Journal of Analytical Methods in Chemistry
Volume 2019 (2019), Article ID 9158942, 18 pages
https://doi.org/10.1155/2019/9158942

Research Article

Chemical Fingerprint and Metabolic Profile Analysis of Tianshu Tablets by Ultra-High Performance Liquid Chromatography/Quadrupole-Time of Flight Mass Spectrometry

Lin Chen,¹ Renhao Chen,¹ Hui Ouyang,² Qi Wang,¹ Zhifeng Li,¹ Yulin Feng,² and Shilin Yang²

¹College of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China
²State Key Laboratory of Innovative Drug and Efficient Energy-Saving Pharmaceutical Equipment, Jiangxi University of Traditional Chinese Medicine, Nanchang 330006, China

Correspondence should be addressed to Zhifeng Li; lizhifeng1976@hotmail.com and Yulin Feng; fengyulin2003@126.com

Received 29 June 2019; Accepted 3 October 2019; Published 23 October 2019

Academic Editor: Eliseo Herrero-Hernández

Copyright © 2019 Lin Chen 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.

In recent years, the chemical fingerprinting of traditional Chinese medicines and the metabolites in these compounds has been a hot topic. In the present study, the chemical fingerprint of Tianshu tablets (TST) and the metabolic characteristics of compounds in rats after intragastric administration were studied by ultra-high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UPLC/Q-TOF MS). In a preliminary study, 77 chemical components in TST were determined by comparison with retention times, accurate molecular mass, and characteristic fragment ions of the known compounds in the literature and some well-known compounds were analyzed in detail, and the fragmentation pathways for parishins B, gastrodin A, and cnidilide or neocnilide were specifically analyzed. After intragastric administration of TST (4 g/kg) to rats, a total of 61 compounds were detected in plasma samples, including 7 prototypes and 54 metabolites. After further analysis, it was found that these metabolites were subjected to glucuronidation, sulfation, methylation, hydroxylation, dehydrogenation, or mixed metabolic processes. Hydroxylation and glucuronidation were finally confirmed as the main metabolic pathways. This is the first research on the chemical fingerprint and metabolites of TST, which lays a foundation for further investigation of TST.

1. Introduction

In recent years, traditional Chinese medicine (TCM) has attracted increasing attention worldwide by virtue of its applications. Da Chuanxiong Formula (DCXF) is a well-known and extensively used traditional Chinese medicine (TCM) decoction for the treatment of migraine caused by blood stasis and wind-heat syndrome. It is composed of two herbs, namely, Chuanxiong (Chuanxiong rhizoma) and Tianma (Gastrodiae rhizoma), with a crude weight ratio of 4 : 1. Tianshu tablets (TST) are a representative DCXF preparation that is widely used in clinics for treating the blood stasis type of headache and migraine [1–3].

Phytochemical and pharmacological investigations of DXCF have shown that phenols, organic acids, phthalides, and nitrogen-containing compounds are the major active ingredients [4]. At present, several qualitative studies on the main components of DCXF have been performed [5–8]. One study used LC-Q-TOF/MS to identify 17 different components in a 50% ethanol extract of DCXF [5]. In one study, three compounds of Chuanxiong and eight components of Tianma were identified by HPLC-DAD-MSn [6]. Two continuous studies showed that 10 different compounds were detected in rat plasma after intragastric administration of DCXF active components, including 6 compounds from Chuanxiong and 4 compounds from Tianma [7, 8]. These four studies were based on samples of a 50% ethanol extract from a 4 : 1 mixture of the two herbs or active ingredients from a single crude herb. In one study, 38 components were identified or preliminarily identified from a Tianshu capsule by means of HPLC, LC-DAD-MSN, and LC-DAD-ESI IT-TOF/MS analysis, although Tianshu tablets and Tianshu capsules are two different dosage forms [9]. This research is still very important because of its different applicability. This study enriches our understanding of the components of DCXF and studies the metabolites of TST for the first time.

In our study, 77 chemical components of TST were preliminarily determined by a comparison with retention time, accurate molecular mass, and characteristic fragment ions of known compounds in the literature. Furthermore, UPLC/Q-TOF MS was used to analyze the plasma of rats after oral administration of TST. A total of 61 compounds were identified or preliminarily identified, including 7 prototypes and 54 metabolites.

2. Experimental

2.1. Chemicals and Materials

Some reference standards (pyroglutamic acid, 5-(hydroxymethyl)furoic acid, and parishin B) for Gastrodia rhizoma were isolated and purified in our laboratory, and other standards (uridine, gastrodin A, and neocnilide) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Purities of the standards were above 98% by HPLC analysis. HPLC-grade acetonitrile, methanol, and formic acid were purchased from Fisher Scientific (MA, USA). Deionized water was prepared by a Milli-Q Water purification system (Millipore, MA, USA). High-purity nitrogen (99.99%) and helium (99.999%) were purchased from Gas Supplies Center of Peking University Health Science Center (Beijing, China).

Gastrodia rhizoma and Chuanxiong rhizoma were purchased from Tian Heng pharmacy (Beijing, China). All herbal materials were authenticated by Professor Bei Wu (Nanchang Institute for Food and Drug Control). TianShu tablets were prepared according to Chinese Pharmacopoeia 2015 Edition [1].

2.2. Animals and Drug

Sprague–Dawley rats (male, 12–14 weeks; 200–240 g) were provided by Hunan SJA Laboratory Animal Co., Ltd. Protocols for all animal experiments were approved. Animals were kept in a controlled environment for 3 days and fasted for 12 h before experiments. TST were dissolved in a 9 g/L NaCl solution (NS) (250 mg/ml) and administered by oral gavage at a dose of 1000 mg/kg (equivalent to 4 g of crude drug per kg) body weight.

2.3. Sample Collection and Pretreatment

After oral administration of TST, blood samples were collected at 30, 60, and 120 min (n = 5) in an Eppendorf tube with heparin sodium and then centrifuged (16000 rpm) at 4°C for 10 min. The supernatant was then separated, and all samples were stored at −80°C immediately until analysis. The protocol for sample preparation is described below: 1 mL plasma was mixed with 5 mL methanol, vortexed for 5 min, and centrifuged at 16000 rpm at 4°C for 20 min. The supernatant was dried with nitrogen gas at 4°C, and 400 μl of 50% methanol was added to reconstitute the residue, which was then centrifuged at 16000 rpm for 10 min at 4°C. The supernatant was transferred to a vial, and 10 μl was injected for LC-MS analysis. All samples were filtered through a membrane (0.22 μm pore size). At the same time, in order to eliminate the influence of matrix, blank plasma was added to participate in the analysis.

