# Chemical Profiling of an Antimigraine Herbal Preparation, Tianshu Capsule, Based on the Combination of HPLC, LC-DAD-MSn, and LC-DAD-ESI-IT-TOF/MS Analyses

Authors: Juanjuan Liang; Huimin Gao; Liangmian Chen; Wei Xiao; Zhenzhong Wang; Yongyan Wang; Zhimin Wang
Journal: Evidence-Based Complementary and Alternative Medicine
 (2014)

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## Abstract

Chemical profiling is always the first task in the standardization and modernization of Traditional Chinese Medicine. HPLC and LC-MS were employed to find out the common chromatographic peaks in various batches of Tianshu Capsule (TSC) and the contribution of the characteristic peaks from individual herbs to the whole chromatographic profile of TSC sample. A total of 38 constituents were identified in TSC sample 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 TSC product, which provides the sufficiently chemical evidence for the global quality evaluation of TSC products.


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## Body

## 1. Introduction

Traditional Chinese Medicine (TCM) is getting more attention all over the world due to its exact clinical practice, especially prescription application, which comprehensively highlights the quintessence of the theory of traditional Chinese medical science.Da Chuan Xiong Fang (DCXF), a well-known and extensively used TCM decoction for the treatment of migraine, first appeared inXuan Ming Lun Fang, a famous formula book written by Wansu Liu who lived in Jin Dynasty (1115–1234). It is composed of two herbs, namely, Chuanxiong (Chuanxiong rhizoma) and Tianma (Gastrodiae rhizoma), with a crude weight ratio of 4 : 1. Eight dosage forms of DCXF such as capsule, tablet, dripping-pill, honeyed pill, oral liquid, and granule, have been authorized to Chinese market. Tianshu Capsule (TSC), as a representative of DCXF preparations, is widely used in clinics for treating the blood stasis type of headache and migraine [1, 2].Phytochemical and pharmacological investigations showed that phenols, organic acids, phthalides, and nitrogen-containing compounds were the major active ingredients of DCXF [3]. Several qualitative analyses have been reported concerning main types of constituents in DCXF [4–7]. One study described the identification of 17 different constituents in the 50% EtOH extract of DCXF by LC-Q-TOF/MS, containing gastrodin, parishin C, ferulic acid, guanosine, adenosine, palmitic acid, and 11 phthalide compounds [4]. Another similar study identified 3 compounds of Chuanxiong (ferulic acid, senkyunolide I, and senkyunolide H) and 8 constituents of Tianma (gastrodin, s-(4-hydroxybenzyl)-glutathione, parishin, parishin B, parishin C, p-hydroxybenzaldehyde, etc.) by using the HPLC-DAD-MSn coupling technique, respectively [5]. Continuous reports from the second research group confirmed that 10 different compounds, including 6 original substances of Chuanxiong and 4 original ones of Tianma, were detected in the rat plasma after the gavage of DCXF active components [6, 7]. However, all these investigations mentioned above were carried out based on the samples of the 50% EtOH extract of the mixture of both herbs (4 : 1) or active components of single crude herb. No systematic reports could be available involving the chemical profiling of the commercial finished products derived from DCXF. TSC was produced from both crude herbs by employing the various pharmaceutical engineering technologies and the complex manufacturing processes such as extraction, concentration, and preparation. The accumulating studies showed that decocting could induce chemical changes of medicinal herbs or combinatorial formula [8]. It is well-known that ferulic acid and some of the phthalides such asZ-ligustilide and dimeric phthalide are unstable at high temperature [9, 10]. Therefore, during the preparation of TSC, these thermolabile components may undertake chemical transformation, consequently leading to the difference of chemical compositions of finished products with DCXF decoction. Understanding the chemical profiles of TSC samples would be helpful in selecting suitable chemical markers for the quality control and pharmacokinetic study. In this work, a combination of HPLC, LC-DAD-MSn, and LC-DAD-ESI-IT-TOF/MS analyses was employed to find out and identify the common components in various batches of commercial TSC samples. The contribution of the characteristic peaks from individual herb to the whole chemical profiling of TSC was also discussed. A total of 38 constituents were identified or tentatively characterized, among which the water-soluble compounds with higher polarity fromGastrodiae rhizoma are detected in TSC samples for the first time.


## 2. Materials and Methods

### 2.1. Materials and Reagents

Five batches of Tianshu Capsules and related crude herbal materials (Chuanxiong rhizoma and Gastrodiae rhizoma) were provided byKanion Pharmaceutical(Lianyungang, China). The reference substances of gastrodin (Lot. 110807-200205), 5-hydroxymethyl-2-furfural (5-HMF, Lot. 111626-201007), ferulic acid (Lot. 0773-9607), andZ-ligustilide (Lot. 111737-201102) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Parishin B (174972-79-3), parishin C (174972-80-6), and parishin (62499-28-9) were from the collection of Dr. Li Wang, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (Dalian, China). The purity for each of these compounds was over 98% by HPLC assay and their structures were shown in Figure 1. HPLC grade methanol (Fisher, Fair Lawn, NJ, USA) and ultrapure water were used for HPLC analyses. All other chemical reagents were of analytical grade from Beijing Chemical Corporation (Beijing, China).Figure 1
The structures of the identified compounds in TSC sample.


### 2.2. Sample Preparation

#### 2.2.1. TSC Sample

1.0 g of pulverized contents of TSC samples was extracted with aqueous methanol (MeOH-H2O, 1 : 1, 25 mL) by ultrasonication (250 w, 40 kHz) for 30 min at room temperature, and the extract was then centrifuged for 10 min at 14800 rpm. A volume of 10 μL of the supernatant was used for HPLC and LC-DAD-MSn analysis.


#### 2.2.2. Extraction of Chuanxiong Rhizoma, Gastrodiae Rhizoma, and DCXF

The ethanolic and aqueous extracts of individual herb (Chuanxiong rhizoma andGastrodiae rhizoma) and the mixture of both herbs (w/w, 4 : 1, DCXF) were prepared according to the manufacturing processes of TSC (Figure S1 in the Supplementary Material available online at http://dx.doi.org/10.1155/2014/580745) described in the current Chinese pharmacopoeia. The ethanolic and aqueous extracts were diluted with aqueous methanol (MeOH-H2O, 1 : 1) to the concentration of 0.05 g·mL−1 and then centrifuged for 10 min at 14800 rpm. Each of the supernatants was used for HPLC and LC-MS analyses.


#### 2.2.3. Reference Solution

Stock solutions with a concentration of about 0.010 mg·mL−1 were prepared by dissolving an accurately weighed amount of each reference substance in aqueous methanol (MeOH-H2O, 1 : 1). The mixture of 7 reference solutions was prepared from the stock solutions.


### 2.3. Qualitative HPLC Analyses of 5 Batches of TSC Samples

The analyses were performed on a Shimadzu HPLC system (Shimadzu, Japan) equipped with a LC-20AT binary pump, a DGU-20A5 degasser, a SIL-20AC autosampler, a CTO-20AC column oven, and a SPD-M20A photodiode array detector. The samples were separated on a Phenomenex Luna C18 column (5 μm, 4.6 × 250 mm). The mobile phase consisted of methanol (A) and water containing 0.1% formic acid (B) using a gradient program as follows: 0 min, 15% A; 5 min, 15% A; 55 min, 95% A; 60 min, 95% A. The flow rate was 1.0 mL·min−1 and the column temperature was set at 30°C. The PDA detector recorded UV spectra in the range from 190 nm to 400 nm and HPLC chromatogram was monitored at 276 nm.


### 2.4. Comparison of Typical TSC Sample and Its Related Crude Herbal Materials as Well as DCXF by HPLC and LC-MS

TSC sample and its related crude herbal material,Chuanxiong rhizomaandGastrodiae rhizomaas well as DXCF, were analyzed under the same chromatographic conditions by HPLC and LC-MS to find the contribution of individual herb to the whole chemical profile of TSC sample. The HPLC system was the same as those in Section 2.3. LC-MS analyses were performed using an Agilent 6130 Quadrupole LC-MS (Agilent, Waldbronn, Germany) connected to an Agilent 1200 HPLC system (Agilent, Waldbronn, Germany). The parameters for MS analysis in the positive and negative ion mode were as follows: nebulizer, 35 psi; ionization voltage, 3500 V; dry temperature, 350°C; flow rate of carrier gas, 9.0 L·min−1. Full-scan mass spectra were acquired in the range of 100–800m/z.


### 2.5. LC-DAD-ESI-IT-MSn Analysis of Typical TSC Sample

To comprehensively identify the chemical constituents in TSC sample by the fragmentation rules, a LC-DAD-ESI-IT-MSn experiment was performed using an Agilent 6320 ion-trap spectrometer (Agilent, Waldbronn, Germany) connected to an Agilent 1200 HPLC system (Agilent, Waldbronn, Germany). The HPLC conditions were the same as those described in Section 2.3. The LC effluent was introduced into an electrospray ionization source after a postcolumn split ratio of 2 : 1. The parameters for MS analysis in the positive ion mode were as follows: nebulizer, 45 psi; ionization voltage, 4000 V; dry temperature, 350°C; flow rate of carrier gas, 12.0 L·min−1. Full-scan mass spectra were acquired in the range of 100–800m/z. The optimized parameters for MS/MS analysis were as follows: collision energy, 1.5 V; nitrogen was used as the collision gas. MSn spectra of pure substances were obtained using the same parameters as mentioned above.


