(36), 575(32), 561(32) 1119(32), 1011(56), 985(41), 967(62), 849(74), 847(88), 577(100), 559(58), 407(50) 997(58), 997(19), 979(34), 845(43), 737(22), 575(100), 573(85), 411(85) 1131(20), 997(75), 979(35), 845(24), 737(17), 575(80), 573(75), 411(100) 1133(14), 1025(41), 999(45), 981(87), 863(100), 861(45), 711(32), 573(41), 411(34) 1005(32), 999(48),981(48), 861(100), 739(68), 573(58), 611(61), 407(35) 1133(32), 999(81), 981(70), 863(43), 861(84), 739(100), 699(38), 577(72), 573(49), 411(39), 407(43) 1133(45), 999(100), 981(48), 863(35), 861(90), 739(83), 587(39), 577(45), 573(59), 411(87), 407(38) 999(78), 981(100), 863(86), 861(76), 739(70), 709(35), 577(38), 573(57), 531(38), 451(30),411(38) 1133(33), 1067(12), 1025(49), 999(21), 981(100), 863(57), 739(12), 737(15), 711(30), 575(48) 1135(54), 1027(74), 1002(42), 983(100), 907(21), 865(63), 863(62), 739(35), 695(32), 577(40), 407(21) 1135(55), 1027(52), 1027(23), 1001(59), 983(96), 865(100), 863(48) , 695(21), 577(46), 575(54) 1135(53), 1027(41), 1001(100), 984(71), 865(79), 863(94), 847(26), 739(50), 701(35), 577(44), 575(65) 1135(55), 1027(64), 1001(50), 983(77), 907(41), 865(50), 863(100), 701(32), 577(55), 575(73), 407(27) 1135(89), 1027(78), 1001(56), 983(44), 907(44), 865(44), 863(44), 739(67), 701(67), 577(33), 575(100) 1135(48),1027(100), 1001(30), 983(83), 965(16), 908(36), 865(52), 739(55), 695(26), 575(31) 1135(100), 1028(72), 983(50), 865(50), 739(39), 737(33)

1421(50), 1313(50), 1295(50), 1149(90), 1007(50), 863(100), 861(50) 1421(67), 1287(100), 1151(67), 1113(33), 863(100), 753(100), 711(67), 637(33), 411(33) 1379(100), 1353(75), 1313(75), 1269(50), 1131(50), 1111(50), 863(75), 857(50), 751 (25) 1421(100), 1089(33), 1013(67), 997(33), 863(67), 711(33), 589(33), 587(67), 575(33), 531(67) 1421(33), 1395(33), 1285(33), 981(100), 863(50), 665 (50), 445(33) 1314(17), 1269(100), 1149(17), 1117(33), 863(67), 817(17), 737(17) , 709(17), 575(17), 453(33) 1441(14), 1421(43), 1269(100), 1151(14), 1107(14), 957(14), 955(29), 863(43), 829(14), 573(14), 531(14) 1371(50), 1269(50), 1143(50), 987(50), 861(50), 711(100), 671(50), 585(50), 575(50), 573(90), 411(50) 1421(100), 1315(64), 1271(67), 1153(74), 1153(33), 1151(48), 1027(38), 865(86), 863(43),739(36), 575(36)

a

Abbreviations: A, apple; C, cranberry extract; D, dark chocolate; G, grape seed extract; J, jujube; L, litchi; M, mangosteen; AL, all tested plants (the number of similar peaks in the sample is listed in parentheses); B1, B2, A2, and C1, procyanidin B-type dimers B1, B2, A-type dimers A2, and trimer C1; 9392

dx.doi.org/10.1021/jf501011y | J. Agric. Food Chem. 2014, 62, 9387−9400

Journal of Agricultural and Food Chemistry

Article

Table 2. continued DP, degree of polymerization; g, galloyl; EC, EA, EG, and EF, epicatechin, epiafzelechin, epigallocatechin, and epifisetinidol, respectively. The signal unit1→unit2 or unit1→A→unit2 expresses the units bonded by B-type (4,8- or 4,6-) bond or A-type (plus additional C2−O−C7− or C2−O−C5−) bond, respectively.

the related propelargonidins and proanthocyanidin-containing EA units are listed above the procyanidins, whereas the related prodelphinidins containing EG units are listed below the procyanidins. Similarly, all of the A-type PAs for each oligomer are listed above the B-type PAs and the galloylated PAs are listed below. The data in Table 1, calculated from eqs 1−5, were found to agree well with experimentally determined [M − H]− values with an error of 4 was negligible. The total concentration of PAs was 48.79 ± 1.95%. Highly accurate masses can be computed for PAs on the basis of the degree of polymerization, the specific flavan-3-ol components, the number of A-type bonds, and the number of galloyls. PAs can be identified by comparing experimentally obtained high-accuracy masses to the computed masses. Identifications can be further confirmed by the analysis of fragments from tandem MS. Conversion of MRRF values from UV absorbance to ion counts with SIM was used for the quantification of individual PAs. Thus, this standardized UHPLC-PDA-ESI/HRMSn profiling method was able to offer identification and quantification of oligomeric PAs in plantderived foods.

MRRF value of 1.00 was assigned to each. The additivity of the molar absorption coefficient makes it possible to quantify most of the PAs using (+)-catechin as a standard with the MRRF values listed above. Unfortunately, even with UHPLC, only a few PAs were well separated and could be quantified on the basis of their UV peak area. Most PAs, when viewed with UV or TIC, had peaks that overlapped (coeluted) with other PAs. Selected ion monitoring (SIM) and multiple reaction monitoring (MRM) are the only methods that allow deconvolution of the overlapping peaks, that is, isolation of the ions of interest.33,34 Consequently, concentrations had to be computed on the basis of ion counts obtained from SIM or MRM as reported in previous studies19,22 The few well-separated absorbance peaks were used to equate the peak area in absorbance to integrated counts of specific ions. In other words, MRRF values based on absorbance were converted to MRRF values based on integrated ion counts. This approach allowed catechin and the MRRF values reported above to be used for computing PA concentrations. Use of MRRFs based on ion counts assumes constant ionization efficiency for all PAs. Unlike absorbance, the ion count of a PA isomer can be expected to be dependent on its structural ionization sensitivity and the mobile phase. The SIM peak intensity might change with the solvent ratio at different retention times, the isomer concentration, and the presence of coeluting PAs (Figure 4). Tests performed with flavan-3-ol monomers and procyanidins B1 and B2 showed the variation in ionization efficiency to be