Evidence-Based Complementary and Alternative Medicine
Volume 2018 (2018), Article ID 2781597, 13 pages
https://doi.org/10.1155/2018/2781597

Research Article

Chemical Profiling of Xueshuan Xinmaining Tablet by HPLC and UPLC-ESI-Q-TOF/MS

Ya-Xin Han,¹ Peng-Fei Wang,2,3 Meng Zhao,2,4 Liang-Mian Chen,² Zhi-Min Wang,² Xiao-Qian Liu,² Hui-Min Gao,² Mu-Xin Gong,¹ Hui Li,² Ji-Zhong Zhu,⁵ and Chuan-Gui Liu⁵

¹School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China
²Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, National Engineering Laboratory for Quality Control Technology of Chinese Herbal Medicine, Beijing 100700, China
³Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
⁴College of Pharmacy, Henan University of Traditional Chinese Medicine, Zhengzhou 450008, China
⁵Jilin Huakang Stock Ltd., Company of Medicines, Dunhua 133700, China

Correspondence should be addressed to Hui-Min Gao; huimin_gao@126.com and Mu-Xin Gong; gongmuxin@126.com

Received 18 April 2018; Accepted 26 September 2018; Published 21 October 2018

Academic Editor: Chang G. Son

Copyright © 2018 Ya-Xin Han et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Xueshuan Xinmaining Tablet (XXT) is a widely used traditional Chinese medicine for the treatment of stroke, chest pain, coronary heart disease, and angina pectoris caused by blood stasis. Having a multiple-component preparation, it is still far from meeting the requirements of modernization and standardization because its detailed chemical basis and action mechanism have not been clarified. In this work, the different batches of XXT samples were analyzed by HPLC and the typical sample was analyzed by UPLC-ESI-Q-TOF/MS to understand its chemical profiling. As a result, 77 chromatographic peaks were detected, among which 63 constituents were identified or tentatively characterized based on the comparison of retention time and UV spectra with authentic compounds as well as by summarized MS fragmentation rules and matching of empirical molecular formula with those of published components. This is the first systematic report on the chemical profiling of the commercial XXT products, which provides the sufficiently chemical evidence for the global quality evaluation of XXT products.

1. Introduction

Xueshuan Xinmaining Tablet (XXT) is a widely used traditional Chinese medicine for the treatment of stroke, chest pain, coronary heart disease, and angina pectoris caused by blood stasis [1]. It is prepared from ten raw materials, including Chuanxiong (Chuanxiong rhizoma), Huaihua (Sophorae flos), Danshen (Salviae miltiorrhizae radix et rhizoma), Shuizhi (Hirudo), Maodongqing (Hairy holly root), Rengong Niuhuang (Bovis calculus artifactus), Rengong Shexiang (Moschus artifactus), Renshen Jingye Zongzaogan (Total ginsenoside of ginseng stems and leaves), Bingpian (Borneolum syntheticum), and Chansu (Bufonis venenum) [2]. Nowadays, several qualitative analyses have been reported concerning volatile constituents by GC-MS [3] and chemical fingerprints by HPLC-ELSD [4] and UPLC-PDA [5]. However, having a multiple-component preparation, it is still far from meeting the requirement of modernization and standardization because its complicated ingredients lead to difficulty in clarifying therapeutic material basis, establishing quality control strategy, or selecting chemical markers for pharmacokinetic study.

Ultraperformance liquid chromatography coupled with electrospray ionization tandem quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q-TOF/MS) has become one of the most effective tools for the on-line structural elucidation of multiple components of TCM, owing to its characteristics of accurate mass measurement, high resolution, and excellent sensitivity. (E represents collision energy) technology provides an automated strategy to decrease analysis time and maximize duty cycles by using parallel alternating scans at low collision energy in the collision cell to obtain precursor ion information or at high collision energy to obtain accurate full-scan mass fragment, precursor ion, and neutral loss information. Therefore, both precursor and fragmentation data in exact mass mode were collected in a single run. This method has been proved to provide excellent chromatographic and MS efficiencies in the previous publications [6]. In the present investigation, a combination of HPLC and UPLC-Q-TOF/MS analyses was employed to find out and identify the common chemical profile in various batches of XXT samples. A total of 63 constituents were identified or tentatively characterized based on the comparison with reference substances, on-line UV spectra, and the fragmentation rules.

2. Materials and Methods

2.1. Materials and Reagents

Ten batches of Xueshuan Xinmaining Tablet (Lot. 160904, 160905, 161001-161008) were provided by Jilin Huakang Stock Ltd., Company of Medicines (Jilin, China). Reference substances of sodium danshensu (Lot. ZZS17032207), rosmarinic acid (Lot. ZZS17032201), salvianolic acid B (Lot. ZZS17032203), dihydrotanshinone (Lot. ZZS17032209), cryptotanshinone (Lot. ZZS17032204), and tanshinone IIA (Lot. ZZS17032206) were purchased from Shanghai ZZBIO Co, (Shanghai, China). Reference substances of neochlorogenic acid (Lot. X-014-140801), isochlorogenic acid B (Lot. Y-069-141122-1), and isochlorogenic acid C (Lot. Y-070-140801) were purchased from Chengdu Herbpurify Co. (Chengdu, China). Reference isochlorogenic acid A (Lot. 20130816) were purchased from Shanghai Yuanye Bio-Technology Co. (Shanghai, China). Reference substances of caffeic acid (Lot. 110885-200102), ginsenoside Rg₁ (Lot. 110703-201027), ginsenoside Re (Lot. 110754-200822), ginsenoside Rb₂ (Lot. 111715-200802), ginsenoside Rb₃ (Lot. 111686-201002), 20S-ginsenoside F₁ (Lot. 111763-200601), 20S-ginsenoside F₂ (Lot. 111764-200601), 20S-ginsenoside Rh₂ (Lot. 111748-200501), cinobufagin (Lot. 110803-200605), and resibufogenin (Lot. 110718-200507) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Gamabufotalin, bufotalin, and bufalin were purified from the fresh toad venom (the purity of gamabufotalin was over 92.9% and the purity of bufotalin and bufalin was over 98% based on the area normalization by HPLC analysis). Ginsenoside Rd, chlorogenic acid, rutin, quercetin, protocatechualdehyde, ursodesoxycholic acid, and chenodeoxycholic acid were from the collection of our laboratory and their purities were over 98%. The structures of all reference compounds were shown in Figure 1.

