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.
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.
Phytochemical and pharmacological investigations showed that phenols, organic acids, phthalides, and nitrogen-containing compounds were the major active ingredients of DCXF [
Five batches of Tianshu Capsules and related crude herbal materials (
The structures of the identified compounds in 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
The ethanolic and aqueous extracts of individual herb (
Stock solutions with a concentration of about 0.010 mg
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
TSC sample and its related crude herbal material,
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
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
Under the HPLC conditions as described in the current Chinese Pharmacopoeia [
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 of
Retention time (
Number |
|
UV |
MW | [M − H]− | [M + H]+ | [M + NH4]+ | [M + Na]+ | [2M + Na]+ | [M + HCOO]− | Tianma | Chuanxiong | Identified compounds |
---|---|---|---|---|---|---|---|---|---|---|---|---|
|
5.403 | 221, 276 | 286 | 304 | 331 | + | − | Gastrodin | ||||
|
7.736 | 230, 283 | 126 | 127 | 144 | 149 | + | − | 5-HMF | |||
|
15.579 | 225 | 460 | 459 | 478 | 483 | + | − | Parishin E/G | |||
|
20.691 | 221, 272 | 728 | 727 | 746 | 751 | + | − | Parishin B | |||
|
22.220 | 221, 274 | 728 | 727 | 746 | 751 | + | − | Parishin C | |||
|
25.080 | 218 | 242 | 241 | 243 | 265 | 507 | − | + | 3-Butyl-3,6,7-trihydroxy-4,5,6,7 | ||
|
25.567 | 220, 236, 323 | 194 | 193 | 195 | 217 | + | + | ferulic acid | |||
|
27.529 | 220, 330 | 226 | 227 | 249 | 475 | − | + | Senkyunolide J/N | |||
|
30.667 | 280 | 224 | 225 | 247 | 471 | − | + | 4,5-Dihydro-3,1′-dihydroxy-3 | |||
|
32.436 | 275 | 206 | 205 | 207 | − | + | 4-hydroxyl-3-butylphthalide | ||||
|
34.299 | 325 | 396 | 397 | + | + | unidentified | |||||
|
38.635 | 220, 270 | 222 | 221 | 223 | 245 | − | + | Senkyunolide D or | |||
|
39.502 | 230, 310 | 206 | 205 | 207 | 229 | 435 | − | + |
|
||
|
39.898 | 280 | 208 | 207 | 209 | 231 | 439 | − | + | Senkyunolide K/G | ||
|
41.465 | 220 | 206 | 205 | 207 | 229 | 435 | − | + | Senkyunolide F | ||
|
42.332 | 220, 240, 325 | 192 | 193 | 215 | 407 | + | + | Senkyunolide A | |||
|
42.956 | 226, 280 | 190 | 191 | 208 | 213 | 403 | − | + | Butylphthalide | ||
|
43.717 | 218, 270 | 204 | 203 | 205 | 227 | 431 | 249 | − | + | Senkyunolide C | |
|
44.147 | 210, 285, 330 | 204 | 203 | 205 | 227 | 431 | 249 | − | + | Senkyunolide E | |
|
44.949 | 220, 270, 330 | 194 | 195 | 217 | 411 | − | + | Cnidilide | |||
|
45.831 | 205, 280, 328 | 190 | 191 | 213 | 403 | − | + | Ligustilide | |||
|
46.394 | 220 | 194 | 195 | 217 | 411 | − | + | Neocnidilide | |||
|
46.653 | 210, 275, 330 | 188 | 189 | 206 | 211 | 399 | − | + | 3-Butylidenephthalide | ||
|
47.231 | 225, 280, 310 | 380 | 381 | 398 | 403 | − | + | Riligustilide | |||
|
50.042 | 276 | 382 | 383 | 399 | 405 | − | + | Senkyunolide P | |||
|
50.476 | 220, 280 | 380 | 381 | 398 | 403 | − | + | 3′,6,8′,3a-Biligustilide | |||
|
50.970 | 220, 280 | 380 | 381 | 398 | 403 | − | + | Tokinolide B | |||
|
51.065 | 230, 279 | 382 | 383 | 400 | 405 | − | + | Unidentified | |||
|
51.962 | 220, 275 | 380 | 381 | 403 | − | + | Levistolide A | ||||
|
52.267 | 220, 285 | 380 | 381 | − | + | 3′ |
|||||
|
52.693 | 220, 280 | 278 | 279 | 301 | 579 | − | + | Senkyunolide M | |||
|
53.134 | 220, 280 | 278 | 279 | 301 | 579 | − | + | Senkyunolide Q | |||
|
53.852 | 225, 278 | 382 | 383 | 400 | 405 | 383 | − | + | Unidentified |
HPLC 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.
