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. Author manuscript; available in PMC: 2020 Apr 20.
Published in final edited form as: J Sep Sci. 2018 Apr 6;41(11):2331–2346. doi: 10.1002/jssc.201800037

Ultra-fast liquid chromatography coupled with electrospray ionization/time-of- flight mass spectrometry for rapid phenolic profiling of red maple (Acer rubrum) leaves

Chunting Li 1, Navindra P Seeram 1
PMCID: PMC7167591  NIHMSID: NIHMS1565854  PMID: 29512337

Abstract

The red maple (Acer rubrum) species is economically important to North America because of its sap which is used to produce maple syrup. In addition, various other red maple plant parts, including leaves, were used as a traditional medicine by the Native Americans. Currently, red maple leaves are being used for nutraceutical and cosmetic applications but there are no published analytical methods for comprehensive phytochemical characterization of this material. Herein, a rapid and sensitive method using liquid chromatography-electrospray ionization/time-of-flight tandem mass spectrometry was developed to characterize the phenolics in a methanol extract of red maple leaves and a proprietary phenolic-enriched red maple leaves extract (Maplifa™). Time-of-flight mass spectrometry and tandem MS experiments led to the identification of 106 phenolic compounds in red maples leaves with the vast majority of these compounds also detected in Maplifa™. The compounds included 68 gallotannins, 25 flavonoids, gallic acid, quinic acid, catechin, epicatechin, and 9 other gallic acid derivatives among which 11 are potentially new and 75 are being reported from red maple for the first time. The developed method to characterize red maple leaves phenolics is rapid and highly sensitive and could aid in future standardization and quality control of this botanical ingredient.

Keywords: Acer rubrum, Maplifa™, Phenolics, Red maple, Tandem mass spectrometry

1. INTRODUCTION

The red maple (Acer rubrum) species is native to eastern North America where it is widely regarded as an economically important agricultural crop mainly for its sap which is used to produce the natural sweetener, maple syrup. Although maple sap/syrup are widely consumed as a food product, various parts of the red maple species, including its buds, flowers, leaves, twigs, and barks, were used as a folk medicine by the Native Americans for the treatment of a variety of ailments [1]. More recently, extracts purified from these red maple plant parts are being investigated for their potential nutraceutical and cosmetic applications based on a wide range of biological properties including antioxidant, α-glucosidase enzyme inhibitory, blood-glucose lowering, and skin lightening cosmetic effects [2–6]. These biological effects have largely been attributed to the phenolic constituents found in the red maple species, in particular, anhydro-1,5-glucitol-core containing gallotannins which are only produced by certain members of the maple (Acer) genus in the entire higher plant kingdom [7]. Our group has conducted extensive previous isolation and structure elucidation studies (by TOF-MS and NMR spectroscopic data) of the leaves, flowers, twigs, and barks of the red maple species which yielded a series of new compounds containing different numbers of galloyl substituents located at different positions on the anhydro-1,5-glucitol-core [8–10]. In addition, the buds of the red maple species have also been reported to contain a variety of phenolic sub-classes, including anhydro-1,5-glucitol-core containing gallotannins [3].

The utilization of well-established analytical methodologies, such as liquid chromatography- electrospray ionization/time-of-flight tandem mass spectrometry (LC-ESI-TOF-MS/MS) have been extensively applied for the structure elucidation of target and/or non-target compounds in botanical extracts [11–15]. Given limited phytochemical compositional data available on the leaves of the red maple species, which are necessary for its future advancement as a botanical raw material or ingredient for dietary supplement and cosmetic applications, herein, we developed a rapid and sensitive ultra-fast liquid chromatography-electrospray ionization/time-of- flight tandem mass spectrometry (UFLC-ESI-TOF-MS/MS) method to identify its phenolic compounds. We also used UFLC-ESI-TOF-MS/MS to evaluate the phenolics present in a proprietary phenolic-enriched red maple leaves extract (Maplifa™) which has been previously studied for its cosmetic applications [5].

2. MATERIALS AND METHODS

2.1. Chemicals

Reference standards for maplexin A, maplexin B, maplexin C, maplexin D, ginnalin A, ginnalin B, ginnalin C, 3,6-di-O-galloyl-1,5-anhydroglucitol, catechin, epicatechin, gallic acid, gallic acid methyl ester, 5-methyl gallic acid methyl ester, and ellagic acid, used for ESI-TOF-MS/MS characterization, were previously isolated and identified by our laboratory [8, 9, 16]. LC-MS grades of acetonitrile and methanol were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Sample preparation

Leaves of the red maple species were collected in the summer and botanically authenticated with a deposited voucher specimen as previously reported by our laboratory [8–10]. Finely powdered air-dried red maple leaves (1.0 g) was extracted by sonication with methanol (10 mL) for 1 hour under room temperature. The extract was stored at 4 °C and filtered (0.22 μm) before analyses.

2.3. Ultra-fast Liquid Chromatography Mass Spectrometry Conditions

UFLC-ESI-TOF-MS/MS analyses were performed on a SHIMADZU Prominence UFLC system (Marlborough, MA, USA) consisting of two LC-20AD pumps, a DGU-20A degassing unit, SIL-20AC auto sampler, CTO-20AC column oven and CBM-20A communication bus module. Chromatographic separation was performed on a 150 mm × 4.6 mm i.d., 5 μm, Prodigy ODS column (Phenomenex, Torrance, CA, USA). The mobile phase consisted of 0.1% formic acid/acetonitrile (A) and 0.1% aqueous formic acid (B) with a gradient elution of 5% A from 0 to 5 min, 5−16% A from 5 to 16 min, and 16−36% A from 16 to 76 min. The column temperature was 40 °C, flow rate was 0.50 mL/min, and injection volume was 4.00 μL. Mass spectrometry was performed using a Triple TOF 4600 system from Applied Biosystems/MDS Sciex (Framingham, MA, USA) coupled with an electrospray ionization (ESI) interface. Nitrogen was used in all cases. In this study, the parameters were optimized as follows: ESI voltage, −4500 V; nebulizer gas, 40; auxiliary gas, 50; curtain gas, 25; turbo gas temperature, 450 °C; declustering potential, −80V; collision energy, 50 eV. Instrument calibration was carried out according to the manufacturer’s instructions. The mass range was set from m/z 100 to 1150. The data were acquired and processed using Analyst TF 1.7 Software.

3. RESULTS AND DISCUSSION

Given previous reports that the phytochemical constituents present in the red maple species are predominantly phenolics, including gallotannins [8–10], methanol was used as the extracting solvent of choice to prepare a phenolic-enriched extract of red maple leaves. A qualitative characterization of the phenolic compounds present in the methanol extract of the red maple leaves was performed in ESI negative ionization mode and all of the compounds showed [M − H]– ions (Table 1). Accurate mass data were then acquired in full scan analysis and product ion mass data were acquired via the Information Dependent Acquisition (IDA) method. A Prodigy ODS column was used and formic acid was introduced into the mobile phase (0.1%) to alleviate peak tailing and to produce better peak shapes. The acidic conditions did not signicantly affect the ionization efficiency of the compounds in negative mode.

