Journal of Chromatography B, 986–987 (2015) 69–84

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Chemical profiling and quantification of Gua-Lou-Gui-Zhi decoction by high performance liquid chromatography/quadrupole-timeof-flight mass spectrometry and ultra-performance liquid chromatography/triple quadrupole mass spectrometry Wen Xu a,b,1 , Mingqing Huang a,b,1 , Huang Li a , Xianwen Chen a , Yuqin Zhang a , Jie Liu a , Wei Xu a,b,∗ , Kedan Chu a,b,∗ , Lidian Chen c a

College of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China Centre of Biomedical Research&Development, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China c College of Rehabilitation Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China b

a r t i c l e

i n f o

Article history: Received 22 September 2014 Accepted 2 February 2015 Available online 8 February 2015 Keywords: Gua-Lou-Gui-Zhi decoction Chemical profiling and quantification Quality control QTOF mass spectrometry QqQ mass spectrometry

a b s t r a c t Gua-Lou-Gui-Zhi decoction (GLGZD) is a classical formula of traditional Chinese medicine, which has been commonly used to treat dysfunction after stroke, epilepsy and spinal cord injury. In this study, a systematic method was established for chemical profiling and quantification analysis of the major constituents in GLGZD. For qualitative analysis, a method of high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (Q-TOF MS) was developed. 106 compounds, including monoterpene glycosides, galloyl glucoses, phenolic acids, flavonoids, gingerols and triterpene saponins were identified or tentatively presumed by comparison with reference standards or literature data. According to the qualitative results, a new quantitative analysis method of ultra-performance liquid chromatography/triple quadrupole mass spectrometry (QqQ-MS) was established. 24 representative compounds were simultaneously detected in 10 batches of GLGZD samples in 7.5 min. The calibration curves for all analytes showed good linearity (r > 0.9959) within the test ranges. The LODs and the LOQs were less than 30.6 and 70.9 ng/mL, respectively. The RSDs of intra- and inter-day precision, repeatability and stability were below 3.64%, 4.85%, 4.84% and 3.87%, respectively. The overall recoveries ranged from 94.94% to 103.66%, with the RSDs within 5.12%. This study established a high sensitive and efficient method for the integrating quality control, including identification and quantification of Chinese medicinal preparation. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Gua-Lou-Gui-Zhi decoction (GLGZD), a classical formula of traditional Chinese medicine (TCM), is consisted of six herbs including Trichosanthis Radix, Cinnamomi Ramulus, Paeoniae Radix Alba, Glycyrrhizae Radix, Zingiberis Rhizoma Recens and Jujubae Fructus, which was first recorded in ‘Essentials from the Golden Cabinet’

∗ Corresponding authors at: College of Pharmacy, Fujian University of Traditional Chinese Medicine, No.1 Qiuyang Road, Shangjie University Town, Fuzhou 350122, China. Tel.: +86 591 22861693; fax: +86 591 22861322. E-mail addresses: [email protected], [email protected] (W. Xu), [email protected] (K. Chu). 1 Co-first authors. http://dx.doi.org/10.1016/j.jchromb.2015.02.002 1570-0232/© 2015 Elsevier B.V. All rights reserved.

in the Eastern Han Dynasty (around 210 AD). It has been widely applied in China to treat dysfunction after stroke (such as muscular spasticity), epilepsy and spinal cord injury [1–3]. Nowaday, GLGZD were primarily used in clinical treatment of cerebrovascular disease and pathological changes of central nerve system, and become a newly hospital preparation (a new drug approved by Fujian State Food and Drug Administration, Approval Number: Min 2013S0001). Recently, we revealed that GLGZD possesses therapeutic effects on focal cerebral ischemia reperfusion injury in middle cerebral artery occlusion (MCAO) rats through regulating the expression of excitatory amino acids and their receptors. In addition, it also had neuroprotective effect on post-stroke spasticity via the modulation of glutamate levels and ␣-amino-3-hydroxy5-methyl-4-isoxazole propionate receptor expression, inhibited lipopolysaccharide (LPS) induced microglial cell motility through

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regulating mitogen-activated protein kinase signaling pathway, and suppressed LPS-induced activation of the toll-like receptor 4/nuclear factor kappa B pathway in BV-2 murine microglial cells [4–8]. High performance liquid chromatography-ultraviolet detection (HPLC-UV) and gas chromatography-mass spectrometry have been reported for the quality control of GLGZD [16–19]. These methods made significant contributions to the quality control of GLGZD, however, they were inclined to determine the limited compounds, and had some shortcomings such as long analysis time and poor LOQs. Major constituents of each component herb in GLGZD have been studied in the past [9–15], but to our best knowledge, few studies on the systematic chemical profile and quantification of GLGZD were reported. Therefore, it is of great significance to develop a method for qualitative and quantitative analyses of the chemical constituents in GLGZD, which is beneficial to investigating the efficacy and evaluating the quality of GLGZD. In recent years, LC combined with quadrupole time-of-flight mass spectrometry (LC–QTOF-MS) has been used to separate and characterize bioactive components in complex matrices with its high resolution and accurate mass measurements, which provide the elemental compositions of unknown peaks with high accuracy (routinely below 10 ppm) [20]. Beside, simultaneous quantification of multi-components has been widely performed in the analysis of TCMs [21]. Ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC–QqQ MS) has been proved to be an ultimate solution to the aforementioned problems, which showed faster analytical speed, narrower chromatographic peaks and higher accuracy [22]. In this paper, an HPLC–QTOF-MS method was established for rapidly separating and reliably identifying the multiple components in GLGZD. A total of 106 compounds in this preparation were identified or tentatively presumed based on their retention time and high resolution MS spectra data under both negative and positive ion modes. Furthermore, quantification of 24 bioactive components was carried out using UPLC–QqQ MS method. It represents a first detailed investigation of the components of GLGZD and provides an applicable method for its quality evaluation.

2. Experimental 2.1. Materials, chemicals and reagents Citrulline, gallic acid, protocatechuic acid, protocatechuic aldehyde, p-hydroxybenzoic acid, catechin, vanillic acid, caffeic acid, puerarin, peoniflorin, ferulic acid, liquiritin, rutin, isoquercitrin, liquirtigenin, kaempferol, cinnamic acid, quercetin, luteolin, glycyrrhizic acid, glycyrrhetinic acid, 6-gingerol, curcumin and luteoloside were bought from the national institute for the control of pharmaceutical and biological products (Beijing, China). Schaftoside, isoliquiritin apioside, isoschaftoside, quercetin-7-O␤-d-glucopyranoside, paeoniflorin sulfonate, 4-hydroxycinnamic acid, 3-hydroxycinnamic acid, taxifolin, pentagalloylglucose, naringin, astragalin, liquiritin apioside, oxypaeoniflorin, neochlorogenic acid, chlorogenic acid, ononin, 8-gingerol, 6-shogaol and licochalcone A were purchased from the Shanghai Tauto Biotech Co., Ltd. (Shanghai, China). Albiflorin, benzoylpaeoniflorin, scopoletin, 2-hydroxycinnamic acid, 2-methoxycinnamic acid, succinic acid, isoliquiritin, isoliquirtigenin, methyl gallate, ethyl gallate, jujuboside A, jujuboside B, formononetin, swertiamarin (IS 1), nicotiflorin (IS 2), and methylparaben (IS 3) were purchased from the Manstie Bio-Technology Co., Ltd. (Chengdu, China). The purity of each reference standard was determined to be higher than 98% by HPLC. All the 6 herbs of GLGZD were purchased from good agricultural practices (GAP) bases approved by State Food and

