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. 2024 May 14;29(10):2300. doi: 10.3390/molecules29102300

Qualitative and Quantitative Analysis of Chemical Components in Yinhua Pinggan Granule with High-Performance Liquid Chromatography Coupled with Q-Exactive Mass Spectrometry

Imranjan Yalkun 1,†, Haofang Wan 1,†, Lulu Ye 1, Li Yu 1, Yu He 1, Chang Li 1,*, Haitong Wan 1,*
Editor: Victoria Samanidou1
PMCID: PMC11124461  PMID: 38792164

Abstract

Yinhua Pinggan Granule (YPG) is an approved compounded traditional Chinese medicine (TCM) prescription for the treatment of cold, cough, viral pneumonia, and related diseases. Due to its complicated chemical composition, the material basis of YPG has not been systematically investigated. In this study, an analytical method based on high-performance liquid chromatography (HPLC) coupled with Q-Exactive mass spectrometry was established. Together with the help of a self-built compound database and Compound Discoverer software 3.1, the chemical components in YPG were tentatively identified. Subsequently, six main components in YPG were quantitatively characterized with a high-performance liquid chromatography–diode array detector (HPLC-DAD) method. As a result, 380 components were annotated, including 19 alkaloids, 8 organic acids, 36 phenolic acids, 27 other phenols, 114 flavonoids, 75 flavonoid glycoside, 72 terpenes, 11 anthraquinones, and 18 other compounds. Six main components, namely, chlorogenic acid, puerarin, 3′-methoxypuerarin, polydatin, glycyrrhizic acid, and emodin, were quantified simultaneously. The calibration curves of all six analytes showed good linearity (R2 > 0.9990) within the test ranges. The precision, repeatability, stability, and recovery values were all in acceptable ranges. In addition, the total phenol content and DPPH scavenging activity of YPG were also determined. The systematic elucidation of the chemical components in YPG in this study may provide clear chemical information for the quality control and pharmacological research of YPG and related TCM compounded prescriptions.

Keywords: Yinhua Pinggan Granule, HPLC–Q-Exactive MS, chemical components, quantitative analysis, quality control of TCM

1. Introduction

Yinhua Pinggan Granule (YPG) is a patent compounded traditional Chinese medicine (TCM) prescription approved by the State Food and Drug Administration of China (No. Z20133007) for the treatment of cold, cough, viral pneumonia, and other related diseases. YPG was originally developed from an ancient TCM formula, Ma-huang-Tang (Ephedra Decoction), which was recorded in the TCM classic Shang Han Lun (Treatise on Febrile Diseases). YPG is composed of six herbs, namely, Lonicerae Japonicae Flos, Puerariae Lobatae Radix, Polygoni Cuspidati Rhizoma Et Radix, Ephedrae Herba, Glycyrrhizae Radix Et Rhizoma, and Armeniacae Semen Amarum, in a ratio of 4:4:4:2:2:1. In recent years, pharmacological and clinical studies of YPG have made effective progress. Previous studies have shown that YPG can inhibit the replication of influenza virus and regulate the occurrence of apoptosis caused by influenza virus infection [1]. In addition, YPG has a significant antiviral effect on H1N1 influenza virus-infected RAW264.7 cells and can protect influenza virus-infected pneumonia mice by reducing their lung injury [2]. At the same time, recent research showed that YPG and its components have significant inhibitory effects on the proliferation of the H1N1 virus [3]. Meanwhile, a randomized, double-blind, parallel, controlled clinical trial program with a total of 240 participants is in progress to test the clinical efficacy of YPG as a complementary therapy against community-acquired drug-resistant bacterial pneumonia [4].

TCM, including YPG, has shown unique advantages in the treatment of influenza virus and related diseases [5,6,7], especially during the COVID-19 pandemic [8,9,10]. However, the chemical complexity of TCM not only leads to great challenges in elucidating the pharmacological mechanisms of TCM but also hinders the deciphering of the material basis and quality control [11]. Therefore, the chemical profiling of compounded TCM prescriptions is of great importance. With its high sensitivity and high resolution, the liquid chromatography–mass spectrometry (LC-MS) technique has been widely applied in the qualitative analysis of TCM products in recent years [12,13].

Considering that most research on YPG has focused on pharmacology and clinical efficacy, the present study aimed to systematically identify and quantitatively characterize the phytochemical constituents of YPG. Based on the HPLC–Q-Exactive MS system, the global qualitative analysis of YPG was carried out, and then the main representative components were simultaneously quantified with HPLC-DAD detection technology in order to provide a theoretical basis for its future clinical application and quality control of YPG.

2. Results and Discussion

2.1. Optimization of Chromatographic Conditions

In order to systematically elucidate the chemical components of YPG, both the extraction method (Table S3 in Supporting Information) and the chromatography conditions were optimized. In particular, three different columns based on C18 packing material were tested, namely, the Welch Ultimate XB-C18 column (Welch, Shanghai, China) (150 mm × 4.0 mm, 3.0 μm), Agilent Poroshell 120 EC-C18 column (Agilent, Santa Clara, CA, USA) (150 mm × 3.0 mm, 2.7 μm), and Capcell Pak C18 MG II (Osaka Soda Co., Ltd., Osaka, Japan) (150 mm × 4.6 mm, 3 μm). Compared with the other chromatographic columns, the MG II column exhibited better separation capacity under optimal conditions. The acetonitrile–water system showed a better resolution and response in the selection of the mobile phase, and the addition of 0.1% or 0.5% formic acid could effectively improve the ionization efficiency and peak shape of some compounds. Finally, the HPLC-UV chromatogram obtained under optimized conditions is shown in Figure 1.

Figure 1.

Figure 1

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The HPLC chromatogram of YPG (wavelength = 254 nm). 1. Chlorogenic acid; 2. puerarin; 3. 3′-methoxypuerarin; 4. polydatin; 5. glycyrrhizic acid; 6. emodin.

2.2. Qualitative Analysis of YPG by HPLC–Q-Exactive MS

Under the optimal chromatographic conditions, the mass spectrometry information of YPG was acquired in both positive and negative modes to cover more components. The base peak chromatograms (BPCs) are presented in Figure 2. Then, as described in Section 3, the data were tentatively annotated with corresponding compounds with the help of Compound Discoverer software according to the values of the accurate molecular weight and patterns of secondary fragmentation [14]. In particular, [M + H]+ and [M − H]− were the common quasi-molecular ions in the positive and negative modes, respectively, from which the chemical formulas were preliminarily inferred, with a mass deviation ≤ 5 ppm. After compound library searching, reference comparison, and fragmentation study, a total of 380 components were tentatively identified in YPG. Among them, 45 compounds were also verified by comparison with reference compounds. The mass spectrometry information of the identified compounds is summarized in Table 1 (for additional information, see Table S1 in Supporting Information).

Figure 2.

Figure 2

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The BPCs of YPG in positive (A) and negative (B) modes.

Table 1.

Mass spectrometry information of chemical components in YPG.