Table 1.

Characterization of phenolic compounds in a methanol extract red maple leaves and Maplifa™ by LC-ESI-TOF-MS/MS

Number tR (min) Formula HR-ESI(-)-MS [M-H]- (m/z exptl (theor)) Error (ppm) Proposed compounds Presence
Extract Maplifa™
1 3.48 C7H12O6 191.0568 (191.0561) 3.6017 173, 155, 137, 127, 109 quinic acid + +
2 4.85 C13H16O10 331.0680 (331.0670) 2.8073 313, 271, 211, 169, 125 galloyl hexoside + +
3 4.98 C14H16O10 343.0687 (343.0670) 4.7485 191, 169, 125 galloyl quinic acid + +
4 5.23 C13H16O9 315.0729 (315.0721) 2.3615 169, 125 maplexin A + +
5 5.49 C13H16O10 331.0679 (331.0670) 2.5052 271, 211, 169, 125 galloyl hexoside + +
6 6.17 C13H16O10 331.0686 (331.0670) 4.6196 271, 211, 169, 125 galloyl hexoside + +
7 6.90 C13H16O9 315.0735 (315.0721) 4.2658 169, 125 maplexin B + +
8 7.06 C14H16O10 343.0686 (343.0670) 4.4580 191, 169, 125 galloyl quinic acid + +
9 7.13 C7H6O5 169.0145 (169.0142) 1.4970 125 gallic acid + +
10 8.18 C13H16O10 331.0684 (331.0670) 4.0155 313, 241, 211, 169, 125 galloyl hexoside + +
11 8.44 C14H16O10 343.0682 (343.0670) 3.2921 191, 173, 169, 125 4-O-galloyl quinic acid + +
12 9.01 C13H16O10 331.0687 (331.0670) 4.9217 313, 241, 169, 125 galloyl hexoside + +
13 9.46 C13H16O9 315.0729 (315.0721) 2.3615 169, 125 galloyl deoxyhexoside + +
14 10.83 C13H16O10 331.0682 (331.0670) 3.4114 169, 125 galloyl hexoside + +
15 11.47 C14H18O10 345.0844 (345.0827) 4.8664 285, 225, 210, 183, 168, 138, 124 methylgalloyl hexoside + −
16 11.60 C14H14O9 325.0577 (325.0565) 3.6736 281, 173, 169, 125 galloyl shikimic acid + +
17 11.83 C13H16O9 315.0725 (315.0721) 1.0920 169, 125 ginnalin B + +
18 12.37 C13H16O9 315.0733 (315.0721) 3.6310 169, 125 ginnalin C + +
19 13.07 C14H18O10 345.0843 (345.0827) 4.5766 183, 168, 124 methylgalloyl hexoside + +
20 13.74 C20H20O14 483.0808 (483.0780) 5.7354 331, 313, 271, 211, 169, 125 digalloyl hexoside + +
21 13.97 C21H20O14 495.0803 (495.0780) 4.5865 343, 325, 191, 173, 169, 125 digalloyl quinic acid + −
22 14.16 C15H20O10 359.0998 (359.0983) 3.9801 197, 182, 167, 153, 138, 123 dimethyl galloyl hexoside + −
23 14.57 C20H20O14 483.0816 (483.0780) 7.3915 331, 271, 211, 169, 125 digalloyl hexoside + +
24 14.61 C14H14O9 325.0577 (325.0565) 3.6736 173, 169, 125 galloyl shikimic acid + +
25 15.15 C8H8O5 183.0305 (183.0298) 3.2942 168, 124 methyl gallic acid + −
26 15.21 C20H20O14 483.0810 (483.0780) 6.1494 331, 313, 271, 211, 169, 125 digalloyl hexoside + +
27 16.02 C21H20O14 495.0817 (495.0780) 7.4143 343, 191, 169, 125 digalloyl quinic acid + +
28 16.18 C14H14O9 325.0583 (325.0565) 5.5194 173, 169, 125 galloyl shikimic acid + +
29 16.79 C20H20O13 467.0842 (467.0831) 2.3236 315, 297, 169, 125 maplexin C + +
30 16.85 C20H20O14 483.0816 (483.0780) 7.3915 465, 331, 313, 271, 211, 169, 125 digalloyl hexoside + +
31 17.39 C21H20O14 495.0806 (495.0780) 5.1924 343, 191, 173, 169, 125 digalloyl quinic acid + +
32 17.68 C20H20O14 483.0812 (483.0780) 6.5635 465, 331, 313, 271, 241, 169, 125 digalloyl hexoside + +
33 18.00 C15H14O6 289.0722 (289.0717) 1.5154 245, 205, 203 catechin + +
34 18.04 C14H10O9 321.0259 (321.0252) 2.1627 169, 125 digallate + +
35 18.42 C8H8O5 183.0313 (183.0298) 7.6550 168, 124 gallic acid methyl ester + +
36 18.55 C20H20O13 467.0862 (467.0831) 6.6055 315, 297, 169, 125 3,6-di-O-galloyl-1,5-anhydroglucitol + +
37 18.90 C20H20O14 483.0813 (483.0780) 6.7705 465, 331, 313, 169, 125 digalloyl hexoside + +
38 19.09 C20H20O13 467.0865 (467.0831) 7.2477 315, 169, 125 digalloyl deoxyhexoside + +
39 19.60 C20H20O13 467.0869 (467.0831) 8.1041 315, 297, 169, 125 maplexin D + +
40 19.92 C21H18O13 477.0704 (477.0674) 6.1530 325, 201, 173, 169, 125 digalloyl shikimic acid + +
41 20.43 C20H20O14 483.0812 (483.0780) 6.5635 331, 313, 169, 125 digalloyl hexoside + +
42 20.95 C15H14O6 289.0724 (289.0717) 2.2072 245, 205, 203 epicatechin + +
43 21.27 C20H20O13 467.0868 (467.0831) 7.8900 423, 315, 169, 152, 125 digalloyl deoxyhexoside + +
44 21.71 C28H24O18 647.0920 (647.0889) 4.6546 495, 343, 191, 173, 169, 125 trigalloyl quinic acid + −
45 21.97 C20H20O13 467.0856 (467.0831) 5.3209 315, 297, 211, 169, 125 ginnalin A + +
46 22.87 C27H24O17 619.0973 (619.0940) 5.2118 467, 315, 297, 169, 125 trigalloyl deoxyhexoside + +
47 23.57 C21H22O13 481.1002 (481.0987) 2.9832 315, 169, 125 galloyl methylgalloyl deoxyhexoside + +
48 23.73 C27H24O17 619.0975 (619.0940) 5.5348 467, 315,169, 125 trigalloyl deoxyhexoside + +
49 24.44 C27H24O17 619.0973 (619.0940) 5.2118 467, 315, 297, 169, 125 trigalloyl deoxyhexoside + +
50 24.86 C26H28O16 595.1333 (595.1304) 4.7738 301, 300, 271, 255, 243, 227, 179, 151 quercetin hexosyl pentoside + +
51 25.66 C28H24O16 615.1035 (615.0991) 7.0577 463, 301, 300, 271, 255, 243, 227, 211, 179, 169, 151, 125 quercetin-3-O-galloyl hexoside + +
52 25.95 C9H10O5 197.0460 (197.0455) 2.2982 182, 167, 123 di-methyl gallic acid + +
53 26.49 C28H24O16 615.1042 (615.0991) 8.1957 463, 301, 300, 271, 255, 243, 227, 211, 179, 169, 151, 125 quercetin-3-O-galloyl hexoside + +
54 27.04 C14H5O8 300.9996 (300.9989) 2.0235 284, 257, 229, 201, 185, 173, 145, ellagic acid + +
55 27.42 C27H30O16 609.1496 (609.1461) 5.7309 301, 300, 271, 255, 243, 151 quercetin-3-O-hexosyl deoxyhexoside + +
56 27.68 C9H10O5 197.0461 (197.0455) 2.8057 182, 167, 123 5-methyl gallic acid methyl ester + +
57 27.81 C22H18O10 441.0857 (441.0827) 6.7545 289, 245, 169, 125 galloyl (epi)catechin + +
58 28.09 C27H24O17 619.0977 (619.0940) 5.8579 467, 315, 297, 169, 125 trigalloyl deoxyhexoside + +
59 28.60 C22H18O10 441.0858 (441.0827) 7.2079 289, 245, 169, 125 galloyl (epi)catechin + +
60 29.18 C28H24O16 615.1021(615.0991) 7.7080 463, 313, 301, 273, 255, 245, 229, 211, 179, 169, 151, 125 quercetin galloyl hexoside + +
61 29.18 C27H24O17 619.0975 (619.0940) 5.5348 467, 315, 297, 169, 125 trigalloyl deoxyhexoside + +
