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

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-M[S.sup.n] [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[degrees]C for 10 min. The supernatant was then separated, and all samples were stored at -80[degrees]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[degrees]C for 20 min. The supernatant was dried with nitrogen gas at 4[degrees]C, and 400 [micro]l of 50% methanol was added to reconstitute the residue, which was then centrifuged at 16000 rpm for 10 min at 4[degrees]C. The supernatant was transferred to a vial, and 10 [micro]l was injected for LC-MS analysis. All samples were filtered through a membrane (0.22 [micro]m 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 [C.sub.18] column (100 mm x 2.1 mm, 1.7 [micro]m, Waters, USA) with a flow rate of 0.3 mL/min at 40[degrees]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[degrees]C; ion spray voltage, 4500 V; gas 1, 50psi; gas 2, 50 psi; curtain gas, 45psi; 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 [+ or -] 50 mDa around the mass defects of the templates and over a mass range of [+ or -] 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[R]1.6 (AB SCIEX, CA, USA), MMDF, and NLF&DPLs of Metabolitepilot[TM] 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.

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.

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].sup.-] ion at m/z 447.1508. Peak X18 produced M[S.sup.2] base peaks at m/z 269.1028 and 161.0449 corresponding to [[M-H-179Da].sup.-]. 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].sup.-] 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).

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].sup.+] 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.

Metabolite M1, which eluted at 9.75 min, formed a molecular ion of [[M-H].sup.+] at m/z 223.0963 corresponding to [C.sub.12][H.sub.14][O.sub.4]. 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].sup.+] at m/z 253.1071 corresponding to [C.sub.13][H.sub.16][O.sub.5]. The characteristic production of m/z 235.0963 and 221.0839 was generated by loss of 18Daand 18 + 14 Da, which implied loss of a [H.sub.2]O group and methyl group. Other product ions were identical to that of M1. Therefore, M2 maybe 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].sup.+] at m/z 303.0535 corresponding to [C.sub.12][H.sub.14]S[O.sub.7]. 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 [H.sub.2]O 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].sup.+] at m/z 399.1285 corresponding to [C.sub.18][H.sub.22][O.sub.10]. 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].sup.+] at m/z 193.1222 corresponding to [C.sub.12][H.sub.16][O.sub.2]. 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].sup.+] at m/z 223.1328 corresponding to [C.sub.13][H.sub.18][O.sub.3]. 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 [H.sub.2]O 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].sup.+] at m/z 372.1475 corresponding to [C.sub.17][H.sub.25][O.sub.6]NS. 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 [H.sub.2]O 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].sup.+] at m/z 399.1285 corresponding to [C.sub.18][H.sub.22][O.sub.10]. 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, [H.sub.2]O 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].sup.+] at m/z 401.1444 corresponding to [C.sub.18][H.sub.24][O.sub.10]. 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 [H.sub.2]O 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].sup.+] at m/z 211.1327 corresponding to [C.sub.12][H.sub.18][O.sub.3]. 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 [H.sub.2]O conjugation. Metabolite M33, which eluted at 9.56 min, formed a molecular ion of [[M+H].sup.+] at m/ z 225.1119 corresponding to [C.sub.12][H.sub.16][O.sub.4]. 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 [H.sub.2]O 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.

https://doi.org/10.1155/2019/9158942

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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Lin Chen [ID], (1) Renhao Chen, (1) Hui Ouyang, (2) Qi Wang, (1) Zhifeng Li [ID], (1) Yulin Feng [ID], (2) and Shilin Yang (2)

(1) College of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China

(2) State 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-Hernandez

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

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

Caption: 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.

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

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

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

Caption: Figure 8: The extract ion chromatograms (EICs) of other metabolites detected.
Table 1: The chemical constituents detected from TST.

Peak       RT                      Formula                    Error
          (min)                                               (ppm)