No. Name RT (min) Formula Ion Type Molecular Ion (m/z) Main Product Ion (m/z)
1 ephedrannin A 3.40 C30H20O11 [M + H]+ 557.1104 395.1281, 215.0650, 177.0543, 145.0284
2 glucose 3.63 C6H12O6 [M + H]+ 181.0705 163.0792, 144.0655, 109.0286, 81.0340
[M − H]− 179.0551 161.0445, 141.0181, 117.0181, 87.0073
3 secologanic acid 3.67 C16H22O10 [M − H]− 373.1134 347.9473, 189.0156, 161.0234, 135.0440
4 D-mannitol 3.82 C6H14O6 [M + H]+ 183.0862 147.0650, 129.0543, 104.1073, 69.0342
5 sucrose 3.86 C12H22O11 [M + H]+ 343.1227 306.1183, 145.0495, 127.0390, 85.0290
[M − H]− 341.1083 261.7968, 179.0559, 113.0230, 59.0125
6 allantoin 3.99 C4H6N4O3 [M + H]+ 159.0512 142.0862, 114.0915, 99.0193, 70.0658
* 7 quinic acid 4.13 C7H12O6 [M + H]+ 193.0706 157.0492, 147.0652, 129.0546, 111.0443
8 salicylic acid 4.14 C7H6O3 [M + H]+ 139.0389 122.0714, 111.0443, 97.0287, 85.0289
9 4-aminophenol 5.72 C6H7NO [M + H]+ 110.0604 87.0046, 81.0340, 78.9949
* 10 citric acid 5.59 C6H8O7 [M + H]+ 193.0343 161.0595, 151.0388, 133.0647, 105.0702
[M − H]− 191.0188 173.0085, 129.0180, 111.0075, 87.0071
11 guanosine 6.34 C10H13N5O5 [M + H]+ 284.0986 258.4957, 152.0566, 135.0302, 110.0351
[M − H]− 282.0843 169.3754, 150.0411, 88.1632, 61.9871
12 adenosine 6.46 C10H13N5O4 [M + H]+ 268.1037 213.3238, 169.7115, 136.0617, 85.0288
* 13 gallic acid 7.23 C7H6O5 [M + H]+ 171.0291 154.0974, 130.0863, 115.0392, 70.0658
14 coumalic acid 7.40 C6H4O4 [M + H]+ 141.0184 113.9639, 90.9481, 72.9378, 56.9430
15 tachioside 8.05 C13H18O8 [M − H]− 301.0926 283.1918, 257.0457, 221.1909, 151.0029
16 isotachioside 8.27 C13H18O8 [M − H]− 301.0926 284.0321, 243.0658, 178.9978, 151.0026
17 ephedroxane 8.27 C11H13NO2 [M + H]+ 192.1019 164.9844, 146.9612, 106.0654, 87.0445
18 quinaldic acid 8.27 C10H7NO2 [M + H]+ 174.0551 146.9612, 128.9507, 105.9351, 55.9352
* 19 hordenine 8.56 C10H15NO [M + H]+ 166.1225 151.0101, 121.0649, 103.0546, 93.0703
20 leonuriside A 8.99 C14H20O9 [M − H]− 331.1035 285.0384, 253.0501, 169.0130, 125.0231
21 4-vinylguaiacol 9.19 C9H10O2 [M + H]+ 151.0755 133.0761, 123.9456, 119.0493, 91.0547
22 7α-morroniside 9.50 C17H26O11 [M − H]− 405.1401 371.0939, 243.0672, 191.0199, 111.0070
23 tetramethylpyrazine 9.83 C8H12N2 [M + H]+ 137.1075 111.0080, 93.0704, 68.9978
24 shuangkangsu 10.49 C20H30O14 [M − H]− 493.1560 447.2225, 431.0965, 269.0450, 169.0130
25 epi-gallocatechin 10.69 C15H14O7 [M + H]+ 307.0807 289.1790, 243.1704, 208.9966, 139.0389
[M − H]− 305.0665 247.5994, 219.0664, 165.0182, 125.0232
26 robinin 10.73 C33H40O19 [M + H]+ 741.2226 678.4389, 579.1688, 381.0964, 297.0752
27 5-(hydroxymethyl)furfural 10.85 C6H6O3 [M + H]+ 127.0393 111.9689, 110.0238, 84.9603, 55.9352
28 N-ethylbenzylamine 10.90 C8H11N [M + H]+ 122.0967 107.0732, 105.0336, 95.0495, 88.0237
29 vicenin-2 11.00 C27H30O15 [M + H]+ 595.1641 433.1127, 415.1019, 313.0700, 283.0596
[M − H]− 593.1510 539.2719, 521.2607, 463.2737, 226.9863
30 4′-hydroxyacetophenone 11.09 C8H8O2 [M + H]+ 137.0599 122.0364, 116.9720, 95.0497, 55.9352
31 6-hydroxykynurenic acid 11.35 C10H7NO4 [M + H]+ 206.0445 178.0497, 148.1121, 117.0699, 90.0797
[M − H]− 204.0293 168.1795, 160.0393, 132.0446, 110.1680
32 chlorogenic acid butyl ester 11.36 C20H26O9 [M − H]− 409.1495 365.0681, 337.0357, 241.0023, 169.0135
33 mirificin-4′-O-glucoside 11.73 C32H38O18 [M + H]+ 711.2118 579.1680, 417.1183, 399.1065, 297.0753
[M − H]− 709.1986 487.1239, 457.1142, 294.0533, 266.0583
34 mandelonitrile 11.82 C8H7NO [M + H]+ 134.0600 106.0654, 91.0544, 79.0548
35 kakkalide 11.83 C28H32O15 [M + H]+ 609.1803 447.1279, 411.1072, 327.0857, 297.0755
[M − H]− 607.1663 588.1254, 487.1243, 309.0403, 281.0458
36 mahuannin A 11.87 C30H24O10 [M − H]− 543.1324 528.5940, 497.2623, 381.1213, 265.0987
37 2,6-dihydroxybenzoic acid 11.92 C17H24O9 [M + H]+ 373.1480 308.2842, 237.3755, 151.0375, 107.0485
38 8-epi-loganic acid 11.96 C16H24O10 [M + H]+ 377.1438 357.1662, 339.1559, 265.0587, 237.0277
[M − H]− 375.1292 315.8566, 265.2063, 201.0163, 113.0231
39 swertiamarin 12.02 C16H22O10 [M − H]− 373.1134 357.0130, 295.0618, 201.0158, 135.0433
40 8-epi-loganin 12.30 C17H26O10 [M − H]− 389.1448 371.8339, 345.1187, 227.0693, 185.0593
41 5-methoxysalicylic acid 12.31 C8H8O4 [M + H]+ 169.0498 151.0391, 128.9508, 111.0444, 93.0339
42 norephedrine 12.65 C9H13NO [M + H]+ 152.1068 134.0964, 117.0700, 115.0545, 91.0547
43 7-epi-vogeloside 12.81 C17H24O10 [M − H]− 387.1295 341.1095, 272.9591, 227.0690, 179.0566
44 loganic acid 12.88 C16H24O10 [M − H]− 375.1292 287.1191, 201.0159, 189.0158, 135.0440
* 45 protocatechuic acid 12.90 C7H6O4 [M + H]+ 155.0339 137.0233, 117.0701, 107.0495, 72.9379
46 polygalin B 13.12 C28H32O15 [M + H]+ 609.1803 555.7846, 447.1268, 285.0752, 270.0516
[M − H]− 607.1663 460.8990, 325.0714, 310.0492, 282.0534
47 lonijaposide B 13.15 C25H32NO12 [M − H]− 537.1833 511.3804, 375.0705, 335.0791, 201.0155
48 3′-hydroxypuerarin 13.17 C21H20O10 [M − H]− 431.0975 415.0348, 311.0557, 283.0609, 255.0659
49 norpseudoephedrine 13.19 C9H13NO [M + H]+ 152.1068 134.0964, 117.0700, 106.0655
50 loganin 13.30 C17H26O10 [M − H]− 389.1448 371.8339, 326.0798, 227.0693, 185.0593
51 secologanoside 13.43 C16H22O11 [M + H]+ 391.1222 239.0796, 241.0385, 163.0388, 151.0389
[M − H]− 389.1083 280.5215, 194.8876, 121.0647, 95.0489
52 leucodelphidin 13.74 C15H14O8 [M − H]− 321.0607 305.2140, 265.0519, 253.0507, 186.9385
53 secologanin 13.83 C17H24O10 [M − H]− 387.1295 /
54 glucoisoliquiritin 14.04 C27H32O14 [M − H]− 579.1715 529.4413, 491.9212, 463.1199, 255.0662
55 glucoliquiritin apioside 14.07 C32H40O18 [M − H]− 711.2134 678.4865, 549.119, 457.1141, 255.0664
* 56 chlorogenic acid 14.15 C16H18O9 [M + H]+ 355.1014 163.0388, 135.0440
[M − H]− 353.0871 191.0553, 161.0234, 135.0440, 127.0385
* 57 neochlorogenic acid 14.22 C16H18O9 [M + H]+ 355.1014 338.1603, 289.0706, 235.0594, 163.0390
* 58 catechin 14.25 C15H14O6 [M + H]+ 291.0857 255.7885, 107.0648, 139.0389, 123.0442
[M − H]− 289.0714 245.0817, 203.0705, 151.0387.123.0439
59 ephedrine 14.36 C10H15NO [M + H]+ 166.1225 148.1119, 133.0886, 117.0700, 91.0548
* 60 cryptochlorogenic acid 14.37 C16H18O9 [M + H]+ 355.1014 337.0915, 235.0589, 205.0494, 163.0388
61 pseudoephedrine 14.43 C10H15NO [M + H]+ 166.1225 148.1119, 133.0887, 117.0701, 91.0546
62 ephedrannin D4 14.44 C30H24O14 [M − H]− 607.1089 563.1151, 487.1202, 413.0903, 267.0680
63 puerarin 6″-O-xyloside 14.61 C26H28O13 [M + H]+ 549.1590 417.1175, 381.0964, 297.0753, 267.0648
[M − H]− 547.1447 437.0846, 295.0609, 277.0504, 267.0661
64 chrysoeriol 7-O-neohesperidoside 14.65 C28H32O15 [M + H]+ 609.1803 447.1284, 429.1182, 327.0859, 285.0755
[M − H]− 607.1663 547.1422, 487.1246, 295.0607, 267.0660
* 65 p-hydroxybenzoic acid 14.70 C7H6O3 [M + H]+ 139.0389 121.0286, 111.0443, 93.0339
* 66 amygdalin 14.72 C20H27NO11 [M + H]+ 458.1647 355.1036, 213.0755, 163.0389, 107.0495
[M − H]− 456.1505 382.6093, 323.0968, 256.1356, 161.0449
67 methyl caffeate 14.83 C10H10O4 [M + H]+ 195.0652 177.0544, 163.0388, 145.0283, 117.0336
68 3,4-dimethyl-5-phenyloxazolidine 14.94 C11H15NO [M + H]+ 178.1227 162.1274, 147.1040, 117.0700, 105.0702
69 apigenin 5-rhamnoside 15.01 C21H20O9 [M + H]+ 417.1167 381.0964, 321.0743, 297.0753, 267.0647
[M − H]− 415.1027 295.0608, 267.0661, 253.0509, 223.0762
* 70 puerarin 15.02 C21H20O9 [M + H]+ 417.1167 399.1072, 381.0956, 363.0844, 255.0646
[M − H]− 415.1035 295.0608, 277.0507, 267.0661
71 mirificin 15.04 C26H28O13 [M + H]+ 549.1590 417.1168, 399.1069, 297.0754, 267.0648
[M − H]− 547.1447 418.4808, 295.0609, 267.0660, 114.2369
72 tectorigenin 7-O-xylosylglucoside 15.07 C27H30O15 [M + H]+ 595.1641 379.0811, 325.0695, 216.0653, 121.0283
[M − H]− 593.1510 495.0386, 473.1082, 310.0499, 282.0529
73 methylephedrine 15.14 C11H17NO [M + H]+ 180.1382 162.1275, 148.1076, 135.0804
74 methylpseudoephedrine 15.18 C11H17NO [M + H]+ 180.1382 162.1275, 147.1042, 135.0803, 117.0700
75 7-O-ethylsweroside 15.26 C18H26O10 [M − H]− 401.1451 325.7480, 269.1024, 253.0505, 178.0263
76 isoviolanthin 15.47 C27H30O14 [M + H]+ 579.1692 417.1190, 399.1072, 297.0753, 267.0649
[M − H]− 577.1554 531.2839, 518.0385, 283.0610, 268.0376
* 77 3′-methoxypuerarin 15.53 C22H22O10 [M + H]+ 447.1277 285.0751, 270.0516, 225.0542, 137.0232
[M − H]− 445.1134 430.0887, 367.1027, 327.1080, 215.0089
* 78 glycitin 15.54 C22H22O10 [M + H]+ 447.1277 429.1190, 411.1062, 327.0855, 297.0754
[M − H]− 445.1135 379.8243, 325.0714, 282.0530, 254.0597
79 benzyl alcohol 15.67 C7H8O [M + H]+ 109.0653 94.0148, 91.0546, 87.0045, 81.0704