62 29.85 C27H24O17 619.0978 (619.0940) 3.7266 467, 449, 315, 297, 169, 125 trigalloyl deoxyhexoside + +
63 29.97 C28H24O16 615.1039(615.0991) 4.7817 463, 313, 301, 273, 255, 245, 229, 211, 179, 169, 151, 125 quercetin galloyl hexoside + +
64 31.90 C20H18O11 433.0808(433.0776) 7.3074 301, 300, 271, 255, 243, 151 quercetin-3-O-pentoside + +
65 32.19 C21H20O11 447.0956 (447.0932) 5.1770 285, 284, 255, 227 kaempferol-3-O-hexoside + +
66 32.51 C15H12O9 335.0430 (335.0408) 6.3998 183, 168, 124 methyl digallate + +
67 32.99 C34H28O21 771.1099 (771.1050) 6.3128 619, 467, 449, 315, 297, 169, 125 tetragalloyl deoxyhexoside + +
68 33.50 C29H22O14 593.0966 (593.0936) 4.9243 441, 289, 271, 245, 203, 169, 125 digalloyl (epi)catechin + +
69 34.30 C23H20O10 455.0987 (455.0983) 0.7234 289, 245, 183, 168, 124 methylgalloyl (epi)catechin + −
70 34.33 C21H20O11 447.0953 (447.0932) 4.5060 301, 300, 271, 255, 243, 227, 151 quercetin-3-O-deoxyhexoside + +
71 34.75 C34H28O21 771.1108 (771.1050) 7.4799 619, 467, 315, 297, 169, 125 tetragalloyl deoxyhexoside + +
72 35.20 C28H26O17 633.1142 (633.1097) 7.0706 467, 315, 297, 169, 125 digalloyl methylgalloyl deoxyhexoside + +
73 35.36 C27H22O15 585.0909 (585.0885) 3.9412 433, 301, 273, 255, 179, 151 quercetin galloyl pentoside + +
74 35.52 C34H28O21 771.1101 (771.1050) 6.5722 619, 467, 315, 297, 169, 125 tetragalloyl deoxyhexoside + +
75 35.87 C28H26O17 633.1113 (633.1097) 2.4901 467, 315, 297, 169, 125 digalloyl methylgalloyl deoxyhexoside + +
76 36.04 C20H18O10 417.0843 (417.0827) 3.7866 285, 284, 255, 227 kaempferol-3-O-pentoside + +
77 36.19 C29H22O14 593.0960 (593.0936) 3.9127 441, 423, 289, 271, 245, 203, 169, 125 digalloyl (epi)catechin + +
78 36.22 C34H28O21 771.1096 (771.1050) 5.9238 619, 467, 315, 297, 169, 125 tetragalloyl deoxyhexoside + +
79 36.73 C28H26O17 633.1140 (633.1097) 6.7547 467, 315, 297, 169, 125 digalloyl methylgalloyl deoxyhexoside + +
80 37.53 C34H28O21 771.1085 (771.1050) 4.4972 619, 467, 315, 297, 169, 125 tetragalloyl deoxyhexoside + +
81 38.56 C34H28O21 771.1098 (771.1050) 6.1831 619, 467, 315, 297, 169, 125 tetragalloyl deoxyhexoside + −
82 39.80 C29H22O14 593.0967 (593.0936) 5.0929 441, 289, 271, 245, 203, 169, 125 digalloyl (epi)catechin + +
83 39.96 C34H26O19 737.1038 (737.0995) 5.7620 585, 433, 301, 273, 255, 179, 151 quercetin digalloyl pentoside + −
84 40.86 C21H20O10 431.1001 (431.0983) 4.0112 285, 284, 255, 227 kaempferol-3-O-deoxyhexoside + +
85 40.93 C29H22O14 593.0969 (593.0936) 5.4301 441, 289, 271, 245, 203, 169, 125 digalloyl (epi)catechin + +
86 40.96 C41H32O25 923.1163 (923.1159) 0.3349 771, 619, 467, 315, 297, 169, 125 pentagalloyl deoxyhexoside + +
87 41.05 C27H22O15 585.0934 (585.0885) 8.2140 433, 301, 300, 271, 255, 179, 151 quercetin-3-O-galloyl pentoside + +
88 41.31 C29H22O14 593.0967 (593.0936) 5.0929 441, 289, 271, 245, 203, 169, 125 digalloyl (epi)catechin + +
89 42.01 C41H32O25 923.1230 (923.1159) 7.5929 771, 619, 467, 315, 297, 169, 125 pentagalloyl deoxyhexoside + +
90 42.05 C35H30O21 785.1248 (785.1206) 5.2446 633, 619, 467, 315, 297, 169, 125 trigalloyl methylgalloyl deoxyhexoside + −
91 42.43 C22H16O13 487.0547 (487.0518) 5.9243 335, 183, 168, 124 methyl trigallate + −
92 42.75 C28H24O15 599.1055 (599.1042) 2.0962 447, 315, 301, 300, 271, 255, 243, 227, 179, 151 quercetin-3-O-galloyl deoxyhexoside + +
93 43.36 C35H30O21 785.1257 (785.1206) 6.3911 633, 619, 467, 315, 297, 169, 125 trigalloyl methylgalloyl deoxyhexoside + −
94 44.31 C16H14O9 349.0574 (349.0565) 2.5615 197, 182, 167, 123 di-methyl digallate + +
95 45.82 C30H24O14 607.1139 (607.1093) 7.5283 561, 455, 399, 289, 245, 203, 183, 125 galloyl methylgalloyl (epi)catechin + −
96 46.52 C28H24O15 599.1088 (599.1042) 7.6044 447, 301, 255, 179, 151 quercetin galloyl deoxyhexoside + +
97 47.31 C28H24O15 599.1078 (599.1042) 3.5559 447, 301, 255, 179, 151 quercetin galloyl deoxyhexoside + +
98 50.70 C36H32O21 799.1406 (799.1363) 5.3404 633, 467, 315, 297, 169, 125 digalloyl dimethylgalloyl deoxyhexoside + −
99 53.04 C35H28O19 751.1214 (751.1152) 8.2505 599, 447, 301, 255, 179, 151 quercetin digalloyl deoxyhexoside + +
100 53.51 C28H24O14 583.1144 (583.1093) 8.6956 431, 297, 285, 255, 227 kaempferol galloyl deoxyhexoside + +
101 53.83 C35H28O19 751.1208 (751.1152) 7.4517 599, 447, 301, 255, 179, 151 quercetin digalloyl deoxyhexoside + +
102 54.47 C28H24O14 583.1133 (583.1093) 6.8092 431, 297, 285, 255, 227, 169, 125 kaempferol galloyl deoxyhexoside + +
103 55.14 C35H28O19 751.1205 (751.1152) 7.0523 599, 447, 301, 255, 179, 151 quercetin digalloyl deoxyhexoside + +
104 57.86 C42H32O23 903.1316 (903.1261) 6.0217 751, 599, 447, 301, 255, 179, 151 quercetin trigalloyl deoxyhexoside + −
105 59.55 C35H28O18 735.1258 (735.1202) 7.4977 583, 431, 297, 285, 255, 227, 169, 125 kaempferol digalloyl deoxyhexoside + +
106 60.13 C36H30O19 765.1347 (765.1308) 3.8471 599, 447, 301, 255, 179, 151 quercetin galloyl methylgalloyl deoxyhexoside + −
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A total of 106 phenolic compounds, including 68 gallotannins, 25 flavonoids, gallic acid, quinic acid, catechin, epicatechin and 9 other gallic acid derivatives, were tentatively characterized (Table 1). Among these, 11 are potentially new compounds and 75 compounds are being reported from the red maple species for the first time (further described below). The general chemical structural skeletons of the compounds are shown in Fig. 1 and the total ion chromatograms (TIC) profiles of the compounds are shown in Fig. 2 (with peaks numbered as shown in Table 1). Accurate mass measurements, retention times (tR), formula, errors and main MS/MS product ions for all of the phenolic compounds are summarized in Table 1. For ease of discussion, the red maple leaves phenolics are classified into general chemical structural classes, namely, gallotannins, flavonoids, and other gallic acid derivatives, as discussed below.