2.4. UPLC/QTOF-MS Conditions

UPLC/QTOF-MS analysis was performed on a Shimadzu LC-30 AD system (Kyoto, Japan) coupled with an AB SCIEX Triple-TOF 5600 mass spectrometer (Foster City, CA, USA). All samples were separated on an Acquity UPLC C18 column (100 mm × 2.1 mm, 1.7 μm, Waters, USA) with a flow rate of 0.3 mL/min at 40°C. The mobile phase consisted of aqueous 0.1% formic acid (A) and 0.1% formic acid in acetonitrile (B). The gradient elution program for TST was set at 0–3.0 min, 5–8% B; 3.0–10.0 min, 8–15% B; 10.0–18 min, 15–20% B; 18–22 min, 20–35% B; 22–37 min, 35–45% B; 37–43 min, 45–95% B; and 43–48 min, 95–95% B. The gradient elution program for the plasma samples was set at 0–2.0 min, 5% B; 2.0–25.0 min, 5–95% B; and 25.0–30.0 min, 95–95%. The equilibration time was 5 min. The conditions for the ion source were as follows: compounds in TST were measured using the total ion chromatograms in negative and positive ion ESI-MS mode in the mass range m/z 50–1250, but plasma samples were analyzed only in positive ion ESI-MS mode. The other operating parameters were optimized as follows: source temperature, 500°C; ion spray voltage, 4500 V; gas 1, 50 psi; gas 2, 50 psi; curtain gas, 45 psi; decluttering potential, 100 V; and collision energy was set to 40 (15) eV.

2.5. Data Process

TST compounds from the extracts and metabolites data were acquired by full scan, which rely on dynamic background subtraction (DBS) and multiple mass defect filtering (MMDF) and includes some compounds with very low concentrations (MDF window was set to ±50 mDa around the mass defects of the templates and over a mass range of ±50 Da around the filter template masses).

Analysis of data on TST compounds in extracts and metabolites was performed using a variety of data mining tools, including extract ion chromatograms (XIC) of PeakView®1.6 (AB SCIEX, CA, USA), MMDF, and NLF&DPLs of Metabolitepilot™ 1.5 (AB SCIEX, Foster City, CA, USA). All compounds were analyzed after removal of the matrix effects.

3. Results and Discussion

3.1. Optimization of LC/MS Conditions

In order to obtain the best analytical data, our analysis builds upon another recent study [10]. The separation conditions, supplements, and chromatographic columns were optimized at the beginning of the experiment. Firstly, in order to obtain sharp peaks and reduce the pressure on the UPLC column, methanol was used as the mobile phase instead of acetonitrile. At the same time, 0.1% formic acid was added to improve peak shape and ionization of the analytes. The gradient was improved, and it was shown that the compounds in TST could be separated within 48 minutes, while plasma samples could be separated within 30 minutes (the specific methods can be found in Section 2.4). In addition, in order to obtain the most abundant mass spectrometry information, the collision energy was optimized. The results showed that when the collision energy rose to 40 eV, the main fragments were seen, but when the energy reached 55 eV, the second order fragments were too fragmented to be easily analyzed. Therefore, a collision energy of 40 eV was selected. As for UPLC/Q-TOF MS, mass spectra were recorded in both positive and negative detection modes.

3.2. UPLC/Q-TOF MS Analysis of TST Extracts

To characterize the chemical constituents of TST, a fast, efficient, and reliable UPLC/Q-TOF MS method was established. By virtue of the high resolution and speed of UPLC and the accurate mass measurement of the TOF MS, a total of 77 compounds were identified. The mass spectra of these components were examined in negative ion mode and positive ion mode. The total ion chromatogram (TIC) of TST in positive and negative ion modes are shown in Figures 1 and 2. Details of the identified components are summarized in Table 1.

Figure 1: MS total ion chromatogram (TIC) of TST fraction by UPLC/Q-TOF MS in negative ion mode.

Figure 2: MS total ion chromatogram (TIC) of TST fraction by UPLC/Q-TOF MS in positive ion mode.

Table 1: The chemical constituents detected from TST.

Peak
RT (min)
Formula
Error (ppm)
[M−H]⁻ (m/z)
Intensity
Product ions
Identification
Structure class
Ref.

Calculated
Measure

X1a
0.98
C₇H₁₂O₆
−1.2
191.0561
191.0559
86129
154.9973, 127.0393, 111.0091, 93.0355, 85.0302, 73.0302
Quinic acid
Organic acids
[10]

X2
1.07
C₆H₈O₇
0.9
191.0197
191.0199
421357
111.0096, 87.0103
Citric acid
Organic acids
[10]

X3b
1.07
C₉H₁₂N₂O₆
−1
243.0623
243.0620
5161
nd
Uridine
Nitrogen-containing compounds

X4b
1.08
C₅H₇NO₃
3
128.0353
128.0357
24352
nd
Pyroglutamic acid
Organic acids

X5b
1.36
C₆H₆O₄
1.1
141.0193
141.0195
3248
nd
5-(Hydroxymethyl)furoic acid
Organic acids
[11]

X6
1.36
C₁₀H₁₃N₅O₅
2.4
282.0844
282.0851
1153
150.0412, 133.0158, 108.0286
Guanosine
Nitrogen-containing compounds

X7
1.63
C₁₃H₁₈O₇
−1.1
285.0980
285.0977
1505
223.0248, 123.0432, 105.0348
Gastrodin
Glycosides
[12]

X8
2.03
C₇H₆O₄
0.7
153.0193
153.0195
16114
109.0300, 91.0193, 65.0057
Protocatechuic acid
Organic acids
[13]

X9
4.02
C₇H₆O₃
2.5
137.0244
137.0248
31464
119.0140, 108.0215, 93.0350, 81.0366, 65.0414
3,4-Dihydroxy benzaldehyde
Phenols
[14]

X10
4.70
C₁₉H₂₄O₁₃
1.7
459.1144
459.1152
283440
423.0939, 397.1138, 173.0088, 61.0444, 129.0184, 111.0088
Parishin E or G
Glycosides
[12]

X11
5.09
C₁₆H₁₈O₉
0.8
353.0878
353.0881
109164
191.0559, 173.0444, 161.0236, 134.0387, 111.0458, 93.0350, 85.0301
4-Caffeoylquinic acid
Organic acids
[15]

X12
5.13
C₁₇H₂₃N₃O₇S
0.5
412.1184
412.1186
11210
306.0753, 272.0898, 179.0459, 160.0077, 143.0461, 128.0348, 99.0559
S-(4-Hydroxybenzyl)-glutathione
Nitrogen-containing compounds
[12]

X13
5.24
C₂₃H₃₃N₃O₁₂S
2.3
574.1712
574.1726
7677
484.1388, 378.0974, 306.0756, 272.0874, 210.0864, 143.0464
S-(4-Hydroxybenzyl)-glutathione glucoside
Nitrogen-containing compounds

X14
5.34
C₉H₁₀O₄
−0.2
181.0506
181.0506
2573
136.9210, 121.0296, 109.0279
4-(2-Hydroxyethoxy)benzoic acid
Organic acids

X15
5.58
C₇H₆O₂
4.3
121.0295
121.0302
50808
92.0266, 77.0403
p-Hydroxybenzaldehyde
Phenols
[14]

X16
5.64
C₉H₈O₄
0.9
179.0350
179.0352
80360
134.0371, 108.0214, 93.0345, 89.0401, 79.0555
Caffeic acid
Organic acids
[13]

X17
5.99
C₈H₈O₄
3
167.0350
167.0355
275549
123.0454, 79.0570
Vanillic acid
Organic acids
[12]