80 methyl,4-hydroxycinnamate 15.68 C10H10O3 [M + H]+ 179.0705 162.1276, 147.1041, 117.0700, 109.0651
81 γ-octalactone 15.94 C8H14O2 [M + H]+ 143.1068 128.9508, 116.9721, 113.9639, 84.9603
82 (6S-9R)-roseoside 15.98 C19H30O8 [M + H]+ 387.2004 369.1341, 297.0762, 267.0641, 151.0388
83 syringin 15.98 C17H24O9 [M + H]+ 373.1480 308.0842, 292.1605, 237.3755, 151.0375
84 5,7-dihydroxyisobenzofuran 15.99 C8H6O4 [M + H]+ 167.0339 148.1120, 133.0885, 117.0700, 111.0442
85 kingiside 16.01 C17H24O11 [M − H]− 403.1240 371.1025, 310.7592, 243.0664, 174.8555
* 86 puerarin-7-O-glucoside 16.04 C27H30O14 [M + H]+ 579.1692 561.1633, 399.1074, 297.0753, 267.0647
87 sweroside 16.09 C16H22O9 [M + H]+ 359.1333 297.8041, 265.6036, 197.0808, 127.0391
* 88 caffeic acid 16.29 C9H8O4 [M + H]+ 181.0494 163.0388, 145.0284, 135.0441, 117.0337
[M − H]− 179.0341 164.0098, 135.0440, 112.1822, 107.0492
89 ephedrannin D1 16.53 C30H24O13 [M + H]+ 593.1279 576.3641, 447.1271, 327.0849, 297.0745
90 5-p-coumaroylquinic acid 16.70 C16H18O8 [M + H]+ 339.1071 266.4322, 245.8672, 147.0439, 119.0494
[M − H]− 337.0930 191.0553, 163.0390, 119.0470, 93.0332
91 loniceracetalide A 16.71 C21H32O11 [M − H]− 459.1869 /
92 piceatannol 3′-O-glucoside 16.77 C20H22O9 [M − H]− 405.1190 359.0753, 243.0659, 201.0549, 159.0440
93 lonijaposide D 16.82 C26H32NO13 [M − H]− 565.1774 550.4224, 519.2438, 445.1137, 325.0718
94 ephedralone 16.90 C11H9NO4 [M + H]+ 220.0602 192.0652, 164.0699, 151.4024, 119.0490
[M − H]− 218.0452 174.0550, 159.0315, 144.0077, 131.0365
95 neochlorogenic acid methyl ester 17.85 C17H20O9 [M + H]+ 369.1173 191.9904, 177.0544, 145.0283, 117.0336
[M − H]− 367.1029 255.0194, 191.0552, 173.0447, 134.0361
96 isoschaftoside 17.86 C26H28O14 [M + H]+ 565.1542 479.5669, 415.1016, 313.0699, 283.0596
[M − H]− 563.1340 341.0680, 311.0559, 283.0609, 149.0235
97 2,6-dihydroxyphenylacetic acid 18.07 C8H8O4 [M + H]+ 169.0498 151.0389, 146.9612, 128.9508, 123.0441
98 schaftoside 18.35 C26H28O14 [M + H]+ 565.1542 520.6823, 433.1122, 313.0700, 283.0597
[M − H]− 563.1340 529.1846, 341.0670, 311.0558, 283.0610
* 99 vanillic acid 18.50 C8H8O4 [M + H]+ 169.0498 151.0388, 146.9612, 128.9508, 123.0442
100 flavoyadorinin B 18.51 C23H24O11 [M + H]+ 477.1379 327.1659, 279.0375, 204.5438, 145.0499
101 ephedrannin D2 18.54 C30H24O13 [M − H]− 591.1150 547.1453, 462.7491, 285.0408, 253.0507
* 102 cinnamic acid 18.59 C9H8O2 [M + H]+ 149.0597 133.0885, 121.0648, 103.0548, 95.0497
103 hydroxyphenylacetic acid 18.65 C8H8O3 [M + H]+ 153.0546 135.1167, 112.0395, 90.9481, 72.9378
104 3-O-caffeoylshikimic acid 18.81 C16H16O8 [M + H]+ 337.0910 181.0494, 163.0388, 145.0283, 95.0495
[M − H]− 335.0771 269.1091, 179.0345, 161.0233, 133.0282
105 leucopelargonidin 18.82 C15H14O6 [M + H]+ 291.0857 273.0753, 207.0647, 147.0440, 139.0389
[M − H]− 289.0714 245.0818, 203.0705, 151.0392, 123.0441
106 licuraside 19.43 C26H30O13 [M − H]− 549.1609 502.1001, 429.1062, 255.0660, 119.0490
107 genistein 7-O-glucoside 20.25 C21H20O10 [M + H]+ 433.1120 271.0595, 215.0700, 137.0231
* 108 liquiritin apioside 20.29 C26H30O13 [M − H]− 549.1609 482.5059, 297.0778, 255.0660, 135.0076
109 isoliquiritin apioside 20.34 C26H30O13 [M − H]− 549.1609 488.6683, 429.1148, 255.0659, 135.0076
* 110 liquiritin 20.38 C21H22O9 [M − H]− 417.1186 402.1664, 373.0210, 255.0662, 119.0490
111 secologanin dimethyl acetal 20.39 C19H30O11 [M − H]− 433.1706 /
112 3-methoxyphenol 20.50 C7H8O2 [M + H]+ 125.0599 102.9706, 97.0287, 84.9602
113 piceid gallate A 20.63 C27H26O13 [M − H]− 557.1293 /
* 114 polydatin 20.92 C20H22O8 [M − H]− 389.1236 227.0707, 185.0598, 159.0808, 143.0491
115 lonicerin 21.02 C27H30O15 [M + H]+ 595.1641 433.1108, 313.0717, 271.0596, 215.0697
[M − H]− 593.1510 430.4479, 329.5679, 285.0396, 227.0705
116 isoliquiritin 21.24 C21H22O9 [M − H]− 417.1186 255.0660, 153.0182, 135.0075, 119.0489
* 117 quercetin 3-glucoside 21.69 C21H20O12 [M − H]− 463.0880 300.0272, 271.0247, 255.0296, 151.0027
118 kaempferol 7-O-glucopyranoside 21.69 C21H20O11 [M + H]+ 449.1072 330.0535, 287.0545, 203.4280, 153.0181
[M − H]− 447.0931 410.9457, 325.0732, 285.0397, 256.0383
119 neoisoliquiritin 22.95 C21H22O9 [M − H]− 417.1186 374.0878, 255.0660, 153.0183, 135.0076
* 120 coumarin 23.54 C9H6O2 [M + H]+ 147.0442 131.9743, 119.0493, 113.9640
* 121 daidzein 23.56 C15H10O4 [M − H]− 253.0502 224.0468, 209.0598, 197.0602, 135.0076
122 rhoifolin 23.76 C27H30O14 [M + H]+ 579.1692 515.2410, 429.1205, 327.0858, 297.0754
* 123 isochlorogenic acid A 23.89 C25H24O12 [M − H]− 515.1185 353.0883, 335.0772, 173.0445, 135.0440
124 reynoutrin 24.65 C20H18O11 [M − H]− 433.0771 /
125 avicularin 25.43 C20H18O11 [M − H]− 433.0771 /
* 126 resveratroloside 25.46 C20H22O8 [M + H]+ 391.1375 229.0856, 211.0759, 135.0440, 107.0495
127 liquiritigenin 7,4′-diglucoside 25.61 C27H32O14 [M + H]+ 581.1848 538.0963, 431.0979, 311.0434, 287.0430
128 centauroside 25.64 C34H46O19 [M − H]− 757.2545 679.1150, 525.1623, 458.1185, 254.0573
129 3,4-dicaffeoylquinic acid 25.75 C25H24O12 [M − H]− 515.1185 437.3583, 353.0874, 191.0552, 135.0440
130 herniarin 26.18 C10H8O3 [M + H]+ 177.0546 149.0597, 145.0283, 117.0336, 89.0390
131 catechin-5-O-β-D-glucopyranoside 26.54 C21H24O11 [M − H]− 451.1243 313.0739, 289.0719, 191.0340, 167.0340
132 vanillin 26.58 C8H8O3 [M + H]+ 153.0546 131.9743, 125.0597, 111.0443, 93.0338
133 4,7-dihydroxyflavone 7-D-glucoside 26.84 C21H20O9 [M + H]+ 417.1167 338.5892, 255.0647, 227.0695, 199.0747
134 methyl chlorogenate 27.00 C17H20O9 [M + H]+ 369.1173 313.0666, 285.0745, 207.0644, 161.0596
135 ketologanin 27.07 C17H24O10 [M + H]+ 389.1433 371.1681, 324.1584, 225.0426, 151.0388
136 naringin 27.96 C27H32O14 [M + H]+ 581.1848 449.1047, 431.0979, 329.0610, 311.0434
137 (E)-aldosecologanin 28.10 C34H46O19 [M − H]− 757.2545 679.1150, 595.2075, 525.1623, 458.1185
138 dihydrocaffeic acid 28.25 C9H10O4 [M + H]+ 183.0649 165.0545, 151.0389, 123.0441, 113.9639
139 p-coumaric acid 28.25 C9H8O3 [M + H]+ 165.0544 137.0597, 133.0283, 109.0650, 79.0547
140 secoxyloganin 28.25 C17H24O11 [M + H]+ 405.1379 373.2119, 309.2449, 165.0545, 151.0389
141 benzoic acid 28.26 C7H6O2 [M + H]+ 123.0441 105.0450, 95.0495, 67.0549
142 1,5-dicaffeoylquinic acid 28.63 C25H24O12 [M − H]− 515.1185 454.9042, 353.0873, 191.0552, 173.0446
143 vogeloside 28.65 C17H24O10 [M + H]+ 389.1433 233.2362, 195.0655, 151.0389, 107.0495
144 3-O-caffeoylquinic acid methyl ester 28.94 C17H20O9 [M + H]+ 369.1173 207.0649, 177.0546, 148.0514, 107.0857
145 quercitrin 30.05 C21H20O11 [M + H]+ 449.1072 330.0535, 287.0545, 269.0448, 153.0181
[M − H]− 447.0931 403.1030, 241.0501, 197.0599, 174.9555
146 4-feruloylquinic acid 30.26 C17H20O9 [M + H]+ 369.1173 239.4636, 207.0649, 177.0539, 148.0516
* 147 naringenin 31.32 C15H12O5 [M − H]− 271.0609 230.0589, 177.0189, 151.0026, 119.0490
148 kuzubutenolide A 31.41 C23H24O10 [M + H]+ 461.1433 299.0909, 253.0853, 193.0497, 107.0494
149 pueroside A 31.42 C29H34O14 [M + H]+ 607.2010 461.1439, 376.1363, 299.0908, 107.0494
150 epicatechingallate 31.75 C22H18O10 [M + H]+ 443.0962 390.0869, 291.0855, 273.0755, 123.0441
151 garbanzol 31.76 C15H12O5 [M + H]+ 273.0752 242.4491, 189.0543, 153.0180, 123.0441
152 chrysoeriol 7-O-glucopyranoside 31.80 C22H22O11 [M + H]+ 463.1220 445.1107, 427.1008, 343.0803, 313.0704
153 sophoraside A 31.92 C24H26O10 [M + H]+ 475.1587 313.1061, 267.1010, 253.0853, 107.0494
[M − H]− 473.1447 377.9086, 311.0924, 267.1024, 252.0786
154 vitexin 32.25 C21H20O10 [M + H]+ 433.1120 415.1018, 397.0909, 313.0698, 283.0597
[M − H]− 431.0975 269.0453, 240.0423, 225.0551, 193.4129
155 5-O-coumaroylcaffeoylquinic acid 32.38 C25H24O11 [M + H]+ 501.1379 483.1254, 320.0835, 255.0652, 163.0388
[M − H]− 499.1244 431.0978, 291.0275, 269.0454, 240.0423
* 156 resveratrol 32.64 C14H12O3 [M + H]+ 229.0856 211.0747, 183.0808, 135.0441, 107.0494
* 157 ferulic acid 32.90 C10H10O4 [M + H]+ 195.0652 177.0544, 163.0389, 138.0661, 107.0494
[M − H]− 193.0498 165.0005, 134.0361, 126.9024, 102.9472
* 158 isoferulic acid 32.91 C10H10O4 [M + H]+ 195.0652 177.0544, 163.0388, 149.0596, 109.0287
[M − H]− 193.0498 161.0233, 149.0236, 134.0363, 121.0281
159 kudzusaponin A1 34.62 C52H84O23 [M − H]− 1075.5314 1029.5265, 763.3842, 603.3890, 485.3619
160 hyperoside 35.15 C21H20O12 [M + H]+ 465.1023 447.1085, 303.0492, 286.0449, 257.0425
161 polygalin A 35.16 C23H24O11 [M + H]+ 477.1379 355.1165, 315.0853, 271.0960, 229.0856
[M − H]− 475.1241 267.0660, 252.0424, 201.9968, 132.0607
162 7-hydroxy-4-methoxy-5-methylcoumarin 35.24 C11H10O4 [M + H]+ 207.0650 189.0544, 161.0599, 150.0261, 123.0807