Drug Administration of China (SFDA), then they were extracted with water, evaporated and freeze-dried to GLGZD powder, which was manufactured in a pharmaceutical manner in Fujian University of TCM Affiliated Second People’s Hospital (Fuzhou, China). Ten batches of GLGZD were collected at different times provided by Fujian University of TCM Affiliated Second People’s Hospital (Fuzhou, China). Methanol, acetonitrile and formic acid were HPLC grade (Merck KGaA, Darmstadt, Germany). Ultra-pure water was prepared using a Milli-Q purification system (Millipore, Bedford, MA, USA). 2.2. Preparation of standard and sample solutions All standard stock solutions were prepared individually at concentrations ranging from 0.33 to 2.31 mg/mL by dissolving accurately weighted amount of reference compound in HPLC grade methanol. An internal standards stock solution was also prepared in a concentration of 4.01 ␮g/mL for swertiamarin, 2.00 ␮g/mL for nicotiflorin and 0.5 ␮g/mL for methylparaben. Then a mixed solution containing all the 24 standards were prepared and serially diluted with 50% methanol–water (v/v) to obtain seven reference solutions with different concentrations used for plotting standard curves. All prepared solutions were stored at 4 ◦ C before analysis. 10 batches of GLGZD samples were ground to fine powder and well mixed. Approximately 0.50 g powder was accurately weighted and ultrasonicated for 30 min with 25 mL 50% methanol–water (v/v) solution. Then, the samples were centrifuged at 16,000 × g for 10 min after replenishment with methanol for the loss. 500 ␮L internal standards working solutions were added to 500 ␮L supernatant. The mixed solution was blended on a vortex mixer, and then filtered through 0.22 ␮m PTFE membrane. All the samples were stored at 4 ◦ C before analysis. 2.3. Chromatographic and mass spectrometric conditions For qualitative analysis, it was performed on a Shimadzu HPLC system (Kyoto, Japan) coupled with a Bruker micrOTOF-Q II mass spectrometer (Bremen, Germany). A LC-20A pump, DGU-20A5 degasser, SIL-20A auto-injector, and CTO-20A column thermostat were included in the HPLC system. Chromatographic separation was carried out on a Shimadzu Inetsil SP C18 (250 mm × 4.6 mm, 5 ␮m), the column temperature was kept at 30 ◦ C. The mobile phase was consisted of 0.1% formic acid aqueous solution (A) and acetonitrile (B) with a gradient elution program as follows: 0–5 min, 5% B; 5–50 min, 5–32% B; 50–65 min, 32–48% B; 65–75 min, 48–57% B; 75–100 min, 57% B; 100–115 min 5–5% B. The flow rate was kept at 0.8 mL/min, and the injecting volume was set at 2 ␮L. The micrOTOF-Q II mass spectrometer was equipped with an electrospray ionization (ESI) source and operated in positive and negative mode. The optimized parameters were as follows: capillary, +3.5 kV (positive mode) and −4.5 kV (negative mode); nebulizer pressure, 2.0 bar; drying gas (N2 ) flow rate, 4.0 L/min; drying gas temperature, 180 ◦ C. Both funnel 1 and 2 were 200.0 Vpp; hexapole Rf, 100.0 Vpp; quadrupole ion energy, 3.0 eV; collision Rf, 150.0 Vpp. The ion transfer time, 80 ␮s; prepulse storage time, 5 ␮s. Argon was applied as the collision gas, and the collision energy was set at 20–50 eV to obtain the fragment ions data. In order to obtain highly accurate mass values, external calibration of QTOF-MS was performed daily before sample injection, and the enhanced quadratic calibration mode was adopted for the calibration curve. For quantitative analysis, the Waters (Milford, MA) Acquity UPLC H-Class system coupled with Xevo TQD QqQ mass spectrometer with ESI was used. Separations were accomplished on ACQUITY UPLC Cortest C18 column (100 mm × 2.1 mm, 1.6 ␮m) at a flow rate of 0.25 mL/min with water (containing 0.1% formic acid) (A) and acetonitrile (B) as mobile phase, and the gradient elution

W. Xu et al. / J. Chromatogr. B 986–987 (2015) 69–84

71

Table 1 The transitions and optimized MS parameters of 24 target markers and 3 internal standards in the UPLC–QqQ MS analysis. Compounds

tR (min)

Precursor ion (m/z)

Daughter ion (m/z)

Cone voltage (V)

Collision energy (eV)

Gallic acid Protocatechuic acid Oxypaeoniflorin Catechin 4-Hydroxybenzoic acid Methyl gallate Vanillic acid Albiflorin Paeoniflorin Pentagalloylglucose Liquiritin Astragalin 3-Hydroxycinnamic acid Isoliquiritin 2-Hydroxycinnamic acid Liquiritigenin Jujuboside A Cinnamic acid Glycyrrhizic acid 6-Gingerol Licochalcone A 8-Gingerol 6-Shogaol Glycyrrhetinic acid Swertiamarin Nicotiflorin Methylparaben

1.20 1.82 2.37 2.48 2.58 2.68 3.06 3.67 3.90 4.06 4.07 4.17 4.22 4.33 4.38 4.56 4.65 4.77 5.02 5.31 5.42 5.73 5.84 6.22 2.77 4.10 4.49

169.01 153.01 495.15 289.07 137.02 183.03 167.03 525.16 525.16 939.11 417.12 447.09 163.03 417.12 163.03 255.06 1205.58 147.04 821.39 293.17 337.14 321.21 275.16 469.33 419.11 593.15 151.03

125.02 109.02 137.02 245.08 93.03 124.01 123.04 121.02 121.02 769.09 255.06 284.03 119.04 255.06 119.04 135.01 1073.53 103.05 351.05 99.07 229.08 127.11 139.11 425.34 179.05 285.04 92.03

30 30 45 40 25 35 30 25 25 50 25 35 20 35 20 30 75 15 75 28 47 25 45 80 20 35 30

15 12 30 15 12 20 10 25 28 32 20 22 12 18 10 15 40 10 35 28 25 15 25 32 12 30 18

program was as follow: 0–0.5 min, 8–10% B; 0.5–2.5 min, 10–15% B; 0.5–2.5 min, 10–15% B; 2.5–4.0 min, 15–60% B; 4.0–5.0 min, 60–95% B; 5.0–6.3 min, 95–95% B, 6.3–7.5 min, 8–8% B. The column temperature was kept at 45 ◦ C. The MS spectra were acquired in multiple reaction monitoring (MRM) mode. Argon was chosen as the collision gas, nitrogen was chosen as the nebulizer gas and heater gas. The MS conditions were optimized as follows: capillary voltage 2.5 kV in negative mode; source temperature, 150 ◦ C; dwell time, 20 ms. The most appropriate precursor ion, daughter ion, cone voltage, collision energy (CE) were adjusted according to each analyte (Table 1).

and accurate masses with data from the corresponding reference standards. The structures of the other 50 compounds were tentatively characterized by comparing their characteristic high resolution mass data with the data from previous publications. All these compounds were divided into 6 types according to their structural characteristics, including monoterpene glycosides, galloyl glucoses, phenolic acids, flavonoids, gingerols, triterpene saponins, and other types. The mass error for molecular ions of all identified compounds was within ±6 ppm, the total ion chromatograms in negative and positive ion modes were displayed in Fig. 2.