Figure 1: Structures of the identified compounds in XXT sample.

HPLC grade acetonitrile and formic acid (Fisher, Fair Lawn, NJ, USA) and ultrapure water were used. All other chemical reagents were of analytical grade from Beijing Chemical Corporation (Beijing, China).

2.2. Reference Solution Preparation

Stock solution with a concentration of about 0.1 g·L⁻¹ was prepared by dissolving an accurately weighed amount of each reference substance in methanol.

2.3. Sample Preparation

2.3.1. XXT Sample

1.0 g (about 2.5 tablets) of pulverized XXT sample was extracted with methanol (25 mL) by ultrasonication (200 w, 53 kHz) for 30 min at room temperature, and the extract was centrifuged for 5 min at 3000 rpm. The supernatant was evaporated at 80°C and the residue was dissolved with aqueous methanol (MeOH-H₂O, 9:1). The obtained solution was centrifuged for 5 min at 12000 rpm. A volume of 10 L of the supernatant was used for HPLC analysis.

1 ml of the sample solution for HPLC analysis was diluted to a volume of 10 mL and used for UPLC-ESI-Q-TOF/MS analysis.

2.3.2. Raw Materials

The ethanolic and aqueous extracts of individual herb (Danshen, Maodongqing, Shuizhi, Chuanxiong, and Huaihua) were prepared according to the manufacturing processes of XXT (Figure S1) described in the current Chinese Pharmacopoeia [2]. The ethanolic and aqueous extracts along with Rengong Shexiang, Rengong Niuhuang, Renshen Jingye Zongzaogan, Bingpian, and Chansu were dissolved with aqueous methanol solution (MeOH-H₂O, 9:1) according to the method described in Section 2.3.1 and used for the comparative analyses on the possible contribution from individual herb to general chromatographic profile of XXT samples.

2.4. Qualitative HPLC Analyses of 10 Batches of XXT Samples

The analyses were performed on a Shimadzu HPLC system (Shimadzu, Japan) equipped with an LC-20AT binary pump, a DGU-20A5 degasser, an SIL-20AC autosampler, a CTO-20AC column oven, and an SPD-M20A photodiode array detector. The samples were separated on a Waters XTerra C₁₈ column (4.6×250mm, 5μm). The mobile phase consisted of water containing 0.1% acetic acid (A) and acetonitrile (B) using a gradient elution program as follows: 0 min, 10% B; 20 min, 25% B; 30 min, 26% B; 45 min, 30% B; 60 min, 60% B; and 95 min, 80% B. The flow rate was 1.0 mL·min⁻¹ and the column temperature was 30°C. The PDA detector recorded UV spectra in the range from 190 nm to 400 nm and HPLC chromatogram was monitored at 251 nm.

2.5. UPLC-ESI-Q-TOF/MS Analysis

To comprehensively identify the chemical constituents in XXT samples, a UPLC-ESI-Q-TOF/MS experiment was performed using a Waters Xevo G2-S spectrometer (Waters, America), connected to a Waters UPLC system (Waters, America). The samples were separated on a Waters ACQUITY BEH C₁₈ column (2.1×50 mm, 1.7 μm). The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile (B) using a gradient elution program as follows: 0 min, 10% B; 4 min, 25% B; 7 min, 25.6% B; 8 min, 36% B; 14 min, 60% B; 18 min, 80% B; and 20 min, 80% B. The mass spectrometer was operated in both positive and negative modes using the following parameters: capillary voltage of 1.8 kV, sample cone voltage of 30 V (ESI+) or 40 V (ESI-), source temperature of 120°C, desolvation temperature of 600°C, and cone gas flow 50 L/h. In mode, the trap collision energy for the low-energy function was set at 6 eV, while the ramp trap collision energy for the high-energy function was set at 25-50 eV. Argon was used as the collision gas for collision-induced dissociation (CID) in and MS² modes. To ensure mass accuracy and reproducibility, the mass spectrometer was calibrated over a range of 100-2000 Da for MS and 50-2000 Da for MS². Leucine-enkephalin (m/z 556.2771 in positive ion mode; m/z 554.2615 in negative ion mode) was used as an external reference for the LockSpray and was infused at a constant flow of 5 μL·min⁻¹. The data were recorded in centroid type during acquisition.

3. Results and Discussion

3.1. Qualitative Analyses of XXT Samples by HPLC

In order to obtain better detection of XXT samples, HPLC conditions were optimized. The acetonitrile-water system displayed more powerful separation ability for the main constituents in all samples than the methanol-water system. When organic acid was added to the mobile phase, the symmetry of most chromatographic peaks was improved. Under optimized conditions, ten batches of XXT samples, together with the reference compounds, were examined and their HPLC chromatograms were shown in Figure 2. High similarity in the number, type, and amount of chemical constituents was observed in the HPLC profile of each individual sample, which suggested the chemical consistency of different batches of XXT samples. The characteristic peak 4 (rutin) represents an important active component in XXT sample with a consistently high content, which was chosen as the reference peak for the calculation of the relative retention time ratio and relative peak area ratio of other common chromatographic peaks. For the validation of the assay procedure, the results of precision and repeatability were indicated by RSDs that were less than 4.9% (n=6) for seventeen peaks (Tables S1~S4). The stability test suggested that these peaks were stable in the sample solution within 24 hr (Tables S5~S6).

Figure 2: HPLC profiles of Xueshuan Xinmaining Tablet samples at 251 nm (from top to bottom: Lot. 160904, 160905, 161001-161008).