163 glycitin-6″-O-xylosyl 36.12 C27H30O14 [M + H]+ 579.1692 433.1124, 337.0699, 313.0699, 283.0596
164 cuspidatumin A 36.14 C14H12O4 [M + H]+ 245.0805 229.0854, 161.0122, 121.0286, 98.9757
[M − H]− 243.0661 225.1119, 207.1026, 174.9554, 146.9600
165 3,5-dicaffeoylquinic acid methyl ester 36.23 C26H26O12 [M + H]+ 531.1486 513.1385, 369.1514, 283.0595, 163.0388
* 166 rutin 36.27 C27H30O16 [M + H]+ 611.1602 465.1010, 303.0493, 257.0441, 229.0495
* 167 taxifolin 36.27 C15H12O7 [M + H]+ 305.0653 287.1236, 269.1125, 227.1023, 191.0814
168 pueroside B 36.31 C30H36O15 [M + H]+ 637.2119 475.1591, 313.1064, 267.1011, 107.0494
169 pueroside C 36.31 C24H26O10 [M + H]+ 475.1587 457.3117, 313.1034, 249.1549, 107.0486
170 macranthoidin B 36.35 C65H106O32 [M + H]+ 1399.6711 1075.5695, 943.5251, 795.2725, 633.2202
[M − H]− 1397.6552 1073.5525, 911.5010, 749.4481, 603.3898
171 kudzusaponin SA2 36.39 C47H76O19 [M + H]+ 945.5027 848.4162, 763.4678, 679.2439, 421.3453
172 macranthoidin A 36.40 C59H96O27 [M + H]+ 1237.6183 1076.5618, 943.5206, 751.4630, 603.2128
[M − H]− 1235.6036 1189.5997, 1073.5534, 911.5006, 749.4482
173 kudzusaponin SA4 36.40 C47H74O20 [M + H]+ 959.4806 892.2470, 764.6243, 615.3878, 421.3457
174 saponin 1 36.43 C58H94O26 [M + H]+ 1207.6077 1075.5693, 913.5162, 751.4610, 603.2120
[M − H]− 1205.5934 881.4901, 749.4479, 603.3898, 471.3479
175 24-hydroxy-licorice-saponin A3 36.45 C48H72O22 [M + H]+ 1001.4559 825.4282, 763.0059, 631.3789, 469.3288
176 3,4-O-dicaffeoylquinic acid methyl ester 36.46 C26H26O12 [M + H]+ 531.1486 319.0808, 271.0598, 177.0545, 163.0388
[M − H]− 529.1344 443.6241, 367.1035, 191.0554, 135.1440
177 isoquercetin 36.47 C21H20O12 [M + H]+ 465.1023 303.0494, 257.0439, 229.0495, 153.0182
178 soyasaponin A3 36.48 C48H78O19 [M + H]+ 959.5183 813.4605, 439.3565, 141.0181, 85.0289
[M − H]− 957.5065 911.5010, 749.4482, 587.3950, 471.3475
179 dipsacoside B 36.49 C53H86O22 [M − H]− 1073.5519 912.0020, 749.4480, 585.3804, 471.3478
180 kudzusaponin B1 36.52 C48H76O21 [M + H]+ 989.4921 843.4330, 681.3870, 469.3314, 141.0181
[M − H]− 987.4794 926.4868, 763.7924, 661.3583, 503.3387
181 saponin 4 36.55 C58H94O27 [M − 2H]2− 610.2908 /
182 licoricesaponin A3 36.57 C48H72O21 [M + H]+ 985.4612 809.4323, 615.3887, 453.3356, 189.1634
[M − H]− 983.4483 943.1790, 821.3969, 645.3637, 351.0566
183 neoliquiritin 36.58 C21H22O9 [M + H]+ 419.1326 315.0854, 257.0803, 217.0483, 124.0392
184 6″-O-malonyldaidzin 36.58 C24H22O12 [M + H]+ 503.1170 480.9303, 392.3837, 255.0647, 199.0751
185 (2E)-1-(2,3-dihydroxy-4-methoxyphenyl)-3-(4-hydroxyphenyl)-2-propen--one 36.58 C16H14O5 [M + H]+ 287.0908 245.0804, 207.0649, 193.0492, 121.0285
[M − H]− 285.0765 270.0532, 177.0185, 150.0311, 108.0206
186 loniceroside D 36.59 C53H86O23 [M + H]+ 1091.5607 1033.7538, 945.5055, 783.4556, 421.3455
[M − H]− 1089.5470 1071.5394, 943.4768, 882.4898, 763.4315
187 akebiasaponin D 36.60 C47H76O18 [M + H]+ 929.5079 767.4589, 635.4064, 437.3408, 189.1637
188 kudzusaponin A2 36.61 C42H68O16 [M + H]+ 829.4560 764.7684, 649.3955, 455.3521, 269.0806
[M − H]− 827.4425 763.3452, 677.4987, 516.0891, 333.8636
189 isorhamnetin 3-O-glucopyranoside 36.62 C22H22O12 [M + H]+ 479.1170 397.5868, 317.0649, 274.0458, 120.0809
* 190 astragalin 36.62 C21H20O11 [M + H]+ 449.1072 409.0180, 346.9581, 287.0544, 252.9790
[M − H]− 447.0931 316.5085, 284.0322, 255.0294, 227.0343
191 7,4′-dihydroxyflavone 36.63 C15H10O4 [M + H]+ 255.0645 227.0696, 199.0752, 137.0234, 91.0546
[M − H]− 253.0502 224.0470, 208.0522, 135.0076, 91.0174
192 isorhamentin 3-O-rutinoside 36.66 C28H32O16 [M + H]+ 625.1743 479.1161, 317.0652, 302.0414, 85.0289
[M − H]− 623.1613 527.7530, 415.1031, 252.0425, 223.0404
* 193 4′-methoxypuerarin 36.67 C22H22O9 [M + H]+ 431.1326 395.1120, 365.1009, 311.0910, 271.0595
194 4,5-O-dicaffeoylquinic acid methyl ester 36.67 C26H26O12 [M − H]− 529.1344 483.1268, 463.2749, 367.1032, 253.0501
195 quercetin 3-O-arabinoside 36.67 C20H18O11 [M + H]+ 435.0917 303.0501, 271.0596, 153.0180, 121.0280
196 loniceroside A 36.69 C52H84O21 [M − H]− 1043.5422 1025.5223, 763.3167, 709.8038, 532.3125
* 197 rhein 36.71 C15H8O6 [M + H]+ 285.0392 269.0440, 257.0428, 151.0385, 121.0283
[M − H]− 283.0246 268.0373, 217.0500, 175.0391, 133.0284
198 3,4,5-tricaffeoylquinic acid 36.72 C34H30O15 [M + H]+ 679.1633 499.1226, 322.2479, 163.0387, 135.0440
[M − H]− 677.1511 515.1179, 353.0875, 173.0446, 135.0440
199 choerospondin 36.74 C21H22O10 [M + H]+ 435.1296 303.0501, 271.0596, 231.0647, 153.0180
200 4,5-dicaffeoylquinic acid 36.75 C25H24O12 [M + H]+ 517.1326 499.1223, 453.8935, 269.0803, 163.0387
201 pollenitin B 36.76 C22H22O12 [M + H]+ 479.1170 412.8673, 317.0651, 302.0415, 274.0472
202 medicarpin3-O-glucoside 36.77 C22H24O9 [M + H]+ 433.1482 312.0939, 271.0596, 214.2812, 153.0182
203 lonfuranacid A 36.77 C12H20O5 [M + H]+ 245.1377 229.0853, 189.1119, 125.0962, 97.1015
204 questin 36.78 C16H12O5 [M + H]+ 285.0751 270.0518, 253.0490, 242.0574, 153.0179
205 Tricin 7-O-glucoside 36.78 C23H24O12 [M + H]+ 493.1326 331.0807, 315.0493, 287.0537, 270.0518
206 tectoridin 36.79 C22H22O11 [M + H]+ 463.1220 301.0700, 286.0467, 258.0517, 153.0181
* 207 liquiritigenin 36.80 C15H12O4 [M + H]+ 257.0805 239.0705, 211.0756, 147.0439, 137.0232
[M − H]− 255.0660 209.0605, 153.0183, 135.0077, 119.0490
208 subproside V 36.80 C54H88O24 [M − H]− 1119.5575 1073.5519, 911.5007, 749.4478, 603.3897
209 loniceroside E 36.81 C53H86O21 [M − H]− 1057.5570 1039.5623, 849.4960, 763.3219, 413.0908
210 kudzusaponin A5 36.82 C48H78O20 [M + H]+ 975.5131 829.4545, 764.4423, 667.4020, 455.3510
211 torachrysone 36.82 C14H14O4 [M + H]+ 247.0960 229.0856, 214.0621, 201.0907, 198.0673
[M − H]− 245.0812 230.0579, 215.0343, 202.0625, 159.0440
212 macranthoside B 36.83 C53H86O22 [M + H]+ 1075.5658 943.5235, 781.4703, 619.4197, 437.3409
213 glycyroside 36.83 C27H30O13 [M + H]+ 563.1743 431.1331, 413.1223, 311.0907, 281.0803
[M − H]− 561.1608 523.2799, 339.0867, 309.0767, 266.0582
214 ohyscion 36.84 C16H12O5 [M + H]+ 285.0751 270.0518, 242.0567, 189.4096, 113.0597
[M − H]− 283.0609 268.0375, 240.0419, 211.0391, 184.0518
215 afzelin 36.88 C21H20O10 [M + H]+ 433.1120 418.8996, 271.0596, 243.0644, 215.0699
[M − H]− 431.0975 269.0454, 240.0424, 225.0552, 152.9942
216 kudzusaponin SA3 36.89 C53H86O23 [M + H]+ 1091.5607 929.5117, 767.4597, 635.4111, 437.3414
[M − H]− 1089.5470 1043.5427, 881.4904, 749.4480, 603.3900
217 22β-acetoxyglycyrrhizin 36.91 C44H64O18 [M + H]+ 881.4143 705.3826, 511.3415, 451.3196, 107.0859
218 kudzusaponin C1 36.93 C54H88O23 [M + H]+ 1105.5767 959.5132, 797.498, 603.4246, 423.3591
219 questinol 36.94 C16H12O6 [M + H]+ 301.0701 286.0460, 269.0439, 167.0338, 134.0362
220 3′-methoxydaidzin 36.96 C22H22O10 [M + H]+ 447.1277 384.1155, 327.0859, 285.0752, 229.0857
221 loniceroside B 36.96 C58H94O25 [M + H]+ 1191.6127 817.3367, 763.4253, 619.4137, 437.3397
222 citreorosein 36.97 C15H10O6 [M + H]+ 287.0542 271.0596, 269.0443, 259.0960, 217.0491
[M − H]− 285.0402 268.0367, 257.0461, 196.0532, 133.0284
223 herbacetin 37.01 C15H10O7 [M + H]+ 303.0492 286.0430, 257.0442, 229.0497, 153.0181
[M − H]− 301.0351 284.0315, 273.0407, 178.9976, 151.0026
224 betulonic acid 37.02 C30H46O3 [M + H]+ 455.3507 409.3467, 388.4104, 203.1793, 189.1635
225 kudzusaponin A3 37.05 C48H78O20 [M + H]+ 975.5131 829.4545, 667.4020, 455.3510, 141.0181
* 226 ononin 37.08 C22H22O9 [M + H]+ 431.1326 269.0805, 254.0569, 213.0910, 107.0494
227 kaempferol 3-O-rutinoside 37.11 C27H30O15 [M + H]+ 595.1641 525.0455, 433.1108, 287.0544, 271.0596
228 isobavachalcone 37.11 C20H20O4 [M + H]+ 325.1429 309.0781, 285.0754, 189.0906, 95.0163
229 liqcoumarin 37.18 C12H10O4 [M + H]+ 219.0648 201.0910, 174.0674, 133.1012, 105.0702
230 isokaempferide 37.19 C16H12O6 [M + H]+ 301.0701 283.0596, 255.0636, 227.0698, 123.1169
231 uralsaponin F 37.21 C44H64O19 [M + H]+ 897.4094 763.6343, 679.2714, 527.3329, 334.7203
232 daidzein 4′,7-diglucoside 37.25 C27H30O14 [M + H]+ 579.1692 503.0054, 447.1283, 285.0753, 229.0858
233 liquoric acid 37.26 C30H44O5 [M + H]+ 485.3245 323.1276, 255.0648, 199.0751, 163.0385
234 isoorientin 37.27 C21H20O11 [M + H]+ 449.1072 330.0535, 287.0545, 153.0181, 135.0439
235 isorhodoptilometrin 37.31 C17H14O6 [M + H]+ 315.0856 300.0623, 272.0670, 153.0185, 95.0858
[M − H]− 313.0715 298.0479, 270.0529, 227.0343, 183.0454
236 chrysophanol 37.34 C15H10O4 [M + H]+ 255.0645 237.0549, 227.0692, 199.0751, 187.0725
237 licoricesaponin G2 37.34 C42H62O17 [M + H]+ 839.4040 663.3734, 487.3410, 469.3306, 141.0181