### 2.6. LC-DAD-ESI-IT-TOF/MS of Analysis of Typical TSC Sample

To confirm the elemental composition of precursor ions and their fragments with high-accurate mass, a LC-ESI-IT-TOF/MS experiment was performed on a Shimadzu LC-MS-IT-TOF instrument equipped with a Shimadzu UFLCXR HPLC system (Shimadzu, Kyoto, Japan). The HPLC system consisted of a CBM-20A controller, two LC-20AD binary pumps, an SPD-M20A diode array detector, an SIL-20AC autosampler, a CTO-20A column oven, and a DGU-20A3 degasser. The HPLC conditions were the same as those for HPLC-DAD-ESI-MSn analysis. The LC effluent was directed into the ESI source as a rough split ratio of 3 : 1. The optimized MS conditions were as follows: positive and negative ion mode; electrospray voltage, +4.5 kV/−3.5 kV; detector voltage, 1.65 kV; curved desolvation line (CDL) temperature, 200°C; heat block temperature, 200°C; nebulizing gas (N2), 1.5 L·min−1; drying gas (N2), 10 L·min−1; scan range,m/z 100–1100 for MS1, 100–800 for MS2, and 100–500 for MS3. The ultrahigh purity argon was used as the collision gas for collision-induced dissociation (CID) experiments, and the collision energy was set at 50% for MS2 and MS3; ion accumulated time was 30 ms. The MSn data were collected in an automatic mode and the software could automatically select precursor ions for MSn analysis according to criteria settings. Accurate mass determination was corrected using the external standard method. The data acquisition and analysis were performed by LC-MS Solution Version 3.6 software (Shimadzu, Kyoto, Japan).


## 2.1. Materials and Reagents

Five batches of Tianshu Capsules and related crude herbal materials (Chuanxiong rhizoma and Gastrodiae rhizoma) were provided byKanion Pharmaceutical(Lianyungang, China). The reference substances of gastrodin (Lot. 110807-200205), 5-hydroxymethyl-2-furfural (5-HMF, Lot. 111626-201007), ferulic acid (Lot. 0773-9607), andZ-ligustilide (Lot. 111737-201102) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Parishin B (174972-79-3), parishin C (174972-80-6), and parishin (62499-28-9) were from the collection of Dr. Li Wang, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (Dalian, China). The purity for each of these compounds was over 98% by HPLC assay and their structures were shown in Figure 1. HPLC grade methanol (Fisher, Fair Lawn, NJ, USA) and ultrapure water were used for HPLC analyses. All other chemical reagents were of analytical grade from Beijing Chemical Corporation (Beijing, China).Figure 1
The structures of the identified compounds in TSC sample.


## 2.2. Sample Preparation

### 2.2.1. TSC Sample

1.0 g of pulverized contents of TSC samples was extracted with aqueous methanol (MeOH-H2O, 1 : 1, 25 mL) by ultrasonication (250 w, 40 kHz) for 30 min at room temperature, and the extract was then centrifuged for 10 min at 14800 rpm. A volume of 10 μL of the supernatant was used for HPLC and LC-DAD-MSn analysis.


### 2.2.2. Extraction of Chuanxiong Rhizoma, Gastrodiae Rhizoma, and DCXF

The ethanolic and aqueous extracts of individual herb (Chuanxiong rhizoma andGastrodiae rhizoma) and the mixture of both herbs (w/w, 4 : 1, DCXF) were prepared according to the manufacturing processes of TSC (Figure S1 in the Supplementary Material available online at http://dx.doi.org/10.1155/2014/580745) described in the current Chinese pharmacopoeia. The ethanolic and aqueous extracts were diluted with aqueous methanol (MeOH-H2O, 1 : 1) to the concentration of 0.05 g·mL−1 and then centrifuged for 10 min at 14800 rpm. Each of the supernatants was used for HPLC and LC-MS analyses.


### 2.2.3. Reference Solution

Stock solutions with a concentration of about 0.010 mg·mL−1 were prepared by dissolving an accurately weighed amount of each reference substance in aqueous methanol (MeOH-H2O, 1 : 1). The mixture of 7 reference solutions was prepared from the stock solutions.


## 2.2.1. TSC Sample

1.0 g of pulverized contents of TSC samples was extracted with aqueous methanol (MeOH-H2O, 1 : 1, 25 mL) by ultrasonication (250 w, 40 kHz) for 30 min at room temperature, and the extract was then centrifuged for 10 min at 14800 rpm. A volume of 10 μL of the supernatant was used for HPLC and LC-DAD-MSn analysis.


## 2.2.2. Extraction of Chuanxiong Rhizoma, Gastrodiae Rhizoma, and DCXF

The ethanolic and aqueous extracts of individual herb (Chuanxiong rhizoma andGastrodiae rhizoma) and the mixture of both herbs (w/w, 4 : 1, DCXF) were prepared according to the manufacturing processes of TSC (Figure S1 in the Supplementary Material available online at http://dx.doi.org/10.1155/2014/580745) described in the current Chinese pharmacopoeia. The ethanolic and aqueous extracts were diluted with aqueous methanol (MeOH-H2O, 1 : 1) to the concentration of 0.05 g·mL−1 and then centrifuged for 10 min at 14800 rpm. Each of the supernatants was used for HPLC and LC-MS analyses.


## 2.2.3. Reference Solution

Stock solutions with a concentration of about 0.010 mg·mL−1 were prepared by dissolving an accurately weighed amount of each reference substance in aqueous methanol (MeOH-H2O, 1 : 1). The mixture of 7 reference solutions was prepared from the stock solutions.


## 2.3. Qualitative HPLC Analyses of 5 Batches of TSC Samples

The analyses were performed on a Shimadzu HPLC system (Shimadzu, Japan) equipped with a LC-20AT binary pump, a DGU-20A5 degasser, a SIL-20AC autosampler, a CTO-20AC column oven, and a SPD-M20A photodiode array detector. The samples were separated on a Phenomenex Luna C18 column (5 μm, 4.6 × 250 mm). The mobile phase consisted of methanol (A) and water containing 0.1% formic acid (B) using a gradient program as follows: 0 min, 15% A; 5 min, 15% A; 55 min, 95% A; 60 min, 95% A. The flow rate was 1.0 mL·min−1 and the column temperature was set at 30°C. The PDA detector recorded UV spectra in the range from 190 nm to 400 nm and HPLC chromatogram was monitored at 276 nm.


## 2.4. Comparison of Typical TSC Sample and Its Related Crude Herbal Materials as Well as DCXF by HPLC and LC-MS

TSC sample and its related crude herbal material,Chuanxiong rhizomaandGastrodiae rhizomaas well as DXCF, were analyzed under the same chromatographic conditions by HPLC and LC-MS to find the contribution of individual herb to the whole chemical profile of TSC sample. The HPLC system was the same as those in Section 2.3. LC-MS analyses were performed using an Agilent 6130 Quadrupole LC-MS (Agilent, Waldbronn, Germany) connected to an Agilent 1200 HPLC system (Agilent, Waldbronn, Germany). The parameters for MS analysis in the positive and negative ion mode were as follows: nebulizer, 35 psi; ionization voltage, 3500 V; dry temperature, 350°C; flow rate of carrier gas, 9.0 L·min−1. Full-scan mass spectra were acquired in the range of 100–800m/z.


## 2.5. LC-DAD-ESI-IT-MSn Analysis of Typical TSC Sample

To comprehensively identify the chemical constituents in TSC sample by the fragmentation rules, a LC-DAD-ESI-IT-MSn experiment was performed using an Agilent 6320 ion-trap spectrometer (Agilent, Waldbronn, Germany) connected to an Agilent 1200 HPLC system (Agilent, Waldbronn, Germany). The HPLC conditions were the same as those described in Section 2.3. The LC effluent was introduced into an electrospray ionization source after a postcolumn split ratio of 2 : 1. The parameters for MS analysis in the positive ion mode were as follows: nebulizer, 45 psi; ionization voltage, 4000 V; dry temperature, 350°C; flow rate of carrier gas, 12.0 L·min−1. Full-scan mass spectra were acquired in the range of 100–800m/z. The optimized parameters for MS/MS analysis were as follows: collision energy, 1.5 V; nitrogen was used as the collision gas. MSn spectra of pure substances were obtained using the same parameters as mentioned above.


## 2.6. LC-DAD-ESI-IT-TOF/MS of Analysis of Typical TSC Sample

To confirm the elemental composition of precursor ions and their fragments with high-accurate mass, a LC-ESI-IT-TOF/MS experiment was performed on a Shimadzu LC-MS-IT-TOF instrument equipped with a Shimadzu UFLCXR HPLC system (Shimadzu, Kyoto, Japan). The HPLC system consisted of a CBM-20A controller, two LC-20AD binary pumps, an SPD-M20A diode array detector, an SIL-20AC autosampler, a CTO-20A column oven, and a DGU-20A3 degasser. The HPLC conditions were the same as those for HPLC-DAD-ESI-MSn analysis. The LC effluent was directed into the ESI source as a rough split ratio of 3 : 1. The optimized MS conditions were as follows: positive and negative ion mode; electrospray voltage, +4.5 kV/−3.5 kV; detector voltage, 1.65 kV; curved desolvation line (CDL) temperature, 200°C; heat block temperature, 200°C; nebulizing gas (N2), 1.5 L·min−1; drying gas (N2), 10 L·min−1; scan range,m/z 100–1100 for MS1, 100–800 for MS2, and 100–500 for MS3. The ultrahigh purity argon was used as the collision gas for collision-induced dissociation (CID) experiments, and the collision energy was set at 50% for MS2 and MS3; ion accumulated time was 30 ms. The MSn data were collected in an automatic mode and the software could automatically select precursor ions for MSn analysis according to criteria settings. Accurate mass determination was corrected using the external standard method. The data acquisition and analysis were performed by LC-MS Solution Version 3.6 software (Shimadzu, Kyoto, Japan).