Figure 1.

Figure 1.

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General structures and substitution patterns of phenolic compounds identified in a methanol extract of red maple leaves.

Figure 2.

Figure 2.

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Total ion chromatogram of a methanol extract of red maple leaves (with peaks numbered as shown in Table 1).

3.1. Characterization of gallotannins

Gallotannins are a subgroup of hydrolyzable tannins composed of galloyl units or their meta-depsidic derivatives bound to diverse polyol-, catechin-, or triterpenoid units [17]. In the present study, 68 gallotannins were identified in red maple leaves including 16 galloyl hexosides, 32 galloyl deoxyhexosides (consisting of 5 galloyl deoxyhexosides, 6 digalloyl deoxyhexosides, 1 methyldigalloyl deoxyhexoside, 6 trigalloyl deoxyhexosides, 3 methyltrigalloyl deoxyhexosides, 6 tetragalloyl deoxyhexosides, 3 methyltetragalloyl deoxyhexosides, and 2 pentagalloyl deoxyhexosides), 9 galloyl catechins, 7 galloyl quinic acid derivatives and 4 galloyl shikimic acid derivatives. The characteristic product ions at m/z 169 ([gallic acid − H]–) and 125 ([gallic acid − H − CO2]–), as well as the loss of 152 Da in the MS/MS spectra supported the presence of galloyl groups in the compounds.

Compound 9 (tR = 7.13 min) exhibited an [M − H]– ion at m/z 169.0142 (C7H6O5). In the MS/MS spectra, it gave a characteristic product ion at m/z 125 corresponding to the loss of CO2 (44 Da). By comparison of the tR, high resolution MS data, and MS/MS spectra data with those of an authentic standard, compound 9 was identified as gallic acid which has previously been reported from the red maple species [8].

3.1.1. Galloyl hexosides

Compounds 2 (tR = 4.85 min), 5 (tR = 5.49 min), 6 (tR = 6.17 min), 10 (tR = 8.18 min), 12 (tR = 9.01 min) and 14 (tR = 10.83 min) all gave the same [M − H]– ion at m/z 331, with molecular formula C13H16O10 provided by TOF-MS. Their MS/MS fragmentation ions at m/z 125 (typical fragment ion for gallic acid due to loss of CO2) and m/z 169 [M − H − 162]− indicated the existence of a galloyl moiety and loss of a hexose moiety. Thus, compounds 2, 5, 6, 10, 12 and 14 were tentatively identified as galloyl hexosides. Notably, 1-O-galloyl-glucoside was previously reported from the red maple species [18].

Compounds 15 (tR = 11.47 min) and 19 (tR = 13.07 min) gave the same [M − H]– ion at m/z 345, with molecular formula C14H18O10 provided by TOF-MS. Their MS/MS fragmentation ions at m/z 183 [M − H − 162]− suggested the existence of a methylgalloyl moiety and the loss of a hexose moiety. The daughter ions at m/z 168 [methyl gallic acid − H − 15]− and m/z 124 [methyl gallic acid − H − 15 − 44]− were attributed to loss of a •CH3 and a CO2 from a methyl gallic acid moiety, respectively. Thus, compounds 15 and 19 were tentatively identified as methylgalloyl hexosides. A methylgalloyl hexoside was previously reported from the red maple species [10]. Similarly, compound 22 (tR = 14.16 min) was tentatively identified as dimethyl galloyl hexoside based on the [M − H]– ion at m/z 359 and MS/MS daughter ion at m/z 197 [M − H − 162]− indicating the existence of a dimethyl galloyl moiety and the loss of a hexose moiety. Due to the sequential losses of two methyl free radicals and a CO2, it formed the MS/MS daughter ions at m/z 182, m/z 167 and m/z 123.