3. Results and discussions

3.1.1. Identification of monoterpene glycosides In the present study, a total of 17 monoterpene glycosides were identified or predicted which originated from Paeoniae Radix Alba (Table 2). The negative ion mode was found to be more suitable for their analyses. Compounds 17, 22, 29, 31 and 74 were identified to be paeoniflorin sulfonate, oxypaeoniflorin, albiflorin, paeoniflorin and benzoylpaeoniflorin by comparison with the reference compounds. To facilitate identification and confirmation of other monoterpene glycosides, the MS fragmentation patterns of above compounds were investigated (take paeoniflorin as an example, Fig. 3A). As for monoterpene glycosides, the losses of CH2 O, CH2 O2 , benzoyl (C7 H6 O2 ) or hydroxybenzoyl (C7 H6 O3 ) and glucose group (Glc, C6 H10 O5 ) were observed clearly in their MS/MS spectra, which yielded product ions of [M−H−CH2 O]− , [M−H−benzoyl]− , [M−H−CH2 O−benzoyl]− , [M−H−benzoyl−Glc]− , [M−H−benzoyl−Glc−CH2 O]− and [benzoic acid−H]− . Consequently, compounds 3, 5, 12, 14, 15, 44, 49, 51, 53 and 76 were tentatively assigned as desbenzoylpaeoniflorin, desbenzoylalbiflorin, oxypaeoniflorin sulfonate, mudanpioside F, isomaltopaeoniflorin sulfonate, 1-O-␤-d-glucopyranosyl-8-Obenzoylpaeonisuffrone, galloylpaeoniflorin, galloylalbiflorin, benzoylpaeoniflorin sulfonate and benzoylalbiflorin by comparing their dissociation pathways with the literatures [23–26], and compounds 6 and 7 were deduced as 1-O-␤-d-glucopyranosylpaeonisuffrone or its isomer, but the positions of substituent group were uncertain [26].

3.1. HPLC–QTOF-MS qualitative analysis of GLGZD In order to improve the resolution and sensitivity, HPLC conditions including type of column, mobile phase system, flow rate and column temperature were optimized. After compared different brands of columns including Waters X-select C18, Dikma diamond C18, Shimadzu Inetsil SP C18 and Phenomenex Luna C18 column, and the Shimadzu Inetsil SP C18 finally was chosen for its successful separation of multi-components. In addition, different kinds of mobile phase, such as acetonitrile and methanol with a variety of modifiers (0.1% formic acid, 0.1% acetic acid and 5 mM ammonium acetate) were tested. The mixture of acetonitrile and 0.1% formic acid water solution was proved to be the suitable mobile phase, which not only improve the compounds resolution, but also inhibit the ionization of the acidic ingredients in GLGZD. Meanwhile, column temperature (25, 30 and 35 ◦ C) and flow rate (0.6, 0.8 and 1.0 mL/min) were studied. Finally, column temperature at 25 ◦ C and 0.8 mL/min flow rate were chosen. For the MS parameters including capillary voltage, and collision energy (CE) were optimized, and both positive and negative ion modes were employed in this work. As can be seen in Table 2 and Fig. 1, a total of 106 compounds were identified or tentatively characterized, 56 of them were identified unambiguously by comparing their retention times

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Table 2 Characterization of compounds in GLGZD by UPLC–QTOF-MS. No. a

tR (min)

Identification

Formula

MS1 (error, ppm)

Fragment ions (m/z)

Source

174.0869 [M−H]− (−2.30) 176.1021 [M+H]+ (−4.46) 341.1088 [M−H]− (2.93) 343.1229 [M+H]+ (−1.46)

131.0824 [M−H−CONH]− , 159.0771[M+H−NH3 ]+

T

221.0667[M−H−C4 H8 O4 ]− , 179.0554[M−H−C6 H10 O5 ]− , 161.0455[M−H−C6 H10 O5 −H2 O]− , 325.1129[M+H−H2 O]+ , 295.1024[M+H−H2 O−CH2 O]+ , 277.0918[M+H−2H2 O−CH2 O]+ , 163.0599[M+H−C6 H10 O5 −H2 O]+ , 145.0495[M+H−C6 H10 O5 −2H2 O]+ 375.1280[M−H]− , 345.1180[M−H−CH2 O]− , 195.0651[M−H−C6 H10 O5 −H2 O]− , 165.0552[M−H−C6 H10 O5 −H2 O−CH2 O]−

G

n.a.

J

375.1285[M−H]− , 345.1182[M−H−CH2 O]−

P

359.1336[M−H]− , 197.0808[M−H−C6 H10 O5 ]− , 179.0561[M−H−C6 H10 O5 −H2 O]−

P

359.1342[M−H]− , 197.0811[M−H−C6 H10 O5 ]− , 179.0565[M−H−C6 H10 O5 −H2 O]−

P

125.0244[M−H−CO2 ]− , 127.0389[M+H−CO2 ]+

P

331.0669[M−H−C6 H10 O5 ]− , 169.0136[M−H−2C6 H10 O5 ]− , 125.0234[M−H−2C6 H10 O5 −CO2 ]− 331.0662[M−H−C6 H10 O5 ]− , 169.0135[M−H−2C6 H10 O5 ]− , 125.0239[M−H−2C6 H10 O5 −CO2 ]− 331.0658[M−H−C6 H10 O5 ]− , 169.0139[M−H−2C6 H10 O5 ]− , 125.0238[M−H−2C6 H10 O5 −CO2 ]− 421.0801[M−H−hydroxybenzoic acid]− , 259.0277[M−H−hydroxybenzoyl−C6 H10 O5 ]− , 213.0222[M−H−hydroxybenzoyl−C6 H10 O5 −CH2 O2 ]− , 137.0244[hydroxybenzoic acid−H]− 109.0295[M−H−CO2 ]− , 111.0439[M+H−CO2 ]+

P

343.1387[M−H]− , 181.0859[M−H−C6 H10 O5 ]− , 151.0753[M−H−C6 H10 O5 −CH2 O]−

P

543.1166[M−H−C6 H10 O5 ]− , 421.0799[M−H−C6 H10 O5 −benzoyl]− , 259.0270[M−H−C6 H10 O5 −benzoyl−C6 H10 O5 ]− , 121.0286[benzoic acid−H]− 191.0556[M−H−caffeoyl]− , 179.0344[caffeic acid−H]− , 173.0450[M−H−caffeoyl−H2 O]− , 135.0446[caffeic acid−H−CO2 ]− , 163.0395[caffeic acid+H−H2 O]+

P

497.1111[M−H−CH2 O2 ]− , 421.0801[M−H−benzoyl]− , 375.0752[M−H−benzoyl−CH2 O2 ]− , 259.0269[M−H−benzoyl−C6 H10 O5 ]− , 213.0224[M−H−benzoyl−C6 H10 O5 −CH2 O2 ]− , 121.0300[benzoic acid−H]− 109.0290[M−H−CO]− , 111.0440[M+H−CO]+

P

191.0557[M−H−caffeoyl]− , 179.0354[caffeic acid−H]− , 173.0451[M−H−caffeoyl−H2 O] − , 161.0231[caffeic acid−H−H2 O]− , 135.0439[caffeic acid−H−CO2 ]− , 163.0395[caffeic acid+H−H2 O]+ 93.0335[M−H−CO2 ]− , 121.0284[M+H−H2 O]+ , 95.0491[M+H−CO2 ]+