## 3. Results and Discussion

### 3.1. Qualitative Analyses of TSC and Its Related Crude Herbal Materials by HPLC and LC-MS

Under the HPLC conditions as described in the current Chinese Pharmacopoeia [1], 5 batches of TSC samples, together with the reference compounds, were examined and their HPLC chromatograms were shown in Figure S2. High similarity in the number, type, and amount of chemical constituents was observed in the HPLC profiles of different batches of TSC samples. General chromatographic profile was obtained by Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine software and characteristic peaks were found in the HPLC profile of each individual sample.In order to identify the origin of these characteristic peaks from individual herbs, a comparative study was carried out by using various extracts of herbs and TSC samples. Accordingly, the possible individual contribution from the corresponding herbs to the general chromatographic profile was found. Compared with the HPLC profiles of the ethanolic and aqueous extracts ofChuanxiong rhizoma and Gastrodiae rhizome (Figure S3), 29 of 38 peaks occurring in HPLC profile of TSC sample were contributed byChuanxiong rhizoma and other 9 peaks came from Gastrodiae rhizome (Figures S4~S10). In addition to the comparison of retention time and on-line UV spectra with those of reference compounds gastrodin, 5-HMF, parishin B, parishin C, parishin, ferulic acid, andZ-ligustilide, the precursor ions obtained by the positive and negative LC-MS (Figure 2) such as [M+H]+, [M+NH4]+, [M+Na]+, and [M-H]- further confirmed the contribution of the characteristic peaks from individual herbs to the chromatographic profile of TSC sample (Table 1).Table 1
Retention time (tR), UV, and LC-MS data of the identified compounds in Tianshu Capsule and related crude materials.
Number
t
R/min
UVλmax⁡/nm
MW
[M − H]−
[M + H]+
[M + NH4]+
[M + Na]+
[2M + Na]+
[M + HCOO]−
Tianma
Chuanxiong
Identified compounds
2
5.403
221, 276
286
304
331
+
−
Gastrodin
3
7.736
230, 283
126
127
144
149
+
−
5-HMF
4
15.579
225
460
459
478
483
+
−
Parishin E/G
5
20.691
221, 272
728
727
746
751
+
−
Parishin B
6
22.220
221, 274
728
727
746
751
+
−
Parishin C
8
25.080
218
242
241
243
265
507
−
+
3-Butyl-3,6,7-trihydroxy-4,5,6,7-tetrahydrophthalide
9
25.567
220, 236, 323
194
193
195
217
+
+
ferulic acid
10
27.529
220, 330
226
227
249
475
−
+
Senkyunolide J/N
11
30.667
280
224
225
247
471
−
+
4,5-Dihydro-3,1′-dihydroxy-3-butylphthalide
13
32.436
275
206
205
207
−
+
4-hydroxyl-3-butylphthalide
14
34.299
325
396
397
+
+
unidentified
16
38.635
220, 270
222
221
223
245
−
+
Senkyunolide D or4,7-dihydroxy-3-butylidenephthalide
17
39.502
230, 310
206
205
207
229
435
−
+
Z-6,7-Epoxyligustilide
18
39.898
280
208
207
209
231
439
−
+
Senkyunolide K/G
19
41.465
220
206
205
207
229
435
−
+
Senkyunolide F
20
42.332
220, 240, 325
192
193
215
407
+
+
Senkyunolide A
21
42.956
226, 280
190
191
208
213
403
−
+
Butylphthalide
22
43.717
218, 270
204
203
205
227
431
249
−
+
Senkyunolide C
23
44.147
210, 285, 330
204
203
205
227
431
249
−
+
Senkyunolide E
24
44.949
220, 270, 330
194
195
217
411
−
+
Cnidilide
26
45.831
205, 280, 328
190
191
213
403
−
+
Ligustilide
27
46.394
220
194
195
217
411
−
+
Neocnidilide
28
46.653
210, 275, 330
188
189
206
211
399
−
+
3-Butylidenephthalide
29
47.231
225, 280, 310
380
381
398
403
−
+
Riligustilide
30
50.042
276
382
383
399
405
−
+
Senkyunolide P
31
50.476
220, 280
380
381
398
403
−
+
3′,6,8′,3a-Biligustilide
32
50.970
220, 280
380
381
398
403
−
+
Tokinolide B
33
51.065
230, 279
382
383
400
405
−
+
Unidentified
34
51.962
220, 275
380
381
403
−
+
Levistolide A
35
52.267
220, 285
380
381
−
+
3′Z-3,3′a,8,6′a-Biligustilide
36
52.693
220, 280
278
279
301
579
−
+
Senkyunolide M
37
53.134
220, 280
278
279
301
579
−
+
Senkyunolide Q
38
53.852
225, 278
382
383
400
405
383
−
+
UnidentifiedHPLC and TIC of typical TSC sample obtained using an Agilent 6130 Quadrupole LC-MS connected to an Agilent 1200 HPLC system. (a) HPLC (276 nm), (b) (+) TIC, and (c) (−) TIC.
(a)
(b)
(c)