X18b
7.31
C₁₉H₂₈O₁₂
0.4
447.1508
447.1510
14270
269.1028, 233.0656, 161.0449, 101.0242, 71.0153
Gastrodin A
Glycosides
[12]

X19
7.68
C₁₇H₁₉N₅O₅
−1.6
372.1313
372.1307
2202
336.1054, 266.0867, 134.0467
p-Hydroxybenzyl adenosine
Nitrogen-containing compounds

X20
7.73
C₈H₈O₃
0.5
151.0401
151.0402
7660
136.0155, 108.0219, 92.0273
Vanilline
Phenols

X21
8.62
C₁₇H₂₀O₉
−0.7
367.1035
367.1032
21071
193.0496, 191.0553, 173.0455, 134.0369, 111.0463, 93.0349, 85.0299
3-Feruloylquinic acid
Organic acids
[16]

X22
8.94
C₉H₁₀O₃
−0.8
165.0557
165.0556
2085
119.0515, 103.0604
L-(-)-Phenyllactic acid
Organic acids

X23b
9.11
C₃₂H₄₀O₁₉
4.9
727.2091
727.2151
122722
459.1156, 441.1045, 423.0937, 397.1142, 369.1188, 217.0496
Parishin B
Glycosides
[13]

X24
9.78
C₃₃H₄₂O₂₀
4
757.2197
757.2227
659
503.1631, 453.0961, 161.0480
Parishin H or M
Glycosides
[12]

X25
10.00
C₁₀H₁₀O₄
0.7
193.0506
193.0508
97733
178.0265, 149.0591, 134.0372
Ferulic acid
Organic acids
[13]

X26
10.02
C₂₁H₂₈O₁₃
0
487.1457
487.1457
4684
441.0958, 423.0934, 397.1169, 161.0430, 111.0091
Parishin O or N
Glycosides
[12]

X27
10.05
C₃₂H₄₀O₁₉
4.9
727.2091
727.2127
122722
459.1156, 441.1045, 423.0937, 397.1142, 369.1188, 161.0453
Parishin C
Glycosides
[12]

X28
10.71
C₁₄H₁₄O₃S
−2.2
261.0591
261.0585
777
205.8269, 167.8694, 137.0057
4,4′-Dihydroxybenzyl sulfoxide
Phenols
[14]

X29
11.74
C₁₁H₁₂O₅
−2.2
223.0612
223.0607
2431
108.0226, 179.0713
Sinapic acid
Organic acids
[13]

X30
12.20
C₇H₆O₃
2.5
137.0244
137.0248
31464
108.0215, 93.0352, 65.0410
p-Hydroxybenzoic acid
Phenols
[14]

X31
13.21
C₄₅H₅₆O₂₅
4.7
995.3038
995.3107
39727
727.2129, 441.1065, 423.0915, 397.1119, 161.0457
Parishin
Glycosides
[12]

X32
13.56
C₂₀H₂₄O₈
0.2
391.1398
391.1399
6858
229.0860, 123.0452, 121.0288, 107.0511, 93.0357, 71.0265
Bis-(4-hydroxybenzyl)-ether-mono-β-D-glucopyranoside
Glycosides
[14]

X33
13.69
C₂₆H₃₀O₁₄
2.9
565.1563
565.1579
811
177.0188, 111.0153
Parishin W
Glycosides
[12]

X34
14.13
C₂₅H₂₄O₁₂
2.6
515.1195
515.1208
91406
353.0879, 335.0781, 191.0558, 179.0345, 173.0450, 161.0240
3,4-Dicaffeoylquinic acid isomer
Organic acids
[15]

X35
16.48
C₂₅H₂₄O₁₂
2.6
515.1195
515.1208
91406
353.0879, 335.0781, 191.0558, 179.0345, 173.0450, 161.0240
3,4-Dicaffeoylquinic acid isomer
Organic acids
[15]

X36
17.42
C₉H₁₂O₂
−0.8
151.0765
151.0763
1891
133.0677, 107.0520, 94.0274, 77.0394
4-(Ethoxymethyl)phenol
Phenols
[14]

X37
23.89
C₁₄H₁₄O₂S
−4
245.0642
245.0632
2498
139.0217
Bis(4-hydroxybenzyl)sulfide
Phenols
[14]

X38
32.90
C₉H₈O₃
−1
163.0401
163.0399
4130
145.0279, 135.0455, 119.0493, 91.0184, 77.0443, 65.0039
p-Hydroxycinnamic acid
Organic acids

X39
39.73
C₁₆H₂₂O₄
−0.2
277.1445
277.1445
5380
233.1544, 206.8262
Dibutyl phthalate
Phenols

Peak
RT (min)
Formula
Error (ppm)
[M+H]⁺ (m/z)
Intensity
Product ions
Identification
Structure class
Ref.

Calculated
Measure

Y1a,b
1.05
C₅H₅N₅
−1.8
136.0618
136.0615
46698
nd
Adenine
Nitrogen-containing compounds

Y2
1.08
C₁₀H₁₃N₅O₄
1.5
268.1040
268.1044
174357
136.0623, 119.0360,
Adenosine
Nitrogen-containing compounds

Y3b
1.09
C₉H₁₁NO₃
−1.9
182.0812
182.0808
34675
nd
Tyrosin
Nitrogen-containing compounds

Y4
8.60
C₂₄H₂₉N₃O₈S
−0.5
520.1748
520.1746
7896
308.0836, 285.0913, 233.0591, 179.0486, 162.0208, 107.0485
(2)-g-L-[N-(4-Hydroxy benzyl)]glutamyl-L-[s-(4-hydroxybenzyl)] cysteinylglycine
Nitrogen-containing compounds

Y5
9.77
C₁₀H₈O₄
−0.8
193.0495
193.0494
14081
178.0257, 150.0323, 133.0281, 122.0361
3-Carboxyethyl-phthalide
Organic acids
[16]

Y6
10.55
C₁₂H₁₈O₅
−0.1
243.1227
243.1227
13011
165.0909, 151.0381, 137.0949
3-Butyl-3-hydroxy-4,5,6,7-tetrahydro-6,7-dihydroxy phthalide
Organic acids

Y7
10.55
C₁₂H₁₆O₄
−0.5
225.1121
225.1119
21174
207.1023, 165.0557, 151.0376, 137.0954
Senkyunolide H or I or ligustilidiol or cis-6,7-dihydroxy-ligustilide
Phthalides
[9]

Y8
11.94
C₁₂H₁₆O₅
−0.8
241.1071
241.1069
1918
150.0677, 107.0497, 71.0498
Senkyunolide R or S
Phthalides

Y9
13.27
C₁₂H₁₆O₃
0.1
209.1172
209.1172
288915
153.0544, 149.0593, 135.0473, 121.1006, 117.0709
Senkyunolide G or K or Z-6,7-epoxyligustilide
Phthalides
[9]

Y10
13.27
C₁₂H₁₈O₄
0.4
227.1278
227.1279
102387
163.1104, 153.0543, 149.0961, 119.0860, 107.0484, 79.054
Senkyunolide J or N or R2
Phthalides
[9]