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
Retention time (
Number |
|
Identified compounds | UV |
[M + Na]+ | [M + H]+ | [2M + Na]+ | Main product ions | [M − H]− | [M + HCOO]− | Main product ions |
---|---|---|---|---|---|---|---|---|---|---|
|
6.3 | Gastrodin glucose (448) | 221, 276 | 471 | 447 | 493 | 323[M – H-C7H8O2]−, | |||
|
6.3 | Gastrodin (286) | 220, 269 | 309 | 185[M + Na-C7H8O2]+ | 285 | 331 | 161[M – H-C7H8O2]−, |
||
|
8.1 | 5-HMF (126) | 230, 283 | 127 | 109[M + H-H2O]+ | 125 | ||||
|
20.3 | Parishin B (728) | 221, 272 | 751 | 483[M + Na-268]+, 215[483-268]+ | 727 | 459, 441, 423, 397, 379, 217 | |||
|
21.8 | Parishin C (728) | 221, 274 | 751 | 483[M + Na-268]+, 215[483-268]+ | 727 | 459, 441, 423, 397, 379, 217 | |||
|
23.4 | Parishin (996) | 221, 271 | 995 | 727[M – H-268]− | |||||
|
26.0 | Ferulic acid (194) | 220, 236, 323 | 195 | 177[M + H-H2O]+, |
|||||
|
27.4 | Senkyunolide J/N (226) | 220, 330 | 249 | 475 | 209[M + H-H2O]+, |
||||
|
30.4 | 4,5-Dihydro-3,1′-dihydroxy |
280 | 247 | 207[M + H-H2O]+, 189[M + H-2H2O]+, 165, 121 | |||||
|
31.6 | Senkyunolide I/H (224) | 276 | 247 | 207[M + H-H2O]+, 189[M + H-2H2O]+, |
|||||
|
37.0 | Unidentified (316) | 230, 276, 280 | 339 | 317 | 299[M + H-H2O]+, 281[M + H-2H2O]+, |
315 | |||
|
37.8 |
|
230 | 229 | 413 | 189[M + Na-H2O]+, 171[189-H2O]+ | ||||
|
38.1 | Senkyunolide K/G (208) | 233, 280 | 231 | 439 | 191[M + H-H2O]+, 173[191 − H2O]+, |
||||
|
40.2 | Senkyunolide A (192) | 280 | 215 | 193 | 407 | 175[M + H-H2O]+, 147, 119, 105 | |||
|
40.7 | Butylphthalide (190) | 232, 275 | 213 | 191 | 403 | 173[M + H-H2O]+, 145, 117 | |||
|
42.6 | Cnidilide (194) | 238, 279, 327 | 217 | ||||||
|
43.3 |
|
281, 327 | 213 | 191 | 403 | 173[M + H-H2O]+, 130 | |||
|
43.5 |
|
237, 260, 312 | 213 | 191 | 403 | 173[M + H-H2O]+, 145[173-28]+ 130, |
|||
|
44.0 | 3-Butylidenephthalide (188) | 230, 277, 326 | 211 | 189 | 171[M + H-H2O]+, 153[171-H2O]+ | ||||
|
47.4 | Riligustilide (380) | 275 | 403 | 381 | 213[2M + Na-190]+, 191, 173 | ||||
|
47.5 | Senkyunolide P (382) | 278 | 405 | 383 | 192[M + H-190]+ | ||||
|
47.8 | 3′,6,8′,3a-Biligustilide (380) | 278, 363 | 403 | 381 | 213[2M + Na-190]+, 191[M + H-190]+, |
||||
|
48.0 | Tokinolide B (380) | 278 | 403 | 381 | 213[2M + Na-190]+, 191[M + H-190]+ | ||||
|
48.2 | Unidentified (382) | 230, 279 | 405 | 383 | 193[M + H-190]+ | ||||