Compounds 20 (tR = 13.74 min), 23 (tR = 14.57 min), 26 (tR = 15.21 min), 30 (tR = 16.85 min), 32 (tR = 17.68 min), 37 (tR = 18.90 min) and 41 (tR = 20.43 min) were tentatively identified as digalloyl hexosides based on their [M − H]− ions at m/z 483 and MS/MS fragment ions at m/z 331 [M − H − 152]− due to the loss of a galloyl group and m/z 169 [M − H − 152 − 162]− due to the loss of a hexose moiety. The typical MS/MS fragmentation ions of the galloyl moiety at m/z 169 and m/z 125 were also observed.

3.1.2. Galloyl deoxyhexosides

Compounds 4 (tR = 5.23 min), 7 (tR = 6.90 min), 13 (tR = 9.46 min), 17 (tR = 11.83 min) and 18 (tR = 12.37 min) all gave [M − H]– ions at m/z 315 with molecular formula of C13H16O9. Their MS/MS fragmentation ions at m/z 169 [M − H − 146]− indicated the existence of a galloyl group and the loss of a deoxyhexose moiety. The typical MS/MS fragmentation ions of galloyl at m/z 169 and m/z 125 were also observed. Thus, compounds 4, 7, 13, 17 and 18 were tentatively identified as galloyl deoxyhexosides. The identification of compounds 4, 7, 17 and 18 were further confirmed as maplexin A, maplexin B, ginnalin B and ginnalin C, respectively, by comparison to corresponding authentic reference standards previously isolated from the red maple species by our laboratory [8, 9, 16].

Compounds 29 (tR = 16.79 min), 36 (tR = 18.55 min), 38 (tR = 19.09 min), 39 (tR = 19.60 min), 43 (tR = 21.27 min) and 45 (tR = 21.97 min) exhibited [M − H]− ions at m/z 467. They were tentatively identified as digalloyl deoxyhexosides based on their MS/MS fragment ions at m/z 315 [M − H − 152]− and m/z 169 [M − H − 152 − 146]− due to the loss of galloyl and deoxyhexose moieties, respectively. The identities of compounds 29, 36, 39, and 45 were confirmed as maplexin C, 3,6-di-O-galloyl-1,5-anhydroglucitol, maplexin D, and ginnalin A, respectively, by comparison to authentic reference standards previously isolated from the red maple species by our laboratory [8, 9].

Compound 47 (tR = 23.57 min) displayed a [M − H]− ion at m/z 481. In the MS/MS spectra, the daughter ion at m/z 315 [M − H − 166]− indicated the loss of a methylgalloyl moiety and the daughter ion at m/z 169 [M − H − 166 − 146]− indicated the loss of a deoxyhexose moiety. The diagnostic fragmentation ions at m/z 169 and m/z 125 confirmed the existence of galloyl moieties. Thus, compound 47 was tentatively identified as galloyl methylgalloyl dexoyhexoside which is potentially a new compound.

Compounds 46, 48, 49, 58, 61 and 62 with tR of 22.87, 23.73, 24.44, 28.09, 29.18 and 29.85 min, respectively, were tentatively identified as isomers of trigalloyl deoxyhexosides. These compounds all gave identical m/z 619 [M − H]− ions with MS/MS product ions at m/z 467 [M − H − 152]− and m/z 315 [M − H − 152 − 152]−, indicating the loss of galloyl moieties. All of the six compounds exhibited typical fragment ions at m/z 125 and m/z 169 [M − H − 152 − 152 − 146]− suggesting the existence of galloyl moieties and the loss of a deoxyhexose moiety. Maplexin E [8] and maplexin F, two trigalloyl deoxyhexosides, were previously reported from the red maple species by our laboratory [9]. These compounds contain 3 galloyl groups located at 3 different positions on the 1,5-anhydro-glucitol core. Since two other trigalloyl deoxyhexosides have previously been reported from Acer ginnala [19], there are at least two potentially new trigalloyl deoxyhexosides among the six trigalloyl deoxyhexosides identified herein.

Compounds 72 (tR = 35.20 min), 75 (tR = 35.87 min) and 79 (tR = 36.73 min) displayed [M − H]− ions at m/z 633. In the MS/MS spectra, the daughter ions at m/z 467 [M − H − 166]−, m/z 315 [M − H − 166 − 152]− and m/z 169 [M − H − 166 − 152 − 146]− indicated the loss of methylgalloyl, galloyl, and deoxyhexose moieties, respectively. The diagnostic fragmentation ions at m/z 169 and m/z 125 indicated the existence of galloyl moieties. Thus, compound 72, 75, and 79 were tentatively identified as digalloyl methylgalloyl dexoyhexosides. Our laboratory has previously isolated and identified three new digalloyl methylgalloyl dexoyhexosides, namely, maplexin G, maplexin H and maplexin I, from the red maple species [9]. Given that these new compounds have not been previously analyzed by MS/MS, a proposed fragmentation pattern is being proposed for maplexin I (Figure 3). In the MS/MS spectrum of maplexin I, fragment ions at m/z 467 [M − H − 166]−, 315 [M − H − 166 − 152]− and m/z 169 [M − H − 166 − 152 − 146]− corresponded to sequential losses of methylgalloyl, galloyl and 1,5-anhydroglucitol groups, respectively. A daughter ion at m/z 297 [M − H − 166 − 170]− was formed from the [M − H]− ion by the loss of a methylgalloyl and a gallic acid and the typical fragment ion at m/z 125 was a product of m/z 169 corresponding to loss of carbon dioxide. Thus, the fragmentation pattern of a digalloyl methylgalloyl dexoyhexoside is being reported herein for the first time.

Figure 3.

Figure 3.

Open in a new tab

Proposed fragmentation pattern of maplexin I.

Compounds 67, 71, 74, 78, 80 and 81 produced pesudomolecular ions [M − H]− at m/z 771 with different tR of 32.99, 34.75, 35.52, 36.22, 37.53 and 38.56 min, respectively. These compounds were tentatively identified as isomers of tetragalloyl deoxyhexosides on the basis of their MS/MS fragment ions at m/z 619 [M − H − 152]−, m/z 467 [M − H − 152 − 152]−, m/z 315 [M − H − 152 − 152 − 152]−, m/z 169 [M − H − 152 − 152 − 152 − 146]−, and m/z 125, suggesting the loss of three galloyl groups, a deoxyhexose, and a CO2, respectively. While tetragalloyl deoxyhexosides have not been previously reported from the red maple species, three tetragalloyl deoxyhexosides have been reported from Acer ginnala [19]. Therefore, among the six tetragalloyl deoxyhexosides tentatively identified herein, there are at least three potentially new compounds. Notably, this is the first evidence of the presence of tetragalloyl deoxyhexosides in the red maple species.

Compounds 90 (tR = 42.05 min) and 93 (tR = 43.36 min) displayed [M − H]− ions at m/z 785. In the MS/MS spectra, the daughter ions at m/z 633 [M − H − 152]−, m/z 467 [M − H − 152 − 166]−, m/z 315 [M − H − 152 − 166 − 152]− and m/z 169 [M − H − 152 − 166 − 152 − 146]− indicated the loss of methylgalloyl, two gallyols and deoxyhexose moieties, respectively. The diagnostic fragmentation ions at m/z 169 and m/z 125 indicated the existence of a galloyl moiety. Thus, compounds 90 and 93 were tentatively identified as trigalloyl methylgalloyl dexoyhexosides which are both potentially new compounds.