X1 (a)    0.98          [C.sub.7][H.sub.12][O.sub.6]          -1.2

X2        1.07           [C.sub.6][H.sub.8][O.sub.7]           0.9

X3 (b)    1.07      [C.sub.9][H.sub.12][N.sub.2][O.sub.6]      -1

X4 (b)    1.08          [C.sub.5][H.sub.7]N[O.sub.3]            3

X5 (b)    1.36           [C.sub.6][H.sub.6][O.sub.4]           1.1

X6        1.36     [C.sub.10][H.sub.13][N.sub.5][O.sub.5]      2.4

X7        1.63          [C.sub.13][H.sub.18][O.sub.7]         -1.1

X8        2.03           [C.sub.7][H.sub.6][O.sub.4]           0.7

X9        4.02           [C.sub.7][H.sub.6][O.sub.3]           2.5

X10       4.70         [C.sub.19][H.sub.24][O.sub.13]          1.7

X11       5.09          [C.sub.16][H.sub.18][O.sub.9]          0.8

X12       5.13     [C.sub.17][H.sub.23][N.sub.3][O.sub.7]S     0.5

X13       5.24    [C.sub.23][H.sub.33][N.sub.3][O.sub.12]S     2.3

X14       5.34          [C.sub.9][H.sub.10][O.sub.4]          -0.2

X15       5.58           [C.sub.7][H.sub.6][O.sub.2]           4.3

X16       5.64           [C.sub.9][H.sub.8][O.sub.4]           0.9

X17       5.99           [C.sub.8][H.sub.8][O.sub.4]            3

X18 (b)   7.31         [C.sub.19][H.sub.28][O.sub.12]          0.4

X19       7.68     [C.sub.17][H.sub.19][N.sub.5][O.sub.5]     -1.6

X20       7.73           [C.sub.8][H.sub.8][O.sub.3]           0.5

X21       8.62          [C.sub.17][H.sub.20][O.sub.9]         -0.7

X22       8.94          [C.sub.9][H.sub.10][O.sub.3]          -0.8

X23 (b)   9.11         [C.sub.32][H.sub.40][O.sub.19]          4.9

X24       9.78         [C.sub.33][H.sub.42][O.sub.20]           4

X25       10.00         [C.sub.10][H.sub.10][O.sub.4]          0.7

X26       10.02        [C.sub.21][H.sub.28][O.sub.13]           0

X27       10.05        [C.sub.32][H.sub.40][O.sub.19]          4.9

X28       10.71        [C.sub.14][H.sub.14][O.sub.3]S         -2.2

X29       11.74         [C.sub.11][H.sub.12][O.sub.5]         -2.2

X30       12.20          [C.sub.7][H.sub.6][O.sub.3]           2.5

X31       13.21        [C.sub.45][H.sub.56][O.sub.25]          4.7

X32       13.56         [C.sub.20][H.sub.24][O.sub.8]          0.2

X33       13.69        [C.sub.26][H.sub.30][O.sub.14]          2.9

X34       14.13        [C.sub.25][H.sub.24][O.sub.12]          2.6

X35       16.48        [C.sub.25][H.sub.24][O.sub.12]          2.6

X36       17.42         [C.sub.9][H.sub.12][O.sub.2]          -0.8

X37       23.89        [C.sub.14][H.sub.14][O.sub.2]S          -4

X38       32.90          [C.sub.9][H.sub.8][O.sub.3]           -1

X39       39.73         [C.sub.16][H.sub.22][O.sub.4]         - 0.2

Y1 (a,b)  1.05           [C.sub.5][H.sub.5][N.sub.5]          -1.8

Y2        1.08     [C.sub.10][H.sub.13][N.sub.5][O.sub.4]      1.5

Y3 (b)    1.09          [C.sub.9][H.sub.11]N[O.sub.3]         -1.9

Y4        8.60     [C.sub.24][H.sub.29][N.sub.3][O.sub.8]S    -0.5

Y5        9.77          [C.sub.10][H.sub.8][O.sub.4]          -0.8

Y6        10.55         [C.sub.12][H.sub.18][O.sub.5]         -0.1

Y7        10.55         [C.sub.12][H.sub.16][O.sub.4]         -0.5

Y8        11.94         [C.sub.12][H.sub.16][O.sub.5]         -0.8

Y9        13.27         [C.sub.12][H.sub.16][O.sub.3]          0.1

Y10       13.27         [C.sub.12][H.sub.18][O.sub.4]          0.4

Y11       16.56         [C.sub.12][H.sub.14][O.sub.3]         -0.6

Y12       16.57         [C.sub.12][H.sub.16][O.sub.4]         -0.9

Y13       17.87         [C.sub.12][H.sub.14][O.sub.4]         -0.2

Y14       18.08         [C.sub.12][H.sub.14][O.sub.3]         -0.1

Y15       19.53         [C.sub.18][H.sub.28][O.sub.8]          0.3

Y16       21.97         [C.sub.12][H.sub.12][O.sub.2]         -0.5

Y17       23.67         [C.sub.12][H.sub.14][O.sub.4]          0.3

Y18       24.29         [C.sub.12][H.sub.12][O.sub.2]           0

Y19       24.50         [C.sub.12][H.sub.14][O.sub.2]         -0.9

Y20       26.27        [C.sub.12][H.sub.15]Cl[O.sub.3]        -0.5

Y21       26.63         [C.sub.12][H.sub.12][O.sub.3]          0.6

Y22       27.74         [C.sub.13][H.sub.16][O.sub.3]         -0.6

Y23       27.90         [C.sub.12][H.sub.12][O.sub.3]          0.6

Y24       28.65         [C.sub.12][H.sub.16][O.sub.2]          0.7

Y25       29.49         [C.sub.12][H.sub.14][O.sub.2]          0.3

Y26       32.06         [C.sub.12][H.sub.12][O.sub.2]         -0.5

Y27 (b)   32.89         [C.sub.12][H.sub.18][O.sub.2]         -0.8

Y28       33.04         [C.sub.12][H.sub.14][O.sub.2]          0.3

Y29       33.64         [C.sub.12][H.sub.12][O.sub.2]         -0.2

Y30       35.24         [C.sub.12][H.sub.20][O.sub.2]         -0.7

Y31       36.15         [C.sub.21][H.sub.32][O.sub.2]          0.9

Y32       36.37         [C.sub.24][H.sub.28][O.sub.5]          0.1

Y33       38.22         [C.sub.24][H.sub.28][O.sub.5]          0.4

Y34       39.08         [C.sub.24][H.sub.32][O.sub.5]         -0.4

Y35       39.48         [C.sub.17][H.sub.24][O.sub.4]          0.8

Y36       41.42         [C.sub.24][H.sub.30][O.sub.4]         -0.1

Y37       41.52         [C.sub.24][H.sub.32][O.sub.4]          0.1

Y38       41.88         [C.sub.24][H.sub.28][O.sub.4]          0.1

Peak      [[M-H].sup.-]    (m/z)     Intensity       Product ions
           Calculated     Measure

X1 (a)      191.0561      191.0559     86129     154.9973, 127.0393,
                                                  111.0091, 93.0355,
                                                   85.0302, 73.0302

X2          191.0197      191.0199    421357      111.0096, 87.0103

X3 (b)      243.0623      243.0620     5161               nd

X4 (b)      128.0353      128.0357     24352              nd

X5 (b)      141.0193      141.0195     3248               nd

X6          282.0844      282.0851     1153      150.0412, 133.0158,
                                                       108.0286

X7          285.0980      285.0977     1505      223.0248, 123.0432,
                                                       105.0348

X8          153.0193      153.0195     16114      109.0300, 91.0193,
                                                       65.0057

X9          137.0244      137.0248     31464     119.0140, 108.0215,
                                                  93.0350, 81.0366,
                                                       65.0414

X10         459.1144      459.1152    283440     423.0939, 397.1138,
                                                  173.0088, 61.0444,
                                                  129.0184, 111.0088