### 3.2. Identification of Chemical Constituents in TSC by LC-DAD-EST-IT-MSn and LC-DAD-ESI-IT-TOF/MS

The combination of LC-DAD-ESI-IT-MSn and LC-DAD-ESI-IT-TOF/MS experiments was employed for the identification of chemical constituents in TSC sample, and, as a result, a total of 38 compounds was identified or tentatively characterized. The structures of the identified compounds are shown in Figure 1 and their chromatographic and mass spectrometric data are shown in Tables 2 and 3. The total ion chromatograms (TICs) of TSC sample are shown in Figures S11 and S12, respectively. Among the identified constituents, 7 compounds (2,3,5,6,7,9, and26) were unambiguously identified as gastrodin, 5-HMF, parishin B, parishin C, parishin, ferulic acid, andZ-ligustilide based on the direct comparison of their retention times, UV spectra, and mass spectra with those of the authentic compounds. Furthermore, 31 compounds were tentatively characterized according to their UV spectra, empirical molecular formula, and mass fragmentation pathways as well as their eluted sequence from ODS column reported in the literature [11–14] and acquired in the present experiments.Table 2
Retention time (tR), UV, and MSn data obtained by LC-DAD-ESI-IT-MSn of the identified compounds in Tianshu Capsule.
Number
t
R/min
Identified compounds
UVλmax⁡/nm
[M + Na]+
[M + H]+
[2M + Na]+
Main product ions
[M − H]−
[M + HCOO]−
Main product ions
1
6.3
Gastrodin glucose (448)
221, 276
471
447
493
323[M – H-C7H8O2]−, 285[M – H-glc]−, 179
2
6.3
Gastrodin (286)
220, 269
309
185[M + Na-C7H8O2]+
285
331
161[M – H-C7H8O2]−, 123[M – H-glc]−
3
8.1
5-HMF (126)
230, 283
127
109[M + H-H2O]+
125
5
20.3
Parishin B (728)
221, 272
751
483[M + Na-268]+, 215[483-268]+
727
459, 441, 423, 397, 379, 217
6
21.8
Parishin C (728)
221, 274
751
483[M + Na-268]+, 215[483-268]+
727
459, 441, 423, 397, 379, 217
7
23.4
Parishin (996)
221, 271
995
727[M – H-268]−
9
26.0
Ferulic acid (194)
220, 236, 323
195
177[M + H-H2O]+, 145[177-CH3OH]+
10
27.4
Senkyunolide J/N (226)
220, 330
249
475
209[M + H-H2O]+, 191[M + H-2H2O]+, 163[M + H-2H2O-28]+
11
30.4
4,5-Dihydro-3,1′-dihydroxy-3-butylphthalide (224)
280
247
207[M + H-H2O]+, 189[M + H-2H2O]+, 165, 121
12
31.6
Senkyunolide I/H (224)
276
247
207[M + H-H2O]+, 189[M + H-2H2O]+, 161[189-H2O]+, 133[189-56]+
15
37.0
Unidentified (316)
230, 276, 280
339
317
299[M + H-H2O]+, 281[M + H-2H2O]+, 271[M + H-H2O-28]+, 243
315
17
37.8
Z-6,7-Epoxyligustilide (206)
230
229
413
189[M + Na-H2O]+, 171[189-H2O]+
18
38.1
Senkyunolide K/G (208)
233, 280
231
439
191[M + H-H2O]+, 173[191 − H2O]+, 149[191-42]+, 135[191-56]+
20
40.2
Senkyunolide A (192)
280
215
193
407
175[M + H-H2O]+, 147, 119, 105
21
40.7
Butylphthalide (190)
232, 275
213
191
403
173[M + H-H2O]+, 145, 117
24
42.6
Cnidilide (194)
238, 279, 327
217
25
43.3
E-Ligustilide (190)
281, 327
213
191
403
173[M + H-H2O]+, 130
26
43.5
Z-Ligustilide (190)
237, 260, 312
213
191
403
173[M + H-H2O]+, 145[173-28]+ 130, 117, 105
28
44.0
3-Butylidenephthalide (188)
230, 277, 326
211
189
171[M + H-H2O]+, 153[171-H2O]+
29
47.4
Riligustilide (380)
275
403
381
213[2M + Na-190]+, 191, 173
30
47.5
Senkyunolide P (382)
278
405
383
192[M + H-190]+
31
47.8
3′,6,8′,3a-Biligustilide (380)
278, 363
403
381
213[2M + Na-190]+, 191[M + H-190]+, 145, 117
32
48.0
Tokinolide B (380)
278
403
381
213[2M + Na-190]+, 191[M + H-190]+
33
48.2
Unidentified (382)
230, 279
405
383
193[M + H-190]+
34
48.5
Levistolide A (380)
276
403
381
213[2M + Na-190]+, 191[M + H-190]+, 145, 117
35
48.8
Senkyunolide O (380)
278
403
381
173[191-H2O]+, 191[M + H-190]+,145, 117
36
49.0
Senkyunolide M (278)
279
301
279
245[M + Na-56]+, 189, 171
37
49.4
Senkyunolide Q (278)
278
301
279
245[M + Na-56]+, 189, 171
38
49.9
Unidentified (382)
225, 278
405
383Table 3
Retention time (tR) and MS data obtained by LC-DAD-ESI-IT-TOF/MS of the identified compounds in the sample of Tianshu Capsule.
Number
t
R/min
Identified compounds
Formula
Mea. mass/m/z
Calc. mass/m/z
Error/ppm
Other precursor ions
Main product ions
3
7.6
5-HMF (126)
C
6
H
6
O
3
127.0384[M + H]+
127.0390[M + H]+
−4.72
149.0240[M + Na]+
5
19.8
Parishin B (728)
C
32
H
40
O
19
727.2123[M − H]−
727.2091[M − H]−
4.40
6
21.5
Parishin C (728)
C
32
H
40
O
19
727.2132[M − H]−
727.2091[M − H]−
5.64
8
24.5
3-Butyl-3,6,7-trihydroxy-4,5,6,7-tetrahydrophthalide (242)
C
12
H
18
O
5
241.1090[M − H]−
241.1081[M − H]−
3.72
223.0885[M – H-H2O]−, 197.1142, 179.1107, 141.0930, 123.0854
243.1220[M + H]+
243.1227[M + H]+
2.88
265.1060[M + Na]+
9
25.3
Ferulic acid (194)
C
10
H
10
O
4
195.0649[M + H]+
195.0652[M + H]+
−1.54
217.0458[M + Na]+
10
27.0
Senkyunolide J/N (226)
C
12
H
18
O
4
227.1259[M + H]+
227.1278[M + H]+
−3.37
249.1082[M + Na]+
209.1177[M + H-H2O]+, 191.1026[M + H-2H2O]+
11
30.3
4,5-Dihydro-3,1′-dihydroxy-3-butylphthalide (224)
C
12
H
1
6
O
4
225.1105[M + H]+
225.1121[M + H]+
−7.11
12
31.6
Senkyunolide I/H (224)
C
12
H
1
6
O
4
225.1105[M + H]+
225.1121[M + H]+
−7.11
15
37.8
Unidentified (316)
C
1
8
H
20
O
5
317.1380[M + H]+
317.1384[M + H]+
−1.26
339.1210[M + Na]+
299.1378[M + H-H2O]+, 271.1343[M  + H-H2O-28]+
16
37.8
4,7-Dihydroxy-3-butylidenephthalide or senkyunolide D (222)
C
12
H
1
4
O
4
223.0986[M + H]+
223.0965[M + H]+
9.41
245.0774[M + Na]+
17
38.7
Z-6,7-Epoxyligustilide (206)
C
12
H
14
O
3
207.1008[M + H]+
207.1016[M + H]+
3.66
229.0824[M + Na]+
189.0856[M + H-H2O]+, 133.0318
18
39.1
Senkyunolide K/G (208)
C
12
H
1
6
O
3
209.1167[M + H]+
209.1172[M + H]+
−2.39
231.0974[M + Na]+
191.1091[M + H-H2O]+, 119.0819
19
40.6
Senkyunolide F (206)
C
12
H
14
O
3
207.1009[M + H]+
207.1016[M + H]+
3.38
229.0815[M + Na]+
189.0911[M + H-H2O]+, 161.0959
205.0874[M − H]−
205.0870[M − H]−
1.95
411.1821[2M − H]−
161.0976[M – H-44]−, 106.0421
20
41.4
Senkyunolide A (192)
C
12
H
1
6
O
2
193.1219[M + H]+
193.1223[M + H]+
−2.07
215.1027[M + Na]+, 407.2196[2M + Na]+
175.1119[M + H-H2O]+, 147.1155, 105.0718
21
42.1
Butylphthalide (190)
C
12
H
1
4
O
2
191.1061[M + H]+
191.1067[M + H]+
−3.14
213.0872[M + Na]+, 403.1888[2M + Na]+
173.0989[M + H-H2O]+, 145.1049[M + H-H2O-28]+
22
42.9
Senkyunolide C (204)
C
12
H
1
2
O
3
205.0847[M + H]+
205.0859[M + H]+
−5.85
227.0685[M + Na]+,  431.1431[2M + Na]+
187.0763[M + H-H2O]+,169.0757[M + H-2H2O]+, 141.0699[169-28]+
23
43.4
Senkyunolide E (204)
C
12
H
1
2
O
3
205.0843[M + H]+
205.0859[M + H]+
−7.80
227.0663[M + Na]+
24
44.1
Cnidilide (194)
C
12
H
1
8
O
2
195.1372[M + H]+
195.1380[M + H]+
−4.10
217.1185[M + Na]+
177.1323[M + H-H2O]+, 149.1340[M + H-H2O-28]+
25
44.1
E-Ligustilide (190)
C
12
H
1
4
O
2
191.1061[M + H]+
191.1067[M + H]+
−3.14
213.0871[M + Na]+
26
45.2
Z-Ligustilide (190)
C
12
H
1
4
O
2
191.1056[M + H]+
191.1067[M + H]+
−5.76
213.0859[M + Na]+, 403.1901[2M + Na]+
173.0984[M + H-H2O]+, 145.0999[M + H-H2O-28]+, 117.0690
27
45.5
Neocnidilide (194)
C
12
H
1
8
O
2
195.1372[M + H]+
195.1380[M + H]+
−4.10
217.1179[M + Na]+
177.1323[M + H-H2O]+, 149.1306[M + H-H2O-28]+, 121.0998
28
45.8
3-Butylidenephthalide (188)
C
12
H
1
2
O
2
189.0907[M + H]+
189.0910[M + H]+
−1.59
211.0720[M + Na]+
171.0817[M + H-H2O]+, 153.0800[M + H-H2O-H2O]+, 143.0882[M + H-H2O-28]+, 129.0724
29
46.2
Riligustilide (380)
C
24
H
28
O
4
381.2058[M + H]+
381.2060[M + H]+
−0.52
403.1872[M + Na]+, 191.1060, 213.0875
30
48.9
Senkyunolide P (382)
383.2220[M + H]+
383.2217[M + H]+
405.2031[M + Na]+
193.1212[M/2 + H]+
31
49.2
3′,6,8′,3a-Biligustilide (380)
C
24
H
28
O
4
381.2058[M + H]+
381.2060[M + H]+
−0.52
403.1885[M + Na]+, 191.1091
32
49.8
Tokinolide B (380)
C
24
H
28
O
4
381.2037[M + H]+
381.2060[M + H]+
−6.03
403.1878[M + Na]+, 191.1062
33
49.9
Unidentified (382)
383.2220[M + H]+
383.2217[M + H]+
405.2040[M + Na]+
193.1214[M/2 + H]+
34
50.3
Levistolide A (380)
C
24
H
28
O
4
381.2064[M + H]+
381.2060[M + H]+
1.05
403.1887[M + Na]+, 191.1054
35
50.8
Senkyunolide O (380)
C
24
H
28
O
4
381.2061[M + H]+
381.2060[M + H]+
0.26
403.1879[M + Na]+, 191.1057
191.1051[M/2 + H]+
36
51.4
Senkyunolide M (278)
279.1582[M + H]+
301.1402[M + Na]+, 579.2952[2M + Na]+
37
52.0
Senkyunolide Q (278)
279.1596[M + H]+
301.1381[M + Na]+, 579.2952[2M + Na]+
38
52.7
Unidentified (382)
383.2217[M + H]+
383.2217[M + H]+
0.00
405.2008[M + Na]+
365.2115, 347.1905, 193.1201[M/2 + H]+


#### 3.2.1.  Fragmentation Characteristic of Reference Compounds

In the present HPLC and MS conditions, characteristic MS adduct ions were observed for phenolic glycosides, organic acid, and phthalide derivatives. Phenolic glycosides and organic acids could be well-detected in positive and negative ionization mode and adduct ions such as [M+H]+, [M+NH4]+, [M+Na]+, and [2M+Na]+ or [M-H]− and [M+HCOO]- were found, whereas phthalides were only detected in positive mode and mainly showed the abundant [M+H]+, [M+Na]+, and [2M+Na]+ ions. The fragmentation characteristic of reference compounds was similar as those described in the literature [11, 14, 15]. For example, gastrodin, the glucoside ofp-hydroxybenzyl alcohol, mainly showed characteristic product ions atm/z123 and 161 corresponding to the elimination of a molecule of glucose and a molecule ofp-hydroxybenzyl alcohol fromm/z 285 [M-H]-, respectively. Parishin B and C, the gastrodin derivatives with citric acid, mainly indicated the fragmentation of the ester glucoside bond and the neutral loss of gastrodin residue (268 Da) corresponding to the ion atm/z 459. The ions atm/z441, 423, 397, and 379 were also observed, which were related to elimination of H2O and CO2 from the tertiary alcoholic hydroxyl group and the free carboxylic groups produced by breaking of the ester glucoside bond. Phthalides mainly displayed two pathways: side-chain cleavage with loss of alkenes and ring-opening followed by elimination of H2O and CO. These series of characteristic ion rules would be beneficial to elucidate the chemical constituents in TSC sample.


#### 3.2.2. Identification of Phenolics Derivatives in TSC Sample

Compounds1 and2 gave the same on-line UV spectrum which is in accordance with that of gastrodin. The structure of2 was identified as gastrodin based on the comparison of retention time, UV spectrum, and characteristic fragment ions with those of authentic compound as well as accurate molecular weight. The molecular weight of1 was deduced as 448 from the sodium adduct ion atm/z 471 [M+Na]+ detected in positive mode and deprotonated molecular ion atm/z 447 [M-H]− in negative mode, respectively. A prominent neutral loss of 162 Da, corresponding to the loss of hexose, and disaccharide residue ions atm/z 323 were observed in MS2 of the [M-H]− ion atm/z 447. The loss of 124 Da was assigned top-hydroxybenzyl alcohol just like the characteristic loss of gastrodin. Therefore, compound1 was identified as elatoside, namely, 6′-(p-hydroxybenzyl methyl)-gastrabiose, which was previously isolated from the rhizome ofG.elata[16].Compounds4,5,6, and7 gave the adduct ions [M+Na]+ and [M+NH4]+ in positive LC-MS and [M-H]− in negative LC-MS experiments as well as UV spectra, which were similar to those of gastrodin derivatives. Compounds5 and6 had the same [M-H]− ions atm/z 727, which produced the prominent fragment ion atm/z 459 in the MS2, due to loss of gastrodin residue (268 Da) as well as other ions atm/z441, 423, 397, and 379. Combining with retention time and addition of reference substances,5 and6 were identified as parishin B and C. Similarly, compound4was assigned to parishin E or its positional isomer parishin G with identical molecular mass of 460 [17]. Compound7 had the molecular mass of 996 and exhibited consecutive loss of gastrodin residue (268 Da), and, therefore, it was identified as parishin, a conjugate of one citric acid and three gastrodins.