Compound 98 (tR = 50.70 min) gave a [M − H]− ion at m/z 799. In the MS/MS spectra, the daughter ions at m/z 633 [M − H − 166]−, m/z 467 [M − H − 166 − 166]−, m/z 315 [M − H − 166 − 166 − 152]− and m/z 169 [M − H − 166 − 166 − 152 − 146]− indicated the loss of two methylgalloyls, a galloyl and deoxyhexose moieties, respectively. The diagnostic fragmentation ions at m/z 169 and m/z 125 indicated the existence of a galloyl moiety. Thus, compound 98 was tentatively identified as a digalloyl di-methylgalloyl dexoyhexoside which is potentially a new compound.

Compounds 86 (tR = 40.96 min) and 89 (tR = 42.01 min) gave the same [M − H]– ion at m/z 923. Based on the MS/MS daughter ions formed by sequential losses of galloyl moieties at m/z 771 [M − H − 152]−, m/z 619 [M − H − 152 − 152]−, m/z 467 [M − H − 152 − 152 − 152]− and m/z 315 [M − H − 152 − 152 − 152 − 152]−, as well as the typical daughter ions at m/z 169 [M − H − 152 − 152 − 152 − 152 − 146]−, and m/z 125, indicating the loss of a deoxyhexose and a CO2, respectively, compounds 86 and 89 were tentatively identified as isomers of pentagalloyl deoxyhexosides. Since a pentagalloyl deoxyhexoside has never been previously reported, compounds 86 and 89 are potentially new compounds.

3.1.3. Galloyl catechins

Compounds 33 (tR = 18.00 min) and 42 (tR = 20.95 min) gave the same [M − H]– ion at m/z 289 with a molecular formula of C15H14O6 and were identified as catechin and epicatechin by comparing their tR, high resolution MS data and MS/MS spectra data with those of authentic reference standards. Their MS/MS spectra showed typical fragment ions at m/z 245 [M − H − CO2]−, m/z 203 ([M − H − CO2 − C2H2O]−) and m/z 205 ([M − H − 2C2H2O]−). Catechin and epicatechin have previously been reported from the red maple species [16].

Compounds 57 (tR = 27.81 min) and 59 (tR = 28.60 min) gave [M − H]– ions at m/z 441 with a molecular formula of C22H18O10. In their MS/MS spectra, fragmentation ions at m/z 289 ([M − H − 152]−) and m/z 245 ([M − H − 152 − 44]−) indicated the loss of a galloyl moiety and the existence of a (epi)catechin moiety. The diagnostic daughter ions at m/z 169 and 125 also indicated the galloyl moiety. Thus, compounds 57 and 59 were tentatively identified as galloyl (epi)catechins. While the (epi)catechin unit cannot be identified as either catechin or epicatechin based on MS data, galloyl epicatechin has previously been isolated and identified (by NMR) from the red maple species [16].

Compounds 69 (tR = 34.30 min) gave a [M − H]– ion at m/z 455. In the MS/MS spectra, fragmentation ions at m/z 289 [M − H − 166]− and m/z 245 [M − H − 166 − 44]− indicated the loss of a methylgalloyl moiety and the existence of a (epi)catechin moiety. The diagnostic daughter ions at m/z 169 and 125 also indicated the galloyl moiety thus compound 69 was tentatively identified as methylgalloyl (epi)catechin.

Compounds 68, 77, 82, 85 and 88, with tR of 33.50, 36.19, 39.80, 40.93 and 41.31 min, respectively, were tentatively identified as isomers of digalloyl-(epi)catechins. These compounds all gave identical m/z 593 [M − H]− ions with MS/MS product ions at m/z 441 [M − H − 152]−, m/z 289 [M − H − 152 − 152]− and m/z 271 [M − H − 170]−, respectively, indicating the loss of two galloyl moieties. All of these five compounds exhibited diagnostic fragment ions at m/z 125 and m/z 169 confirming the galloyl moiety, as well as typical daughter ions at m/z 289 and m/z 245 corresponding to the (epi)catechin moiety.

Compounds 95 (tR = 45.82 min) displayed a [M − H]− ion at m/z 607. In the MS/MS spectra, the daughter ions at m/z 455 [M − H − 152]−, m/z 289 [M − H− 152 − 166]− and m/z 245 [M − H − 152 − 166 − 44]− indicated the loss of galloyl, methylgalloyl and (epi)catechin moieties, respectively. Therefore, compound 95 was tentatively identified as galloyl methylgalloyl (epi)catechin.

3.1.4. Galloyl quinic acids

Compound 1 (tR = 3.48 min) gave a [M − H]– ion at m/z 191.0568 (C7H12O6). In the MS/MS spectra, it gave product ions at m/z 173 due to the loss of H2O (18 Da) and m/z 127 [M − H − CO − 2H2O]–. Therefore, compound 1 was identified as quinic acid [14] and this is the first report of its occurrence in the red maple species.

Compounds 3 (tR = 4.98 min), 8 (tR = 7.06 min) and 11 (tR = 8.44 min), all gave deprotonated ions at m/z 343, with a molecular formula of C14H16O10. In the MS/MS spectra, they showed diagnostic [quinic acid – H]– ions at m/z 191 [M − H − 152]− and loss of 152 Da (galloyl), as well as diagnostic fragment ions at m/z 125 and m/z 169 for a galloyl moiety suggesting that these compounds are galloyl quinic acids. Based on the intense characteristic [quinic acid − H − H2O]– fragmental ion at m/z 173 [20], compound 11 was identified as 4-O-galloyl quinic acid. The presence of this compound in the red maple species is in agreement with the previous report [10].

Compounds 21, 27 and 31 produced a pesudomolecular ion [M − H]− at m/z 495 with different tR at 13.97, 16.02 and 17.39 min, respectively. In their MS/MS spectra, fragmentation ions at m/z 343 [M − H − 152]− and m/z 191 [M − H − 152 − 152]− indicated the loss of two galloyl moieties. The diagnostic fragment ion at m/z 191 indicated the existence of quinic acid. Thus, compounds 21, 27 and 31 were tentatively identified as digalloyl quinic acid isomers.

Compound 44 (tR = 21.71 min) gave a [M − H]– ion at m/z 647. Based on the MS/MS daughter ions formed by sequential losses of three galloyl moieties at m/z 495 [M − H − 152]−, m/z 343 [M − H − 152 − 152]− and m/z 191 [M − H − 152 − 152 − 152]−, as well as typical daughter ions at m/z 191, m/z 169 and m/z 125 indicating the existence of quinic acid and galloyl moieties, compound 44 was tentatively identified as trigalloyl quinic acid.