To identify the origin of these characteristic peaks from individual raw material, a comparative study was carried out by using various extract of raw materials and XXT samples. Considering the chromatogram recorded at the wavelength of 251 nm, main chromatographic peaks in the HPLC profile were attributed to the following five raw materials: Danshen, Huaihua, Maodongqing, Chuanxiong, and Chansu (Figure S2). The contribution of other raw materials was not manifested, either because of the absence of UV absorption of active compounds or due to the existence of no potent components at the detection condition. More information is expected to further confirm the contribution of each raw material to the general chromatographic profile of XXT sample.

3.2. Identification of Chemical Constituents in XXT Sample by UPLC-ESI-Q-TOF/MS

analyses in the positive ion mode and negative ion mode were selected for obtaining extensive structural information via collision-induced dissociation. For the purpose of determining the optimized CE to generate fragment ions for structural elucidation and characterization, MS/MS experiments at different CE values were performed. Under optimized parameters, the total ion chromatogram of XXT samples are shown in Figure 3.

Figure 3: TICs of Xueshuan Xinmaining Tablet in the negative and positive modes.

The fragmentation behaviors of reference compounds were investigated, and these rules were applied for the structural elucidation of their derivatives with the same basic skeleton. A total of 63 compounds in XXT sample were detected and tentatively characterized by comparing the retention times, MS fragmentation behavior, and literature information. Among these compounds, there were twenty saponins, fifteen phenolic acids, ten quinones, eight steroids, four bile acids, four flavonoids, one amino acid, and one lactone. Their chemical structures are provided in Figure 1. For better understanding of the fragmentation behavior, 63 compounds were divided into several groups and deduced as follows. The chromatographic and mass data of the identified constituents were summarized in Table 1.

Table 1: Retention time (), UV, and MS data of the identified compounds in XXT samples.

No.
/min
UV 
Identified compounds
Formula
[M+H]⁺/[M+Na]⁺
Calc.mass
Δppm
[M-H]⁻
Calc.mass
Δppm
[M+HCOO]⁻/[2M-H]⁻
Calc.mass
Δppm