#### 3.2.3. Identification of Phthalide Derivatives in TSC Sample

The molecular formula of compound8, detected from individual herbChuanxiong rhizoma, was calculated as C12H18O5 by [M+H]+ ion atm/z 243.1220 and [M-H]− ion atm/z 241.1090 in its LC-IT-TOF/MS experiment. It had the same molecular weight of 242 as those of known compounds, senkyunolide L and 3-butyl-3,6,7-trihydroxy-4,5,6,7-tetrahydrophthalide, present inChuanxiong rhizoma; however, compound8 could not be senkyunolide L (C12H15ClO3) because both had the different element composition [18]. The fragmentation ion of8 atm/z 197.1142, which was derived from a retro-Diels-Alder cleavage of the [M-H-H2O]− ion atm/z 223.0885, suggested that the structure of8 was proposed as 3-butyl-3,6,7-trihydroxy-4,5,6,7-tetrahydrophthalide.Compound10 displayed [M+H]+ ion atm/z 227.1259, suggesting the molecular formula of C12H18O4. The fragment ions atm/z 209 and 191 in the MS2, indicating consecutive loss of H2O, supported that10 was a dihydroxylated derivative of ligustilide. UV and MS data of10 were in accordance with those of senkyunolide J and senkyunolide N, but the stereochemistry information of two hydroxy groups could not be provided. Thus,10 was tentatively assigned as one of senkyunolide J and senkyunolide N. Compounds11 and12 gave the same [M-H]− ions atm/z 224 and their molecular formula was established as C12H18O4 according to the [M+H]+ ion atm/z 225.1105. However, both constituents had different fragmentation rules. The MS2 of11 displayed the fragment ions atm/z 207, 189 and the characteristic ion atm/z 165, which were similar to those of 4,5-dihydro-3,1′-dihydroxy-3-butylphthalide [15], whereas12 showed elimination of two H2O followed by side-chain cleavage. Therefore,11 was deduced as 4,5-dihydro-3,1′-dihydroxy-3-butylphthalide and12 could be one of senkyunolide I and senkyunolide H [14, 15].Compounds13,17, and19 have the same molecular formula of C12H14O3 and were tentatively characterized as 4-hydroxy-3-butylphthalide,Z-6,7-epoxyligustilide, and senkyunolide F. Compared with monohydroxy phthalide derivatives13 and19, the instable structure of17 resulted in the ion atm/z 189 as base peak observed in MS1, suggesting loss of H2O from the [M-H]− ion atm/z 207. Senkyunolide A20 andZ-ligustilide26 as main constituents were identified and butylphthalide21, senkyunolide C22, senkyunolide E23,E-ligustilide24, and 3-butylidenephthalide28 were characterized as minor constituents. For the compounds20,21,24, and26, the neutral loss of CO (28 Da) and side-chain cleavage (56 Da) from [M+H-H2O]+ were the common fragmentation rules.Compound16 (C12H14O3) was tentatively proposed to 4,7-dihydroxy-3-butylidenephthalide or senkyunolide D. Compound18 was assigned as senkyunolide K or senkyunolide G. These compounds and their isomers could not be differentiated by available MS data.Five dimeric ligustilides29,31,32,34, and35, which produced the protonated ion [M+H]+ atm/z 381, were detected in extracted ion chromatograms. They showed the base peak atm/z 403 [M+Na]+ in MS1 and the fragment ion atm/z 191 [M+H-190]+ as base peak in MS2 by the loss of a RDA fragment (145 Da) followed by the loss of H2O and HCOOH (46 Da). Comprehensively considering the information described in the literature [14], they were tentatively identified as riligustilide, 3′,6,8′,3a-biligustilide, tokinolide B, levistolide A, and senkyunolide O, respectively. Three compounds, indicating the protonated ion [M+H]+ atm/z383, were detected in extracted ion chromatograms; however, only senkyunolide P was previously reported inChuanxiong Rhizoma. Thus, compound30 was tentatively characterized as senkyunolide P and the other compounds (33 and38) were still indefinite based on the available information.In the modernization of TCM, chemical profiling is always the first task. It is of importance for development of the suitable quality standard and control strategy, study of pharmacokinetics, and interpretation of therapeutic character of TCM [19, 20]. As a marketed product in China, the quality control system of TSC is still to be improved and its pharmacokinetics and action mechanism are not completely clear. Usually, gastrodin, ferulic acid, and 6,7-dihydroxyligustilide are selected as marker compounds for the quality control or the pharmacokinetic study of Tianshu Capsule [1, 21, 22]. The present investigation provides more chemical information for the selection of the marker compounds and the improvement of quality control. It also tells the pharmacokinetic scientists and ethnopharmacologists which compounds in TSC samples are worth further evaluation. In addition to gastrodin and ferulic acid, more efforts should be made to the group of phthalide derivatives such as major constituents, senkyunolide I/H12, 4-hydroxy-3-butylphthalide13, senkyunolide A20, andZ-ligustilide26 as well as their dimers. Additionally, it is worth noting that 5-HMF3, a potent toxic compound, was found as main peak in the chemical profile of TSC samples. This compound originated from the crude materialGastrodiae rhizomaand its content in TSC samples was up to 2.67 mg·g−1 [23]. If it is necessary to develop its limit standard in TSC product or not, more study are expected. Also, ligustrazine is not detected in TSC sample, even in the extracted ion chromatogram, which could result from its very low amount in crude herbal material (about 0.01~0.02%), although, actually, it is often considered as one of active components inChuanxiong rhizoma.


## 3.1. Qualitative Analyses of TSC and Its Related Crude Herbal Materials by HPLC and LC-MS

Under the HPLC conditions as described in the current Chinese Pharmacopoeia [1], 5 batches of TSC samples, together with the reference compounds, were examined and their HPLC chromatograms were shown in Figure S2. High similarity in the number, type, and amount of chemical constituents was observed in the HPLC profiles of different batches of TSC samples. General chromatographic profile was obtained by Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine software and characteristic peaks were found in the HPLC profile of each individual sample.In order to identify the origin of these characteristic peaks from individual herbs, a comparative study was carried out by using various extracts of herbs and TSC samples. Accordingly, the possible individual contribution from the corresponding herbs to the general chromatographic profile was found. Compared with the HPLC profiles of the ethanolic and aqueous extracts ofChuanxiong rhizoma and Gastrodiae rhizome (Figure S3), 29 of 38 peaks occurring in HPLC profile of TSC sample were contributed byChuanxiong rhizoma and other 9 peaks came from Gastrodiae rhizome (Figures S4~S10). In addition to the comparison of retention time and on-line UV spectra with those of reference compounds gastrodin, 5-HMF, parishin B, parishin C, parishin, ferulic acid, andZ-ligustilide, the precursor ions obtained by the positive and negative LC-MS (Figure 2) such as [M+H]+, [M+NH4]+, [M+Na]+, and [M-H]- further confirmed the contribution of the characteristic peaks from individual herbs to the chromatographic profile of TSC sample (Table 1).Table 1
Retention time (tR), UV, and LC-MS data of the identified compounds in Tianshu Capsule and related crude materials.
Number
t
R/min
UVλmax⁡/nm
MW
[M − H]−
[M + H]+
[M + NH4]+
[M + Na]+
[2M + Na]+
[M + HCOO]−
Tianma
Chuanxiong
Identified compounds
2
5.403
221, 276
286
304
331
+
−
Gastrodin
3
7.736
230, 283
126
127
144
149
+
−
5-HMF
4
15.579
225
460
459
478
483
+
−
Parishin E/G
5
20.691
221, 272
728
727
746
751
+
−
Parishin B
6
22.220
221, 274
728
727
746
751
+
−
Parishin C
8
25.080
218
242
241
243
265
507
−
+
3-Butyl-3,6,7-trihydroxy-4,5,6,7-tetrahydrophthalide
9
25.567
220, 236, 323
194
193
195
217
+
+
ferulic acid
10
27.529
220, 330
226
227
249
475
−
+
Senkyunolide J/N
11
30.667
280
224
225
247
471
−
+
4,5-Dihydro-3,1′-dihydroxy-3-butylphthalide
13
32.436
275
206
205
207
−
+
4-hydroxyl-3-butylphthalide
14
34.299
325
396
397
+
+
unidentified
16
38.635
220, 270
222
221
223
245
−
+
Senkyunolide D or4,7-dihydroxy-3-butylidenephthalide
17
39.502
230, 310
206
205
207
229
435
−
+
Z-6,7-Epoxyligustilide
18
39.898
280
208
207
209
231
439
−
+
Senkyunolide K/G
19
41.465
220
206
205
207
229
435
−
+
Senkyunolide F
20
42.332
220, 240, 325
192
193
215
407
+
+
Senkyunolide A
21
42.956
226, 280
190
191
208
213
403
−
+
Butylphthalide
22
43.717
218, 270
204
203
205
227
431
249
−
+
Senkyunolide C
23
44.147
210, 285, 330
204
203
205
227
431
249
−
+
Senkyunolide E
24
44.949
220, 270, 330
194
195
217
411
−
+
Cnidilide
26
45.831
205, 280, 328
190
191
213
403
−
+
Ligustilide
27
46.394
220
194
195
217
411
−
+
Neocnidilide
28
46.653
210, 275, 330
188
189
206
211
399
−
+
3-Butylidenephthalide
29
47.231
225, 280, 310
380
381
398
403
−
+
Riligustilide
30
50.042
276
382
383
399
405
−
+
Senkyunolide P
31
50.476
220, 280
380
381
398
403
−
+
3′,6,8′,3a-Biligustilide
32
50.970
220, 280
380
381
398
403
−
+
Tokinolide B
33
51.065
230, 279
382
383
400
405
−
+
Unidentified
34
51.962
220, 275
380
381
403
−
+
Levistolide A
35
52.267
220, 285
380
381
−
+
3′Z-3,3′a,8,6′a-Biligustilide
36
52.693
220, 280
278
279
301
579
−
+
Senkyunolide M
37
53.134
220, 280
278
279
301
579
−
+
Senkyunolide Q
38
53.852
225, 278
382
383
400
405
383
−
+
UnidentifiedHPLC and TIC of typical TSC sample obtained using an Agilent 6130 Quadrupole LC-MS connected to an Agilent 1200 HPLC system. (a) HPLC (276 nm), (b) (+) TIC, and (c) (−) TIC.
(a)
(b)
(c)