X11         353.0878      353.0881    109164     191.0559, 173.0444,
                                                 161.0236, 134.0387,
                                                  111.0458, 93.0350,
                                                       85.0301

X12         412.1184      412.1186     11210     306.0753, 272.0898,
                                                 179.0459, 160.0077,
                                                 143.0461, 128.0348,
                                                       99.0559

X13         574.1712      574.1726     7677      484.1388, 378.0974,
                                                 306.0756, 272.0874,
                                                  210.0864, 143.0464

X14         181.0506      181.0506     2573      136.9210, 121.0296,
                                                       109.0279

X15         121.0295      121.0302     50808       92.0266, 77.0403

X16         179.0350      179.0352     80360     134.0371, 108.0214,
                                                  93.0345, 89.0401,
                                                       79.0555

X17         167.0350      167.0355    275549      123.0454, 79.0570

X18 (b)     447.1508      447.1510     14270     269.1028, 233.0656,
                                                 161.0449, 101.0242,
                                                       71.0153

X19         372.1313      372.1307     2202      336.1054, 266.0867,
                                                       134.0467

X20         151.0401      151.0402     7660      136.0155, 108.0219,
                                                       92.0273

X21         367.1035      367.1032     21071     193.0496, 191.0553,
                                                 173.0455, 134.0369,
                                                  111.0463, 93.0349,
                                                       85.0299

X22         165.0557      165.0556     2085       119.0515, 103.0604

X23 (b)     727.2091      727.2151    122722     459.1156, 441.1045,
                                                 423.0937, 397.1142,
                                                  369.1188, 217.0496

X24         757.2197      757.2227      659      503.1631, 453.0961,
                                                       161.0480

X25         193.0506      193.0508     97733     178.0265, 149.0591,
                                                       134.0372

X26         487.1457      487.1457     4684      441.0958, 423.0934,
                                                 397.1169, 161.0430,
                                                       111.0091

X27         727.2091      727.2127    122722     459.1156, 441.1045,
                                                 423.0937, 397.1142,
                                                  369.1188, 161.0453

X28         261.0591      261.0585      777      205.8269, 167.8694,
                                                       137.0057

X29         223.0612      223.0607     2431       108.0226, 179.0713

X30         137.0244      137.0248     31464      108.0215, 93.0352,
                                                       65.0410

X31         995.3038      995.3107     39727     727.2129, 441.1065,
                                                 423.0915, 397.1119,
                                                       161.0457

X32         391.1398      391.1399     6858      229.0860, 123.0452,
                                                 121.0288, 107.0511,
                                                   93.0357, 71.0265

X33         565.1563      565.1579      811       177.0188, 111.0153
                                                 353.0879, 335.0781,

X34         515.1195      515.1208     91406     191.0558, 179.0345,
                                                  173.0450, 161.0240

X35         515.1195      515.1208     91406     353.0879, 335.0781,
                                                 191.0558, 179.0345,
                                                  173.0450, 161.0240

X36         151.0765      151.0763     1891      133.0677, 107.0520,
                                                   94.0274, 77.0394

X37         245.0642      245.0632     2498            139.0217

X38         163.0401      163.0399     4130      145.0279, 135.0455,
                                                  119.0493, 91.0184,

X39         277.1445      277.1445     5380        77.0443, 65.0039
                                                  233.1544, 206.8262

Y1 (a,b)    136.0618      136.0615     46698              nd

Y2          268.1040      268.1044    174357     136.0623, 119.0360,

Y3 (b)      182.0812      182.0808     34675              nd

Y4          520.1748      520.1746     7896      308.0836, 285.0913,
                                                 233.0591, 179.0486,
                                                  162.0208, 107.0485

Y5          193.0495      193.0494     14081     178.0257, 150.0323,
                                                  133.0281, 122.0361

Y6          243.1227      243.1227     13011     165.0909, 151.0381,
                                                       137.0949

Y7          225.1121      225.1119     21174     207.1023, 165.0557,
                                                  151.0376, 137.0954

Y8          241.1071      241.1069     1918      150.0677, 107.0497,
                                                       71.0498

Y9          209.1172      209.1172    288915     153.0544, 149.0593,
                                                 135.0473, 121.1006,
                                                       117.0709

Y10         227.1278      227.1279    102387     163.1104, 153.0543,
                                                 149.0961, 119.0860,
                                                   107.0484, 79.054

Y11         207.1016      207.1015    1327567    189.0917, 165.0550,
                                                 146.0732, 133.0653,
                                                  119.0841, 105.0693

Y12         225.1121      225.1119     39845     165.0533, 133.0658,
                                                  128.0619, 91.0532,
                                                       77.0402

Y13         223.0965      223.0966     23855     177.0921, 149.0591,
                                                 121.0308, 103.0523,
                                                       77.0401

Y14         207.1016      207.1015    425060     189.0901, 165.0558,
                                                 161.0948, 128.0620,
                                                  105.0701, 91.0549,
                                                       77.0393

Y15         373.1857      373.1858     45450     211.1335, 193.1228,
                                                 147.1172, 105.0702,
                                                       79.0577

Y16         189.0910      189.0910    151946     128.0623, 115.0544,
                                                  105.0702, 91.0551,
                                                       77.0388

Y17         223.0965      223.0966     43428     177.0899, 167.0387,
                                                 149.0227, 121.0278,
                                                   91.0541, 77.0382

Y18         189.0910      189.0910    305525     152.0611, 128.0618,
                                                 115.0529, 105.0341,
                                                   91.0537, 77.0394

Y19         191.1067      191.1067    340079      117.0688, 91.0560,
                                                       77.0396

Y20         243.0783      243.0781     21244     207.1020, 189.0907,
                                                 161.0364, 133.0640,
                                                  119.0840, 91.0533,

Y21         205.0859      205.0860     90627     187.0745, 168.0574,
                                                 144.0573, 131.0493,
                                                 115.0541, 103.0552,
                                                   91.0533, 77.0401