Y11
16.56
C₁₂H₁₄O₃
−0.6
207.1016
207.1015
1327567
189.0917, 165.0550, 146.0732, 133.0653, 119.0841, 105.0693
3-Butylidene-6-hydroxy-5,6-dihy-drophthalide or senkyunolide F or chuanxiongol
Phthalides
[9]

Y12
16.57
C₁₂H₁₆O₄
−0.9
225.1121
225.1119
39845
165.0533, 133.0658, 128.0619, 91.0532, 77.0402
Senkyunolide H or I or ligustilidiol or cis-6,7-dihydroxy-ligustilide
Phthalides
[9]

Y13
17.87
C₁₂H₁₄O₄
−0.2
223.0965
223.0966
23855
177.0921, 149.0591, 121.0308, 103.0523, 77.0401
Senkyunolide D or 4,7-dihydroxy-3-butylphthalide
Phthalides
[9]

Y14
18.08
C₁₂H₁₄O₃
−0.1
207.1016
207.1015
425060
189.0901, 165.0558, 161.0948, 128.0620, 105.0701, 91.0549, 77.0393
3-Butylidene-6-hydroxy-5,6-dihy-drophthalide or senkyunolide F or chuanxiongol
Phthalides
[9]

Y15
19.53
C₁₈H₂₈O₈
0.3
373.1857
373.1858
45450
211.1335, 193.1228, 147.1172, 105.0702, 79.0577
Ligusticoside A
Glycosides
[17]

Y16
21.97
C₁₂H₁₂O₂
−0.5
189.0910
189.0910
151946
128.0623, 115.0544, 105.0702, 91.0551, 77.0388
Butylidenephthalide isomer
Phthalides
[9]

Y17
23.67
C₁₂H₁₄O₄
0.3
223.0965
223.0966
43428
177.0899, 167.0387, 149.0227, 121.0278, 91.0541, 77.0382
Senkyunolide D or 4,7-dihydroxy-3-butylphthalide
Phthalides
[9]

Y18
24.29
C₁₂H₁₂O₂
0
189.0910
189.0910
305525
152.0611, 128.0618, 115.0529, 105.0341, 91.0537, 77.0394
Butylidenephthalide isomer
Phthalides
[9]

Y19
24.50
C₁₂H₁₄O₂
−0.9
191.1067
191.1067
340079
117.0688, 91.0560, 77.0396
3-Butylphthalide or Z-ligustilide or E-ligustilide
Phthalides
[9]

Y20
26.27
C₁₂H₁₅ClO₃
−0.5
243.0783
243.0781
21244
207.1020, 189.0907, 161.0364, 133.0640, 119.0840, 91.0533,
Senkyunolide L
Phthalides
[18]

Y21
26.63
C₁₂H₁₂O₃
0.6
205.0859
205.0860
90627
187.0745, 168.0574, 144.0573, 131.0493, 115.0541, 103.0552, 91.0533, 77.0401
Senkyunolide B or C
Phthalides
[9]

Y22
27.74
C₁₃H₁₆O₃
−0.6
221.1172
221.1171
4804
175.1090, 105.0691
2-(1-Oxopentyl)-benzoic
Organic acids

Y23
27.90
C₁₂H₁₂O₃
0.6
205.0859
205.0860
90627
187.0745, 168.0574, 144.0573, 131.0493, 115.0541, 103.0552, 91.0533, 77.0401
Senkyunolide B or C
Phthalides
[9]

Y24
28.65
C₁₂H₁₆O₂
0.7
193.1223
193.1225
2273337
147.1167, 137.0590, 119.0498, 105.0699, 91.0544, 77.0389, 65.0383
Senkyunolide A
Phthalides
[9]

Y25
29.49
C₁₂H₁₄O₂
0.3
191.1067
191.1067
3489472
153.0704, 145.1007, 135.0440, 117.0695, 91.0546, 77.0391
3-Butylphthalide or Z-ligustilide or E-ligustilide
Phthalides
[9]

Y26
32.06
C₁₂H₁₂O₂
−0.5
189.0910
189.0910
39396
152.0611, 128.0615, 115.0545, 91.0554
Butylidenephthalide isomer
Phthalides
[9]

Y27b
32.89
C₁₂H₁₈O₂
−0.8
195.1380
195.1378
686098
177.1344, 149.1309, 79.0550
Cnidilide or neocnilide
Phthalides
[9]

Y28
33.04
C₁₂H₁₄O₂
0.3
191.1067
191.1067
637725
173.0961, 145.1008, 130.0773, 117.0694, 91.0548, 77.0392
3-Butylphthalide or Z-ligustilide or E-ligustilide
Phthalides
[9]

Y29
33.64
C₁₂H₁₂O₂
−0.2
189.0910
189.0910
401466
152.0627, 128.0622, 115.0540, 91.0550
Butylidenephthalide isomer
Phthalides
[9]

Y30
35.24
C₁₂H₂₀O₂
−0.7
197.1536
197.1535
21102
119.0862, 95.0856, 91.0555, 81.0697, 67.0552,
3,7-Dimethyl-3-acetate-1,6-octadiene-3-ol acetate
Others

Y31
36.15
C₂₁H₃₂O₂
0.9
317.2475
317.2478
12477
281.2253, 211.1524, 187.1501, 159.1179, 149.1329, 145.0997, 117.0717, 81.0720
Pregnenolone
Others

Y32
36.37
C₂₄H₂₈O₅
0.1
397.2010
397.2010
71592
191.1064, 173.0954, 155.0852, 145.1003, 128.0625, 117.0715, 105.0724, 91.0544
Chuanxiongnolide A or B
Others
[19]

Y33
38.22
C₂₄H₂₈O₅
0.4
397.2010
397.2010
66600
361.1802, 333.1842, 307.1708, 291.1401, 279.1403, 261.1264,2 17.0999, 191.1070, 173.0972, 91.0553
Chuanxiongnolide A or B
Others
[19]

Y34
39.08
C₂₄H₃₂O₅
−0.4
401.2323
401.2321
20533
383.2246, 191.1061, 163.1084, 149.0594, 145.1037, 135.0439
Chuanxiongdiolide R2 or chuanxiongdiolide B
Others
[20]

Y35
39.48
C₁₇H₂₄O₄
0.8
293.1747
293.1750
22471
175.1115, 151.0754, 137.0605, 111.0415, 91.0547, 83.0488
Senkyunolide M or Q
Phthalides
[9]

Y36
41.42
C₂₄H₃₀O₄
−0.1
383.2217
383.2217
1160225
191.1068, 173.0958, 163.1120, 149.0601, 135.0440, 91.0546, 79.0566
Senkyunolide P or 3,8-dihydro-diligustilide or angelicide or Z,Z′−3,3′ −8,8′-diligustilide
Phthalides
[9]

Y37
41.52
C₂₄H₃₂O₄
0.1
385.2373
385.2374
627373
367.2247, 349.2092, 321.2178, 293.1915, 193.1229, 175.1113147.1159, 137.0587, 119.0871, 93.0687
Chuanxiongdiolide A
Others
[20]

Y38
41.88
C₂₄H₂₈O₄
0.1
381.2060
381.2061
4796897
191.1070, 173.0955, 163.1126, 149.0596, 135.0437, 91.0551, 79.0549
Levistolide A or senkyunolide O or tokinolide B or riligustilide
Phthalides
[9]

a“X” in negative ion mode and “Y” in negative-positive mode. bCompared with reference standards.