## 3.2. Identification of Chemical Constituents in TSC by LC-DAD-EST-IT-MSn and LC-DAD-ESI-IT-TOF/MS

The combination of LC-DAD-ESI-IT-MSn and LC-DAD-ESI-IT-TOF/MS experiments was employed for the identification of chemical constituents in TSC sample, and, as a result, a total of 38 compounds was identified or tentatively characterized. The structures of the identified compounds are shown in Figure 1 and their chromatographic and mass spectrometric data are shown in Tables 2 and 3. The total ion chromatograms (TICs) of TSC sample are shown in Figures S11 and S12, respectively. Among the identified constituents, 7 compounds (2,3,5,6,7,9, and26) were unambiguously identified as gastrodin, 5-HMF, parishin B, parishin C, parishin, ferulic acid, andZ-ligustilide based on the direct comparison of their retention times, UV spectra, and mass spectra with those of the authentic compounds. Furthermore, 31 compounds were tentatively characterized according to their UV spectra, empirical molecular formula, and mass fragmentation pathways as well as their eluted sequence from ODS column reported in the literature [11–14] and acquired in the present experiments.Table 2
Retention time (tR), UV, and MSn data obtained by LC-DAD-ESI-IT-MSn of the identified compounds in Tianshu Capsule.
Number
t
R/min
Identified compounds
UVλmax⁡/nm
[M + Na]+
[M + H]+
[2M + Na]+
Main product ions
[M − H]−
[M + HCOO]−
Main product ions
1
6.3
Gastrodin glucose (448)
221, 276
471
447
493
323[M – H-C7H8O2]−, 285[M – H-glc]−, 179
2
6.3
Gastrodin (286)
220, 269
309
185[M + Na-C7H8O2]+
285
331
161[M – H-C7H8O2]−, 123[M – H-glc]−
3
8.1
5-HMF (126)
230, 283
127
109[M + H-H2O]+
125
5
20.3
Parishin B (728)
221, 272
751
483[M + Na-268]+, 215[483-268]+
727
459, 441, 423, 397, 379, 217
6
21.8
Parishin C (728)
221, 274
751
483[M + Na-268]+, 215[483-268]+
727
459, 441, 423, 397, 379, 217
7
23.4
Parishin (996)
221, 271
995
727[M – H-268]−
9
26.0
Ferulic acid (194)
220, 236, 323
195
177[M + H-H2O]+, 145[177-CH3OH]+
10
27.4
Senkyunolide J/N (226)
220, 330
249
475
209[M + H-H2O]+, 191[M + H-2H2O]+, 163[M + H-2H2O-28]+
11
30.4
4,5-Dihydro-3,1′-dihydroxy-3-butylphthalide (224)
280
247
207[M + H-H2O]+, 189[M + H-2H2O]+, 165, 121
12
31.6
Senkyunolide I/H (224)
276
247
207[M + H-H2O]+, 189[M + H-2H2O]+, 161[189-H2O]+, 133[189-56]+
15
37.0
Unidentified (316)
230, 276, 280
339
317
299[M + H-H2O]+, 281[M + H-2H2O]+, 271[M + H-H2O-28]+, 243
315
17
37.8
Z-6,7-Epoxyligustilide (206)
230
229
413
189[M + Na-H2O]+, 171[189-H2O]+
18
38.1
Senkyunolide K/G (208)
233, 280
231
439
191[M + H-H2O]+, 173[191 − H2O]+, 149[191-42]+, 135[191-56]+
20
40.2
Senkyunolide A (192)
280
215
193
407
175[M + H-H2O]+, 147, 119, 105
21
40.7
Butylphthalide (190)
232, 275
213
191
403
173[M + H-H2O]+, 145, 117
24
42.6
Cnidilide (194)
238, 279, 327
217
25
43.3
E-Ligustilide (190)
281, 327
213
191
403
173[M + H-H2O]+, 130
26
43.5
Z-Ligustilide (190)
237, 260, 312
213
191
403
173[M + H-H2O]+, 145[173-28]+ 130, 117, 105
28
44.0
3-Butylidenephthalide (188)
230, 277, 326
211
189
171[M + H-H2O]+, 153[171-H2O]+
29
47.4
Riligustilide (380)
275
403
381
213[2M + Na-190]+, 191, 173
30
47.5
Senkyunolide P (382)
278
405
383
192[M + H-190]+
31
47.8
3′,6,8′,3a-Biligustilide (380)
278, 363
403
381
213[2M + Na-190]+, 191[M + H-190]+, 145, 117
32
48.0
Tokinolide B (380)
278
403
381
213[2M + Na-190]+, 191[M + H-190]+
33
48.2
Unidentified (382)
230, 279
405
383
193[M + H-190]+
34
48.5
Levistolide A (380)
276
403
381
213[2M + Na-190]+, 191[M + H-190]+, 145, 117
35
48.8
Senkyunolide O (380)
278
403
381
173[191-H2O]+, 191[M + H-190]+,145, 117
36
49.0
Senkyunolide M (278)
279
301
279
245[M + Na-56]+, 189, 171
37
49.4
Senkyunolide Q (278)
278
301
279
245[M + Na-56]+, 189, 171
38
49.9
Unidentified (382)
225, 278
405
383Table 3
Retention time (tR) and MS data obtained by LC-DAD-ESI-IT-TOF/MS of the identified compounds in the sample of Tianshu Capsule.
Number
t
R/min
Identified compounds
Formula
Mea. mass/m/z
Calc. mass/m/z
Error/ppm
Other precursor ions
Main product ions
3
7.6
5-HMF (126)
C
6
H
6
O
3
127.0384[M + H]+
127.0390[M + H]+
−4.72
149.0240[M + Na]+
5
19.8
Parishin B (728)
C
32
H
40
O
19
727.2123[M − H]−
727.2091[M − H]−
4.40
6
21.5
Parishin C (728)
C
32
H
40
O
19
727.2132[M − H]−
727.2091[M − H]−
5.64
8
24.5
3-Butyl-3,6,7-trihydroxy-4,5,6,7-tetrahydrophthalide (242)
C
12
H
18
O
5
241.1090[M − H]−
241.1081[M − H]−
3.72
223.0885[M – H-H2O]−, 197.1142, 179.1107, 141.0930, 123.0854
243.1220[M + H]+
243.1227[M + H]+
2.88
265.1060[M + Na]+
9
25.3
Ferulic acid (194)
C
10
H
10
O
4
195.0649[M + H]+
195.0652[M + H]+
−1.54
217.0458[M + Na]+
10
27.0
Senkyunolide J/N (226)
C
12
H
18
O
4
227.1259[M + H]+
227.1278[M + H]+
−3.37
249.1082[M + Na]+
209.1177[M + H-H2O]+, 191.1026[M + H-2H2O]+
11
30.3
4,5-Dihydro-3,1′-dihydroxy-3-butylphthalide (224)
C
12
H
1
6
O
4
225.1105[M + H]+
225.1121[M + H]+
−7.11
12
31.6
Senkyunolide I/H (224)
C
12
H
1
6
O
4
225.1105[M + H]+
225.1121[M + H]+
−7.11
15
37.8
Unidentified (316)
C
1
8
H
20
O
5
317.1380[M + H]+
317.1384[M + H]+
−1.26
339.1210[M + Na]+
299.1378[M + H-H2O]+, 271.1343[M  + H-H2O-28]+
16
37.8
4,7-Dihydroxy-3-butylidenephthalide or senkyunolide D (222)
C
12
H
1
4
O
4
223.0986[M + H]+
223.0965[M + H]+
9.41
245.0774[M + Na]+
17
38.7
Z-6,7-Epoxyligustilide (206)
C
12
H
14
O
3
207.1008[M + H]+
207.1016[M + H]+
3.66
229.0824[M + Na]+
189.0856[M + H-H2O]+, 133.0318
18
39.1
Senkyunolide K/G (208)
C
12
H
1
6
O
3
209.1167[M + H]+
209.1172[M + H]+
−2.39
231.0974[M + Na]+
191.1091[M + H-H2O]+, 119.0819
19
40.6
Senkyunolide F (206)
C
12
H
14
O
3
207.1009[M + H]+
207.1016[M + H]+
3.38
229.0815[M + Na]+
189.0911[M + H-H2O]+, 161.0959
205.0874[M − H]−
205.0870[M − H]−
1.95
411.1821[2M − H]−
161.0976[M – H-44]−, 106.0421
20
41.4
Senkyunolide A (192)
C
12
H
1
6
O
2
193.1219[M + H]+
193.1223[M + H]+
−2.07
215.1027[M + Na]+, 407.2196[2M + Na]+
175.1119[M + H-H2O]+, 147.1155, 105.0718
21
42.1
Butylphthalide (190)
C
12
H
1
4
O
2
191.1061[M + H]+
191.1067[M + H]+
−3.14
213.0872[M + Na]+, 403.1888[2M + Na]+
173.0989[M + H-H2O]+, 145.1049[M + H-H2O-28]+
22
42.9
Senkyunolide C (204)
C
12
H
1
2
O
3
205.0847[M + H]+
205.0859[M + H]+
−5.85
227.0685[M + Na]+,  431.1431[2M + Na]+
187.0763[M + H-H2O]+,169.0757[M + H-2H2O]+, 141.0699[169-28]+
23
43.4
Senkyunolide E (204)
C
12
H
1
2
O
3
205.0843[M + H]+
205.0859[M + H]+
−7.80
227.0663[M + Na]+
24
44.1
Cnidilide (194)
C
12
H
1
8
O
2
195.1372[M + H]+
195.1380[M + H]+
−4.10
217.1185[M + Na]+
177.1323[M + H-H2O]+, 149.1340[M + H-H2O-28]+
25
44.1
E-Ligustilide (190)
C
12
H
1
4
O
2
191.1061[M + H]+
191.1067[M + H]+
−3.14
213.0871[M + Na]+
26
45.2
Z-Ligustilide (190)
C
12
H
1
4
O
2
191.1056[M + H]+
191.1067[M + H]+
−5.76
213.0859[M + Na]+, 403.1901[2M + Na]+
173.0984[M + H-H2O]+, 145.0999[M + H-H2O-28]+, 117.0690
27
45.5
Neocnidilide (194)
C
12
H
1
8
O
2
195.1372[M + H]+
195.1380[M + H]+
−4.10
217.1179[M + Na]+
177.1323[M + H-H2O]+, 149.1306[M + H-H2O-28]+, 121.0998
28
45.8
3-Butylidenephthalide (188)
C
12
H
1
2
O
2
189.0907[M + H]+
189.0910[M + H]+
−1.59
211.0720[M + Na]+
171.0817[M + H-H2O]+, 153.0800[M + H-H2O-H2O]+, 143.0882[M + H-H2O-28]+, 129.0724
29
46.2
Riligustilide (380)
C
24
H
28
O
4
381.2058[M + H]+
381.2060[M + H]+
−0.52
403.1872[M + Na]+, 191.1060, 213.0875
30
48.9
Senkyunolide P (382)
383.2220[M + H]+
383.2217[M + H]+
405.2031[M + Na]+
193.1212[M/2 + H]+
31
49.2
3′,6,8′,3a-Biligustilide (380)
C
24
H
28
O
4
381.2058[M + H]+
381.2060[M + H]+
−0.52
403.1885[M + Na]+, 191.1091
32
49.8
Tokinolide B (380)
C
24
H
28
O
4
381.2037[M + H]+
381.2060[M + H]+
−6.03
403.1878[M + Na]+, 191.1062
33
49.9
Unidentified (382)
383.2220[M + H]+
383.2217[M + H]+
405.2040[M + Na]+
193.1214[M/2 + H]+
34
50.3
Levistolide A (380)
C
24
H
28
O
4
381.2064[M + H]+
381.2060[M + H]+
1.05
403.1887[M + Na]+, 191.1054
35
50.8
Senkyunolide O (380)
C
24
H
28
O
4
381.2061[M + H]+
381.2060[M + H]+
0.26
403.1879[M + Na]+, 191.1057
191.1051[M/2 + H]+
36
51.4
Senkyunolide M (278)
279.1582[M + H]+
301.1402[M + Na]+, 579.2952[2M + Na]+
37
52.0
Senkyunolide Q (278)
279.1596[M + H]+
301.1381[M + Na]+, 579.2952[2M + Na]+
38
52.7
Unidentified (382)
383.2217[M + H]+
383.2217[M + H]+
0.00
405.2008[M + Na]+
365.2115, 347.1905, 193.1201[M/2 + H]+