J

1

3.93

Citrulline

C6 H13 N3 O3

2

4.03

Gentiobiose

C12 H22 O11

3

6.26

Desbenzoylpaeoniflorin

C16 H24 O10

4a

7.04

Succinic acid

C4 H6 O4

5

8.48

Desbenzoylalbiflorin

C16 H24 O10

6

9.55

1-O-␤-d-glucopyranosylpaeonisuffrone or isomer

C16 H24 O9

7

10.51

1-O-␤-d-glucopyranosylpaeonisuffrone or isomer

C16 H24 O9

8a

11.77

Gallic acid

C7 H6 O5

9

13.14

1 -O-galloylsucrose or isomer

C19 H26 O15



10

13.78

6 -O-galloylsucrose or isomer

C19 H26 O15

11

15.39

6-O-galloylsucrose or isomer

C19 H26 O15

12

18.58

Oxypaeoniflorin sulfonate

C23 H28 O14 S

13a

19.89

Protocatechuic acid

C7 H6 O4

14

20.95

Mudanpioside F

C16 H24 O8

15

21.52

Isomaltopaeoniflorin sulfonate

C29 H38 O18 S

16a

21.85

Neochlorogenic acid

C16 H18 O9

17a

22.51

Paeoniflorin sulfonate

C23 H28 O13 S

18a

24.57

Protocatechuic aldehyde

C7 H6 O3

19a

25.75

Chlorogenic acid

C16 H18 O9

20a

26.21

p-Hydroxybenzoic acid

C7 H6 O3

421.1340 [M+HCOO]− (0.00) 377.1433 [M+H]+ (−2.39) 117.0193 [M−H]− (4.27) 119.0334 [M+H]+ (−3.36) 421.1354 [M+HCOO]− (3.32) 377.1460 [M+H]+ (2.65) 405.1405 [M+HCOO]− (3.46) 361.1510 [M+H]+ (4.71) 405.1400 [M+HCOO]− (2.22) 361.1510 [M+H]+ (4.71) 169.0142 [M−H]− (2.96) 171.0286 [M+H]+ (−1.17) 493.1184 [M−H]− (−0.61) 493.1186 [M−H]− (−0.2) 493.1194 [M−H]− (1.42) 559.1125 [M−H]− (1.61) 561.1284 [M+H]+ (2.14) 153.0192 [M−H]− (2.61) 155.0329 [M+H]+ (−4.52) 389.1435 [M+HCOO]− (−1.80) 705.1711 [M−H]− (2.27) 729.1663 [M+Na]+ (−1.10) 353.0883 [M−H]− (4.53) 355.1034 [M+H]+ (3.10) 543.1183 [M−H]− (3.13) 545.1310 [M+H]+ (−2.38) 137.0239 [M−H]− (4.38) 139.0395 [M+H]+ (4.32) 353.0881 [M−H]− (3.97) 355.1032 [M+H]+ (2.53) 137.0237 [M−H]− (2.92) 139.0388 [M+H]+ (−0.72)

P

P P P

PJ

J

P

TPJ

W. Xu et al. / J. Chromatogr. B 986–987 (2015) 69–84

73

Table 2 (Continued ) No.

tR (min)

Identification

Formula

a

21

27.04

Methyl gallate

C8 H8 O5

22a

27.95

Oxypaeoniflorin

C23 H28 O12

23a

29.00

Catechin

C15 H14 O6

24a

30.65

Vanillic acid

C8 H8 O4

25a

30.85

2-Methoxycinnamic acid

C10 H10 O3

26

31.17

Glucoliquiritin apioside

C32 H40 O18

27a

32.07

Caffeic acid

C9 H8 O4

28a

32.36

Puerarin

C21 H20 O9

29a

33.13

Albiflorin

C23 H28 O11

30

33.75

Vicenin-2

C27 H30 O15

31a

35.31

Paeoniflorin

C23 H28 O11

32a

36.96

Ethyl gallate

C9 H10 O5

33a

37.08

Schaftoside

C26 H28 O14

34a

38.94

Isoschaftoside

C26 H28 O14

35a

39.51

4-Hydroxycinnamic acid

C9 H8 O3

36

40.32

Naringenin 7-O-(2-␤-d-apiofuranosyl)-␤d-glucopyranoside

C26 H30 O13

37

41.15

Neoliquiritin

C21 H22 O9

MS1 (error, ppm) −

183.0301 [M−H] (3.82) 185.0438 [M+H]+ (−3.24) 495.1501 [M−H]− (0.81) 519.1461 [M+Na]+ (−2.12)

289.0717 [M−H]− (3.81) 291.0867 [M+H]+ (1.37) 167.0346 [M−H]− (4.79) 169.0487 [M+H]+ (−4.73) 177.0548 [M−H]− (1.13) 179.0709 [M+H]+ (3.91) 711.2126 [M−H]− (−0.56) 735.2114 [M+Na]+ (1.09) 179.0345 [M−H]− (1.68) 181.0510 [M+H]+ (4.97) 415.1045 [M−H]− (3.85) 417.1189 [M+H]+ (2.16) 525.1608 [M+HCOO]− (1.14) 481.1694 [M+H]+ (−2.08) 593.1517 [M−H]− (2.87) 595.1661 [M+H]+ (0.67) 525.1615 [M+HCOO]− (2.48) 481.1699 [M+H]+ (−1.04)

197.0449 [M−H]− (2.53) 199.0611 [M+H]+ (5.02) 563.1415 [M−H]− (3.55) 565.1559 [M+H]+ (1.42) 563.1413 [M−H]− (3.2) 565.1555 [M+H]+ (0.71) 163.0398 [M−H]− (5.52) 165.0549 [M+H]+ (1.82) 549.1614 [M−H]− (2.19) 551.1787 [M+H]+ (5.08)

417.1196 [M−H]− (3.84) 419.1322 [M+H]+ (−3.34)

Fragment ions (m/z)

Source −

−

168.0053[M−H−CH3 ] , 140.0104[M−H−CH3 −CO] , 124.0154[M−H−CH3 −CO2 ]− , 167.0338[M+H−H2 O]+

P

465.1394[M−H−CH2 O]− , 345.1189[M−H−CH2 O−hydrobenzoyl]− , 333.0980[M−H−C6 H10 O5 ]− , 195.0656[M−H−C6 H10 O5 −hydrobenzoyl]− , 165.0549[M−H−C6 H10 O5 −hydrobenzoyl−CH2 O]− , 137.0240[hydroxybenzoic acid−H]− 245.0817[M−H−CO2 ]− , 179.0349[M−H−C6 H6 O2 ]− , 165.0179[M−H−C7 H8 O2 ]− , 245.0808[M+H−CH2 O2 ]+ , 167.0339[M+H−C7 H8 O2 ]+

P

152.0117[M−H−CH3 ]− , 123.0447[M−H−CO2 ]− , 108.0213[M−H−CH3 −CO2 ]− , 125.0597[M+H−CO2 ]+

T

145.0288[M−H−CH3 ]− , 133.0654[M−H−CO2 ]− , 135.0804[M+H−CO2 ]+

C

549.1602[M−H−C6 H10 O5 ]− , 417.1180[M−H−C6 H10 O5 −C5 H8 O4 ]− , 255.0651[M−H−2C6 H10 O5 −C5 H8 O4 ]− , 419.0338[M+H−C6 H10 O5 −C5 H8 O4 ]+ , 257.0809[M+H−2C6 H10 O5 −C5 H8 O4 ]+ 135.0451[M−H−CO2 ]− , 117.0340[M−H−CO2 −H2 O]− , 163.0383[M+H−H2 O]+ , 145.0290[M+H−2H2 O]+ , 135.0440[M+H−CH2 O2 ]+

G

295.0601[M−H−C4 H8 O4 ]− , 277.0495[M−H−C4 H8 O4 −H2 O]− , 267.0651[M−H−C4 H8 O4 −CO]− , 399.1074[M+H−H2 O]+ , 381.0968[M+H−2H2 O]+ , 297.0757[M+H−C4 H8 O4 ]+ 479.1546[M−H]− , 449.1444[M−H−CH2 O]− , 357.1180[M−H−benzoyl]− , 327.1080[M−H−benzoyl−CH2 O]− , 121.0293[benzoic acid−H]− 503.1184[M−H−C3 H6 O3 ]− , 473.1078[M−H−C4 H8 O4 ]− , 551.1759[M+H−CO2 ]+ , 505.1340[M−H−C3 H6 O3 ]+ , 475.1234[M+H−C4 H8 O4 ]+