Through analysis, it was found that the 77 compounds contained 19 organic acids, 9 nitrogen-containing compounds, 11 glucosides, 8 phenols, 24 phthalides, and 6 other compounds. The numbering information of these compounds is shown in Figure 3.

Figure 3: Information about classification of compounds in TST.

3.2.1. Chemical Fingerprint of TST in Negative Ion Modes

According to the literature, the main components of Tianma are phenols and organic acids [9]. However, there are also glycosides in the components of Tianma [12]. Many characteristic components of Tianma were analyzed and identified in the negative ion mode. Because the structures of organic acids and phenols are relatively simple, the characteristic glycoside compounds X18 and X23 were identified here and the chromatographic and spectral data for compounds X18 and X23 were preliminarily characterized by referring to the literature and reference materials.

Peak X18 gave an [M−H]⁻ ion at m/z 447.1508. Peak X18 produced MS² base peaks at m/z 269.1028 and 161.0449 corresponding to [M-H-179 Da]⁻. This suggests that Peak X18 may contain a glucose group and a fructose group. Therefore, we deduced that the molecular structure likely contains sucrose. According to literature reports [12] and reference standards, we identified Peak X18 as gastrodin A. Peak X23 gave a [M-H]⁻ ion at m/z 727.2091 and had characteristic fragment ions at m/z 459.1156, 441.1045, 423.0937, 397.1142, 369.1188, and 217.0496. Based on previous studies [12] and a reference standard, Peak X23 was identified as parishin B. The characteristic fragmentation patterns of gastrodin A and parishin B are described in Figures 4(a) and 4(b).

(a)

(b)

(c)

Figure 4: MS/MS spectra and the proposed fragmentation pathways. (a) Parishin B in negative ion mode. (b) Gastrodin A in negative ion mode. (c) Cnidilide or neocnilide in positive ion mode.

3.2.2. Chemical Fingerprint of TST in Positive Ion Modes

The analysis of the positive ion mode results showed that the characteristic components of Chuanxiong, including phthalides, were present. Here, compound Y27 was selected for analysis, and the chromatographic and spectral data of this compound were analyzed by comparison with the literature and reference materials. The cleavage pathway of phenyl peptides in Chuanxiong was also analyzed.

Peak Y27 gave a [M+H]⁺ ion at m/z 195.1378 and fragment ions at m/z 177.1344, 149.1309, and 107.0550. According to previous literature reports [9] and a reference standard, we identified peak Y27 as cnidilide or neocnilide. The characteristic fragmentation pattern of Y27 is shown in Figure 4(c)

According to our analysis, the main components of Tianma in negative ion mode were organic acids, phenols, and glycosides, with mainly phthalides detected in positive ion mode. The specific pyrolysis fragments were similar to the standards.

3.3. Detection and Identification of the Metabolites of TST in Rat Plasma

In order to identify as many potential pharmacologically active compounds as possible in TST, metabolic profiling of TST in rat plasma was performed. Compounds absorbed in vivo can be further metabolized by a variety of enzymes through oxidation, hydrolyzation, methylation, glucuronidation, and sulfation. Only peaks that were detected in the dosed plasma samples but not in blank samples were considered as probable metabolites. The mass spectra of the metabolites were examined in positive ion mode. These were further analyzed by using Peakview 1.2 to identify expected and unexpected metabolites from different metabolic pathways, and their structures were identified by tandem MS. We selected senkyunolide D or 4,7-dihydroxy-3-butylphthalide and senkyunolide A as examples of the structural identification process. The metabolites of these compounds and others are summarized in Table 2, and their TIC and extract ion chromatogram (EICs) are shown in Figures 5–8.

Table 2: Metabolites identified in plasma of rats after oral administration by UPLC/Q-TOF MS.

No.
Parent compounds
Metabolic pathways
Formula
tR (min)
[M+H]⁺ (m/z)
Error (ppm)
Product ions

Calculated
Measure

1
4,7-Dihydroxy-3-butylphthalide, senkyunolide D
Prototype
C₁₂H₁₄O₄
9.75
223.0965
223.0963
−1
177.0899, 167.0387, 149.0226, 121.0278, 91.0541

2
4,7-Dihydroxy-3-butylphthalide, senkyunolide D
Hydroxyl and methyl conjugation
C₁₃H₁₆O₅
10.78
253.1071
253.1071
0.2
235.0963, 221.0829, 202.0596, 193.0489, 179.0332, 175.0379, 150.0301, 121.0268

3
4,7-Dihydroxy-3-butylphthalide, senkyunolide D
Sulfate conjugation
C₁₂H14SO₇
9.2
303.0533
303.0535
0.5
285.0430, 205.0858, 187.0753, 177.0904, 149.0244, 121.0283, 91.0534

4
4,7-Dihydroxy-3-butylphthalide, senkyunolide D
Glucuronide conjugation
C₁₈H₂₂O₁₀
10.6
399.1286
399.1285
−0.3
223.0969, 205.0847, 177.0883, 167.0331, 149.0233, 121.0315

5
4,7-Dihydroxy-3-butylphthalide, senkyunolide D
Hydrogenation and glucuronide conjugation
C₁₈H₂₄O₁₀
8.67
401.1442
401.1444
0.3
225.4427, 207.1017, 189.0924, 172.0884, 165.0548, 141.0170, 119.0851, 113.0288

6
4,7-Dihydroxy-3-butylphthalide, senkyunolide D
Hydroxyl and glucuronide conjugation
C₁₈H₂₂O₁₁
8.92
415.1235
415.1238
0.7
227.0584, 221.0824, 167.0331

7
3-Butyl-3-hydroxy-4,5,6,7-tetrahy-dro-6,7-dihydroxyphthalide
Prototype
C₁₂H₁₈O₅
7.76
243.1227
243.1225
−0.7
165.0909, 151.0414, 137.0951, 123.0431, 107.0499, 91.0546, 85.0648

8
3-Butyl-3-hydroxy-4,5,6,7-tetrahy-dro-6,7-dihydroxyphthalide
Methyl conjugation
C₁₃H₂₀O₅
10.01
257.1384
257.1381
−1
221.1211, 207.0993, 165.0913, 137.0951, 123.0434

9
3-Butyl-3-hydroxy-4,5,6,7-tetrahy-dro-6,7-dihydroxyphthalide
Cystein conjugation
C₁₅H₂₃O₆NS
7.67
346.1319
346.1319
0
328.1222, 310.1111, 264.1056, 238.0916, 223.0771, 207.1018, 165.0923, 137.0955

10
3-Butyl-3-hydroxy-4,5,6,7-tetrahy-dro-6,7-dihydroxyphthalide
Oxidation and cystein conjugation
C₁₅H₂₃O₇NS
9.47
362.1268
362.1267
−0.3
327.0911, 247.1337, 229.1216, 151.0746

11
3-Butyl-3-hydroxy-4,5,6,7-tetrahy-dro-6,7-dihydroxyphthalide
N-Acetyl-L-cysteine conjugation
C₁₇H₂₅O₈NS
5.97
404.1374
404.1372
−0.5
205.0833, 171.1364