### 3.2.1.  Fragmentation Characteristic of Reference Compounds

In the present HPLC and MS conditions, characteristic MS adduct ions were observed for phenolic glycosides, organic acid, and phthalide derivatives. Phenolic glycosides and organic acids could be well-detected in positive and negative ionization mode and adduct ions such as [M+H]+, [M+NH4]+, [M+Na]+, and [2M+Na]+ or [M-H]− and [M+HCOO]- were found, whereas phthalides were only detected in positive mode and mainly showed the abundant [M+H]+, [M+Na]+, and [2M+Na]+ ions. The fragmentation characteristic of reference compounds was similar as those described in the literature [11, 14, 15]. For example, gastrodin, the glucoside ofp-hydroxybenzyl alcohol, mainly showed characteristic product ions atm/z123 and 161 corresponding to the elimination of a molecule of glucose and a molecule ofp-hydroxybenzyl alcohol fromm/z 285 [M-H]-, respectively. Parishin B and C, the gastrodin derivatives with citric acid, mainly indicated the fragmentation of the ester glucoside bond and the neutral loss of gastrodin residue (268 Da) corresponding to the ion atm/z 459. The ions atm/z441, 423, 397, and 379 were also observed, which were related to elimination of H2O and CO2 from the tertiary alcoholic hydroxyl group and the free carboxylic groups produced by breaking of the ester glucoside bond. Phthalides mainly displayed two pathways: side-chain cleavage with loss of alkenes and ring-opening followed by elimination of H2O and CO. These series of characteristic ion rules would be beneficial to elucidate the chemical constituents in TSC sample.


### 3.2.2. Identification of Phenolics Derivatives in TSC Sample

Compounds1 and2 gave the same on-line UV spectrum which is in accordance with that of gastrodin. The structure of2 was identified as gastrodin based on the comparison of retention time, UV spectrum, and characteristic fragment ions with those of authentic compound as well as accurate molecular weight. The molecular weight of1 was deduced as 448 from the sodium adduct ion atm/z 471 [M+Na]+ detected in positive mode and deprotonated molecular ion atm/z 447 [M-H]− in negative mode, respectively. A prominent neutral loss of 162 Da, corresponding to the loss of hexose, and disaccharide residue ions atm/z 323 were observed in MS2 of the [M-H]− ion atm/z 447. The loss of 124 Da was assigned top-hydroxybenzyl alcohol just like the characteristic loss of gastrodin. Therefore, compound1 was identified as elatoside, namely, 6′-(p-hydroxybenzyl methyl)-gastrabiose, which was previously isolated from the rhizome ofG.elata[16].Compounds4,5,6, and7 gave the adduct ions [M+Na]+ and [M+NH4]+ in positive LC-MS and [M-H]− in negative LC-MS experiments as well as UV spectra, which were similar to those of gastrodin derivatives. Compounds5 and6 had the same [M-H]− ions atm/z 727, which produced the prominent fragment ion atm/z 459 in the MS2, due to loss of gastrodin residue (268 Da) as well as other ions atm/z441, 423, 397, and 379. Combining with retention time and addition of reference substances,5 and6 were identified as parishin B and C. Similarly, compound4was assigned to parishin E or its positional isomer parishin G with identical molecular mass of 460 [17]. Compound7 had the molecular mass of 996 and exhibited consecutive loss of gastrodin residue (268 Da), and, therefore, it was identified as parishin, a conjugate of one citric acid and three gastrodins.


### 3.2.3. Identification of Phthalide Derivatives in TSC Sample

The molecular formula of compound8, detected from individual herbChuanxiong rhizoma, was calculated as C12H18O5 by [M+H]+ ion atm/z 243.1220 and [M-H]− ion atm/z 241.1090 in its LC-IT-TOF/MS experiment. It had the same molecular weight of 242 as those of known compounds, senkyunolide L and 3-butyl-3,6,7-trihydroxy-4,5,6,7-tetrahydrophthalide, present inChuanxiong rhizoma; however, compound8 could not be senkyunolide L (C12H15ClO3) because both had the different element composition [18]. The fragmentation ion of8 atm/z 197.1142, which was derived from a retro-Diels-Alder cleavage of the [M-H-H2O]− ion atm/z 223.0885, suggested that the structure of8 was proposed as 3-butyl-3,6,7-trihydroxy-4,5,6,7-tetrahydrophthalide.Compound10 displayed [M+H]+ ion atm/z 227.1259, suggesting the molecular formula of C12H18O4. The fragment ions atm/z 209 and 191 in the MS2, indicating consecutive loss of H2O, supported that10 was a dihydroxylated derivative of ligustilide. UV and MS data of10 were in accordance with those of senkyunolide J and senkyunolide N, but the stereochemistry information of two hydroxy groups could not be provided. Thus,10 was tentatively assigned as one of senkyunolide J and senkyunolide N. Compounds11 and12 gave the same [M-H]− ions atm/z 224 and their molecular formula was established as C12H18O4 according to the [M+H]+ ion atm/z 225.1105. However, both constituents had different fragmentation rules. The MS2 of11 displayed the fragment ions atm/z 207, 189 and the characteristic ion atm/z 165, which were similar to those of 4,5-dihydro-3,1′-dihydroxy-3-butylphthalide [15], whereas12 showed elimination of two H2O followed by side-chain cleavage. Therefore,11 was deduced as 4,5-dihydro-3,1′-dihydroxy-3-butylphthalide and12 could be one of senkyunolide I and senkyunolide H [14, 15].Compounds13,17, and19 have the same molecular formula of C12H14O3 and were tentatively characterized as 4-hydroxy-3-butylphthalide,Z-6,7-epoxyligustilide, and senkyunolide F. Compared with monohydroxy phthalide derivatives13 and19, the instable structure of17 resulted in the ion atm/z 189 as base peak observed in MS1, suggesting loss of H2O from the [M-H]− ion atm/z 207. Senkyunolide A20 andZ-ligustilide26 as main constituents were identified and butylphthalide21, senkyunolide C22, senkyunolide E23,E-ligustilide24, and 3-butylidenephthalide28 were characterized as minor constituents. For the compounds20,21,24, and26, the neutral loss of CO (28 Da) and side-chain cleavage (56 Da) from [M+H-H2O]+ were the common fragmentation rules.Compound16 (C12H14O3) was tentatively proposed to 4,7-dihydroxy-3-butylidenephthalide or senkyunolide D. Compound18 was assigned as senkyunolide K or senkyunolide G. These compounds and their isomers could not be differentiated by available MS data.Five dimeric ligustilides29,31,32,34, and35, which produced the protonated ion [M+H]+ atm/z 381, were detected in extracted ion chromatograms. They showed the base peak atm/z 403 [M+Na]+ in MS1 and the fragment ion atm/z 191 [M+H-190]+ as base peak in MS2 by the loss of a RDA fragment (145 Da) followed by the loss of H2O and HCOOH (46 Da). Comprehensively considering the information described in the literature [14], they were tentatively identified as riligustilide, 3′,6,8′,3a-biligustilide, tokinolide B, levistolide A, and senkyunolide O, respectively. Three compounds, indicating the protonated ion [M+H]+ atm/z383, were detected in extracted ion chromatograms; however, only senkyunolide P was previously reported inChuanxiong Rhizoma. Thus, compound30 was tentatively characterized as senkyunolide P and the other compounds (33 and38) were still indefinite based on the available information.In the modernization of TCM, chemical profiling is always the first task. It is of importance for development of the suitable quality standard and control strategy, study of pharmacokinetics, and interpretation of therapeutic character of TCM [19, 20]. As a marketed product in China, the quality control system of TSC is still to be improved and its pharmacokinetics and action mechanism are not completely clear. Usually, gastrodin, ferulic acid, and 6,7-dihydroxyligustilide are selected as marker compounds for the quality control or the pharmacokinetic study of Tianshu Capsule [1, 21, 22]. The present investigation provides more chemical information for the selection of the marker compounds and the improvement of quality control. It also tells the pharmacokinetic scientists and ethnopharmacologists which compounds in TSC samples are worth further evaluation. In addition to gastrodin and ferulic acid, more efforts should be made to the group of phthalide derivatives such as major constituents, senkyunolide I/H12, 4-hydroxy-3-butylphthalide13, senkyunolide A20, andZ-ligustilide26 as well as their dimers. Additionally, it is worth noting that 5-HMF3, a potent toxic compound, was found as main peak in the chemical profile of TSC samples. This compound originated from the crude materialGastrodiae rhizomaand its content in TSC samples was up to 2.67 mg·g−1 [23]. If it is necessary to develop its limit standard in TSC product or not, more study are expected. Also, ligustrazine is not detected in TSC sample, even in the extracted ion chromatogram, which could result from its very low amount in crude herbal material (about 0.01~0.02%), although, actually, it is often considered as one of active components inChuanxiong rhizoma.