/nm

1
1.03
282
danshensu

197.0446
197.0450
-2.5
/395.0974
395.0978
-1.0
DS

2
1.22
295
neochlorogenic acid

353.0869
353.0873
-1.1

DS/MDQ

3
1.59

suberyl arginine

331.1977/
331.1981
-1.2
329.1819
329.1825
-1.8

CS

4
1.83
320
chlorogenic acid

355.1026/
355.1029
-0.8
353.0876
353.0873
0.8
/707.1840
707.1823
2.4
DS/MDQ

5
1.83
220, 318
protocatechuic aldehyde

137.0235/
137.0239
-2.9

DS

6
1.99
233
cryptochlorogenic acid

/377.0854

353.0871

DS/MDQ

7
2.28
232, 318
caffeic acid

179.0341
179.0344
-1.7

DS

8
3.41
244, 329
unidentified

/465.1384

441.1395

9
3.68
254, 352
rutin

611.1618/
611.1612
1.0
609.1458
609.1456
0.3
/1219.3020
1219.2989
2.5
HH

10
4.31
264, 343
kaempferol-3-O-

595.1652/
595.1663
-1.8
593.1505
593.1506
-0.1

HH

rutinoside

/617.1472
617.1482
-1.6

11
4.35
244, 327
isochlorogenic acid B

517.1331/
517.1346
-2.9
515.1188
515.1190
-0.4

DS/MDQ

/539.1177
539.1165
2.2

12
4.39
244, 327
salvianolic acid H

539.1149/
539.1190
-7.6
537.1031
537.1033
-0.4

DS

13
4.42
252, 339
isorhamnetin 3-O-rutinoside

625.1754/
625.1769
-2.4
623.1616
623.1612
0.6

HH

/647.1578
647.1588
-1.5

14
4.50
329
isochlorogenic acid A

/539.1154
539.1165
-2.0
515.1193
515.1190
0.6

DS/MDQ

15
4.90
244, 326
isochlorogenic acid C

517.1359/
517.1346
2.5
515.1192
515.1190
0.4

DS/MDQ

/539.1176
539.1165
2.0

16
5.02
244, 329
rosmarinic acid

359.0766
359.0767
-0.3
/719.1610
719.1612
-0.3
DS

17
5.13
250, 306
salvianolic acid G

341.0666/
341.0661
1.5

DS

18
5.17
251, 317
salvianolic acid A

493.1134
493.1135
0.2

DS

19
5.28

gamabufotalin

403.2483/
403.2484
-0.2

447.2381/
447.2383
-0.4
CS

20
5.32

bufarenogin

417.2273/
417.2277
-1.0

CS

21
5.43
276
4-hydroxyl-3-butylphthalide

207.1024/
207.1021
1.4

CX

22
5.53
232
salvianolic acid B

717.1475
717.1456
2.6

DS

23
6.17

ginsenoside Rg₁

/823.4792
823.4820
-3.4

845.4905/
845.4899
0.7
RSZZG

24
6.24

ginsenoside Re

/969.5378
969.5399
-2.2
945.5450
945.5423
2.9
991.5548/
991.5478
7.1
RSZZG

25
6.44

arenobufagin

417.2271/
417.2277
-1.4
415.2111
415.2121
-2.4
461.2168/
461.2175
-1.5
CS

26
6.45

quercetin

303.0506/
303.0505
0.3
301.0346
301.0348
-0.7

HH

27
6.71

unidentified

765.2371

28
6.99

unidentified

/677.4221

699.4323/

29
7.17
245, 328
hellebrigenin

417.2274
417.2277
-0.7

461.2173/
461.2175

CS

30
7.25

unidentified

677.1508

31
7.49

tanshindiol A

313.1079/
313.1076
1.0

DS

/335.0901
335.0895
1.8

32
8.14

ethyl lithospermate

567.1498/
567.1503
-0.9
565.1342
565.1346
-0.1

DS

/589.1320
589.1322
-0.3

33
8.49

unidentified

/755.1589

731.1622

34
8.65

unidentified

/1117.5740

1139.5891/

35
9.01

20S-glc-ginsenoside Rf

/985.5332
985.5348
-1.6

1007.5457/
1007.5427
3.0
RSZZG

36
9.15

beads ginseng saponins F₁

/985.5319
985.5348
-2.9

1007.5457/
1007.5427
3.0
RSZZG

37
9.26

notoginsenoside R₃

/985.5339
985.5348
-0.9

1007.5459/
1007.5427
3.2
RSZZG

38
9.35

unidentified

/951.4901

973.5042/

39
9.52

unidentified

/538.2797

514.2904

/1029.5789

40
9.64

ginsenoside Rf

801.4985/
801.5000
-1.9

845.4923/
845.4899
2.8
RSZZG

/823.4802
823.4820
-2.2

41
9.72

bufotalin

445.2588/
445.2590
-0.4

489.2489/
489.2488
0.2
CS

42
9.93

notoginsenoside R₂

/793.4705
793.4714
-1.1

815.4818/
815.4793
2.5
RSZZG

43
10.07

ginsenoside F₃

/793.4700
793.4714
-1.8

815.4820/
815.4793
3.3
RSZZG

44
10.10

ginsenoside Rc

/1101.5831
1101.5821
0.9

1123.5947/
1123.5900
4.2
RSZZG

45
10.20

ginsenoside Rg₂

/807.4908
807.4871
4.3

829.4968/
829.4949
2.3
RSZZG

46
10.31

ginsenoside Rb₂

/1101.5798
1101.5821
-2.1

1123.5952/
1123.5900
4.6
RSZZG

47
10.38

ginsenoside Rb₃

/1101.5797
1101.5821
-2.2

1123.5940/
1123.5900
3.6
RSZZG

48
10.49

unidentified

466.3162/

464.3032

49
10.75

20S-ginsenoside F₁

/661.4288
661.4292
-0.6

683.4393/
683.4370
3.4
RSZZG

50
10.73

bufalin

387.2531/
387.2535
-1.0

CS

51
10.86

unidentified

/969.5361
969.5399
-3.9

991.5539/
991.5478
6.2

52
10.99

unidentified

503.3368/

501.3224

53
11.27

ginsenoside Rd

/969.5374
969.5399
-2.6

991.5509/
991.5478
3.1
RSZZG

54
11.61

gypenoside IX

/939.5284
939.5293
-1.0

961.5390/
961.5372
1.9
RSZZG

55
11.68

resibufogenin

385.2385/
385.2379
1.6

CS

56
11.71

cinobufagin

443.2435/
443.2434
0.2

CS

57
11.79

cholic acid

/431.2778
431.2773
1.2
407.2803
407.2797
1.5
453.2857/
453.2852
1.1
RGNH

/815.5698
815.5673
3.1

58
12.03

ursodeoxycholic acid

391.2851
391.2848
0.8
437.2902/
437.2903
1.4
RGNH

/783.5796
783.5775
2.6

/783.5778
783.5775
0.4

59
12.41

1-oxo tanshinone IIA

309.1134/
309.1127
2.3

DS

60
12.59

ginsenoside Rk₁

/789.4742
789.4765
-2.9

811.4871/
811.4844
3.3
RSZZG

61
12.66

20S-ginsenoside F₂

/807.4860
807.4871
-1.4

829.4964/
829.4949
1.8
RSZZG

62
12.89

neotanshinone D

337.1431/
337.1440
-2.7

DS

63
13.25

dihydrotanshinone

279.1033/
279.1021
4.3

DS

/301.0849
301.0841
2.7

64
13.52

20R-ginsenoside Rg₃

/807.4858
807.4871
-2.1

829.4963/
829.4949
1.7
RSZZG

65
13.71

20S-ginsenoside Rg₃

/807.4852
807.4871
-2.4

829.4968/
829.4949
2.3
RSZZG

66
13.76

tetrahydro-tanshinone I

281.1189/
281.1178
3.9

DS

/303.1004
303.0997
2.3

67
13.85

hyodeoxycholic acid

391.2852
391.2848
1.0
437.2903/
437.2903
0.0
RGNH

/783.5792
783.5775
2.2

68
13.92

unidentified

503.3368/

501.3222

/1003.6544

69
13.98

methyl tanshinonate

339.1240/
339.1232
2.4

DS

/361.1054
361.1052
1.4

70
14.15

chenodeoxycholic acid

391.2848
391.2848
0.0
437.2901/
437.2903
-0.5
RGNH

/783.5789
783.5775
1.8

71
15.05

cryptotanshinone

297.1508/
297.1491
5.7

DS

/319.1313
319.1310
0.9

72
15.62

ilexoside A/D

/789.4742
789.4765
-2.9

811.4847/
811.4844
0.4
MDQ

73
15.69
293
methylene

279.1030/
279.1021
3.2

DS

tanshinquinone

/301.0848
301.0841
2.3

74
16.03

20S-ginsenoside Rh₂

667.4425/
667.4421
0.6
RSZZG

75
16.39

unidentified

496.3405/

540.3302/

76
16.81
267
tanshinone IIA

295.1361/
295.1334
9.1

DS

/317.1165
317.1154
3.5

77
17.31

miltirone

283.1709/
283.1698
3.9

DS

/305.1525
305.1517
2.6

Danshen, Huaihua, Maodongqing, Chansu, Chuanxiong, Renshen Jingye Zongzaogan, and Rengong Niuhuang are abbreviated as DS, HH, MDQ, CS, CX, RSZZG, and RGNH, respectively.

Saponin Compounds. . Saponin compounds have no UV absorption, but they could be well detected in both positive and negative ionization modes. Usually, the adduct ions such as [M+Na]⁺ (ESI+) or [M+HCOO]⁻ (ESI-) were detected in MS spectrum. In MS/MS spectrum, a series of fragmentation ions were observed due to the neutral loss of glucose, rhamnose, arabinose, or xylose (see Figure 4). These fragmentation rules are in accordance with the previous data [7, 8]. Totally, 20 ginsenoside saponins were identified, and they are deduced from the raw material, total ginsenoside of ginseng stems and leaves. Among the identified saponins, eight compounds (23, 24, 46, 47, 49, 53, 61, and 74) were unambiguously identified as ginsenoside Rg₁, ginsenoside Re, ginsenoside Rb₂, ginsenoside Rb₃, 20S-ginsenoside F₁, ginsenoside Rd, 20S-ginsenoside F₂, and 20S-ginsenoside Rh₂. Unknown ginsenoside saponins followed the similar dissociation pathways. For the ginsenosides of protopanaxadiol, the ions at m/z 407, 425, and 443 could be found in the positive ion mode. Meanwhile the ginsenosides of protopanaxatriol showed the ions at m/z 405, 423, and 441 in the positive ion mode.