G

479.1559[M−H]− , 449.1440[M−H−CH2 O]− , 357.1182[M−H−benzoyl]− , 327.1085[M−H−CH2 O−benzoyl]− , 195.0655[M−H−benzoyl−C6 H10 O5 ]− , 165.0555[M−H−benzoyl−C6 H10 O5 −CH2 O]− , 121.0294[benzoic acid−H] − 169.0140[M−H−C2 H4 ]− , 124.0168[M−H−C3 H5 O2 ]− , 171.0288[M+H−C2 H4 ]+ , 127.0389[M+H−C3 H4 O2 ]+ , 109.0283[M+H−C3 H4 O2 −H2 O]+

P

545.1285[M−H−H2 O]− , 503.1177[M−H−C2 H4 O2 ]− , 473.1076[M−H−C3 H6 O3 ]− , 443.0969[M−H−C4 H8 O4 ]− , 413.0867[M−H−C5 H10 O5 ]− , 383.0767[M−H−C6 H12 O6 ]− , 353.0663[M−H−C7 H14 O7 ]− , 445.1129[M+H−C4 H8 O4 ]+ 473.1064[M−H−C3 H6 O3 ]− , 443.0972[M−H−C4 H8 O4 ]− , 413.0875[M−H−C5 H10 O5 ]− , 383.0762[M−H−C6 H12 O6 ]− , 353.0666[M−H−C7 H14 O7 ]− , 445.1128[M+H−C3 H6 O3 −CH2 O]+ 119.0498[M−H−CO2 ]− , 121.0647[M+H−CO2 ]+

G

417.1180[M−H−C5 H8 O4 ]− , 255.0656[M−H−C5 H8 O4 −C6 H10 O5 ]− , 135.0085[M−H−C5 H8 O4 −C6 H10 O5 −C8 H8 O]− , 119.0490[M−H−C5 H8 O4 −C6 H10 O5 −C7 H4 O3 ]− , 419.0336[M+H−C5 H8 O4 ]+ , 257.0808[M+H−C5 H8 O4 −C6 H10 O5 ]+ 255.0654[M−H−C6 H9 O5 ]− , 257.0809[M−H−C6 H9 O5 ]+

G

P

J

P

G

P

G

C

G

74

W. Xu et al. / J. Chromatogr. B 986–987 (2015) 69–84

Table 2 (Continued ) No.

tR (min)

Identification

Formula

MS1 (error, ppm) −

a

38

41.20

Scopoletin

C10 H8 O4

39a

41.54

Ferulic Acid

C10 H10 O4

40a

41.79

Liquirtin apioside

C26 H30 O13

41a

42.03

Liquiritin

C21 H22 O9

417.1202 [M−H]− (5.27) 419.1322 [M+H]+ (−3.34)

42a

42.14

Rutin

C27 H30 O16

43a

42.33

Quercetin-7-Oglucopyranoside

C21 H20 O12

44

42.58

1-O-␤-d-glucopyranosyl-8-Obenzoylpaeonisuffrone

C23 H28 O10

45a

43.17

3-Hydroxycinnamic acid

C9 H8 O3

46a

43.66

Taxifolin

C15 H12 O7

609.1463 [M−H]− (2.13) 611.1606 [M+H]+ (0.00) 463.0884 [M−H]− (2.81) 465.1037 [M+H]+ (2.15) 509.1663 [M+HCOO]− (1.96) 465.1765 [M+H]+ (−1.51) 163.0395 [M−H]− (3.68) 165.0551 [M+H]+ (3.03) 303.0509 [M−H]− (3.30) 305.0649 [M+H]+ (−1.97)

47a

43.87

Isoquercitrin

C21 H20 O12

48a

44.96

Pentagalloylglucose

C41 H32 O26

49

45.80

Galloylpaeoniflorin or isomer

C30 H32 O15

50

45.95

Liquiritigenin-7, 4-diglucoside

C27 H32 O14

51

46.54

Galloylalbiflorin or isomer

C30 H32 O15

52a

46.98

Naringin

C27 H32 O14

53

47.26

Benzoylpaeoniflorin sulfonate

C30 H32 O14 S

54a

47.33

2-Hydroxycinnamic acid

C9 H8 O3

191.0344 [M−H] (3.14) 193.0494 [M+H]+ (−0.52) 193.0503 [M−H]− (4.14) 195.0648 [M+H]+ (−1.54) 549.1613 [M−H]− (2.00) 551.1753 [M+H]+ (−1.09)

463.0872 [M−H]− (0.22) 465.1020 [M+H]+ (−1.51) 939.1105 [M−H]− (0.75) 963.1060 [M+Na]+ (−1.45)

631.1655 [M−H]− (−0.32) 633.1822 [M+H]+ (1.42) 579.1723 [M−H]− (2.59) 581.1845 [M+H]+ (−3.27) 631.1665 [M−H]− (1.27) 633.1822 [M+H]+ (1.42) 579.1723 [M−H]− (2.59) 581.1869 [M+H]+ (0.86) 647.1422 [M−H]− (−1.08) 9.1592[M+H]+ (1.07) 163.0398 [M−H]− (5.52) 165.0549 [M+H]+ (1.82)

Fragment ions (m/z)

Source −

−

176.0114[M−H−CH3 ] , 148.0166[M−H−CH3 −CO] , 178.0258[M+H−CH3 ]+ , 150.0298[M+H−CH3 −CO]+

J

178.0260[M−H−CH3 ]− , 149.0597[M−H−COOH]− , 134.0362[M−H−CH3 −CO2 ]− , 177.0533[M+H−H2 O]+

TC

417.1180[M−H−C5 H8 O4 ]− , 255.0656[M−H−C5 H8 O4 −C6 H10 O5 ]− , 153.0188[M−H−C5 H8 O4 −C6 H10 O5 −C8 H8 O+H2 O]− , 135.0086[M−H−C5 H8 O4 −C6 H10 O5 −C8 H8 O]− , 119.0497[M−H−C5 H8 O4 −C6 H10 O5 −C7 H4 O3 ]− , 257.0808[M+H−C5 H8 O4 −C6 H10 O5 ]+ 255.0648[M−H−C6 H10 O5 ]− , 153.0181[M−H−C6 H10 O5 −C8 H8 O+H2 O]− , 135.0078[M−H−C6 H10 O5 −C8 H8 O]− , 119.0493[M−H−C6 H10 O5 −C7 H4 O3 ]− , 257.0812[M+H−C6 H10 O5 ]+ , 137.0235[M+H−C6 H10 O5 −C8 H8 O]+ 300.0281[M−H−C12 H21 O9 ]− , 301.0350[M−H−C12 H20 O9 ]− , 303.0496[M+H−C12 H20 O9 ]+

G

300.0275[M−H−C6 H11 O5 ]− , 301.0348[M−H−C6 H10 O5 ]− , 303.0499[M+H−C6 H10 O5 ]+

TG

463.1591 [M−H]− , 359.1342[M−H−benzoyl]− , 121.0293[benzoic acid−H]−

P

119.0497[M−H−CO2 ]− , 121.0645[M+H−CO2 ]+

C

285.0380[M−H−H2 O]− , 275.0536[M−H−CO]− , 259.0574[M−H−CO2 ]− , 241.0494[M−H−H2 O−CO2 ]− , 217.0497[M−H−CO2 −C2 H2 O]− , 177.0178[M−H−C6 H6 O3 ]− , 151.0076[M−H−C8 H8 O3 ]− , 125.0234[M−H−C9 H6 O4 ]− , 287.0550[M+H−H2 O]+ , 179.0338[M+H−C6 H6 O3 ]+ , 153.0186[M+H−C8 H8 O3 ]+ 300.0274[M−H−C6 H11 O5 ]− , 301.0345[M−H−C6 H10 O5 ]− , 303.0494[M+H−C6 H10 O5 ]+