|
48.5 | Levistolide A (380) | 276 | 403 | 381 | 213[2M + Na-190]+, 191[M + H-190]+, |
||||
|
48.8 | Senkyunolide O (380) | 278 | 403 | 381 | 173[191-H2O]+, 191[M + H-190]+, |
||||
|
49.0 | Senkyunolide M (278) | 279 | 301 | 279 | 245[M + Na-56]+, 189, 171 | ||||
|
49.4 | Senkyunolide Q (278) | 278 | 301 | 279 | 245[M + Na-56]+, 189, 171 | ||||
|
49.9 | Unidentified (382) | 225, 278 | 405 | 383 |
Retention time (
Number |
|
Identified compounds | Formula | Mea. mass/ |
Calc. mass/ |
Error/ppm | Other precursor ions | Main product ions |
---|---|---|---|---|---|---|---|---|
|
7.6 | 5-HMF (126) |
|
127.0384[M + H]+ | 127.0390[M + H]+ | −4.72 | 149.0240[M + Na]+ | |
|
19.8 | Parishin B (728) |
|
727.2123[M − H]− | 727.2091[M − H]− | 4.40 | ||
|
21.5 | Parishin C (728) |
|
727.2132[M − H]− | 727.2091[M − H]− | 5.64 | ||
|
24.5 | 3-Butyl-3,6,7-trihydroxy-4,5,6,7-tetrahydrophthalide (242) |
|
241.1090[M − H]− | 241.1081[M − H]− | 3.72 | 223.0885[M – H-H2O]−, 197.1142, | |
243.1220[M + H]+ | 243.1227[M + H]+ | 2.88 | 265.1060[M + Na]+ | |||||
|
25.3 | Ferulic acid (194) |
|
195.0649[M + H]+ | 195.0652[M + H]+ | −1.54 | 217.0458[M + Na]+ | |
|
27.0 | Senkyunolide J/N (226) |
|
227.1259[M + H]+ | 227.1278[M + H]+ | −3.37 | 249.1082[M + Na]+ | 209.1177[M + H-H2O]+, |
|
30.3 | 4,5-Dihydro-3,1′-dihydroxy-3-butylphthalide (224) |
|
225.1105[M + H]+ | 225.1121[M + H]+ | −7.11 | ||
|
31.6 | Senkyunolide I/H (224) |
|
225.1105[M + H]+ | 225.1121[M + H]+ | −7.11 | ||
|
37.8 | Unidentified (316) |
|
317.1380[M + H]+ | 317.1384[M + H]+ | −1.26 | 339.1210[M + Na]+ | 299.1378[M + H-H2O]+, |
|
37.8 | 4,7-Dihydroxy-3-butylidenephthalide or senkyunolide D (222) |
|
223.0986[M + H]+ | 223.0965[M + H]+ | 9.41 | 245.0774[M + Na]+ | |
|
38.7 |
|
|
207.1008[M + H]+ | 207.1016[M + H]+ | 3.66 | 229.0824[M + Na]+ | 189.0856[M + H-H2O]+, 133.0318 |
|
39.1 | Senkyunolide K/G (208) |
|
209.1167[M + H]+ | 209.1172[M + H]+ | −2.39 | 231.0974[M + Na]+ | 191.1091[M + H-H2O]+, 119.0819 |
|
40.6 | Senkyunolide F (206) |
|
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 | ||||
|
41.4 | Senkyunolide A (192) |
|
193.1219[M + H]+ | 193.1223[M + H]+ | −2.07 | 215.1027[M + Na]+, |
175.1119[M + H-H2O]+, 147.1155, |
|
42.1 | Butylphthalide (190) |
|
191.1061[M + H]+ | 191.1067[M + H]+ | −3.14 | 213.0872[M + Na]+, |
173.0989[M + H-H2O]+, |
|
42.9 | Senkyunolide C (204) |
|