12
3-Butyl-3-hydroxy-4,5,6,7-tetrahy-dro-6,7-dihydroxyphthalide
Desat and S-GSH conjugation
C₂₂H₃₃O₁₁N3S
4.39
548.1909
548.1921
2.3
473.1636, 419.1465, 205.0860

13
3-Butyl-3-hydroxy-4,5,6,7-tetrahy-dro-6,7-dihydroxyphthalide
H₂O conjugation
C₁₂H₂₀O₆
4.65
261.1333
261.1320
−4.6
261.1310

14
3-Carboxyethyl-phthalide
Prototype
C₁₀H₈O₄
7.28
193.0495
193.0490
−2.7
178.0257, 150.0323, 133.0277, 122.0361, 105.0338, 77.0388

15
3-Carboxyethyl-phthalide
Methyl conjugation
C₁₁H₁₀O₄
11.52
207.0652
207.0646
−3
147.0441, 131.0502, 103.0546, 91.0533

16
3-Carboxyethyl-phthalide
Glucuronide conjugation
C₁₆H₁₆O₁₀
5.51
369.0816
369.0822
1.5
193.0493

17
3-Carboxyethyl-phthalide
Hydroxyl and glucuronide conjugation
C₁₆H₁₆O₁₁
5.72
385.0765
385.0758
−2.1
209.0455

18
3-Carboxyethyl-phthalide
Hydrogenation
C₁₀H₁₀O₄
7.64
195.0652
195.0651
−0.6
177.0547, 149.0609, 145.0276, 134.0354, 117.0309, 89.0395

19
Cnidilide, neocnilide
Prototype
C₁₂H₁₈O₂
16.14
195.1380
195.1377
−1.5
177.1350, 149.1348, 125.0599, 107.0873, 97.0640, 91.0550, 79.0543

20
Cnidilide, neocnilide
Methyl conjugation
C₁₃H₂₀O₂
15.63
209.1536
209.1537
0.4
193.0211, 167.1088, 153.0917, 121.0648, 68.9961

21
Cnidilide, neocnilide
Acetyl conjugation
C₁₄H₂₀O₃
12.45
237.1485
237.1483
−1.1
177.0257

22
Cnidilide, neocnilide
Hydroxyl and acetyl conjugation
C₁₄H₂₀O₄
12.46
253.1434
253.1435
0.4
235.1340, 193.0856, 157.1012, 135.0816

23
Cnidilide, neocnilide
Oxidation and cystein conjugation
C₁₅H₂₃O₄NS
10.07
314.1421
314.1424
1.1
268.1343, 193.1185

24
Cnidilide, neocnilide
Hydroxyl and glucuronide conjugation
C₁₈H₂₆O₉
9.62
387.1650
387.1650
0.1
211.1330, 193.1223, 175.1129, 147.1168, 121.0368

25
Cnidilide, neocnilide
2Hydroxyl and glucuronide conjugation
C₁₈H₂₆O₁₀
8.02
403.1599
403.1598
−0.2
227.1287, 209.1174, 191.1065, 171.1373, 163.1123, 153.0549, 145.1025, 141.0186, 135.1164, 121.0995

26
Cnidilide, neocnilide
2Hydroxyl conjugation
C₁₂H₁₈O₄
8.68
227.1278
227.1279
0.7
191.1057, 163.1115, 153.0554, 145.1001, 135.0444, 105.0705, 91.0541

27
Senkyunolide A
Prototype
C₁₂H₁₆O₂
14.81
193.1223
193.1222
−0.5
175.1118, 147.1170, 137.0580, 119.0848, 105.0710, 91.0556, 77.0393

28
Senkyunolide A
Hydroxyl and methyl conjugation
C₁₃H₁₈O₃
15.85
223.1329
223.1326
−1.2
205.1224, 191.1060, 149.0235, 135.0429, 121.0279, 105.0697, 91.0542, 77.0397

29
Senkyunolide A
Hydroxyl and acetylcysteine conjugation
C₁₇H₂₅O₆NS
10.35
372.1475
372.1475
−0.1
330.1375, 284.1322, 267.1048.239.0756, 209.1169, 191.1074, 162.0210, 153.0540, 130.0492

30
Senkyunolide A
Carboxyl and glucuronide conjugation
C₁₈H₂₂O₁₀
10.6
399.1286
399.1285
−0.3
223.0969, 205.0847, 177.0883, 159.0293, 149.0233, 131.0840, 85.0275

31
Senkyunolide A
2Hydroxyl and glucuronide conjugation
C₁₈H₂₄O₁₀
8.67
401.1442
401.1444
0.3
225.1127, 207.1017, 189.0924, 172.0884, 165.0548, 141.0170, 119.0851, 113.0288, 85.0265, 73.0295

32
Senkyunolide A
H₂O conjugation
C₁₂H₁₈O₃
10.54
211.1329
211.1327
−0.9
193.1225, 175.1096, 147.1156, 129.0700, 105.0693, 93.0692, 79.0548

33
Senkyunolide A
2Hydroxyl conjugation
C₁₂H₁₆O₄
9.56
225.1121
225.1119
−1
207.1013, 189.0914, 165.0537, 133.0637, 105.0706, 91.0536, 81.0713

34
Senkyunolide G, senkyunolide K,Z-6,7-epoxyligustilide
Prototype
C₁₂H₁₆O₃
12.79
209.1172
209.1173
0.3
153.0686, 149.0594, 145.0984, 135.0472, 105.0693, 91.0562, 77.0409

35
Senkyunolide G, senkyunolide K,Z-6,7-epoxyligustilide
Methyl conjugation
C₁₃H₁₈O₃
15.85
223.1329
223.1326
−1.2
191.1060, 173.0946, 149.0235, 145.1014, 135.0429, 105.0697, 91.0542, 79.0551

36
Senkyunolide G, senkyunolide K,Z-6,7-epoxyligustilide
Acetyl conjugation
C₁₄H₁₈O₄
15.99
251.1278
251.1276
−0.6
177.1261, 149.0593, 69.0014, 57.0752

37
Senkyunolide G, senkyunolide K,Z-6,7-epoxyligustilide
Hydroxyl and acetyl conjugation
C₁₄H₁₈O₅
12.27
267.1227
267.1226
−0.4
249.1137, 193.0479, 189.0582, 135.0435, 119.0846

38
Senkyunolide G, senkyunolide K,Z-6,7-epoxyligustilide
Taurine conjugation
C₁₅H₂₁O₅NS
9.02
328.1213
328.1214
0.1
207.1015, 189.0911, 165.0541, 161.0955, 147.0814, 133.0644, 119.0859, 91.0538

39
Senkyunolide G, senkyunolide K,Z-6,7-epoxyligustilide
N-Acetyl-L-cysteine conjugation
C₁₇H₂₃O₆NS
11.15
370.1319
370.1320
0.3
282.1162, 264.1083, 207.1015, 189.0911, 165.0544, 147.0785, 133.0652, 119.0858, 91.0546