PJ

787.0997[M−H−C7 H4 O4 ]− , 769.0901[M−H−C7 H6 O5 ]− , 617.0766[M−H−C7 H6 O5 −C7 H4 O4 ]− , 465.0667[M−H−C7 H6 O5 −2C7 H4 O4 ]− , 447.0567[M−H−C7 H6 O5 −2C7 H4 O4 −H2 O]− , 295.0449[M−H−2C7 H6 O5 −2C7 H4 O4 ]− , 169.0136[gallic acid−H]− 613.1554[M−H−H2 O]− , 509.1288[M−H−C7 H6 O2 ]− , 491.1183[M−H−C7 H6 O2 −H2 O]− , 169.0135[gallic acid−H]−

P

417.1181[M−H−C6 H10 O5 ]− , 255.0652[M−H−2C6 H10 O5 ]− , 135.0087[M−H−2C6 H10 O5 −C8 H8 O]− , 119.0505[M−H−2C6 H10 O5 −C7 H4 O3 ]− , 257.0802[M+H−2C6 H10 O5 ]+ 613.1552[M−H−H2 O]− , 509.1288[M−H−C7 H6 O2 ]− , 491.1183[M−H−C7 H6 O2 −H2 O]− , 169.0139[gallic acid−H]−

G

459.1285[M−H−C4 H8 O4 ]− , 271.0601[M−H−C12 H20 O9 ]− , 273.0757[M+H−C12 H20 O9 ]+

GJ

525.1617[M−H−benzoyl]− , 259.0276[M−H−benzoyl−C6 H10 O5 ]− , 213.0222[M−H−benzoyl−C6 H10 O5 −CH2 O2 ]− , 121.0294[benzoic acid−H]− 119.0498[M−H−CO2 ]− , 121.0648[M+H−CO2 ]+

P

G

TGJ

TGJ

P

P

C

W. Xu et al. / J. Chromatogr. B 986–987 (2015) 69–84

75

Table 2 (Continued ) No.

tR (min)

Identification

Formula

MS1 (error, ppm) −

a

55

48.52

Astragalin

C21 H20 O11

56a

49.87

Luteoloside

C21 H20 O11

57a

51.53

Isoliquiritin apioside (Licuraside)

C26 H30 O13

58

52.26

Neolicuraside

C26 H30 O13

549.1609 [M−H]− (1.27) 551.1751 [M+H]+ (−1.45)

59a

53.27

Isoliquiritin

C21 H22 O9

417.1181 [M−H]− (0.24) 419.1317 [M+H]+ (−4.53)

60

54.14

Neoisoliquiritin

C21 H22 O9

61a

54.58

Ononin

C22 H22 O9

62

56.12

Licochalcone B

C16 H14 O5

63a

57.32

Liquiritigenin

C15 H12 O4

64a

57.97

Kaempferol

C15 H10 O6

65a

58.04

Cinnamic acid

C9 H8 O2

66

58.13

Uralsaponin C

C42 H64 O16

417.1185 [M−H]− (1.2) 419.1322 [M+H]+ (−3.34) 475.1239 [M+HCOO]− (1.05) 431.1340 [M+H]+ (0.93) 285.0756 [M−H]− (−0.35) 287.0900 [M+H]+ (−4.88) 255.0661 [M−H]− (3.92) 257.0814 [M+H]+ (2.33) 285.0391 [M−H]− (−0.70) 287.0551 [M+H]+ (0.35) 147.0444 [M−H]− (2.72) 149.0596 [M+H]+ (−0.67) 823.4119 [M−H]− (1.09) 825.4255 [M+H]+ (−1.45)

67

58.42

Biochanin A

C16 H12 O5

68

58.71

22-Hydroxyl-glycyrrhizin or isomer

C42 H62 O17

69

59.50

22-Hydroxyl-Licorice saponin G2

C42 H62 O18

70a

59.72

Quercetin

C15 H10 O7

447.0936 [M−H] (3.36) 449.1086 [M+H]+ (1.78) 447.0933 [M−H]− (2.68) 449.1067 [M+H]+ (−2.45) 549.1609 [M−H]− (1.27) 551.1755 [M+H]+ (−0.73)

283.0610 [M−H]− (3.18) 285.0745 [M+H]+ (−4.21) 837.3900 [M−H]− (−0.36) 839.4031 [M+H]+ (−3.34)

853.3839 [M−H]− (−1.52) 855.4011 [M+H]+ (0.35) 301.0357 [M−H]− (4.98) 303.0492 [M+H]+ (−2.31)

Fragment ions (m/z)

Source −

−

285.0399[M−H−C6 H10 O5 ] , 284.0321[M−H−C6 H11 O5 ] , 287.0556[M+H−C6 H10 O5 ]+

T

285.0397[M−H−C6 H10 O5 ]− , 284.0321[M−H−C6 H11 O5 ]− , 287.0550[M+H−C6 H10 O5 ]−

T

417.1177[M−H−C5 H8 O4 ]− , 255.0659[M−H−C5 H8 O4 −C6 H10 O5 ]− , 153.0189[M−H−C5 H8 O4 −C6 H10 O5 −C8 H8 O+H2 O]− , 135.0085[M−H−C5 H8 O4 −C6 H10 O5 −C8 H8 O]− , 119.0498[M−H−C5 H8 O4 −C6 H10 O5 −C7 H4 O3 ]− , 257.0807[M+H−C5 H8 O4 −C6 H10 O5 ]− 417.1175[M−H−C5 H8 O4 ]− , 255.0655[M−H−C5 H8 O4 −C6 H10 O5 ]− , 153.0188[M−H−C5 H8 O4 −C6 H10 O5 −C8 H8 O+H2 O]− , 135.0088[M−H−C5 H8 O4 −C6 H10 O5 −C8 H8 O]− , 119.0494[M−H−C5 H8 O4 −C6 H10 O5 −C7 H4 O3 ]− , 257.0808[M+H−C5 H8 O4 −C6 H10 O5 ]+ 255.0650[M−H−C6 H10 O5 ]− , 153.0180[M−H−C6 H10 O5 −C8 H8 O+H2 O]− , 135.0087[M−H−C6 H10 O5 −C8 H8 O]− , 119.0492[M−H−C6 H10 O5 −C7 H4 O3 ]− , 257.0815[M+H−C6 H10 O5 ]+ 255.0650[M−H−C6 H10 O5 ]− , 135.0088[M−H−C6 H10 O5 −C8 H8 O]− , 119.0495[M−H−C6 H10 O5 −C7 H4 O3 ]− , 257.0815[M+H−C6 H10 O5 ]+ 267.0659[M−H−C6 H10 O5 ]− , 252.0418[M−H−C6 H10 O5 −CH3 ]− , 269.0802[M+H−C6 H10 O5 ]+ , 254.0573[M+H−C6 H10 O5 −CH3 ]− , 270.0522[M−H−CH3 ]− , 150.0315[M−H−CH3 −C7 H4 O2 ]− , 121.0285 [M−H−C9 H8 O3 ]− , 245.0522808[M+H−CH3 ]−

G

153.0192[M−H−C8 H8 O+H2 O]− , 135.0086[M−H−C8 H8 O]− , 119.0501[M−H−C7 H4 O3 ]− , 137.0230[M+H−C8 H10 O]+

G

267.0294[M−H−H2 O]− , 255.0304[M−H−CH2 O]− , 239.0342[M−H−H2 O−CO]− , 227.0356[M−H−CH2 O−CO]− , 153.0181[M−H−C8 H4 O2 ]− , 133.0283[M−H−C7 H4 O4 ]− , 269.0444[M+H−H2 O]+ , 241.0493[M+H−CH2 O2 ]+ 103.0542[M−H−CO2 ]− , 105.0698[M+H−CO2 ]+