205.0847[M + H]+ | 205.0859[M + H]+ | −5.85 | 227.0685[M + Na]+, |
187.0763[M + H-H2O]+, |
|
43.4 | Senkyunolide E (204) |
|
205.0843[M + H]+ | 205.0859[M + H]+ | −7.80 | 227.0663[M + Na]+ | |
|
44.1 | Cnidilide (194) |
|
195.1372[M + H]+ | 195.1380[M + H]+ | −4.10 | 217.1185[M + Na]+ | 177.1323[M + H-H2O]+, |
|
44.1 |
|
|
191.1061[M + H]+ | 191.1067[M + H]+ | −3.14 | 213.0871[M + Na]+ | |
|
45.2 |
|
|
191.1056[M + H]+ | 191.1067[M + H]+ | −5.76 | 213.0859[M + Na]+, |
173.0984[M + H-H2O]+, |
|
45.5 | Neocnidilide (194) |
|
195.1372[M + H]+ | 195.1380[M + H]+ | −4.10 | 217.1179[M + Na]+ | 177.1323[M + H-H2O]+, |
|
45.8 | 3-Butylidenephthalide (188) |
|
189.0907[M + H]+ | 189.0910[M + H]+ | −1.59 | 211.0720[M + Na]+ | 171.0817[M + H-H2O]+, |
|
46.2 | Riligustilide (380) |
|
381.2058[M + H]+ | 381.2060[M + H]+ | −0.52 | 403.1872[M + Na]+, |
|
|
48.9 | Senkyunolide P (382) | 383.2220[M + H]+ | 383.2217[M + H]+ | 405.2031[M + Na]+ | 193.1212[M/2 + H]+ | ||
|
49.2 | 3′,6,8′,3a-Biligustilide (380) |
|
381.2058[M + H]+ | 381.2060[M + H]+ | −0.52 | 403.1885[M + Na]+, |
|
|
49.8 | Tokinolide B (380) |
|
381.2037[M + H]+ | 381.2060[M + H]+ | −6.03 | 403.1878[M + Na]+, |
|
|
49.9 | Unidentified (382) | 383.2220[M + H]+ | 383.2217[M + H]+ | 405.2040[M + Na]+ | 193.1214[M/2 + H]+ | ||
|
50.3 | Levistolide A (380) |
|
381.2064[M + H]+ | 381.2060[M + H]+ | 1.05 | 403.1887[M + Na]+, |
|
|
50.8 | Senkyunolide O (380) |
|
381.2061[M + H]+ | 381.2060[M + H]+ | 0.26 | 403.1879[M + Na]+, |
191.1051[M/2 + H]+ |
|
51.4 | Senkyunolide M (278) | 279.1582[M + H]+ | 301.1402[M + Na]+, |
||||
|
52.0 | Senkyunolide Q (278) | 279.1596[M + H]+ | 301.1381[M + Na]+, |
||||
|
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]+ |
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 [
Compounds
Compounds
The molecular formula of compound
Compound
Compounds
Compound
Five dimeric ligustilides
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 [
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.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This research work was supported by China Postdoctoral Science Foundation (2012M510731) and State Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Process (SKL2010M0204). Meanwhile great thanks are due to Dr. Feng Ji from Shimadzu (China) Co., Ltd., Beijing Office, for measuring the LC-DAD-ESI-IT-TOF/MS experiments.