Y22         221.1172      221.1171     4804       175.1090, 105.0691

Y23         205.0859      205.0860     90627     187.0745, 168.0574,
                                                 144.0573, 131.0493,
                                                 115.0541, 103.0552,
                                                   91.0533, 77.0401

Y24         193.1223      193.1225    2273337    147.1167, 137.0590,
                                                 119.0498, 105.0699,
                                                  91.0544, 77.0389,
                                                       65.0383

Y25         191.1067      191.1067    3489472    153.0704, 145.1007,
                                                 135.0440, 117.0695,
                                                   91.0546, 77.0391

Y26         189.0910      189.0910     39396     152.0611, 128.0615,
                                                  115.0545, 91.0554

Y27 (b)     195.1380      195.1378    686098     177.1344, 149.1309,
                                                       79.0550

                                                 173.0961, 145.1008,
Y28         191.1067      191.1067    637725     130.0773, 117.0694,
                                                   91.0548, 77.0392

Y29         189.0910      189.0910    401466     152.0627, 128.0622,
                                                  115.0540, 91.0550

                                                  119.0862, 95.0856,
Y30         197.1536      197.1535     21102      91.0555, 81.0697,
                                                       67.0552,

Y31         317.2475      317.2478     12477     281.2253, 211.1524,
                                                 187.1501, 159.1179,
                                                 149.1329, 145.0997,
                                                  117.0717, 81.0720

Y32         397.2010      397.2010     71592     191.1064, 173.0954,
                                                 155.0852, 145.1003,
                                                 128.0625, 117.0715,
                                                  105.0724, 91.0544
                                                 361.1802, 333.1842,
                                                 307.1708, 291.1401,

Y33         397.2010      397.2010     66600     279.1403, 261.1264,2
                                                  17.0999, 191.1070,
                                                  173.0972, 91.0553

Y34         401.2323      401.2321     20533     383.2246, 191.1061,
                                                 163.1084, 149.0594,
                                                  145.1037, 135.0439

Y35         293.1747      293.1750     22471     175.1115, 151.0754,
                                                 137.0605, 111.0415,
                                                   91.0547, 83.0488

Y36         383.2217      383.2217    1160225    191.1068, 173.0958,
                                                 163.1120, 149.0601,
                                                  135.0440, 91.0546,
                                                       79.0566

Y37         385.2373      385.2374    627373     367.2247, 349.2092,
                                                 321.2178, 293.1915,
                                                      193.1229,
                                                  175.1113147.1159,
                                                 137.0587, 119.0871,
                                                       93.0687

Y38         381.2060      381.2061    4796897    191.1070, 173.0955,
                                                 163.1126, 149.0596,
                                                  135.0437, 91.0551,
                                                       79.0549

Peak           Identification          Structure     Ref.
                                         class

X1 (a)          Quinic acid          Organic acids   [10]

X2              Citric acid             Organic      [10]
                                         acids

X3 (b)            Uridine              Nitrogen-
                                      containing
                                       compounds

X4 (b)       Pyroglutamic acid          Organic
                                         acids

X5 (b)       5-(Hydroxymethyl)          Organic      [11]
                furoic acid              acids

X6               Guanosine             Nitrogen-
                                      containing
                                       compounds

X7               Gastrodin            Glycosides     [12]

X8          Protocatechuic acid         Organic      [13]
                                         acids

X9             3,4-Dihydroxy            Phenols      [14]
                benzaldehyde

X10           Parishin E or G         Glycosides     [12]

X11        4-Caffeoylquinic acid        Organic      [15]
                                         acids

X12         S-(4-Hydroxybenzyl)-       Nitrogen-     [12]
                glutathione           containing
                                       compounds

X13         S-(4-Hydroxybenzyl)-       Nitrogen-
           glutathione glucoside      containing
                                       compounds

X14         4-(2-Hydroxyethoxy)         Organic
                benzoic acid             acids

X15                  P-                 Phenols      [14]
            Hydroxybenzaldehyde

X16             Caffeic acid            Organic      [13]
                                         acids

X17            Vanillic acid            Organic      [12]
                                         acids

X18 (b)         Gastrodin A           Glycosides     [12]

X19           p-Hydroxybenzyl          Nitrogen-
                 adenosine            containing
                                       compounds

X20              Vanilline              Phenols

X21        3-Feruloylquinic acid        Organic      [16]
                                         acids

X22       L-(-)-Phenyllactic acid       Organic
                                         acids

X23 (b)          Parishin B           Glycosides     [13]

X24           Parishin H or M         Glycosides     [12]

X25             Ferulic acid            Organic      [13]
                                         acids

X26           Parishin O or N         Glycosides     [12]

X27              Parishin C           Glycosides     [12]

X28         4,4'-Dihydroxybenzyl        Phenols      [14]
                 sulfoxide

X29             Sinapic acid            Organic      [13]
                                         acids

X30           p-Hydroxybenzoic          Phenols      [14]
                    acid

X31               Parishin            Glycosides     [12]

X32               Bis-(4-             Glycosides     [14]
              hydroxybenzyl)-
            ether-mono-[beta]-D-
              glucopyranoside

X33              Parishin W           Glycosides     [12]

X34         3,4-Dicaffeoylquinic        Organic      [15]
                acid isomer              acids

X35         3,4-Dicaffeoylquinic        Organic
                acid isomer              acids       [15]

X36           4-(Ethoxymethyl)          Phenols      [14]
                   phenol

X37         Bis(4-hydroxybenzyl)        Phenols      [14]
                  sulfide

X38          p-Hydroxycinnamic          Organic
                    acid                 acids

X39          Dibutyl phthalate          Phenols

Y1 (a,b)          Adenine              Nitrogen-
                                      containing
                                       compounds

Y2               Adenosine             Nitrogen-
                                      containing
                                       compounds

Y3 (b)            Tyrosin              Nitrogen-
                                      containing
                                       compounds