Figure 4: Proposed fragmentation pathway of ginsenoside Rb₃.

Ginsenoside Rg₁ showed [M+Na]⁺ ion at m/z 823.4824 (C₄₂H₇₂O₁₄Na) in the positive ion mode and [M+HCOO]⁻ ion at m/z 845.4895 (C₄₃H₇₃O₁₆) in the negative ion mode. The fragmentation ions at m/z 621.4368 and 603.4259 were produced from the protonated ion [M+H]⁺ by losing glucose and the further loss of H₂O. In the negative MS/MS analysis, [M-H]⁻ at m/z 799.4838 gave the characteristic ion at m/z 637.4307 [M-H-glc]⁻and 475.3773[M-H-2glc]⁻. Compounds 23 and 40 showed the quasimolecular ions at m/z 823 [M+Na]⁺ and 845 [M+HCOO]⁻ in the full mass spectrum. Compound 23 has the same retention time and MS data as those of reference ginsenoside Rg₁ and it was identified as ginsenoside Rg₁. As for compound 40, although the fragmentation ions at m/z 405, 423, or 441 could not be detected in the MS/MS spectrum, it was temporarily identified as ginsenoside .

Ginsenoside Re showed [M+Na]⁺ ion at m/z 969.5427 (C₄₈H₈₂O₁₈Na) in the positive ion mode and [M+HCOO]⁻ ion at m/z 991.5482 (C₄₉H₈₃O₂₀) in the negative ion mode. The fragmentation ions at m/z 945.5428 [M-H]⁻, 799.4845 [M-H-rha]⁻, 783.4893 [M-H-glc]⁻, and 637.1430 [M-H-rha-glc]⁻ were found in MS/MS spectrum in the negative ion mode. Compound 24 showed similar retention time and MS data as those of reference ginsenoside Re and it was identified as ginsenoside Re.

As for reference ginsenoside Rb₂, [M+Na]⁺ ion at m/z 1101.5868 (C₅₃H₉₀O₂₂Na) in the positive ion mode and [M+HCOO]⁻ ion at m/z 1123.5908 (C₅₄H₉₁O₂₄) in the negative ion mode are shown in the full mass spectrum. The fragmentation ions at m/z 945.5425 [M-H-ara]⁻ and 783.4889 [M-H-ara-glc]⁻ were detected in MS². Reference ginsenoside Rb₃ (Figure 4) gave the adduct ions at m/z 1101.5844 [M+Na]⁺ and 1123.5903 [M+HCOO]⁻. The fragmentation ions at m/z 945.5430 [M-H-xyl]⁻, 783.4881 [M-H-xyl-glc]⁻, and m/z 621.4352[M-H-xyl-2glc]⁻ were observed in MS².

Compounds 44, 46, and 47 have the same molecular formula of C₅₃H₉₀O₂₂ according to the quasimolecular ions at m/z 1101[M+Na]⁺ and 1123 [M+HCOO]⁻. Compounds 46 and 47 were unambiguously identified as ginsenoside Rb₂ and ginsenoside Rb₃ by comparing the retention time and MS data as those of references. Compound 44 was tentatively deduced as ginsenoside Rc.

20S-ginsenoside F₁ gave the adduct ions at m/z 661.4300 [M+Na]⁺ (C₃₆H₆₂O₉Na) and 683.4375 [M+HCOO]⁻ (C₃₇H₆₃O₁₁Na). In the negative MS/MS experiment, the fragmentation ion at m/z 475.3785 [M-H-glc]⁻ was detected. Compound 49 showed similar retention time and MS data as those of reference 20S-ginsenoside F₁.

Reference ginsenoside Rd showed the adduct ions at m/z 969.5379 [M+Na]⁺ and 991.5495 [M+HCOO]⁻ in the full mass spectrum. The fragmentation ions at m/z 783.4892 [M-H-glc]⁻, 621.4368 [M-H-2glc]⁻, and 459.3842 [M-H-3glc]⁻ were produced by losing glucose. Compound 53 was identified as ginsenoside Rd by displaying the same retention time and MS information.

As for reference 20S-ginsenoside F₂, it showed the quasimolecular ions at m/z 807.4890 [M+Na]⁺ and 829.4949 [M+HCOO]⁻. The deprotonated ion peak at m/z 783.4881 [M-H]⁻ produced the fragmentation ion at m/z 621.4355 [M-H-glc]⁻ in MS². Compounds 45, 61, 64, and 65 gave the same adduct ions [M+Na]⁺ in positive MS and [M+HCOO]⁻ in negative MS, indicating the molecular formula as C₄₂H₇₂O₁₃. Compound 45 was deduced as protopanaxatriol type and the latter three compounds were deduced as protopanaxadiol type on the base of the fragmentation rules described above. Among them, compound 61 has similar ions in full mass spectrum and MS/MS experiment as those of reference 20S-ginsenoside F₂ and was unambiguously identified as 20S-ginsenoside F₂. The deprotonated ion of 45 at m/z 783.4910 [M-H]⁻ indicated the ion at m/z 475.3791 [M-H-rha-glc]⁻, supporting that 45 was ginsenoside Rg₂. Compounds 64 and 65 displayed same MS data; however, unfortunately, MS² information could not be detected and they were tentatively identified as 20R-ginsenoside Rg₃ and 20S-ginsenoside Rg₃.

Reference ginsenoside Rh₂ showed the ions at m/z 1245.8995 [2M+H]⁺, 645.4348 [M+Na]⁺, and 667.4437[M+HCOO]⁻. Compound 74 was identified as 20S-ginsenoside Rh₂ by comparing the retention time and MS data.