TGJ

647.3789[M−H−C6 H8 O6 ]− , 471.3468[M−H−2C6 H8 O6 ]− , 351.0554[2glucuronic acid−H−H2 O]− , 193.0345[glucuronic acid−H]− , 649.3946[M+H−C6 H8 O6 ]+ , 473.3625[M+H−2C6 H8 O6 ]+ , 455.3519[M+H−2C6 H8 O6 −H2 O]+ , 437.3414[M+H−2C6 H8 O6 −2H2 O]+ 268.0366[M−H−CH3 ]− , 270.0522[M+H−CH3 ]+

G

661.3582[M−H−C6 H8 O6 ]− ,485.3216[M−H−2C6 H8 O6 ]− , 351.0555[2glucuronic acid−H−H2 O]− , 193.0346[glucuronic acid−H]− , 663.3740[M+H−C6 H8 O6 ]+ , 487.3417[M+H−2C6 H8 O6 ]+ , 469.3312[M+H−2C6 H8 O6 −H2 O]+ , 451.3206[M+H−2C6 H8 O6 −2H2 O]+ , 433.3101[M+H−2C6 H8 O6 −3H2 O]+ , 423.3257[M+H−2C6 H8 O6 −2H2 O−CO]+ 677.3531[M−H−C6 H8 O6 ]− , 351.0555[2glucuronic acid−H−H2 O]− , 193.0346[glucuronic acid−H]− , 679.3685[M+H−C6 H8 O6 ]+ , 503.3367[M+H−2C6 H8 O6 ]+ , 485.3261[M+H−2C6 H8 O6 −H2 O]+ 273.0398[M−H−CO]− , 229.0469[M−H−CO−CO2 ]− , 178.9985[M−H−C7 H6 O2 ]− , 151.0031[M−H−C8 H7 O3 ]− , 121.0294[M−H−C8 H4 O5 ]− , 285.0399[M+H−H2 O]+ , 257.0450[M+H−H2 O−CO]+ ,229.0501[M+H−H2 O−2CO]+

G

G

G

G

G

G

C

G

G

TGJ

76

W. Xu et al. / J. Chromatogr. B 986–987 (2015) 69–84

Table 2 (Continued ) No. a

tR (min)

Identification

Formula

MS1 (error, ppm) −

71

59.75

Luteolin

C15 H10 O6

285.0404 [M−H] (3.86) 287.0538 [M+H]+ (−4.18)

72

60.14

Licoricesaponin H2

C42 H62 O16

73

60.62

Licoricesaponine A or isomer

C48 H72 O21

821.3950 [M−H]− (−0.49) 823.4119 [M+H]+ (1.09) 983.4477 [M−H]− (−0.51) 985.4622 [M+H]+ (−1.62)

74a

61.180

Benzoylpaeoniflorin

C30 H32 O12

629.1871 [M+HCOO]− (1.11) 607.1795 [M+Na]+ (1.65)

75

62.23

Licoricesaponine A or isomer

C48 H72 O21

983.4462 [M−H]− (−2.03) 985.4648 [M+H]+ (1.01)

76

62.740

Benzoylalbiflorin

C30 H32 O12

77

62.85

22-Hydroxyl-glycyrrhizin or isomer

C42 H62 O17

629.1865 [M+HCOO]− (0.16) 607.1792 [M+Na]+ (1.32) 837.3901 [M−H]− (−0.24) 839.4038 [M+H]+ (−2.50)

78

63.29

24-Hydroxyl-licorice E2

C42 H60 O17

835.3755 [M−H]− (1.08) 837.3903 [M+H]+ (0.00)

79

63.46

22-Acetoxyl-glycyrrhizin

C44 H64 O18

80a

64.20

Jujuboside A

C58 H94 O26

879.3992 [M−H]− (−1.82) 881.4167 [M+H]+ (0.23) 1251.5967 [M+HCOO]− (−2.96)

81

64.470

Dihydroapigenin

C15 H12 O5

82

65.89

Licorice-saponine G2 or isomer

C42 H62 O17

83

66.11

Licorice-saponine E2

C42 H60 O16

84

66.57

Licorice-saponine G2 or isomer

C42 H62 O17

271.0612 [M−H]− (4.06) 273.0763 [M+H]+ (2.20) 837.3906 [M−H]− (0.36) 839.4052 [M+H]+ (−0.83)

819.3793 [M−H]− (−0.49) 821.3964 [M+H]+ (1.22) 837.3911 [M−H]− (0.96) 839.4050 [M+H]+ (−1.07)

Fragment ions (m/z)

Source −

−

267.00300[M−H−C2 H2 O] , 243.0285[M−H−C2 H2 O] , 241.0495[M−H−CO2 ]− , 217.0506[M−H−C3 O2 ]− , 199.0394[M−H−C2 H2 O−CO2 ]− , 175.0401[M−H−C3 O2 −C2 H2 O]− , 151.0035[M−H−C8 H6 O2 ]− ,133.0290[M−H−C7 H4 O4 ]− , 269.0450[M+H−H2 O]+ , 241.0501[M+H−H2 O−CO]+ , 153.0188[M+H−C8 H6 O2 ]+ , 135.0441[M+H−C7 H4 O4 ]+ 645.3633[M−H−C6 H8 O6 ]− , 351.0555[2glucuronic acid−H−H2 O]− , 193.0346[glucuronic acid−H]− , 647.3789[M+H−C6 H8 O6 ]+ , 471.3469[M+H−2C6 H8 O6 ]+ , 453.3362[M+H−2C6 H8 O6 −H2 O]+ 821.3954[M−H−C6 H10 O5 ]− , 351.0558[2glucuronic acid−H−H2 O]− , 193.0344[glucuronic acid−H]− , 647.3789[M+H−C6 H10 O5 −C6 H8 O6 ]+ , 471.3468[M+H−C6 H10 O5 −2C6 H8 O6 ]+ , 453.3365[M+H−C6 H10 O5 −2C6 H8 O6 −H2 O]+ 583.1810[M−H]− , 553.1699[M−H−CH2 O]− , 535.1599[M−H−CH2 O−H2 O]− , 431.1345[M−H−benzoyl]− , 165.0558[M−H−C6 H10 O5 −2benzoyl−CH2 O]− , 121.0290[benzoic acid−H]− 821.3957[M−H−C6 H10 O5 ]− , 351.0558[2glucuronic acid−H−H2 O]− , 193.0341[glucuronic acid−H]− , 647.3780[M+H−C6 H10 O5 −C6 H8 O6 ]+ , 471.3461[M+H−C6 H10 O5 −2C6 H8 O6 ]+ , 453.3365[M+H−C6 H10 O5 −2C6 H8 O6 −H2 O]+ 583.1812[M−H]− , 553.1693[M−H−CH2 O]− , 535.1598[M−H−CH2 O−H2 O]− , 121.0291 [benzoic acid−H]−