Y4             (2)-g-L-[N-(4-          Nitrogen-
              Hydroxy benzyl)]        containing
             glutamyl-L-[s-(4-         compounds
              hydroxybenzyl)]
              cysteinylglycine

Y5            3-Carboxyethyl-           Organic      [16]
                 phthalide               acids

Y6           3-Butyl-3-hydroxy-         Organic
            4,5,6,7-tetrahydro-          acids
               6,7-dihydroxy
                 phthalide

Y7          Senkyunolide H or I       Phthalides     [9]
          or ligustilidiol or cis-
               6,7-dihydroxy-
                ligustilide

Y8          Senkyunolide R or S       Phthalides

Y9          Senkyunolide G or K       Phthalides     [9]
                 or Z-6,7-
              epoxyligustilide

Y10        Senkyunolide J or N or     Phthalides     [9]
                     R2

Y11           3-Butylidene-6-         Phthalides     [9]
             hydroxy-5,6-dihy-
              drophthalide or
             senkyunolide F or
                chuanxiongol

Y12         Senkyunolide H or I       Phthalides     [9]
          or ligustilidiol or cis-
               6,7-dihydroxy-
                ligustilide

Y13          Senkyunolide D or        Phthalides     [9]
              4,7-dihydroxy-3-
               butylphthalide

Y14           3-Butylidene-6-         Phthalides     [9]
             hydroxy-5,6-dihy-
              drophthalide or
             senkyunolide F or
                chuanxiongol

Y15           Ligusticoside A         Glycosides     [17]

Y16         Butylidenephthalide       Phthalides     [9]
                   isomer

Y17          Senkyunolide D or        Phthalides     [9]
              4,7-dihydroxy-3-
               butylphthalide

Y18         Butylidenephthalide       Phthalides     [9]
                   isomer

Y19        3-Butylphthalide or Z-     Phthalides     [9]
             ligustilide or E-
                ligustilide

Y20            Senkyunolide L         Phthalides     [18]

Y21         Senkyunolide B or C       Phthalides     [9]

Y22           2-(1-Oxopentyl)-          Organic
                  benzoic                acids

Y23         Senkyunolide B or C       Phthalides     [9]

Y24            Senkyunolide A         Phthalides     [9]

Y25        3-Butylphthalide or Z-     Phthalides     [9]
             ligustilide or E-
                ligustilide

Y26         Butylidenephthalide       Phthalides     [9]
                   isomer

Y27 (b)         Cnidilide or          Phthalides     [9]
                 neocnilide

           3-Butylphthalide or Z-     Phthalides     [9]
Y28          ligustilide or E-
                ligustilide

Y29         Butylidenephthalide       Phthalides     [9]
                   isomer

              3,7-Dimethyl-3-           Others
Y30        acetate-1,6-octadiene-
                3-ol acetate

Y31             Pregnenolone            Others

Y32          Chuanxiongnolide A         Others       [19]
                    or B

Y33          Chuanxiongnolide A         Others       [19]
                    or B

Y34         Chuanxiongdiolide R2        Others       [20]
            or chuanxiongdiolide
                     B

Y35         Senkyunolide M or Q       Phthalides     [9]

Y36        Senkyunolide P or 3,8-     Phthalides     [9]
           dihydro-diligustilide
              or angelicide or
              Z,Z'-3,3' -8,8'-
               diligustilide

Y37         Chuanxiongdiolide A         Others       [20]

Y38           Levistolide A or        Phthalides     [9]
             senkyunolide O or
              tokinolide B or
               riligustilide

(a) "X" in negative ion mode and "Y" in negative-positive mode. (b)
Compared with reference standards.

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

No.       Parent compounds         Metabolic pathways

1         4,7-Dihydroxy-3-             Prototype
          butylphthalide,
           senkyunolide D

2         4,7-Dihydroxy-3-        Hydroxyl and methyl
          butylphthalide,             conjugation
           senkyunolide D

3         4,7-Dihydroxy-3-        Sulfate conjugation
          butylphthalide,
           senkyunolide D

4         4,7-Dihydroxy-3-            Glucuronide
          butylphthalide,             conjugation
           senkyunolide D

5         4,7-Dihydroxy-3-         Hydrogenation and
          butylphthalide,             glucuronide
           senkyunolide D             conjugation

6         4,7-Dihydroxy-3-            Hydroxyl and
          butylphthalide,             glucuronide
           senkyunolide D             conjugation

7        3-Butyl-3-hydroxy-            Prototype
      4,5,6,7-tetrahy-dro-6,7-
         dihydroxyphthalide

8        3-Butyl-3-hydroxy-        Methyl conjugation
      4,5,6,7-tetrahy-dro-6,7-
         dihydroxyphthalide

9        3-Butyl-3-hydroxy-       Cystein conjugation
      4,5,6,7-tetrahy-dro-6,7-
         dihydroxyphthalide

10       3-Butyl-3-hydroxy-      Oxidation and cystein
      4,5,6,7-tetrahy-dro-6,7-        conjugation
         dihydroxyphthalide

11       3-Butyl-3-hydroxy-       N-Acetyl-L-cysteine
      4,5,6,7-tetrahy-dro-6,7-        conjugation
         dihydroxyphthalide

12       3-Butyl-3-hydroxy-         Desat and S-GSH
      4,5,6,7-tetrahy-dro-6,7-        conjugation
         dihydroxyphthalide

13       3-Butyl-3-hydroxy-      [H.sub.2]O conjugation
      4,5,6,7-tetrahy-dro-6,7-
         dihydroxyphthalide

14        3-Carboxyethyl-              Prototype
             phthalide

15        3-Carboxyethyl-          Methyl conjugation
             phthalide

16        3-Carboxyethyl-             Glucuronide
             phthalide                conjugation

17        3-Carboxyethyl-             Hydroxyl and
             phthalide                glucuronide
                                      conjugation