Compounds 35, 36, and 37 have the same molecular formula of C₄₈H₈₂O₁₉ and were tentatively characterized as 20S-glc-ginsenoside , beads ginseng saponin F₁, and notoginsenoside R₃. They gave [M+Na]⁺ ions at m/z 985.5332, 985.5319, and 985.5339 in the positive ion mode and [M+HCOO]⁻ ions at m/z 1007.5457, 1007.5457, and 1007.5459 in the negative ion mode. In their MS/MS experiments, the fragmentation ions of 35 at m/z 799.4843 and 637.4273 were produced from the ion at m/z 961.5391 [M-H]⁻ by loss of glucose unit. Similarly, the fragmentation ions of 36 and 37 at m/z 799 and 637 were yielded. The retention order of three chromatographic peaks is determined according to the description in the literature [9].

The chemical formulas of compounds 42 and 43 are calculated as C₄₁H₇₀O₁₃ based on the ions at m/z 793.4705 [M+Na]⁺ in the positive ion mode and 815.4818 [M+HCOO]⁻ in the negative ion mode. Both compounds showed the fragmentation ions at m/z 405, 423, or 441 indicating that they are due to protopanaxatriol type. In MS/MS analysis, the ions at m/z 637.4326 [M-H-xyl]⁻ and 475.3794 [M-H-xyl-glc]⁻ for compound 42, as well as 637.4319 [M-H-ara]⁻ and 475.3788 [M-H-ara-glc]⁻ for compound 43, were detected from the deprotonated ions at m/z 769.4753 [M-H]⁻ and 769.4758 [M-H]⁻, respectively. Compounds 42 and 43 were identified as notoginsenoside R₂ and ginsenoside F₃.

Compound 54 showed the quasimolecular ions at m/z 939.5284 [M+Na]⁺ and 961.5390 [M+HCOO]⁻, indicating the molecular formula as C₄₇H₈₀O₁₇. The fragmentation ions 783.4901 [M-H-xyl]⁻ and 621.4363 [M-H-xyl-glc]⁻ were detected, supporting that 54 was gypenoside IX.

Compound 60 showed the quasimolecular ions at m/z 789.4742 [M+Na]⁺ and 811.4871 [M+HCOO]⁻ and the molecular formula was calculated as C₄₂H₇₀O₁₂. In the MS/MS analysis, the deprotonated ion at m/z 765.4792[M-H]⁻ gave the ions at m/z 603.4261 [M-H-glc]⁻ and 441.3730 [M-H-2glc]⁻. Further loss of H₂O yielded the ions at m/z 423 and 405. It was identified as ginsenoside Rk₁.

Compound 34 showed [M+Na]⁺ ion at m/z 1117.5740 and [M+HCOO]⁻ ion at m/z 1139.5891, suggesting the molecule formula of C₅₃H₉₀O₂₃. Besides, it showed [M-H]⁻ ion at m/z 1093.5826 and fragment ions at m/z 961 [M-H- xyl/ara]⁻, 799 [M-H-xyl/ara-glc]⁻, and 637 [M-H-xyl/ara-2glc]⁻, concerning consecutive loss of 132, 294, and 456. These data were in accordance with those of floral ginsenoside P, floranotoginsenoside D, notoginsenoside FT₃, or yesanchinoside H [9], but its structure was not accurately determined based on the limited information.

Compound 72 showed [M+Na]⁺ ion at m/z 789.4742 and [M+HCOO]⁻ ion at m/z 811.4847, indicating the molecule formula as C₄₂H₇₀O12. Besides, the ions at m/z 603.4251 [M-H-162]⁻ and 471.3466 [M-H-162-132]⁻ in the MS/MS analysis suggested that the glycoside chain of 72 consisted of a molecule of glucose and a molecule of arabinose or xylose. It could be one of ilexoside A or ilexoside D described in the literature [10].

Flavonoids. Flavonoids always present high sensitivity in the UV spectra. They could be well detected in both positive and negative ionization modes. Rutin, which has UV absorption at 255 and 353 nm, showed the protonated ion at m/z 611.1656 [M+H]⁺ and deprotonated ion at m/z 609.1461 [M-H]⁻ as well as the ion at m/z 1219.3008 [2M-H]⁻. Quercetin, with UV absorption at 253 and 356 nm, displayed the deprotonated ion at m/z 301.0376 [M-H]⁻. Four compounds (9, 10, 13, and 26) were determined as flavonoid derivatives due to the typical UV absorption. Especially, compound 9 was one of main constituents of XXT. Compounds 9 and 26 were unambiguously identified as rutin and quercetin based on the direct comparison of their UV spectra, and mass spectra with those of the authentic compounds. Compound 10 has UV absorption at 264 and 343 nm. The molecular formula was calculated as C₂₇H₃₀O₁₅ by the quasimolecular ion at m/z 595.1652 [M+H]⁺, 617.1472 [M+Na]⁺, and 593.1505 [M-H]⁻. Other fragment ions at m/z 449.1083 [M+H-rha]⁺ and 287.1055 [M+H-rha-glc]⁺ were detected. Therefore, compound 10 was identified as kaempferol-3-O-rutinoside. Compound 13 has UV absorption at 252 and 339 nm. The molecular formula was calculated as C₂₈H₃₂O₁₆ by the quasimolecular ion at m/z 625.1754 [M+H]⁺, 647.1578 [M+Na]⁺, and 623.1616 [M-H]⁻, suggesting it was isorhamnetin 3-O-rutinoside [11].

Phenolic acids. Phenolic acids are the main constituents of XXT. This category of compounds was primarily derived from the ingredient herb, Salviae miltiorrhizae radix et rhizoma and Hairy holly root. In this study, 14 phenolic acids were found, among which 10 compounds (1, 2, 4, 5, 7, 11, 14, 15, 16, and 22) were identified as danshensu, neochlorogenic acid, chlorogenic acid, protocatechuic aldehyde, caffeic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, rosmarinic acid, and salvianolic acid B by using reference substances. Other compounds (12, 17, 18, and 32) were tentatively characterized by comparing UV absorption, retention time, molecular formula, and mass fragmentation pathways [12–14].