40
Senkyunolide G, senkyunolide K,Z-6,7-epoxyligustilide
Hydrogenation and glucuronide conjugation
C₁₈H₂₆O₉
9.62
387.1650
387.1650
0.1
211.1330, 193.1223, 175.1129, 147.1168, 121.0638, 91.0546, 79.0562

41
Senkyunolide G, senkyunolide K,Z-6,7-epoxyligustilide
Hydroxyl and acetylcysteine conjugation
C₁₇H₂₅O₇NS
8.29
388.1425
388.1422
−0.6
207.1010, 164.0390, 122.0273, 105.0347, 79.0529

42
Senkyunolide G, senkyunolide K,Z-6,7-epoxyligustilide
Hydroxyl and glucuronide conjugation
C₁₈H₂₄O₁₀
8.67
401.1442
401.1444
0.3
267.1015, 225.1127, 207.1017, 189.0924, 172.0844, 141.0170, 113.0228, 85.0265, 73.0295

43
Senkyunolide G, senkyunolide K,Z-6,7-epoxyligustilide
Carboxyl and glucuronide conjugation
C₁₈H₂₂O₁₁
8.92
415.1235
415.1238
0.7
227.0584, 221.0824

44
Senkyunolide G, senkyunolide K,Z-6,7-epoxyligustilide
Desat and S-GSH conjugation
C₂₂H₃₁O₉N₃S
9.17
514.1854
514.1854
0
439.1552, 385.1429, 282.1160, 207.1017, 189.0921, 179.0484, 162.0221, 144.0103, 116.0174, 76.0218

45
Senkyunolide G, senkyunolide K,Z-6,7-epoxyligustilide
S-GSH conjugation
C₂₂H₃₃O₉N₃S
8.61
516.2010
516.2010
−0.1
441.1694, 387.1593, 284.1315, 209.1173, 191.1055, 162.0212, 144.0109, 116.0175, 84.0447

46
Senkyunolide G, senkyunolide K,Z-6,7-epoxyligustilide
Hydrogenation
C₁₂H₁₈O₃
10.54
211.1329
211.1327
−0.9
193.1225, 175.1096, 151.0737, 147.1156, 129.0700, 121.0641, 105.0693, 93.0693, 91.0546, 77.0398

47
Senkyunolide G, senkyunolide K,Z-6,7-epoxyligustilide
Hydroxyl and demethyl conjugation
C₁₂H₁₄O₄
9.75
223.0965
223.0963
−1
177.0906, 149.0590, 145.0980, 105.0329, 91.0556, 77.0394

48
Senkyunolide G, senkyunolide K,Z-6,7-epoxyligustilide
H₂O conjugation
C₁₂H₁₈O₄
8.68
227.1278
227.1279
0.7
209.1169, 191.1057, 163.1115, 153.0554, 145.1001, 135.0444, 105.0705, 91.0541, 77.0388, 65.0402, 55.0198

49
Senkyunolide G, senkyunolide K,Z-6,7-epoxyligustilide
Demethyl and carboxyl conjugation
C₁₂H₁₄O₅
9.79
239.0914
239.0915
0.4
221.0816, 193.0885, 179.0336, 165.0173, 161.0227, 128.0633, 109.0292, 77.0376

50
Senkyunolide G, senkyunolide K,Z-6,7-epoxyligustilide
2hydrogenation and 2hydroxyl conjugation
C₁₂H₁₈O₅
7.76
243.1227
243.1225
−0.7
225.1135, 207.1017, 179.1084, 165.0909, 151.0414, 137.0951, 123.0431, 95.0486

51
Senkyunolide G, senkyunolide K,Z-6,7-epoxyligustilide
Aromatic hydrocarbon oxidation
C₁₃H₁₈O₅
11.38
255.1227
255.1222
−2.1
195.1024, 135.0798, 131.0870

52
Senkyunolide J,N,R2
Prototype
C₁₂H₁₈O₄
8.68
227.1278
227.1279
0.7
163.1115, 153.0554, 145.1001, 107.0705, 91.0541, 79.0544, 65.0402, 55.0198

53
Senkyunolide J,N,R2
Methyl conjugation
C₁₃H₂₀O₄
11.42
241.1434
241.1433
−0.6
209.1147, 191.1067, 163.1089, 153.0559, 121.0652, 93.0717, 85.0663, 79.0663, 57.0698

54
Senkyunolide J,N,R2
Hydroxyl and methyl conjugation
C₁₃H₂₀O₅
10.01
257.1384
257.1381
−1
221.1211, 207.0993, 165.0913, 161.0984, 137.0951, 123.0434, 93.0699, 79.0549, 67.0538

55
Senkyunolide J,N,R2
Glycine conjugation
C₁₄H₂₁O₅N
7.49
284.1493
284.1495
0.8
238.1464, 209.1195, 191.1065, 163.1151, 153.0546, 135.1157, 117.0704, 91.0562, 76.0410, 57.0711

56
Senkyunolide J,N,R2
Cystein conjugation
C₁₅H₂₃O₅NS
8.48
330.1370
330.1375
1.7
284.1324, 209.1176, 191.1066, 163.1120, 153.0544, 145.1015, 121.1008, 93.0695, 85.0650

57
Senkyunolide J,N,R2
Taurine conjugation
C₁₄H₂₃O₆NS
7.56
334.1319
334.1319
0
209.1147, 181.1211, 153.0555, 126.0211, 108.0102, 91.0549

58
Senkyunolide J,N,R2
Oxidation and cystein conjugation
C₁₅H₂₃O₆NS
7.67
346.1319
346.1319
0
328.1222, 310.1111, 264.1056, 238.0916, 207.1018, 195.0853, 165.0923, 137.0955

59
Senkyunolide J,N,R2
N-Acetyl-L-cysteine conjugation
C₁₇H₂₅O₇NS
8.29
388.1425
388.1422
−0.6
207.1010, 164.0390, 107.0491, 122.0273, 105.0347

60
Senkyunolide J,N,R2
Glucuronide conjugation
C₁₈H₂₆O₁₀
8.02
403.1599
403.1598
−0.2
227.1287, 209.1174, 191.1065, 163.1123, 153.0549, 145.1025, 135.1164, 121.0995, 93.0712

61
Senkyunolide J,N,R2
S-GSH conjugation
C₂₂H₃₅O₁₀N₃S
4.96
534.2116
534.2113
−0.6
459.1795, 405.1696, 387.1582, 369.1490, 341.1469, 302.1438, 284.1303, 284.1303, 241.0913, 209.1157, 191.1075

Figure 5: MS total ion chromatogram (TIC) of metabolites by UPLC/Q-TOF MS.

Figure 6: Metabolites of M1 and extract ion chromatogram (EIC).

Figure 7: Metabolites of M27 and extract ion chromatogram (EIC).

Figure 8: The extract ion chromatograms (EICs) of other metabolites detected.