3.1.5. Galloyl shikimic acid derivatives

Compounds 16, 24 and 28 all produced the same pesudomolecular ion [M − H]− at m/z 325 (molecular formula C14H14O9 provided by TOF-MS) with different tR at 11.60, 14.61 and 16.18 min, respectively. These compounds were tentatively identified as isomers of galloyl shikimic acid derivatives based on their product ions in MS/MS at m/z 173 [M − H − 152]− indicating the loss of 152 Da and the existence of a shikimic acid moiety, as well as diagnostic galloyl daughter ions at m/z 169 and m/z 125. These MS data were in agreement with the presence of monogalloyl shikimic acid derivatives [13].

Compound 40 (tR = 19.92 min) gave a deprotonated ion at m/z 477, with a molecular formula of C21H18O13. The MS/MS product ions at m/z 325 [M − H − 152]− and m/z 173 [M − H − 152 − 152]− indicated the loss of two galloyl moieties and the existence of a shikimic acid moiety. The compound also exhibited the diagnostic daughter ions at m/z 169 and m/z 125 for a galloyl moiety and thus, was tentatively identified as digalloyl shikimic acid.

3.2. Characterization of flavonoids

In the present study, 25 flavonoids were identified based on previously reported MS/MS fragmentation patterns for flavonoids [15]. The compounds constituted 19 flavonoids based on the quercetin aglycon and 6 flavonoids based on the kaempferol aglycon. The flavonoids included flavonoid glycosides and flavonoid galloyl glycosides.

Compound 70 (tR = 34.33 min) displayed a pesudomolecular ion [M − H]− at m/z 447. The characteristic fragment ions of quercetin at m/z 301, m/z 255 and m/z 151 [15] were observed in their MS/MS spectra. The loss of 146 Da was associated with a deoxyhexose moiety and thus, compound 70 was tentatively identified as quercetin deoxyhexoside. According to the previous study [21], quercetin flavonoids substituted at the 3-OH position should yield a high intensity radical aglycone ion at m/z 300. The intense typical product ion at m/z 300 was observed in the MS/MS spectra of compound 70 suggesting that it was quercetin-3-O-deoxyhexoside. Notably, quercetin-3-O- rhamnopyranoside was previously reported from the red maple species [16].

Compound 64 (tR = 31.90 min) displayed a deprotonated ion at m/z 433 and was tentatively identified as quercetin-3-O-pentoside based on the characteristic fragment ions of quercetin at m/z 301, m/z 255 and m/z 151, the diagnostic daughter ion of 3-OH substituted quercetin at m/z 300, and the loss of 132 Da corresponding to a pentose moiety. Notably, quercetin-3-O-arabinoside has previously been reported from the red maple species [3].

Compounds 50 (tR = 24.86 min) and 55 (tR = 27.42 min) gave [M − H]− ions at m/z 595 and 609. Their MS/MS spectra showed the intense quercetin aglycone ions at m/z 301, suggesting the loss of pentosyl-hexosyl (294 Da) and deoxyhexosyl-hexosyl (308 Da) groups, respectively. A high intensity ion at m/z 300 was observed in the MS/MS spectra of compound 55. Thus, compounds 50 and 55 were identified as quercetin pentosyl hexoside and quercetin-3-O-deoxyhexosyl hexoside, respectively. Notably, quercetin-3-O-rhamnoglucoside was previously reported from the red maple species [22].

Compounds 92, 96 and 97 all produced the same [M − H]− ion at m/z 599 with different tR of 42.75, 46.52 and 47.31 min, respectively. In their MS/MS spectra, they all showed a similar fragment ion at m/z 447 which was produced by the loss of a galloyl group (152 Da). In addition, the characteristic fragment ions of quercetin at m/z 301, m/z 255, and m/z 151 were observed. Thus, these compounds were tentatively identified as quercetin galloyl deoxyhexosides. A high intensity radical aglycone ion at m/z 300 was found in the MS/MS spectra of compound 92 indicating that it was quercetin-3-O-galloyl deoxyhexoside. Notably, quercetin-3-O-(3”-O-galloyl)-rhamnopyranoside and quercetin-3-O-(2”-O-galloyl)-rhamnopyranoside were previously reported from the red maple species [16].

Compounds 51 (tR = 25.66 min), 53 (tR = 26.49 min), 60 (tR = 29.18 min) and 63 (tR = 29.97 min) all exhibited [M − H]− ions at m/z 615. Based on the MS/MS daughter ions corresponding to loss of a galloyl moiety and a hexose moiety at m/z 463 [M − H − 152]− and m/z 301 [M − H − 152 − 162]−, respectively, as well as the typical daughter ions at m/z 301, m/z 255 and m/z 151, indicating the existence of a quercetin moiety, compounds 51, 53, 60 and 63 were tentatively identified as quercetin galloyl hexosides. Compounds 51 and 53 were further tentatively identified as quercetin-3-O-galloyl hexosides by their high intensity radical aglycone ion at m/z 300 in MS/MS spectra. Notably, a quercetin-O-galloyl-hexoside was previously reported in the red maple species [3].

Compounds 73 (tR = 35.36 min) and 87 (tR = 41.05 min), produced the same [M − H]− ions at m/z 585 and were tentatively identified as quercetin galloyl pentosides by their MS/MS product ions at m/z 433 [M − H − 152]− and m/z 301 [M − H − 152 − 132]− indicating the loss of a galloyl and a pentose moiety, as well as the typical daughter ions at m/z 301, m/z 255 and m/z 151, indicating the existence of a quercetin moiety. Compound 87 was further tentatively identified as quercetin-3-O-galloyl pentoside based on the intense MS/MS product ion at m/z 300.

Compounds 99 (tR = 53.04 min), 101 (tR = 53.83 min) and 103 (tR = 55.14 min) all gave [M − H]− ions at m/z 751. The MS/MS product ions at m/z 599 [M − H − 152]−, m/z 447 [M − H − 152 − 152]− and m/z 301 [M − H − 152 − 152 − 146]−, suggested the loss of two galloyl moieties and a deoxyhexose moiety. The typical daughter ions at m/z 301, m/z 255 and m/z 151 indicated the existence of a quercetin moiety. Thus, compounds 99, 101 and 103 were tentatively identified as quercetin digalloyl deoxyhexosides.

Compound 83 (tR = 39.96 min) gave a deprotonated ion at m/z 737. The MS/MS product ions at m/z 585 [M − H − 152]−, m/z 433 [M − H − 152 − 152]− and m/z 301 [M − H − 152 − 152 − 132]− indicated the loss of two galloyls and a pentose moiety, respectively. It also exhibited diagnostic daughter ions at m/z 301, m/z 255 and m/z 151 corresponding to quercetin. Thus, compound 83 was tentatively identified as quercetin digalloyl pentoside.

Compound 106 (tR = 60.13 min) gave a deprotonated ion at m/z 765. The MS/MS product ions at m/z 599 [M − H − 166]−, m/z 447 [M − H − 166 − 152]− and m/z 301 [M − H − 166 − 152 − 146]− indicated the loss of a methylgalloyl, a galloyl and a deoxyhexose moiety, respectively. It also exhibited the diagnostic quercetin daughter ions at m/z 301, m/z 255 and m/z 151. Thus, compound 106 was tentatively identified as quercetin galloyl methylgalloyl deoxyhexoside.