Danshensu has UV absorption at 279 nm and retention time at 1.03 min. It showed the precursor ion at m/z 197.0443 and the molecular formula was calculated as C₉H₁₀O₅. In the full mass spectrum, the [2M-H]⁻ ion at m/z 395.07949 was found. The MS/MS spectrum of [M-H]⁻ exhibited an obvious fragment ion, [M-H-H₂O]⁻ at m/z 179.0338, and further loss of COOH obtained ion at m/z 135.0438. Compound 1 was identified as danshensu.

Neochlorogenic acid has UV absorption at 323 nm. In the full mass spectrum, the deprotonated ion at m/z 353.0869 [M-H]⁻ was found. The MS/MS spectrum of [M-H]⁻ showed ions at m/z 191.0548, 179.0336, and 135.0438. Compound 2 was identified as neochlorogenic acid.

Chlorogenic acid has UV absorption at 325 nm and showed the quasimolecular ion at m/z 353.0872 [M-H]⁻ and 707.1482 [2M-H]⁻. The fragment ion at m/z 191.0553 was yielded by losing C₉H₆O₃. Compound 4 was identified as chlorogenic acid.

Protocatechuic aldehyde has UV absorption at 229, 278, and 310 nm. The [M-H]⁻ ion at m/z 137.0236 was detected. In the MS/MS analysis, the ion at m/z 108.0204 was yielded by losing CHO and further losing oxygen produced from the ion at m/z 92.0256. Compound 5 was identified as protocatechuic aldehyde.

Caffeic acid has UV absorption at 238 and 322 nm. It showed [M-H]⁻ ion at m/z 179.0339 in the full mass spectrum and the ion at m/z 135.0438 by losing CO₂ in the MS/MS experiment. Compound 7 was identified as caffeic acid.

The parent ions of three compounds (11, 14, and 15) at m/z 515 [M-H]⁻ and 517 [M+H]⁺ were easily located in the chromatogram of XXT, suggesting the molecular formula of C₂₅H₂₄O₁₂. They were assigned as dicaffeoylquinic acids by comparison with retention times. Besides, they gave the same fragment ions with those of chlorogenic acid, such as m/z 191, 179, 173, and 135. in MS/MS experiments. The fragmentation pathways are in accordance with those described in the literature [15]. Therefore, compounds 11, 14, and 15 were isochlorogenic acid B, A, and C.

Rosmarinic acid included a caffeic acid moiety and a molecule of danshensu. It showed [M-H]⁻ ion at m/z 359.0759. The fragment ions at m/z 197.040 and 179.0332 were yielded by losing caffeic acid or danshensu moiety in MS². Besides, the ion at m/z 161.0231 was yielded by further loss of H₂O. Compound 16 was identified as rosmarinic acid.

Salvianolic acid B showed [M-H]⁻ ion at m/z 717.1464. Besides, the fragment ions at m/z 519.0934 ([M-H-C₉H₁₀O₅]⁻), 339.0504, and 321.0392 were detected in the MS/MS analysis. The characteristic fragmentation pathway of losing C₉H₁₀O₅ is in accordance with those described in the literature [16]. Based on the retention time and the mass fragmentation pathway, compound 22 was identified as salvianolic acid B.

Compound 12 has UV absorption at 244 and 327 nm. The molecular formula was calculated as C₂₇H₂₂O₁₂ on the basis of the quasimolecular ions at m/z 539.1149 [M+H]⁺ and 537.1031 [M-H]⁻. In the MS/MS experiment, it indicated the characteristic ion at m/z 339.0500 [M-H-C₉H₁₀O₅]⁻, supporting that compound 12 was salvianolic acid H.

Compound 17 has UV absorption at 250 and 306 nm. It showed the ion at m/z 341.0666 [M+H]⁺. In the MS/MS analysis, the ions at m/z 295.0617 and 279.0659 were yielded by losing CH₂O₂ and CH₂O₃. Compound 17 was identified as salvianolic acid G.

Compound 18 has UV absorption at 251 and 317 nm. It showed [M-H]⁻ ion at m/z 493.1134 and the molecular formula was calculated as C₂₆H₂₂O₁₀. In the MS/MS analysis, the fragment ion at m/z 295.0604 [M-H-C₉H₁₀O₅]⁻ was detected, suggesting compound 18 was salvianolic acid A.

Compound 32 showed the ions at m/z 567.1498 [M+H]⁺, 589.1320 [M+Na]⁺, and 565.1342 [M-H]⁻ in MS spectrum, revealing molecular formula of C₂₉H₂₆O₁₂. Besides, fragment ion at m/z 369.0971 [M-H-C₉H₁₀O₅]⁻ was observed. Compound 32 was identified as ethyl lithospermate.

Bufadienolides. The MS/MS behaviors of bufadienolides have been extensively described [17, 18]. Briefly, for bufadienolides with only hydroxyl substituents, the fragmentation was characterized by successive eliminations of H₂O and CO molecules, and the profile of MS/MS product ions was correlated with the number of hydroxyl groups. If a C-16 acetoxyl group was present, the fragmentation of [M+H]⁺ ions was triggered by initial loss of 60 Da (HOAc). The elimination of CO was significant for bufadienolides with a 19-formyl group, and the 19-hydroxyl group could be characterized by the loss of 30 Da (HCHO). These fragmentation rules were applied to the identification of bufadienolides in XXT sample. As shown in Table 1, eight bufadienolides were screened from XXT, six of which were unambiguously identified as gamabufotalin (19), arenobufagin (25), bufotalin (41), bufalin (50), resibufogenin (55), and cinobufagin (56) by comparison with reference substances isolated from toad venom. The other two bufadienolides were tentatively identified as bufarenogin (20) and hellebrigenin (29) [18, 19]. Taking gamabufotalin as a case, the fragmentation rules of bufadienolides were explained (Figure 5). Gamabufotalin showed UV absorption at 295 nm. In its full mass spectrum, [M+H]⁺ ion at m/z 403.2486 was found and the fragment ions at m/z 385.2366 [M+H-H₂O]⁺, 367.2262 [M+H-2H₂O]⁺, and 349.2169 [M+H-3H₂O]⁺ were detected in MS².