[M − H]− 837.3906 763.7259, 724.4032, 351.0571, 193.0345
238 kudzusaponin SA1 37.35 C42H68O15 [M + H]+ 813.4611 764.7317, 439.3548, 141.0181, 95.0860
[M − H]− 811.4482 765.4406, 603.3923, 432.7037, 283.0584
239 1,4-dicaffeoylquinic acid 37.38 C25H24O12 [M + H]+ 517.1326 460.9336, 414.0990, 269.0804, 213.0906
240 ethyl caffeate 37.38 C11H12O4 [M + H]+ 209.0806 163.0388, 145.1011, 135.0441, 117.0337
[M − H]− 207.0654 179.0341, 161.0233, 135.0441, 121.0284
241 apigenin 7-glucoside 37.41 C21H20O10 [M + H]+ 433.1120 379.0797, 337.0701, 313.0699, 271.0596
[M − H]− 431.0975 311.0562, 269.0453, 225.0554, 152.9949
* 242 daidzin 37.42 C21H20O9 [M + H]+ 417.1167 387.2100, 297.0747, 255.0648, 199.0753
243 2,5-dimethyl-7-hydroxychromenone 37.46 C11H10O3 [M + H]+ 191.0699 151.0387, 131.0856, 107.0861, 95.0860
244 prunetin 37.46 C16H12O5 [M + H]+ 285.0751 253.0489, 242.0572, 211.0750, 151.0390
[M − H]− 283.0609 268.0380, 240.0423, 197.0600, 168.0650
245 glabrolide 37.47 C30H44O4 [M + H]+ 469.3298 233.1540, 175.1479, 135.1167, 107.0858
246 pinocembrin 37.53 C15H12O4 [M + H]+ 257.0805 239.0701, 229.0850, 211.0752, 147.0440
247 polygonin B 37.54 C26H26O13 [M + H]+ 547.1430 299.0909, 284.0674, 239.0702, 163.0385
* 248 glycyrrhizic acid 37.56 C42H62O16 [M + H]+ 823.4086 647.3788, 471.3457, 453.3356
[M − H]− 821.3959 763.8063, 469.3315, 351.0569, 193.0347
249 echinatin 37.56 C16H14O4 [M + H]+ 271.0960 254.2115, 147.0438, 137.0596, 123.0441
[M − H]− 269.0816 251.0708, 225.0552, 151.0030, 119.0490
250 hesperetin 37.62 C16H14O6 [M + H]+ 303.0853 258.0517, 153.0183, 106.0866, 88.0762
[M − H]− 301.0714 273.0774, 255.0296, 230.0580, 183.0447
251 (S)-naringenin 37.62 C15H12O5 [M + H]+ 273.0752 189.0543, 153.0180, 123.0441
252 puerol B 37.64 C18H16O5 [M + H]+ 313.1063 267.1011, 253.0854, 107.0495
[M − H]− 311.0922 296.0689, 267.1026, 252.0789, 161.0233
253 coumestrol 37.72 C15H8O5 [M + H]+ 269.0437 254.0572, 241.0492, 213.0543, 185.0595
[M − H]− 267.0297 251.0709, 225.0550, 181.0649, 151.0026
254 polygonin A 37.73 C25H24O13 [M + H]+ 533.1273 488.1885, 360.1438, 285.0753, 270.0517
[M − H]− 531.1141 341.9318, 253.0502, 229.0135, 191.0555
255 tricin 37.73 C17H14O7 [M + H]+ 331.0804 315.0492, 302.0406, 270.0519, 73.0291
[M − H]− 329.0664 271.0247, 211.1332, 171.1017
256 neobavaisoflavone 37.76 C20H18O4 [M + H]+ 323.1272 308.0663, 267.0647, 255.0648, 239.0698
257 biochanin 37.76 C16H12O5 [M + H]+ 285.0751 270.0516, 253.0491, 225.0540, 137.0233
[M − H]− 283.0609 268.0377, 240.0423, 224.0474, 135.0075
258 gancaonin V 37.76 C19H20O4 [M + H]+ 313.1425 281.1162, 244.0359, 153.0181
[M − H]− 311.1285 296.0687, 267.1025, 252.0789, 161.0232
259 6,7-dimethoxycoumarin 37.76 C11H10O4 [M + H]+ 207.0650 189.1636, 175.0388, 148.0517, 91.0547
260 puerol A 37.78 C17H14O5 [M + H]+ 299.0908 284.0674, 256.0726, 239.0698, 95.0163
[M − H]− 297.0764 281.0457, 256.0376, 239.0346, 151.0025
261 biapigenin 37.78 C30H18O10 [M + H]+ 539.0958 522.9716, 387.0856, 286.0465, 184.0731
[M − H]− 537.0822 521.0623, 417.0622, 375.0506, 331.0608
262 2-methoxy-6-acetyl-7-methyljuglone 37.79 C14H12O5 [M + H]+ 261.0754 243.0648, 215.0699, 200.0466, 187.0754
[M − H]− 259.0608 243.1414, 231.0657, 216.0422, 188.0471
263 kudzusaponin SB1 37.79 C53H86O22 [M + H]+ 1075.5658 943.5235, 751.4603, 437.3405, 189.1636
[M − H]− 1073.5519 911.5006, 749.4489, 603.3903, 471.3480
264 diosmetin 37.81 C16H12O6 [M + H]+ 301.0701 286.0468, 258.0523, 241.0491, 88.0762
[M − H]− 299.0557 284.0325, 256.0372, 227.0344, 151.0030
265 (-)-epiafzelechin 37.82 C15H14O5 [M + H]+ 275.0906 257.0795, 217.0492, 189.0544, 107.0495
[M − H]− 273.0766 258.0532, 230.0579, 215.0343, 135.0076
266 licoricesaponin E2 37.85 C42H60O16 [M + H]+ 821.3938 764.7926, 451.3198, 173.1327, 121.1012
267 methyl glycyrrhizate 37.86 C43H64O16 [M + H]+ 837.4255 764.7705, 663.3716, 469.3308, 141.0181
268 licoisoflavanone 37.88 C20H18O6 [M + H]+ 355.1169 337.1062, 299.0546, 179.0337, 123.0441
[M − H]− 353.1027 335.0921, 312.0276, 217.0863, 189.0913
269 3-methoxyherbacetin 37.89 C16H12O7 [M + H]+ 317.0647 302.0411, 237.0383, 153.0181, 127.0391
[M − H]− 315.0506 300.0272, 272.0323, 188.0482, 112.9845
270 erybacin B 37.89 C19H18O5 [M + H]+ 327.1222 271.0597, 117.0367, 95.0163, 77.0059
[M − H]− 325.1076 309.2072, 297.0051, 197.1174, 171.1016
271 soyasaponin I 37.91 C48H78O18 [M + H]+ 943.5241 797.4680, 764.6400, 423.3611, 85.0289
[M − H]− 941.5101 912.5775, 763.8070, 615.3933, 438.3518
272 licoricesaponin B2 37.93 C42H64O15 [M + H]+ 809.4296 633.3988, 439.3564, 285.2223, 107.0859
[M − H]− 807.4168 763.8304, 520.9705, 351.0565, 193.0345
273 ephedrannin B 37.94 C30H20O10 [M + H]+ 541.1106 415.0806, 389.1013, 171.0287, 153.0181
[M − H]− 539.0981 521.2609, 507.2097, 396.8802, 266.9637
274 medicarpin 37.97 C16H14O4 [M + H]+ 271.0960 253.0497, 229.0855, 197.0594, 121.0285
* 275 kaempferol 37.99 C15H10O6 [M + H]+ 287.0542 271.0556, 254.0524, 226.0577, 153.0181
[M − H]− 285.0402 268.0364, 257.0451, 241.0497, 211.0396
276 glyasperin D 38.04 C22H26O5 [M + H]+ 371.1844 315.1218, 303.1219, 167.0701, 123.0441
277 isoliquiritigenin 38.09 C15H12O4 [M + H]+ 257.0805 239.0698, 211.0755, 147.0440, 137.0232
[M − H]− 255.0660 153.0182, 135.0077, 119.0489, 91.0175
278 3′-hydroxydaidzein 38.10 C15H10O5 [M + H]+ 271.0594 253.0492, 243.0647, 215.0702, 153.0180
279 kaikasaponin III 38.15 C48H78O17 [M + H]+ 927.5280 767.4596, 635.4124, 437.3406, 203.1794
280 3,4,3′,4′-tetrahydroxychalcone 38.16 C15H12O5 [M + H]+ 273.0752 245.0811, 171.0285, 153.0181, 123.0442
281 araboglycyrrhizin 38.21 C41H62O14 [M + H]+ 779.4183 /
282 macranthoside A 38.22 C47H76O17 [M + H]+ 913.5130 781.4694, 617.4044, 423.3610, 141.0180
[M − H]− 911.5004 749.4489, 603.3895, 471.3479, 423.3271
283 hydnocarpin 38.23 C25H20O9 [M + H]+ 465.1173 447.1065, 286.0468, 257.0440, 147.0438
[M − H]− 463.1033 447.2420, 285.0402, 255.0293, 208.9755
284 puerariafuran 38.24 C16H12O5 [M + H]+ 285.0751 270.0512, 253.0493, 242.0569, 211.0754
285 vestitol 38.29 C16H16O4 [M + H]+ 273.1118 255.1017, 227.1794, 137.0233, 121.0285
286 homobutein 38.31 C16H14O5 [M + H]+ 287.0908 269.0440, 241.0491, 185.0592, 151.0389
287 glycycoumarin 38.34 C21H20O6 [M + H]+ 369.1324 351.1228, 297.0746, 193.0494, 165.0545
288 licoricesaponin J2 38.34 C42H64O16 [M + H]+ 825.4243 764.8267, 455.3507, 189.1634, 141.0181
[M − H]− 823.4119 763.2627, 473.1696, 351.0565, 193.0342
289 licoricesaponin C2 38.35 C42H62O15 [M + H]+ 807.4139 764.8302, 678.4443, 631.3784, 437.3406
[M − H]− 805.4016 763.3167, 453.3408, 351.0559, 193.0349
290 3′-hydroxy-4′-O-methylglabridin 38.39 C21H22O5 [M − H]− 353.1393 /
291 blumenol A 38.39 C13H20O3 [M + H]+ 225.1483 210.1245, 167.9932, 114.0913, 95.0860
292 dihydrodaidzein 38.40 C15H12O4 [M + H]+ 257.0805 239.0690, 229.0851, 211.0744, 147.0439
* 293 formononetin 38.44 C16H12O4 [M + H]+ 269.0803 254.0567, 213.0907, 118.0414, 95.0859
[M − H]− 267.0660 252.0423, 225.0553, 195.0443, 132.0204
294 lupiwighteone 38.44 C20H18O5 [M + H]+ 339.1219 322.2484, 283.0594, 271.0597, 209.1646
* 295 quercetin 38.48 C15H10O7 [M + H]+ 303.0492 285.0393, 257.0439, 229.0493, 153.0181
[M − H]− 301.0351 283.0246, 255.0298, 227.0342, 138.0312
296 glycyuralin E 38.52 C21H22O6 [M + H]+ 371.1480 353.1372, 339.1213, 285.0749, 167.0695
[M − H]− 369.1341 311.0558, 229.0865, 206.0213, 139.0390
297 estradiol 38.53 C18H24O2 [M + H]+ 273.1845 255.1007, 248.4772, 153.0180, 119.0856
298 licoflavone A 38.57 C20H18O4 [M + H]+ 323.1272 280.0719, 267.0648, 254.0570, 239.0700
299 irisolidone 38.67 C17H14O6 [M + H]+ 315.0856 297.0751, 226.0619, 199.0751, 153.0182
[M − H]− 313.0715 295.0610, 270.0479, 224.0468, 167.2795
300 1-methoxyphaseollidin 38.68 C21H22O5 [M + H]+ 355.1532 299.0548, 221.1169, 165.0546, 123.0441
[M − H]− 353.1393 338.1162, 292.0359, 253.0505, 150.0311
301 cupressuflavone 38.69 C30H18O10 [M + H]+ 539.0958 497.0887, 403.0439, 377.0645, 335.0543
[M − H]− 537.0822 521.2611, 505.2242, 375.0520, 266.9636
302 licoarylcoumarin 38.69 C21H20O6 [M + H]+ 369.1324 313.0699, 271.0596, 243.0647, 147.0439
303 isoformononetin 38.71 C16H12O4 [M + H]+ 269.0803 251.0697, 241.0828, 237.0537, 107.0855
[M − H]− 267.0660 252.0424, 241.0503, 197.0604, 96.9588
304 kakkasaponin I 38.72 C47H76O16 [M − H]− 895.5067 877.5569, 763.6689, 678.9240, 509.4025
305 β-amyrone 38.76 C30H48O [M + H]+ 425.3767 /
306 tuberosin 38.82 C20H18O5 [M − H]− 337.1080 309.0397, 281.0454, 254.0585, 203.1068
307 glicophenone 38.83 C20H22O6 [M + H]+ 359.1482 301.0710, 283.0596, 175.0389, 153.0545
[M − H]− 357.1341 247.0974, 232.0737, 189.0186, 109.0282