## 3.2.1.  Fragmentation Characteristic of Reference Compounds

In the present HPLC and MS conditions, characteristic MS adduct ions were observed for phenolic glycosides, organic acid, and phthalide derivatives. Phenolic glycosides and organic acids could be well-detected in positive and negative ionization mode and adduct ions such as [M+H]+, [M+NH4]+, [M+Na]+, and [2M+Na]+ or [M-H]− and [M+HCOO]- were found, whereas phthalides were only detected in positive mode and mainly showed the abundant [M+H]+, [M+Na]+, and [2M+Na]+ ions. The fragmentation characteristic of reference compounds was similar as those described in the literature [11, 14, 15]. For example, gastrodin, the glucoside ofp-hydroxybenzyl alcohol, mainly showed characteristic product ions atm/z123 and 161 corresponding to the elimination of a molecule of glucose and a molecule ofp-hydroxybenzyl alcohol fromm/z 285 [M-H]-, respectively. Parishin B and C, the gastrodin derivatives with citric acid, mainly indicated the fragmentation of the ester glucoside bond and the neutral loss of gastrodin residue (268 Da) corresponding to the ion atm/z 459. The ions atm/z441, 423, 397, and 379 were also observed, which were related to elimination of H2O and CO2 from the tertiary alcoholic hydroxyl group and the free carboxylic groups produced by breaking of the ester glucoside bond. Phthalides mainly displayed two pathways: side-chain cleavage with loss of alkenes and ring-opening followed by elimination of H2O and CO. These series of characteristic ion rules would be beneficial to elucidate the chemical constituents in TSC sample.


## 3.2.2. Identification of Phenolics Derivatives in TSC Sample

Compounds1 and2 gave the same on-line UV spectrum which is in accordance with that of gastrodin. The structure of2 was identified as gastrodin based on the comparison of retention time, UV spectrum, and characteristic fragment ions with those of authentic compound as well as accurate molecular weight. The molecular weight of1 was deduced as 448 from the sodium adduct ion atm/z 471 [M+Na]+ detected in positive mode and deprotonated molecular ion atm/z 447 [M-H]− in negative mode, respectively. A prominent neutral loss of 162 Da, corresponding to the loss of hexose, and disaccharide residue ions atm/z 323 were observed in MS2 of the [M-H]− ion atm/z 447. The loss of 124 Da was assigned top-hydroxybenzyl alcohol just like the characteristic loss of gastrodin. Therefore, compound1 was identified as elatoside, namely, 6′-(p-hydroxybenzyl methyl)-gastrabiose, which was previously isolated from the rhizome ofG.elata[16].Compounds4,5,6, and7 gave the adduct ions [M+Na]+ and [M+NH4]+ in positive LC-MS and [M-H]− in negative LC-MS experiments as well as UV spectra, which were similar to those of gastrodin derivatives. Compounds5 and6 had the same [M-H]− ions atm/z 727, which produced the prominent fragment ion atm/z 459 in the MS2, due to loss of gastrodin residue (268 Da) as well as other ions atm/z441, 423, 397, and 379. Combining with retention time and addition of reference substances,5 and6 were identified as parishin B and C. Similarly, compound4was assigned to parishin E or its positional isomer parishin G with identical molecular mass of 460 [17]. Compound7 had the molecular mass of 996 and exhibited consecutive loss of gastrodin residue (268 Da), and, therefore, it was identified as parishin, a conjugate of one citric acid and three gastrodins.


## 3.2.3. Identification of Phthalide Derivatives in TSC Sample

The molecular formula of compound8, detected from individual herbChuanxiong rhizoma, was calculated as C12H18O5 by [M+H]+ ion atm/z 243.1220 and [M-H]− ion atm/z 241.1090 in its LC-IT-TOF/MS experiment. It had the same molecular weight of 242 as those of known compounds, senkyunolide L and 3-butyl-3,6,7-trihydroxy-4,5,6,7-tetrahydrophthalide, present inChuanxiong rhizoma; however, compound8 could not be senkyunolide L (C12H15ClO3) because both had the different element composition [18]. The fragmentation ion of8 atm/z 197.1142, which was derived from a retro-Diels-Alder cleavage of the [M-H-H2O]− ion atm/z 223.0885, suggested that the structure of8 was proposed as 3-butyl-3,6,7-trihydroxy-4,5,6,7-tetrahydrophthalide.Compound10 displayed [M+H]+ ion atm/z 227.1259, suggesting the molecular formula of C12H18O4. The fragment ions atm/z 209 and 191 in the MS2, indicating consecutive loss of H2O, supported that10 was a dihydroxylated derivative of ligustilide. UV and MS data of10 were in accordance with those of senkyunolide J and senkyunolide N, but the stereochemistry information of two hydroxy groups could not be provided. Thus,10 was tentatively assigned as one of senkyunolide J and senkyunolide N. Compounds11 and12 gave the same [M-H]− ions atm/z 224 and their molecular formula was established as C12H18O4 according to the [M+H]+ ion atm/z 225.1105. However, both constituents had different fragmentation rules. The MS2 of11 displayed the fragment ions atm/z 207, 189 and the characteristic ion atm/z 165, which were similar to those of 4,5-dihydro-3,1′-dihydroxy-3-butylphthalide [15], whereas12 showed elimination of two H2O followed by side-chain cleavage. Therefore,11 was deduced as 4,5-dihydro-3,1′-dihydroxy-3-butylphthalide and12 could be one of senkyunolide I and senkyunolide H [14, 15].Compounds13,17, and19 have the same molecular formula of C12H14O3 and were tentatively characterized as 4-hydroxy-3-butylphthalide,Z-6,7-epoxyligustilide, and senkyunolide F. Compared with monohydroxy phthalide derivatives13 and19, the instable structure of17 resulted in the ion atm/z 189 as base peak observed in MS1, suggesting loss of H2O from the [M-H]− ion atm/z 207. Senkyunolide A20 andZ-ligustilide26 as main constituents were identified and butylphthalide21, senkyunolide C22, senkyunolide E23,E-ligustilide24, and 3-butylidenephthalide28 were characterized as minor constituents. For the compounds20,21,24, and26, the neutral loss of CO (28 Da) and side-chain cleavage (56 Da) from [M+H-H2O]+ were the common fragmentation rules.Compound16 (C12H14O3) was tentatively proposed to 4,7-dihydroxy-3-butylidenephthalide or senkyunolide D. Compound18 was assigned as senkyunolide K or senkyunolide G. These compounds and their isomers could not be differentiated by available MS data.Five dimeric ligustilides29,31,32,34, and35, which produced the protonated ion [M+H]+ atm/z 381, were detected in extracted ion chromatograms. They showed the base peak atm/z 403 [M+Na]+ in MS1 and the fragment ion atm/z 191 [M+H-190]+ as base peak in MS2 by the loss of a RDA fragment (145 Da) followed by the loss of H2O and HCOOH (46 Da). Comprehensively considering the information described in the literature [14], they were tentatively identified as riligustilide, 3′,6,8′,3a-biligustilide, tokinolide B, levistolide A, and senkyunolide O, respectively. Three compounds, indicating the protonated ion [M+H]+ atm/z383, were detected in extracted ion chromatograms; however, only senkyunolide P was previously reported inChuanxiong Rhizoma. Thus, compound30 was tentatively characterized as senkyunolide P and the other compounds (33 and38) were still indefinite based on the available information.In the modernization of TCM, chemical profiling is always the first task. It is of importance for development of the suitable quality standard and control strategy, study of pharmacokinetics, and interpretation of therapeutic character of TCM [19, 20]. As a marketed product in China, the quality control system of TSC is still to be improved and its pharmacokinetics and action mechanism are not completely clear. Usually, gastrodin, ferulic acid, and 6,7-dihydroxyligustilide are selected as marker compounds for the quality control or the pharmacokinetic study of Tianshu Capsule [1, 21, 22]. The present investigation provides more chemical information for the selection of the marker compounds and the improvement of quality control. It also tells the pharmacokinetic scientists and ethnopharmacologists which compounds in TSC samples are worth further evaluation. In addition to gastrodin and ferulic acid, more efforts should be made to the group of phthalide derivatives such as major constituents, senkyunolide I/H12, 4-hydroxy-3-butylphthalide13, senkyunolide A20, andZ-ligustilide26 as well as their dimers. Additionally, it is worth noting that 5-HMF3, a potent toxic compound, was found as main peak in the chemical profile of TSC samples. This compound originated from the crude materialGastrodiae rhizomaand its content in TSC samples was up to 2.67 mg·g−1 [23]. If it is necessary to develop its limit standard in TSC product or not, more study are expected. Also, ligustrazine is not detected in TSC sample, even in the extracted ion chromatogram, which could result from its very low amount in crude herbal material (about 0.01~0.02%), although, actually, it is often considered as one of active components inChuanxiong rhizoma.


## 4. Conclusions

In this study, HPLC analysis was employed to find out the common chromatographic peak in various batches of TSC samples. The contribution of the characteristic peaks from individual herbs to the whole chromatographic profile was discussed based on comparative HPLC and LC-MS analyses. A total of 38 constituents were identified 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 provided the good basis for monitoring the manufacturing processes and improving quality control of TSC products.



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*Source: 580745-2014-07-20.xml*