18        3-Carboxyethyl-            Hydrogenation
             phthalide

19     Cnidilide, neocnilide           Prototype

20     Cnidilide, neocnilide       Methyl conjugation

21     Cnidilide, neocnilide       Acetyl conjugation

22     Cnidilide, neocnilide      Hydroxyl and acetyl
                                      conjugation

23     Cnidilide, neocnilide     Oxidation and cystein
                                      conjugation
                                      Hydroxyl and

24     Cnidilide, neocnilide          glucuronide
                                      conjugation

25     Cnidilide, neocnilide         2Hydroxyl and
                                      glucuronide
                                      conjugation

26     Cnidilide, neocnilide           2Hydroxyl
                                      conjugation

27         Senkyunolide A              Prototype

28         Senkyunolide A         Hydroxyl and methyl
                                      conjugation

29         Senkyunolide A             Hydroxyl and
                                     acetylcysteine
                                      conjugation

30         Senkyunolide A             Carboxyl and
                                      glucuronide
                                      conjugation

31         Senkyunolide A            2Hydroxyl and
                                      glucuronide
                                      conjugation

32         Senkyunolide A        [H.sub.2]O conjugation

33         Senkyunolide A              2Hydroxyl
                                      conjugation

34        Senkyunolide G,              Prototype
       senkyunolide K,Z-6,7-
          epoxyligustilide

35        Senkyunolide G,          Methyl conjugation
       senkyunolide K,Z-6,7-
          epoxyligustilide

36        Senkyunolide G,          Acetyl conjugation
       senkyunolide K,Z-6,7-
          epoxyligustilide

37        Senkyunolide G,         Hydroxyl and acetyl
       senkyunolide K,Z-6,7-          conjugation
          epoxyligustilide

38        Senkyunolide G,         Taurine conjugation
       senkyunolide K,Z-6,7-
          epoxyligustilide

39        Senkyunolide G,         N-Acetyl-L-cysteine
       senkyunolide K,Z-6,7-          conjugation
          epoxyligustilide

40        Senkyunolide G,          Hydrogenation and
       senkyunolide K,Z-6,7-          glucuronide
          epoxyligustilide            conjugation

41        Senkyunolide G,             Hydroxyl and
       senkyunolide K,Z-6,7-         acetylcysteine
          epoxyligustilide            conjugation

42        Senkyunolide G,             Hydroxyl and
       senkyunolide K,Z-6,7-          glucuronide
          epoxyligustilide            conjugation

43        Senkyunolide G,             Carboxyl and
       senkyunolide K,Z-6,7-          glucuronide
          epoxyligustilide            conjugation

44        Senkyunolide G,           Desat and S-GSH
       senkyunolide K,Z-6,7-          conjugation
          epoxyligustilide

45        Senkyunolide G,          S-GSH conjugation
       senkyunolide K,Z-6,7-
          epoxyligustilide

46        Senkyunolide G,            Hydrogenation
       senkyunolide K,Z-6,7-
          epoxyligustilide

47        Senkyunolide G,             Hydroxyl and
       senkyunolide K,Z-6,7-      demethyl conjugation
          epoxyligustilide

48        Senkyunolide G,        [H.sub.2]O conjugation
       senkyunolide K,Z-6,7-
          epoxyligustilide

49        Senkyunolide G,             Demethyl and
       senkyunolide K,Z-6,7-      carboxyl conjugation
          epoxyligustilide

50        Senkyunolide G,          2hydrogenation and
       senkyunolide K,Z-6,7-           2hydroxyl
          epoxyligustilide            conjugation

51        Senkyunolide G,               Aromatic
       senkyunolide K,Z-6,7-          hydrocarbon
          epoxyligustilide             oxidation

52      Senkyunolide J,N,R2            Prototype

53      Senkyunolide J,N,R2        Methyl conjugation

54      Senkyunolide J,N,R2       Hydroxyl and methyl
                                      conjugation

55      Senkyunolide J,N,R2       Glycine conjugation

56      Senkyunolide J,N,R2       Cystein conjugation

57      Senkyunolide J,N,R2       Taurine conjugation

58      Senkyunolide J,N,R2      Oxidation and cystein
                                      conjugation

59      Senkyunolide J,N,R2       N-Acetyl-L-cysteine
                                      conjugation

60      Senkyunolide J,N,R2           Glucuronide
                                      conjugation

61      Senkyunolide J,N,R2        S-GSH conjugation

No.         Formula           tR     [[M+H].sup.+]
                             (min)    Calculated

1     [C.sub.12][H.sub.14]   9.75      223.0965
           [O.sub.4]

2     [C.sub.13][H.sub.16]   10.78     253.1071
           [O.sub.5]

3     [C.sub.12][H.sub.14]    9.2      303.0533
           S[O.sub.7]

4     [C.sub.18][H.sub.22]   10.6      399.1286
           [O.sub.10]

5     [C.sub.18][H.sub.24]   8.67      401.1442
           [O.sub.10]

6     [C.sub.18][H.sub.22]   8.92      415.1235
           [O.sub.11]

7     [C.sub.12][H.sub.18]   7.76      243.1227
           [O.sub.5]

8     [C.sub.13][H.sub.20]   10.01     257.1384
           [O.sub.5]

9     [C.sub.15][H.sub.23]   7.67      346.1319
          [O.sub.6]NS

10    [C.sub.15][H.sub.23]   9.47      362.1268
          [O.sub.7]NS

11    [C.sub.17][H.sub.25]   5.97      404.1374
          [O.sub.8]NS

12    [C.sub.22][H.sub.33]   4.39      548.1909
      [O.sub.11][N.sub.3S]

13    [C.sub.12][H.sub.20]   4.65      261.1333
           [O.sub.6]

14    [C.sub.10][H.sub.8]    7.28      193.0495
           [O.sub.4]

15    [C.sub.11][H.sub.10]   11.52     207.0652
           [O.sub.4]

16    [C.sub.16][H.sub.16]   5.51      369.0816
           [O.sub.10]

17    [C.sub.16][H.sub.16]   5.72      385.0765
           [O.sub.11]