Figure 5: Proposed fragmentation pathway of gamabufotalin.

Bile acids. Bile acid derivatives are in lack of conjugated system and their UV absorption is not obvious. However, they always present high sensitivity in the negative ion mode. The deprotonated ion [M-H]⁻ and adduct ion [M+HCOO]⁻ were obviously detected in MS spectrum. The loss of side chain was commonly observed in MS². Four bile acid derivatives were detected from XXT sample, two of which were unambiguously identified as ursodeoxycholic acid (58, UDCA) and chenodeoxycholic acid (70, CDCA) based on the direct comparison of reference substances. The other two compounds were tentatively identified as cholanic acid (57) and hyodeoxycholic acid (67). They are derived from the raw material, Rengong Niuhuang (Bovis calculus artifactus) [20].

Ursodeoxycholic acid showed the ions at m/z 391.2851 [M-H]⁻, 437.2916 [M+HCOO]⁻, and 783.5778 [2M-H]⁻ in the negative ion mode. In the positive ion mode, the ion at m/z 357.2807 [M+H-2H₂O]⁺ was assigned as the loss of two molecules of H₂O. The ion at m/z 321.2587 represented its side chain loss in MS². Compound 58 was identified as UDCA.

CDCA showed the ions at m/z 391.2872 [M-H]⁻, 437.2907[M+HCOO]⁻, and 783.5798 [2M-H]⁻ in the negative ion mode. In the positive ion mode, the loss of H₂O unit yielded [M+H-2H₂O]⁺ ion at m/z 357.2809. The occurrence of the ion at m/z 321.2586 in MS² was due to side chain loss. Compound 70 was identified as CDCA.

Compound 57 showed [M+Na]⁺ ion at m/z 431.2778 in positive mode, and [M-H]⁻ ion at m/z 407.2803, [M+HCOO]⁻ ion at m/z 453.2857, and [2M-H]⁻ ion at m/z 815.5698 in the negative mode. The ions representing a series of H₂O loss was observed, such as 373.2749 [M+H-2H₂O]⁺ and 355.2649 [M+H-3H₂O]⁺. It was tentatively characterized as cholanic acid.

Compound 67 showed [M-H]⁻ ion at m/z 391.2852, [M+HCOO]⁻ ion at m/z 437.2903, and [2M-H]⁻ at m/z 783.5792 in the full mass spectrum. The molecular formula was calculated as C₂₄H₄₀O₄. It was tentatively deduced as hyodeoxycholic acid.

Quinones derivatives. Quinones derivatives are another kind of active constituents from Danshen and they were easily detected in XXT sample. Compounds 63, 71, and 76 were identified as dihydrotanshinone, cryptotanshinone, and tanshinone II A by comparison with reference substances. They displayed similar fragmentation pathways concerning successive eliminations of H₂O and CO molecules. Tanshinone IIA was used as an example to illustrate the fragmentation pathway of quinones constituents as shown in Figure 6. Based on these rules as well as empirical molecular formula, 7 compounds were characterized as tanshindiol A 31, 1-oxo tanshinone IIA 59, neotanshinone D 62, tetrahydrotanshinone I 66, methyltanshinonate 69, methylenetanshinquinone 73, and miltirone 77. [12, 16, 20, 21].

Figure 6: Proposed fragmentation pathway of tanshinone IIA.

Phthalide derivative. Compound 21 has UV absorption at 276 nm. Its molecular formula was calculated as C₁₂H₁₄O₃ on the basis of [M+H]⁺ ion at m/z 207.1024. The above data were in accordance with those of 4-hydroxyl-3-butylphthalide in the literature [22]. Compound 21 was tentatively assigned as 4-hydroxyl-3-butylphthalide. It could be derived from individual herb Chuanxiong rhizome [23].

Among the identified compounds, most constituents were derived from the raw materials, Danshen and Renshen Jingye Zongzaogan, and a small proportion of compounds were considered from Huaihua, Maodongqing, Chuanxiong, Chansu, and Rengong Niuhuang. The other ingredients, Rengong Shexiang, Bingpian, and Shuizhi, were not characterized in the present HPLC and UPLC-QTOF/MS condition. This could be related to the prescription of raw materials and the manufacturing process employed. Usually, muscone, one of active constituents in Rengong Shexiang and borneol are detected by GC or GC-MS [3]. They are not easily detected in the liquid chromatography. Additionally, it is worth noting that no chemical information of Shuizhi, a famous traditional Chinese medicine originated from animal source, was found, although small molecule compounds with diverse structures have been reported from this drug [24]. More effort is made to explain its prescription role in XXT product.

4. Conclusion

In this work, HPLC analysis was employed to find out the common chromatographic peak in various batches of XXT samples and UPLC-Q-TOF/MS was used for the identification of main constituents in the typical XXT sample. As a result, a total of 63 constituents including twenty saponins, four flavonoids, fifteen phenolic acids, eight steroids, four bile acids, ten quinones, and other two compounds were identified or tentatively characterized based on the comparison of retention time and UV spectra with authentic compounds as well as by summarized MS fragmentation rules and matching empirical molecular formula with those of published components. The present investigation clearly understood the nonvolatile constituents in XXT and provided good basis for further study on the active substances and quality control of this preparation.

Data Availability

HPLC and UPLC-ESI-Q-TOF/MS data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This work was financially supported by NSFC (81773897), Standardization Project of Chinese Medicines (ZYBZH-C-JL-25), and the Fundamental Research Funds for the Central Public Welfare Research Institutes (QZPT004). The first author is thankful to Beijing Municipal Education Commission for providing a bachelor degree project (JWX201707).

Supplementary Materials

The pharmaceutical manufacture process of XXT described in current Chinese Pharmacopoeia is shown in Figure S1. HPLC of XXT sample and extract of each raw material at 251 nm are shown Figure S2. Relative retention time ratio and relative area ratio of common characteristic peaks in precision, repeatability, and stability test for the HPLC method validation are shown in Tables S1~S6. (Supplementary Materials)

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