308 7,4′-dihydroxy-3′-methoxyisoflavan 38.83 C16H16O4 [M + H]+ 273.1118 245.1898, 163.0750, 137.0596, 123.0442
[M − H]− 271.0973 241.0499, 225.0550, 197.0596, 181.0652
309 2′,3′-dihydro-7,7′-dihydroxy-5′-methoxy-2′,2′-dimethyl[3,6′-bi-4H-1-benzopyran]-4-one 38.89 C21H20O6 [M + H]+ 369.1324 313.0699, 285.0752, 270.0518, 243.0648
[M − H]− 367.1182 337.0717, 309.0403, 256.0376, 203.0708
310 3,4-didehydroglabridin 38.98 C20H18O4 [M + H]+ 323.1272 267.0648, 255.0647, 239.0698, 95.0163
[M − H]− 321.1129 277.0503, 265.0505, 252.0424, 149.0598
311 glyasperin C 38.98 C21H24O5 [M + H]+ 357.1689 301.1063, 221.1165, 165.0546, 123.0441
[M − H]− 355.1547 298.0483, 229.0865, 174.0313, 125.0232
312 neouralenol 39.01 C20H18O7 [M + H]+ 371.1119 315.0856, 268.2631, 183.0287, 165.0181
[M − H]− 369.0976 351.0870, 310.0444, 283.0975, 193.0135
313 phaseol 39.05 C20H16O5 [M + H]+ 337.1065 319.0956, 283.0596, 255.0646, 163.0388
314 eurycarpin A 39.08 C20H18O5 [M + H]+ 339.1219 322.2490, 293.0592, 163.0388, 114.0915
[M − H]− 337.1080 293.1182, 268.0376, 224.0470, 135.0077
315 glyurallin A 39.10 C21H20O5 [M − H]− 351.1236 335.0564, 323.0929, 308.0317, 191.0711
316 sophoraisoflavone A 39.10 C20H16O6 [M + H]+ 353.1014 335.0906, 325.1064, 283.02599, 191.0343
317 licocoumarone 39.11 C20H20O5 [M + H]+ 341.1379 323.1265, 267.0648, 209.1646, 114.0915
[M − H]− 339.1233 296.0677, 268.0377, 219.0656, 119.0490
318 dehydrovomifoliol 39.18 C13H18O3 [M + H]+ 223.1326 135.1167, 107.0858, 81.0704
[M − H]− 221.1177 205.1224, 164.0829, 148.0516, 118.5610
319 fallacinol 39.20 C16H12O6 [M + H]+ 301.0701 283.0598, 269.0440, 227.0701, 199.0752
[M − H]− 299.0557 284.0317, 255.0649, 240.0422, 212.0468
320 genkwanin 39.23 C16H12O5 [M + H]+ 285.0751 270.0519, 253.0494, 225.0542, 137.0233
[M − H]− 283.0609 268.0378, 240.0423, 186.6367, 118.3947
321 kanzonol U 39.25 C19H16O4 [M + H]+ 309.1115 /
322 2,3-dehydrokievitone 39.26 C20H18O6 [M + H]+ 355.1169 337.1066, 229.0854, 179.0338, 123.0442
[M − H]− 353.1027 284.0319, 243.1021, 216.0419, 201.0915
323 2,3,4-trimethyl-5-phenyloxazolidine 39.28 C12H17NO [M + H]+ 192.1382 133.1011, 119.0493, 91.0547
324 pratensein 39.29 C16H12O6 [M + H]+ 301.0701 283.0598, 269.0440, 227.0701, 199.0752
325 lupenone 39.32 C30H48O [M + H]+ 425.3767 /
326 corylifol B 39.35 C20H20O5 [M + H]+ 341.1379 267.0648, 209.1646, 114.0916
[M − H]− 339.1233 269.0453, 233.0818, 187.1117, 167.0340
327 4-O-methylglabridin 39.36 C21H22O4 [M + H]+ 339.1586 322.2483, 209.1644, 114.0916, 95.0163
328 luteone 39.43 C20H18O6 [M + H]+ 355.1169 338.3415, 299.0540, 267.0284, 239.0334
[M − H]− 353.1027 257.0063, 227.0702, 165.0179, 125.0232
329 glyinflanin H 39.43 C19H16O4 [M + H]+ 309.1115 291.1940, 223.0596, 113.0600, 95.0163
330 butyl octyl phthalate 39.44 C20H30O4 [M − H]− 333.2062 293.0450, 281.0451, 252.0419, 201.0916
331 glycyrrhetic acid 3-O-glucuronide 39.45 C36H54O10 [M + H]+ 647.3769 453.3359, 357.2422, 285.2203, 121.1012
[M − H]− 645.3641 580.9614, 521.2628, 469.3308, 322.6431
332 glyasperin A 39.46 C25H26O6 [M − H]− 421.1654 403.9289, 353.1024, 312.0273, 280.0371
333 1-methoxyphaseollin 39.47 C21H20O5 [M − H]− 351.1236 294.4448, 243.1023, 227.0710, 125.0232
334 licochalcone D 39.53 C21H22O5 [M + H]+ 355.1532 338.3410, 311.0542, 193.0494, 135.0440
335 wighteone 39.54 C20H18O5 [M + H]+ 339.1219 321.2453, 311.0548, 209.1647, 114.0916
[M − H]− 337.1080 321.0765, 309.1127, 253.0500, 209.0596
336 2′-O-demethylbidwillol B 39.54 C19H18O4 [M + H]+ 311.1269 293.1166, 278.0932, 263.0694, 95.0163
337 glycyrol 39.63 C21H18O6 [M + H]+ 367.1168 337.0697, 227.0702, 167.0337, 91.0547
[M − H]− 365.1026 335.0560, 307.0247, 295.0245, 254.0220
338 3-hydroxyglabrol 39.65 C25H28O5 [M − H]− 407.1863 387.2756, 371.2437, 150.9878, 93.0001
339 kumatakenin 39.72 C17H14O6 [M + H]+ 315.0856 255.0647, 227.0699, 153.0180, 60.0452
[M − H]− 313.0715 295.0606, 283.0609, 267.0663, 239.0712
340 eriodictyol 39.73 C15H12O6 [M + H]+ 289.0697 271.0959, 229.0855, 163.0388, 153.0181
[M − H]− 287.0559 272.0326, 258.0119, 216.0419, 155.1432
341 licoflavone B 39.74 C25H26O4 [M + H]+ 391.1897 358.2020, 323.1262, 267.0647, 195.0430
342 gancaonin U 39.76 C24H28O4 [M − H]− 379.1908 /
343 dehydroglyceollin I 39.78 C20H16O4 [M + H]+ 321.1115 306.0873, 187.0752, 159.0803, 147.0439
[M − H]− 319.0970 303.0658, 289.0504, 243.0657, 161.0233
344 tectorigenin 39.80 C16H12O6 [M + H]+ 301.0701 283.0598, 255.0646, 227.0698, 199.0748
[M − H]− 299.0557 284.0317, 267.0301, 240.0422, 212.0468
345 derrone 39.87 C20H16O5 [M + H]+ 337.1065 309.1118, 267.0650, 225.0545, 91.0549
[M − H]− 335.0921 319.0606, 305.0436, 278.3866, 158.8393
346 abyssinone II 39.91 C20H20O4 [M + H]+ 325.1429 269.0804, 241.0850, 135.0440, 95.0163
[M − H]− 323.1283 308.1031, 201.0914, 187.0761, 135.0441
347 corylin 39.96 C20H16O4 [M + H]+ 321.1115 306.0870, 279.0649, 265.0488, 137.0232
348 ephedradine A 39.98 C28H36N4O4 [M + H]+ 493.2800 465.2870, 394.2122, 219.1489, 120.0809
349 kudzusapogenol A 40.02 C30H50O5 [M − H]− 489.3575 /
350 kanzonol Y 40.04 C25H30O5 [M − H]− 409.2015 391.2520, 373.2436, 235.0971, 177.0912
351 kanzonol W 40.11 C20H16O5 [M + H]+ 337.1065 321.1119, 281.0443, 253.0488, 163.0388
[M − H]− 335.0921 320.0677, 291.1024, 199.0758, 135.0078
352 isoglycyrol 40.13 C21H18O6 [M + H]+ 367.1168 349.1074, 325.0702, 291.0630, 167.0338
[M − H]− 365.1026 349.0708, 309.0393, 216.0423, 192.0055
353 licoisoflavone B 40.17 C20H16O6 [M + H]+ 353.1014 311.0558, 299.0544, 153.0180, 95.0163
[M − H]− 351.0869 337.0660, 283.0974, 241.0864, 199.0756
354 6,8-diprenylgenistein 40.26 C25H26O5 [M + H]+ 407.1844 339.1198, 283.0596, 237.0534, 91.0547
[M − H]− 405.1704 387.2755, 371.2439, 281.0460, 150.9878
355 parvisoflavone A 40.29 C20H16O6 [M + H]+ 353.1014 335.0906, 325.1068, 191.0328, 153.0180
356 licoricidin 40.30 C26H32O5 [M + H]+ 425.2315 369.1328, 313.0703, 175.0388, 139.0389
357 kanzonol C 40.31 C25H28O4 [M + H]+ 393.2051 376.1539, 329.0271, 268.0656, 215.0684
* 358 emodin 40.43 C15H10O5 [M + H]+ 271.0594 243.0650, 229.0493, 197.0596, 173.0591
[M − H]− 269.0450 241.0503, 225.0551, 197.0597, 181.0647
359 apigenin 40.43 C15H10O5 [M + H]+ 271.0594 229.0495, 201.0543, 173.0597, 91.0548
[M − H]− 269.0450 241.0501, 225.0551, 210.0314, 181.0644
360 genistein 40.44 C15H10O5 [M + H]+ 271.0594 243.0642, 229.0495, 201.0543, 371.0596
[M − H]− 269.0450 241.0504, 225.0550, 197.0597, 181.0647
361 lupalbigenin 40.45 C25H26O5 [M + H]+ 407.1844 373.1034, 283.0597, 213.0541, 149.0232
362 angustone A 40.51 C25H26O6 [M − H]− 421.1654 404.9251, 352.0951, 269.0453, 201.0913
363 dehydroglyasperin D 40.59 C22H24O5 [M + H]+ 369.1688 313.0700, 295.0597, 197.0441, 179.0337
[M − H]− 367.1545 351.9669, 322.9651, 269.0455, 240.0420
364 corymbosin 40.63 C19H18O7 [M + H]+ 359.1118 329.0648, 313.0695, 269.0804, 95.0163
365 euchrenone a5 40.64 C25H26O4 [M + H]+ 391.1897 358.2020, 267.0647, 239.0701, 149.0236
[M − H]− 389.1751 319.0978, 298.0473, 266.0580, 195.1691
366 paratocarpin L 40.75 C25H28O5 [M − H]− 407.1863 /
367 diisobutyl phthalate 40.79 C16H22O4 [M + H]+ 279.1583 167.0340, 149.0232, 121.0284, 57.0706
[M − H]− 277.1438 245.3889, 193.7951, 134.0361, 121.0283
368 glyurallin B 40.79 C25H26O6 [M + H]+ 423.1793 /
369 angustone B 40.82 C25H24O6 [M + H]+ 421.1637 365.1014, 309.0388, 281.0439, 140.0342
[M − H]− 419.1497 402.9280, 375.0866, 363.0872, 308.0323
370 licoagrocarpin 40.93 C21H22O4 [M + H]+ 339.1586 /
371 palmitic acid 41.04 C16H32O2 [M − H]− 255.2322 170.3186, 162.0524, 116.9273, 74.0233
372 2′-hydroxyisolupalbigenin 41.11 C25H26O6 [M − H]− 421.1654 404.9261, 363.0872, 227.0711, 193.0862
373 butesuperin A 41.15 C26H22O8 [M + H]+ 463.1375 445.1275, 283.0597, 255.0647, 161.0594
* 374 luteolin 41.16 C15H10O6 [M − H]− 285.0402 268.9432, 257.0451, 242.0536, 196.0504
375 sophoracoumestan A 41.19 C20H14O5 [M + H]+ 335.0909 320.0672, 307.0952, 292.0722, 137.0237
* 376 glycyrrhetic acid 41.41 C30H46O4 [M + H]+ 471.3456 317.2107, 269.0803, 189.1636, 121.1013
377 8-prenylphaseollinisoflavan 41.74 C25H28O4 [M + H]+ 393.2051 339.0701, 269.0807, 167.0337, 149.0232
[M − H]− 391.1912 289.1443, 271.1335, 187.0393, 119.0490
378 stearic acid 41.75 C18H36O2 [M − H]− 283.2635 /
379 puerarol 41.75 C25H24O5 [M + H]+ 405.1683 319.0950, 281.0439, 209.0591, 171.0138
[M − H]− 403.1546 387.2754, 371.2439, 333.0764, 150.9877
380 hederagenin 41.79 C30H48O4 [M + H]+ 473.3615 310.8371, 189.1640, 133.1015, 59.0162
[M − H]− 471.3470 429.2146, 403.1549, 319.0597, 280.0376
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*: Verified with its reference standard.