18    [C.sub.10][H.sub.10]   7.64      195.0652
           [O.sub.4]

19    [C.sub.12][H.sub.18]   16.14     195.1380
           [O.sub.2]

20    [C.sub.13][H.sub.20]   15.63     209.1536
           [O.sub.2]

21    [C.sub.14][H.sub.20]   12.45     237.1485
           [O.sub.3]

22    [C.sub.14][H.sub.20]   12.46     253.1434
           [O.sub.4]

23    [C.sub.15][H.sub.23]   10.07     314.1421
          [O.sub.4]NS

24    [C.sub.18][H.sub.26]   9.62      387.1650
           [O.sub.9]

25    [C.sub.18][H.sub.26]   8.02      403.1599
           [O.sub.10]

26    [C.sub.12][H.sub.18]   8.68      227.1278
           [O.sub.4]

27    [C.sub.12][H.sub.16]   14.81     193.1223
           [O.sub.2]

28    [C.sub.13][H.sub.18]   15.85     223.1329
           [O.sub.3]

29    [C.sub.17][H.sub.25]   10.35     372.1475
          [O.sub.6]NS

30    [C.sub.18][H.sub.22]   10.6      399.1286
           [O.sub.10]

31    [C.sub.18][H.sub.24]   8.67      401.1442
           [O.sub.10]

32    [C.sub.12][H.sub.18]   10.54     211.1329
           [O.sub.3]

33    [C.sub.12][H.sub.16]   9.56      225.1121
           [O.sub.4]

34    [C.sub.12][H.sub.16]   12.79     209.1172
           [O.sub.3]

35    [C.sub.13][H.sub.18]   15.85     223.1329
           [O.sub.3]

36    [C.sub.14][H.sub.18]   15.99     251.1278
           [O.sub.4]

37    [C.sub.14][H.sub.18]   12.27     267.1227
           [O.sub.5]

38    [C.sub.15][H.sub.21]   9.02      328.1213
          [O.sub.5]NS

39    [C.sub.17][H.sub.23]   11.15     370.1319
          [O.sub.6]NS

40    [C.sub.18][H.sub.26]   9.62      387.1650
           [O.sub.9]

41    [C.sub.17][H.sub.25]   8.29      388.1425
          [O.sub.7]NS

42    [C.sub.18][H.sub.24]   8.67      401.1442
           [O.sub.10]

43    [C.sub.18][H.sub.22]   8.92      415.1235
           [O.sub.11]

44    [C.sub.22][H.sub.31]   9.17      514.1854
      [O.sub.9][N.sub.3]S

45    [C.sub.22][H.sub.33]   8.61      516.2010
      [O.sub.9][N.sub.3]S

46    [C.sub.12][H.sub.18]   10.54     211.1329
           [O.sub.3]

47    [C.sub.12][H.sub.14]   9.75      223.0965
           [O.sub.4]

48    [C.sub.12][H.sub.18]   8.68      227.1278
           [O.sub.4]

49    [C.sub.12][H.sub.14]   9.79      239.0914
           [O.sub.5]

50    [C.sub.12][H.sub.18]   7.76      243.1227
           [O.sub.5]

51    [C.sub.13][H.sub.18]   11.38     255.1227
           [O.sub.5]

52    [C.sub.12][H.sub.18]   8.68      227.1278
           [O.sub.4]

53    [C.sub.13][H.sub.20]   11.42     241.1434
           [O.sub.4]

54    [C.sub.13][H.sub.20]   10.01     257.1384
           [O.sub.5]

55    [C.sub.14][H.sub.21]   7.49      284.1493
           [O.sub.5]N

56    [C.sub.15][H.sub.23]   8.48      330.1370
          [O.sub.5]NS

57    [C.sub.14][H.sub.23]   7.56      334.1319
          [O.sub.6]NS

58    [C.sub.15][H.sub.23]   7.67      346.1319
          [O.sub.6]NS

59    [C.sub.17][H.sub.95]   8.29      388.1425
          [O.sub.7]NS

60    [C.sub.18][H.sub.26]   8.02      403.1599
           [O.sub.10]

61    [C.sub.22][H.sub.35]   4.96      534.2116
      [O.sub.10][N.sub.3]S

No.    (m/z)     Error          Product ions
      Measure    (ppm)

1     223.0963    -1        177.0899, 167.0387,
                            149.0226, 121.0278,
                                  91.0541

2     253.1071    0.2       235.0963, 221.0829,
                            202.0596, 193.0489,
                            179.0332, 175.0379,
                             150.0301, 121.0268

3     303.0535    0.5       285.0430, 205.0858,
                            187.0753, 177.0904,
                            149.0244, 121.0283,
                                  91.0534

4     399.1285   -0.3       223.0969, 205.0847,
                            177.0883, 167.0331,
                             149.0233, 121.0315

5     401.1444    0.3       225.4427, 207.1017,
                            189.0924, 172.0884,
                            165.0548, 141.0170,
                             119.0851, 113.0288

6     415.1238    0.7       227.0584, 221.0824,
                                  167.0331

7     243.1225   -0.7       165.0909, 151.0414,
                            137.0951, 123.0431,
                             107.0499, 91.0546,
                                  85.0648

8     257.1381    -1        221.1211, 207.0993,
                            165.0913, 137.0951,
                                  123.0434

9     346.1319     0        328.1222, 310.1111,
                            264.1056, 238.0916,
                            223.0771, 207.1018,
                             165.0923, 137.0955

10    362.1267   -0.3       327.0911, 247.1337,
                             229.1216, 151.0746

11    404.1372   -0.5        205.0833, 171.1364

12    548.1921    2.3       473.1636, 419.1465,
                                  205.0860

13    261.1320   -4.6             261.1310

14    193.0490   -2.7       178.0257, 150.0323,
                            133.0277, 122.0361,

15    207.0646    -3         105.0338, 77.0388
                            147.0441, 131.0502,
                             103.0546, 91.0533