Metabolite M1, which eluted at 9.75 min, formed a molecular ion of [M+H]⁺ at m/z 223.0963 corresponding to C₁₂H₁₄O₄. M1 was found to have major fragment ions in common with senkyunolide D, so M1 is most likely senkyunolide D or 4,7-dihydroxy-3-butylphthalide. Metabolite M2, which eluted at 10.78 min, formed a molecular ion of [M+H]⁺ at m/z 253.1071 corresponding to C₁₃H₁₆O₅. The characteristic production of m/z 235.0963 and 221.0839 was generated by loss of 18 Da and 18 + 14 Da, which implied loss of a H₂O group and methyl group. Other product ions were identical to that of M1. Therefore, M2 may be a metabolite of senkyunolide D after hydroxyl and methyl conjugation. Metabolite M3, which eluted at 9.2 min, formed a molecular ion of [M+H]⁺ at m/z 303.0535 corresponding to C₁₂H₁₄SO₇. Its major fragment ions at m/z 285.0430 and 205.0858 were generated by the loss of 18 Da and 18 + 80 Da, which implied a H₂O group and sulfate group. Other product ions were identical to that of M1. Therefore, M3 may be a metabolite of senkyunolide D following sulfation. Metabolite M4, which eluted at 10.6 min, formed a molecular ion of [M+H]⁺ at m/z 399.1285 corresponding to C₁₈H₂₂O₁₀. Its major fragment ion (m/z 223.0969) was generated by a loss of 176 Da, which implied a glucuronide group. Other product ions were identical to that of M1. Therefore, M4 might be a metabolite of senkyunolide D after glucuronidation. Metabolites M5 and M6 appear to correspond to M4 plus 2 Da or 16 Da, respectively. The product ions m/z 225.4427 and m/z 227.0584 have both lost 176 Da. Therefore, M5 might be a metabolite of senkyunolide D after hydrogenation and glucuronidation, while M6 might be a metabolite of senkyunolide D after hydroxylation and glucuronidation (metabolites of M1 and extract ion chromatograms (EICs) are shown in Figure 6).

Metabolite M27, which eluted at 14.81 min, formed a molecular ion of [M+H]⁺ at m/z 193.1222 corresponding to C₁₂H₁₆O₂. A major fragment ion was shared with senkyunolide A, suggesting that M27 is prototype of senkyunolide A. Metabolite M28, which eluted at 14.81 min, formed a molecular ion of [M+H]⁺ at m/z 223.1328 corresponding to C₁₃H₁₈O₃. Its major fragment ions m/z 205.1224 and 191.1060 were generated by loss of 18 Da and 18 + 14 Da, which implied loss of a H₂O group and methyl group. Other product ions were identical to that of M27. Based on the possible metabolic reactions, M28 might be a metabolite of senkyunolide A after hydroxylation and methylation. Metabolite M29, which eluted at 10.35 min, formed a molecular ion of [M+H]⁺ at m/z 372.1475 corresponding to C₁₇H₂₅O₆NS. Its major fragment ions m/z 209.1269 and 191.1074 were generated by loss of 163 Da and 163 + 18 Da, which implied loss of an acetylcysteine group and H₂O group. Other product ions were identical to that of M27. Therefore, M29 may be a metabolite of senkyunolide A after hydroxyl and acetylcysteine conjugation. Metabolite M30, which eluted at 10.60 min, formed a molecular ion of [M+H]⁺ at m/z 399.1285 corresponding to C₁₈H₂₂O₁₀. Its major fragment ions m/z 223.0969, 205.0847, and 177.0883 were generated by the loss of 176 Da, 176 + 18 Da, and 176 + 18 + 28 Da, which implied loss of a glucuronide group, H₂O group, and CO group. Other product ions were identical to that of M27. Therefore, M30 may be a metabolite of senkyunolide A following carboxylation and glucuronidation. Metabolite M31, which eluted at 8.67 min, formed a molecular ion of [M+H]⁺ at m/z 401.1444 corresponding to C₁₈H₂₄O₁₀. Its major fragment ions m/z 225.1127, 207.1017, and 189.0924 were generated by loss of 176 Da, 176 + 18 Da, and 176 + 18 + 18 Da, which implied loss of a glucuronide group and two H₂O groups. Other product ions were identical to that of M27. Therefore, M31 might be a metabolite of senkyunolide A after 2 hydroxylation events and glucuronidation. Metabolite M32, which eluted at 10.54 min, formed a molecular ion of [M+H]⁺ at m/z 211.1327 corresponding to C₁₂H₁₈O₃. Its major fragment ions were m/z 193.1225 and 175.1096. An m/z of 193.1225 (loss of 18 Da) corresponds to senkyunolide A, suggesting that M32 might be a metabolite of senkyunolide A after H₂O conjugation. Metabolite M33, which eluted at 9.56 min, formed a molecular ion of [M+H]⁺ at m/z 225.1119 corresponding to C₁₂H₁₆O₄. Its major fragment ions m/z 207.1013 and 189.0914 were generated by loss of 18 Da and 18 + 18 Da, which implied loss of one or two H₂O groups. Other product ions were identical to that of M27. Therefore, M33 might be a metabolite of senkyunolide A after 2 hydroxylation events (metabolites of M27 and extracted ion chromatograms (EICs) are shown in Figure 7).

Sixty-one metabolites were identified in rat plasma. Through the analysis of these 61 metabolites, it was found that hydroxylation and glucuronidation were the main metabolic ways following oral administration of TST. From the identified metabolites, it can be speculated that after absorption of TST by human blood, most of the compounds undergo hydroxylation and glucuronidation, which allow TST to play a positive role in the treatment of migraine and blood stasis headaches. This provides a basis for follow-up research on the medical uses of TST. At the same time, from the information obtained on the metabolites, it can be seen that the main metabolites in positive ion mode of TST are concentrated as chuanxiong lactones, but there are no effective metabolites from Tianma. It is possible that Tianma metabolites are mainly present in the negative ion mode of plasma or in feces, urine, and bile, which requires further study.

4. Conclusion

In this study, UPLC/Q-TOF MS was used to comprehensively determine the chemical fingerprint and metabolic profile of TST after intragastric administration. In the analysis of the chemical constituents of TST, 77 compounds were identified, including 39 compounds identified in negative ion mode and 38 compounds identified in positive ion mode. In order to elucidate the mass spectrometric pyrolysis law of the main compounds in TST, gastrodin A, parishin B, and cnidilide or neocnilide were specifically analyzed, and the results were completely consistent with the results in reference standards and the reported literature. And 61 metabolites of TST in rat plasma were detected, which were mainly metabolites of 7 compounds. Two prototypes (senkyunolide D or 4,7-dihydroxy-3-butylphthalide and senkyunolide A) and their metabolites were analyzed in detail, which showed hydroxylation and glucuronidation were the main metabolic pathways following oral administration. This study expanded our understanding of the chemical constituents of TST, studied its metabolic spectrum for the first time, and clarified its main metabolic pathway in plasma, which will lay the foundation for follow-up studies of the pharmacological mechanism of TST.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare no conflicts of interest in publication of this study.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (no. 81560638), Young Scientists Training Project of Jiangxi Province (no. 20162BCB23003), Jiangxi Province 5511 R&D Projects (no. 20165BCB19009), Jiangxi Province Talent Project (no. [2016]332), Nanchang Innovative Talent Team (no. [2018]274), and Research Projects of Traditional Chinese Medicine of Jiangxi Provincial Health Department (no. 2016A018).

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