In general, the 380 proposed chemical components of YPG can be classified into 9 categories based on chemical structure, including 19 alkaloids, 8 organic acids, 36 phenolic acids, 27 other phenols, 114 flavonoids, 75 flavonoid glycoside,72 terpenes, 11 anthraquinones and 18 other compounds (Figure 3a). While in the term of plant source, 114 compounds are from Glycyrrhizae Radix Et Rhizoma, 93 from Lonicerae Japonicae Flos, 76 from Puerariae Lobatae Radix, 47 from Ephedrae Herba, 42 from Polygoni Cuspidati Rhizoma Et Radix, and 8 from Armeniacae Semen Amarum (Figure 3b).

Figure 3.

Figure 3

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(a) The distribution of plant sources tentatively identified in YPG; (b) the types of compounds tentatively identified in YPG.

2.3. Identification of Several Specific Compounds in YPG

Based on the global identification of the components in YPG, the chemical structures of some compounds were inferred from the MS2 fragment ions, the proposed fragmentation pathways, and published references.

2.3.1. Phenolic Acids

Chlorogenic acid and its isomers, including neochlorogenic acid, cryptochlorogenic acid, etc., are typical phenolic acids found in YPG with high contents [15,16]. As shown in Figure 4, in the positive ion mode, the chlorogenic acid molecule is ionized to produce an [M + H]+ peak at m/z 355.1014. It is observed that fragmentation occurred via the neutral loss of quinic acid (C7H11O6), resulting in the formation of a product ion at m/z 163.0388. Alternatively, the [M + H]+ ion may lose a neutral molecule of C8H12O7 to generate a product ion at m/z 135.0440.

Figure 4.

Figure 4

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MS/MS spectrum of [M + H]+ ions and plausible fragmentation pathway of chlorogenic acid (positive ion mode).

2.3.2. Flavonoids

In YPG, 114 flavonoids were identified and were the most abundant compound type. As the most basic natural product in plant medicine, flavonoids have a wide variety of biological activities and efficacies [17,18]. In this study, puerarin was taken as an example to illustrate its fragmentation pathway. As a representative compound with high content in YPG, puerarin was observed in multiple characteristic fragmentation patterns in secondary mass spectrometry (Figure 5). The [M − H]− ion (m/z 415.1035) of puerarin loses a C4H8O4 moiety to generate a product ion at m/z 295.0608. Further neutral loss of H2O (18 Da) and CO (28 Da) then produced the fragment ions at m/z 277.0507 and 267.0661, respectively.

Figure 5.

Figure 5

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MS/MS spectrum of [M − H]− ions and plausible fragmentation pathway of puerarin (negative ion mode).

2.3.3. Alkaloids

The alkaloids in YPG are mainly ephedra alkaloids and isomers from Herba Ephedrae [19]. As a representative compound, the fragmentation pathway of methylephedrine is discussed herein. As shown in Figure 6, based on the protonated methylephedrine ion at m/z 180.1382, three characteristic fragment ions were observed at m/z 162.1275, 148.1076, and 138.0804, which can be attributed to [M + H − H2O]+, [M + H − H2O − CH2]+, and [M + H − C2H7N]+, respectively.

Figure 6.

Figure 6

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MS/MS spectrum of [M + H]+ ions and plausible fragmentation pathway of methylephedrine (positive ion mode).

2.3.4. Terpenoids

A total of 72 terpenoids were tentatively identified in YPG, of which triterpenoid saponins were mainly concentrated in Lonicera Japonica Thunb, Puerariae Lobatae Radix, and Glycyrrhizae Radix. Glycyrrhizic acid is the main active ingredient of Glycyrrhizae Radix, and it possesses various pharmacological effects, such as detoxification and anti-inflammatory activities [20,21,22]. As shown in Figure 7, the [M + H]+ ion of glycyrrhizic acid at m/z 823.4086 subsequently loses two gluconic acid moieties to form fragment ions at m/z 647.3788 and 471.3457. The latter ion can further lose one molecule of H2O to produce a product ion at m/z 453.3356.

Figure 7.

Figure 7

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MS/MS spectrum of [M + H]+ ions and plausible fragmentation pathway of glycyrrhizic acid (positive ion mode).

2.4. Quantitative Analysis, Total Phenolic Content, and DPPH Radical Scavenging Activity of YPG

In addition to the qualitative analysis of YPG, the contents of six main components in YPG, namely, chlorogenic acid, puerarin, 3′-methoxypuerarin, polydatin, glycyrrhizic acid, and emodin, were also determined with the help of the HPLC-DAD method. The method validation results in Table 2 reveal that all analytes showed good linear regression in the range of 0.01–2.00 mg/mL (R2 ≥ 0.9990). The limits of detection (LODs) were 0.38–2.13 μg/mL, and the limits of quantitation (LOQs) ranged between 1.14 and 6.47 μg/mL. The repeatability RSD was 1.18–2.94%, and the intermediate precision was 2.00–3.89%, indicating that the method has good precision. The RSD of stability was less than 3.60%, indicating that the six components were stable under storage conditions. The method also showed satisfactory accuracy, with recovery values ranging from 96.79% to 103.13%, and the RSD was less than 3.15% (data shown in Table S2 in Supporting Information).

Table 2.

Quantitative method validation results of YPG.