16    369.0822    1.5             193.0493

17    385.0758   -2.1             209.0455

18    195.0651   -0.6       177.0547, 149.0609,
                            145.0276, 134.0354,
                             117.0309, 89.0395

19    195.1377   -1.5       177.1350, 149.1348,
                            125.0599, 107.0873,
                         97.0640, 91.0550, 79.0543

20    209.1537    0.4       193.0211, 167.1088,
                            153.0917, 121.0648,
                                  68.9961

21    237.1483   -1.1             177.0257

22    253.1435    0.4       235.1340, 193.0856,
                             157.1012, 135.0816

23    314.1424    1.1        268.1343, 193.1185
                            211.1330, 193.1223,

24    387.1650    0.1       175.1129, 147.1168,
                                  121.0368

25    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    227.1279    0.7       191.1057, 163.1115,
                            153.0554, 145.1001,
                            135.0444, 105.0705,
                                  91.0541

27    193.1222   -0.5       175.1118, 147.1170,
                            137.0580, 119.0848,
                             105.0710, 91.0556,
                                  77.0393

28    223.1326   -1.2       205.1224, 191.1060,
                            149.0235, 135.0429,
                            121.0279, 105.0697,
                              91.0542, 77.0397

29    372.1475   -0.1       330.1375, 284.1322,
                             267.1048.239.0756,
                            209.1169, 191.1074,
                            162.0210, 153.0540,
                                  130.0492

30    399.1285   -0.3       223.0969, 205.0847,
                            177.0883, 159.0293,
                            149 0233, 131 0840,
                                  85.0275

31    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    211.1327   -0.9       193.1225, 175.1096,
                            147.1156, 129.0700,
                             105.0693, 93.0692,
                                  79.0548

33    225.1119    -1        207.1013, 189.0914,
                            165.0537, 133.0637,
                             105.0706, 91.0536,
                                  81.0713

34    209.1173    0.3       153.0686, 149.0594,
                            145.0984, 135.0472,
                             105.0693, 91.0562,
                                  77.0409

35    223.1326   -1.2       191.1060, 173.0946,
                            149.0235, 145.1014,
                            135.0429, 105.0697,
                              91.0542, 79.0551

36    251.1276   -0.6       177.1261, 149.0593,
                              69.0014, 57.0752

37    267.1226   -0.4       249.1137, 193.0479,
                            189.0582, 135.0435,
                                  119.0846

38    328.1214    0.1       207.1015, 189.0911,
                            165.0541, 161.0955,
                            147.0814, 133.0644,
                             119.0859, 91.0538

39    370.1320    0.3       282.1162, 264.1083,
                            207.1015, 189.0911,
                            165.0544, 147.0785,
                            133.0652, 119.0858,
                                  91.0546

40    387.1650    0.1       211.1330, 193.1223,
                            175.1129, 147.1168,
                             121.0638, 91.0546,
                                  79.0562

41    388.1422   -0.6       207.1010, 164.0390,
                            122.0273, 105.0347,
                                  79.0529

42    401.1444    0.3       267.1015, 225.1127,
                            207.1017, 189.0924,
                            172.0844, 141.0170,
                             113.0228, 85.0265,
                                  73.0295

43    415.1238    0.7        227.0584, 221.0824

44    514.1854     0        439.1552, 385.1429,
                            282.1160, 207.1017,
                            189.0921, 179.0484,
                            162.0221, 144.0103,
                             116.0174, 76.0218

45    516.2010   -0.1       441.1694, 387.1593,
                            284.1315, 209.1173,
                            191.1055, 162.0212,
                            144.0109, 116.0175,
                                  84.0447

46    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    223.0963    -1        177.0906, 149.0590,
                            145.0980, 105.0329,
                              91.0556, 77.0394

48    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    239.0915    0.4       221.0816, 193.0885,
                            179.0336, 165.0173,
                            161.0227, 128.0633,
                             109.0292, 77.0376

50    243.1225   -0.7       225.1135, 207.1017,
                            179.1084, 165.0909,
                            151.0414, 137.0951,
                             123.0431, 95.0486

51    255.1222   -2.1       195.1024, 135.0798,
                                  131.0870

52    227.1279    0.7       163.1115, 153.0554,
                            145.1001, 107.0705,
                         91.0541, 79.0544, 65.0402,
                                  55.0198

53    241.1433   -0.6       209.1147, 191.1067,
                            163.1089, 153.0559,
                             121.0652, 93.0717,
                         85.0663, 79.0663, 57.0698

54    257.1381    -1        221.1211, 207.0993,
                            165.0913, 161.0984,
                            137.0951, 123.0434,
                         93.0699, 79.0549, 67.0538

55    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    330.1375    1.7       284.1324, 209.1176,
                            191.1066, 163.1120,
                            153.0544, 145.1015,
                             121.1008, 93.0695,
                                  85.0650

57    334.1319     0        209.1147, 181.1211,
                            153.0555, 126.0211,
                             108.0102, 91.0549

58    346.1319     0        328.1222, 310.1111,
                            264.1056, 238.0916,
                            207.1018, 195.0853,
                             165.0923, 137.0955

59    388.1422   -0.6       207.1010, 164.0390,
                            107.0491, 122.0273,
                                  105.0347

60    403.1598   -0.2       227.1287, 209.1174,
                            191.1065, 163.1123,
                            153.0549, 145.1025,
                            135.1164, 121.0995,
                                  93.0712

61    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 3: Information about classification of compounds in TST.

Group

Organic acids         19
Nitrogen-containing    9
  compounds
Glycosides            11
Phenols                8
Phthalides            24
Others compounds       6
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Title Annotation:Research Article
Author:Chen, Lin; Chen, Renhao; Ouyang, Hui; Wang, Qi; Li, Zhifeng; Feng, Yulin; Yang, Shilin
Publication:Journal of Analytical Methods in Chemistry
Geographic Code:9CHIN
Date:Nov 1, 2019
Words:9793
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