Compound 104 (tR = 57.86 min) displayed an [M − H]– ion at m/z 903. This compound was tentatively identified as quercetin trigalloyl deoxyhexoside based on the MS/MS daughter ions formed by sequential losses of three galloyls and a deoxyhexose moiety at m/z 751 [M − H − 152]−, m/z 599 [M − H − 152 − 152]−, m/z 447 [M − H − 152 − 152 − 152]− and m/z 301 [M − H − 152 − 152 − 152 − 146]−, respectively, as well as the typical daughter ions at m/z 301, m/z 255 and m/z 151 indicating the existence of quercetin moiety [15].

Compound 84 (tR = 40.86 min) displayed a pesudomolecular ion [M − H]− at m/z 431. The characteristic fragment ions of kaempferol [15] at m/z 285, m/z 255 and m/z 227 were observed in the MS/MS spectra and the loss of 146 Da was associated with a deoxyhexose moiety. Thus, compound 84 was tentatively identified as kaempferol deoxyhexoside. The diagnostic daughter ion of 3-OH substituted kaempferol at m/z 284 was observed in the MS/MS spectra suggesting that compound 84 was kaempferol-3-O-deoxyhexoside. Notably, kaempferol-3-O-rhamnoside was previously reported from the red maple species [22].

Compounds 65 (tR = 32.19 min) and 76 (tR = 36.04 min) displayed deprotonated ions at m/z 447 and m/z 417. In their MS/MS spectra, the characteristic fragment ions at m/z 285, m/z 284, m/z 255 and m/z 227 indicated a 3-OH substituted kaempferol moiety, and the loss of 162 Da and 132 Da suggested the existence of a hexose moiety for compound 65 and a pentose moiety for compound 76, respectively. Therefore, compounds 65 and 76 were tentatively identified as kaempferol-3-O-hexoside and kaempferol-3-O-pentoside, respectively. Notably, kaempferol-3-O-glucoside and kaempferol-3-O-galactoside were previously reported from the red maple species [22].

Compounds 100 (tR = 53.51 min) and 102 (tR = 54.47 min) both gave similar deprotonated ion at m/z 583. The MS/MS product ions at m/z 431 [M − H − 152]− and m/z 285 [M − H − 152 − 146]− indicated the loss of a galloyl and a deoxyhexose moiety, respectively. The compounds also exhibited diagnostic daughter ions for kaempferol at m/z 285, m/z 255 and m/z 227 and thus, compounds 100 and 102 were tentatively identified as kaempferol galloyl deoxyhexosides.

Compound 105 (tR = 59.55 min) displayed a [M − H]– ion at m/z 735 and was tentatively identified as kaempferol digalloyl deoxyhexoside based on the MS/MS daughter ions formed by losses of two galloyls and a deoxyhexose moiety at m/z 583 [M − H − 152]−, m/z 431 [M − H − 152 − 152]− and m/z 285 [M − H − 152 − 152 − 146]−, respectively, as well as the typical daughter ions at m/z 285, m/z 255 and m/z 227 indicating the existence of kaempferol moiety.

3.3. Characterization of other gallic acid derivatives

Compound 54 (tR = 27.04 min) gave a [M − H]− ion at m/z 300.9996, with a molecular formula of C14H5O8 provided by TOF-MS and was identified as ellagic acid. In its MS/MS spectrum, the characteristic fragment ions at m/z 284, 257, 229, 201, 185, 173 and 145 were observed, which were identical with an authentic reference standard of ellagic acid. Notably, ellagic acid was previously reported in the red maple species [22].

Compounds 25 (tR = 15.15 min) and 35 (tR = 18.42 min) exhibited deprotonated [M − H]− ions at m/z 183, with a molecular formula of C8H8O5 provided by TOF-MS. In the MS/MS spectrum, the characteristic fragment ions at m/z 168 [M − H − 15]− and m/z 124 [M − H − 15 − 44]− were due to the loss of a methyl free radical and a CO2, respectively. These compounds were tentatively identified as isomers of methyl gallic acid. Notably, two methyl gallic acids were previously reported from the red maple [8, 16]. The identification of compound 35 was further confirmed as gallic acid methyl ester after comparing its data with that of a corresponding authentic reference standard.

Compounds 52 (tR = 25.95 min) and 56 (tR = 27.68 min) gave [M − H]− ions at m/z 197, with a molecular formula of C9H10O5 provided by TOF-MS. In the MS/MS spectrum, the characteristic fragment ions at m/z 182 [M − H − 15]−, m/z 167 [M − H − 15 − 15]− and m/z 123 [M − H − 15 − 15 − 44]− were due to the loss of two methyl radicals and a CO2. They were tentatively identified as isomers of di-methyl gallic acid which were in agreement with the presence of di-methyl gallic acid from the red maple species [8]. By comparison of tR, high resolution MS data and MS/MS spectra data with those of an authentic standard, compound 56 was identified as 5-methyl gallic acid methyl ester.

Similarly, according to this fragmentation pathway, compounds 34, 66, 91, and 94 with tR of 18.04, 32.51, 42.43 and 44.31 min, respectively, were tentatively identified as digallate, methyl digallate, methyl trigallate and di-methyl digallate, respectively. Notably, a digallate and a di-methyl digallate were previously reported from the red maple species [22].

3.4. LC-ESI-TOF-MS/MS analyses of Maplifa™

Maplifa™ is a proprietary phenolic-enriched extract of red maple leaves which has previously been studied for its cosmetic potential [5]. As shown in Table 1, LC-ESI-TOF-MS/MS analyses revealed that Maplifa ™ was very similar to the methanol extract of red maple leaves and contained the vast majority of the identified compounds except for compounds 15, 21, 22, 25, 44, 69, 81, 83, 90, 91, 93, 95, 98, 104 and 106.

4. CONCLUDING REMARKS

In summary, a rapid and reliable method employing UFLC-ESI-TOF-MS/MS was developed for the identification of phenolic compounds in red maple leaves. Based on accurate mass measurement and characteristic fragmentation ions, 106 phenolic compounds, including 68 galloyl tannins, 25 flavonoids, gallic acid, quinic acid, catechin, epicatechin and 9 other gallic acid derivatives, were identified. Among these, 11 compounds are potentially new and 75 compounds are being reported in the red maple species for the first time. The UFLC-ESI-TOF-MS/MS method established herein could be used for future characterization and standardization of this botanical material used as an ingredient in nutraceutical and/or cosmetic formulations.

ACKNOWLEDGEMENT

Research reported in this publication was made possible by the use of equipment and services available through the RI-INBRE Centralized Research Core Facility which is supported by the Institutional Development Award (IDeA) Network for Biomedical Research Excellence from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103430.

Non-standard abbreviations

UFLC

ultra-fast liquid chromatography

IDA

information dependent acquisition

TIC

total ion chromatogram

tR

retention time

Footnotes

CONFLICT OF INTEREST STATEMENT

The authors have declared no conflicts of interest.

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