Main
Components		 Calibration
Curve		 Linear
Range
(mg/mL)		 R2 LOD (μg/mL) LOQ (μg/mL) Repeatability RSD (%) Stability RSD (%) Intermediate Precision
(%, n = 12)
chlorogenic acid y = 12,757x – 376.29 0.05–2.00 0.9991 2.13 6.47 1.18% 1.37% 2.82%
puerarin y = 37,995x + 303.18 0.05–1.60 0.9990 1.85 5.61 1.23% 2.60% 2.00%
3′-methoxypuerarin y = 25,755x + 64.034 0.04–1.50 0.9998 1.26 3.83 2.94% 1.09% 2.81%
polydatin y = 9872.5x – 89.081 0.03–1.00 0.9992 1.25 3.80 2.27% 3.10% 3.45%
glycyrrhizic acid y = 7190.5x + 101.33 0.02–1.00 0.9994 0.77 2.33 2.03% 1.30% 3.89%
emodin y = 33,371x + 138.83 0.01–0.50 0.9992 0.38 1.14 1.26% 3.59% 3.53%
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As presented in Table 3 and Figure 8, chlorogenic acid (34.15 ± 1.25 mg/g) and puerarin (28.30 ± 1.09 mg/g) were found as the most abundant compounds in YPG, followed by 3′-methoxypuerarin (9.63 ± 0.12 mg/g), polydatin (10.83 ± 0.57 mg/g), glycyrrhizic acid (3.33 ± 0.56 mg/g), and emodin (4.14 ± 0.34 mg/g). The contents of the six compounds were stable in three batches of YPG samples, which suggested that the six compounds may be used as quality markers of YPG.

Table 3.

Quantitative analysis, total phenol content, and DPPH radical scavenging results of YPG.

Main Components Sample
S1 (mg/g)		 Sample
S2 (mg/g)		 Sample
S3 (mg/g)		 Content (mg/g) Scavenging Percentage of
DPPH Radical (%)
chlorogenic acid 33.16 33.72 35.56 34.15 ± 1.25 59.2
puerarin 27.30 28.15 29.46 28.30 ± 1.09 49.6
3′-methoxypuerarin 9.73 9.66 9.50 9.63 ± 0.12 58.9
polydatin 10.29 10.76 11.43 10.83 ± 0.57 58.0
glycyrrhizic acid 3.18 2.85 3.94 3.33 ± 0.56 54.6
emodin 4.15 4.48 3.80 4.14 ± 0.34 30.3
total phenol 144.11 147.89 147.99 144.66 ± 2.21 /
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Figure 8.

Figure 8

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Quantitative analysis results of six main components in YPG.

Considering that the six compounds are phenols with antioxidative and radical scavenging activities, total phenolic contents and DPPH scavenging capacities were also determined. The total phenolic contents of three batches of YPG were calculated as 144.66 ± 2.21 mg/g with the Folin–Ciocalteu method (Table 3). In addition, the results of an online HPLC-based DPPH radical quenching assay revealed that all six main components were good radical scavengers with scavenging percentages ranging from 30.3% to 59.2% (Table 3 and Figure S1 in Supporting Information). Emodin exhibited a lower capacity against the DPPH radical compared with the other five compounds.

3. Materials and Methods

3.1. Reagents and Materials

YPG (Batch No. 200404) was provided by Shaanxi Dongke Pharmaceutical Co., Ltd. (Xianyang, China). Chlorogenic acid (≥95%), gallic acid (≥95%), 2,2-diphenyl-1-picrylhydrazyl (≥97%), and sodium carbonate anhydrous (≥99.8%) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); 3′-methoxypuerarin (≥98%) and polydatin (≥98%) were purchased from Chengdu Efa Biotechnology Co., Ltd. (Chengdu, China); puerarin (≥98%) and Folin–Ciocalteu reagent (>99.5%) were purchased from Shanghai Yien Chemical Technology Co., Ltd. (Shanghai, China); glycyrrhizic acid (≥98%) was purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China); emodin (≥99%) was obtained from China Institute for Food and Drug Control (Beijing, China). LC-MS-grade methanol, acetonitrile, and formic acid for HPLC analysis and LC-MS analysis were purchased from Tedia Co. (Fairfield, OH, USA). The water used was obtained from a Milli-Q water purification system (Bedford, MA, USA).

3.2. Sample Preparation

First, 100.00 mg of YPG was accurately weighed and added to 50% methanol (v/v, 1 mL). After extraction in an ultrasonic bath (300 W, 40 kHz, 50 °C) for 30 min, the extracted solution was centrifuged (13,000 rpm, 10min) and filtered through a 0.22 μm membrane. The obtained solution was kept at 4 °C until analysis.

3.3. Preparation of Standard Solutions

Six reference standards (chlorogenic acid 20.00 mg, puerarin 16.00 mg, 3′-methoxypuerarin 15.00 mg, polydatin 10.00 mg, glycyrrhizic acid 10.00 mg, emodin 5.00 mg) were accurately weighed and dissolved in 10 mL of 50% (v/v) methanol to prepare the respective stock solutions. The stock solutions were mixed and diluted with 50% methanol to prepare a series of mixed reference solutions in certain concentrations. All standard solutions were prepared in a 10 mL dark-brown volumetric flask and stored in a refrigerator at 4 °C before use.

3.4. Qualitative Analysis with HPLC–Q-Exactive MS

HPLC analysis was performed on a Thermo U3000 HPLC system (Thermo, San Jose, CA, USA). A CAPCELL PAK C18 MG II (150 mm × 4.6 mm, 3 μm; Osaka Soda, Osaka, Japan) column was used. The mobile phase consisted of 0.5% formic acid (A) and acetonitrile (B), with the elution gradient as follows: 0–10 min, 5–20% B; 10–30 min, 20–35% B; 30–32 min, 35–55% B; 32–35 min, 55–95% B; 35–42 min, 95% B, flow rate 0.4 mL/min. The column temperature was maintained at 35 °C, and the injection volume was 5 μL.

The Q-Exactive mass spectrometer (Thermo, San Jose, USA) equipped with a heated electrospray ion source (HESI) was operated in both positive and negative modes. The MS parameters were set as follows: scanning mode, Full MS/ddMS2; nebulizer voltage, 2.5 kV; sheath gas, 50 arb; aux gas, 14 arb; capillary temperature 320 °C; probe heater temperature, 300 °C; scanning range, m/z 100–1500. For different compounds, the collision energy was 30 and 40 eV. Instrument control and data acquisition were achieved using Xcalibur 2.3.1 (Thermo, San Jose, USA).

The raw data were processed with Compound Discoverer 3.1 (CD, Thermo, San Jose, USA) using a self-built compound library and workflow. The compound information was collected from public databases, including PubChem (https://pubchem.ncbi.nlm.nih.gov/), CAS SciFinder (https://scifindern.cas.org/), and CNKI (https://www.cnki.net/). Then, the original data of MS detection were matched with the self-built database in Compound Discoverer 3.1. The workflow included peak extraction, normalization, and compound annotation, and the specific parameters in the process were as follows: retention time range, 0–42 min; mass range, 100–1500 Da; positive adducts, [M + H]+, [2M + H]+; negative adducts, [M − H]−, [2M − H]−, [M − 2H]2−; mass tolerance, 5.0 ppm; S/N threshold, 3; minimum peak intensity, 100000.

3.5. Quantitative Analysis by HPLC-DAD

For quantitative analysis, an Agilent 1260 system equipped with a G7112C Quat Pump, a G7129A Vial sampler, and a G7117C diode array detector (DAD) was used (Agilent, Santa Clara, CA, USA). The chromatographic parameters were the same as described in Section 3.4. The chromatograms were recorded at 254 nm.

3.6. Quantitative Analysis Method Validation

In order to verify the applicability of the established method, the linearity, limits of quantitation (LOQs), limits of detection (LODs), repeatability, precision, stability, and recovery were validated. In the linear relationship experiment, the standard solution with a certain concentration gradient was used to draw the curve of the peak area (y) of the standard and the corresponding concentration (x, mg/mL). In the precision test, the standard solution was analyzed 6 times continuously, the intra-day precision was analyzed, and the intermediate precision was analyzed by different analysts at different times. The sample solution was analyzed at 0, 2, 4, 8, and 12 h in the stability test. In the repeatability test, the contents of the six target compounds in the sample solution were quantified. At the same time, the standard substance of each analyte was added to the sample according to 80%, 100%, and 120% of its content in the sample solution to prepare the sample solution for the recovery test. Three replicates were required for each spiked amount, and the recovery rate was calculated as described previously [16].

3.7. Determination of Total Phenolic Contents

3.7.1. Total Phenol Standard Curve Drawing

The total phenolic contents were measured according to a previously reported Folin–Ciocalteu reagent-based method, with gallic acid as a reference substance [23,24]. The absorbance was measured at 760 nm with an ultraviolet spectrophotometer. With absorbance as y, and total phenol mass concentration as x, the standard curve was drawn. The linear regression equation of gallic acid was y = 0.0965x + 0.0277 (R2 = 0.9992), and the linear range was 0.01–0.50 mg/mL.

3.7.2. Total Phenol Content in the Sample

Different batches of YPG samples were taken, and the solution was prepared according to the above method. The determination was repeated three times, and the content of each sample was calculated according to the linear regression equation.

3.8. In Vitro Antioxidant Activity Evaluation—DPPH Radical Scavenging Activity

The DPPH free radical scavenging activities of the six components in YPG were evaluated with an HPLC-based method, as previously reported [14]. The scavenging percentages were calculated using the following equation:

Scavenging percentage (%) = (Aoriginal − ADPPH)/Aoriginal × 100%

where Aoriginal stands for the absolute HPLC peak area of each ingredient, and ADPPH stands for the HPLC peak area of each ingredient after YPG reacted with DPPH.

4. Conclusions

In this study, a set of integrated methods, which include the qualitative HPLC–Q-Exactive MS method and the quantitative HPLC-DAD method, were established to profile the chemical properties of YPG from a macroscopic and systematic view. In the qualitative analysis part, 380 components were tentatively identified in YPG with the help of a self-built compound database and Compound Discoverer software. In the quantitative analysis part, the contents of six main components in YPG were determined with a validated method. These results may provide clear chemical information for the quality control and pharmacological research of YPG. This study may also contribute as a valuable reference for research on other complex TCM compounded prescriptions.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/molecules29102300/s1: Table S1: Mass spectrometric information of chemical components of Yinhua Pinggan Granule; Table S2: Recovery of six representative components. Table S3: The contents of representative components of YPG under different extraction conditions. Figure S1. The HPLC chromatograms at 254 nm of YPG before and after the reaction with DPPH. Refs. [25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60] are cited in the Supplementary Materials.

molecules-29-02300-s001.zip (4.8MB, zip)

Author Contributions

Conceptualization, C.L.; methodology, I.Y. and L.Y. (Li Yu); validation, H.W. (Haofang Wan), L.Y. (Lulu Ye) and L.Y. (Li Yu); formal analysis, H.W. (Haofang Wan); investigation, I.Y., H.W. (Haofang Wan) and L.Y. (Lulu Ye); resources, H.W. (Haitong Wan); data curation, I.Y. and C.L.; writing—original draft preparation, I.Y. and H.W. (Haofang Wan); writing—review and editing, C.L., Y.H. and H.W. (Haitong Wan); supervision, C.L., Y.H. and H.W. (Haitong Wan); project administration, C.L.; funding acquisition, C.L. and H.W. (Haitong Wan). All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in article and Supplementary Materials. Additional data that support the findings of this study are also available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was funded by the National Natural Science Foundation of China [81930111] and the Key Laboratory of TCM Encephalopathy of Zhejiang Province [2020E10012].

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

molecules-29-02300-s001.zip (4.8MB, zip)

Data Availability Statement

The data presented in this study are available in article and Supplementary Materials. Additional data that support the findings of this study are also available on request from the corresponding author.

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