T

661.3585[M−H−C6 H8 O6 ]− , 485.3219[M−H−2C6 H8 O6 ]− , 351.0551[2glucuronic acid−H−H2 O]− , 193.0342[glucuronic acid−H]− , 663.3748[M+H−C6 H8 O6 ]+ , 487.3412[M+H−2C6 H8 O6 ]+ , 469.3315[M+H−2C6 H8 O6 −H2 O]+ , 451.3209[M+H−2C6 H8 O6 −2H2 O]+ , 433.3104[M+H−2C6 H8 O6 −3H2 O]+ , 423.3250[M+H−2C6 H8 O6 −2H2 O−CO]+ 659.3425[M−H−C6 H8 O6 ]− , 351.0558[M−H−C30 H41 O4 −H2 O]− , 193.0340[M−H−C36 H50 O10 ]− , 661.3582[M+H−C6 H8 O6 ]− , 485.3260[M+H−2C6 H8 O6 ]− , 467.3155[M+H−2C6 H8 O6 −H2 O]− , 437.3050[M+H−2C6 H8 O6 −H2 O−CH2 O]+ , 449.3055[M+H−2C6 H8 O6 −2H2 O]+ 703.3688[M−H−C6 H8 O6 ]− , 351.0554[2glucuronic acid−H−H2 O]− , 193.0344[glucuronic acid−H]− , 705.3844[M+H−C6 H8 O6 ]+ , 529.3523[M+H−2C6 H8 O6 ]+ , 511.3418[M+H−2C6 H8 O6 −H2 O]+ 1073.5476[M−H−C5 H8 O4 ]− , 911.4971[M−H−C5 H8 O4 −C6 H10 O5 ]− , 749.4440[M−H−C5 H8 O4 −2C6 H10 O5 ]− , 603.3858[M−H−C5 H8 O4 −2C6 H10 O5 −C6 H10 O4 ]− , 179.0547[glucose−H]− 177.0182[M−H−C6 H6 O]− , 165.0184[M−H−C7 H6 O]− , 151.0025[M−H−C8 H8 O]− , 179.0338[M+H−C6 H6 O]− , 167.0335[M+H−C7 H6 O]−

G

661.3578[M−H−C6 H8 O6 ]− ,485.3210[M−H−2C6 H8 O6 ]− , 351.0559[2glucuronic acid−H−H2 O]− , 193.0342[glucuronic acid−H]− , 663.3740[M+H−C6 H8 O6 ]+ , 487.3422[M+H−2C6 H8 O6 ]+ , 469.3321[M+H−2C6 H8 O6 −H2 O]+ , 439.3212[M+H−2C6 H8 O6 −H2 O−CH2 O]+ 643.3477[M−H−C6 H8 O6 ]− , 351.0555[2glucuronic acid−H−H2 O]− , 193.0345[glucuronic acid−H]− , 645.3633[M+H−C6 H8 O6 ]+ , 469.3310[M+H−2C6 H8 O6 ]+ , 451.3201[M+H−2C6 H8 O6 −H2 O]+ 661.3587[M−H−C6 H8 O6 ]− , 485.3215[M−H−2C6 H8 O6 ]− , 351.0550[2glucuronic acid−H−H2 O]− , 193.0348[glucuronic acid−H]− , 663.3747[M+H−C6 H8 O6 ]+ , 487.3415[M+H−2C6 H8 O6 ]+ , 469.3310[M+H−2C6 H8 O6 −H2 O]+ , 439.3201[M+H−2C6 H8 O6 −H2 O−CH2 O]+

G

G

G

P

G

P

G

G

J

P

G

G

W. Xu et al. / J. Chromatogr. B 986–987 (2015) 69–84

77

Table 2 (Continued ) No.

tR (min)

Identification

Formula

MS1 (error, ppm)

a

85

67.34

Jujuboside B

C52 H84 O21

1089.5442 [M+HCOO]− (−3.12)

86a

68.21

Glycyrrhizic acid

C42 H62 O16

821.3965 [M−H]− (1.34) 823.4085 [M+H]+ (−3.04)

87

68.53

Licorice saponine B2

C42 H64 O15

88a

69.40

Formononetin

C16 H12 O4

89

69.53

Uralsaponin B or isomer

C42 H62 O16

90a

69.77

Isoliquiritigenin

C15 H12 O4

91

71.77

Licorice saponine J2

C42 H64 O16

92a

73.31

6-Gingerol

C17 H26 O4

93

74.03

Uralsaponin B or isomer

C42 H62 O16

94

74.53

22-Dehydroxyl-uralsaponin C

C42 H64 O15

95

76.21

Glycyrrhisoflavanone

C21 H20 O6

96

78.63

Unidentified

C15 H22 O4

97

79.17

Licoricone

C22 H22 O6

98

79.89

3 -Hydroxy-4 -Ometthylglabridin

C20 H18 O6

99a

79.94

Curcumin

C21 H20 O6

807.4179 [M−H]− (2.23) 809.4296 [M+H]+ (−2.59) 267.0664 [M−H]− (4.87) 269.0804 [M+H]+ (−1.49) 821.3971 [M−H]− (2.07) 823.4097 [M+H]+ (−1.58) 255.0658 [M−H]− (2.74) 257.0803 [M+H]+ (−1.94) 823.4111 [M−H]− (0.12) 825.4249 [M+H]+ (−2.18) 293.1757 [M−H]− (3.41) 317.1712 [M+Na]+ (−3.47) 821.3945 [M−H]− (−1.1) 823.4082 [M+H]+ (−3.4) 807.4163 [M−H]− (0.25) 831.4141 [M+Na]+ (0.48) 367.1187 [M−H]− (3.00) 369.1345 [M+H]+ (3.52) 265.1447 [M−H]− (4.90) 267.1581 [M+H]+ (−3.37) 381.1345 [M−H]− (3.41) 383.1474 [M+H]+ (−3.91) 353.1028 [M−H]− (2.55) 355.1165 [M+H]+ (−3.10) 367.1187 [M−H]− (3.00) 369.1323 [M+H]+ (−2.44)

100

80.16

Glycycoumarin

C21 H20 O6

101

80.34

4 -O-Methylglabridin

C21 H22 O4

102

80.56

Gancaonin C

C20 H18 O6

367.1191 [M−H]− (4.09) 369.1320 [M+H]+ (−3.25) 337.1446 [M−H]− (3.56) 339.1581 [M+H]+ (−2.65) 353.1027 [M−H]− (2.27) 355.1169 [M+H]+ (−1.97)

Fragment ions (m/z)

Source −

911.4975[M−H−C5 H8 O4 ] , 749.4367[M−H−C5 H8 O4 −C6 H10 O5 ]− , 603.3869[M−H−C5 H8 O4 −C6 H10 O5 −C6 H10 O4 ]− , 131.0324[glucose−H−H2 O−CH2 O]− 759.3909[M−H−CO2 −H2 O]− , 645.3596[M−H−C6 H8 O6 ]− , 469.3298[M−H−2C6 H8 O6 ]− , 351.0554[2glucuronic acid−H−H2 O]− , 193.0342[glucuronic acid−H]− , 647.3789[M+H−C6 H8 O6 ]+ , 471.3468[M+H−2C6 H8 O6 ]+ , 453.3363[M+H−2C6 H8 O6 −H2 O]+ 631.3840[M−H−C6 H8 O6 ] − , 351.0551[2glucuronic acid−H−H2 O] − , 193.0341[glucuronic acid−H]− , 633.3997[M+H−C6 H8 O6 ] + , 457.3676[M+H−2C6 H8 O6 ] + , 439.3570[M+H−2C6 H8 O6 −H2 O] + 252.0419[M−H−CH3 ]− , 254.0573[M+H−CH3 ]+

J

645.3636[M−H−C6 H8 O6 ]− , 351.0553[2glucuronic acid−H−H2 O]− , 193.0347[glucuronic acid−H]− , 647.3789[M+H−C6 H8 O6 ]+ , 471.3469[M+H−2C6 H8 O6 ]+ , 453.3362[M+H−2C6 H8 O6 −H2 O]+ 153.0187[M−H−C8 H8 O+H2 O]− , 135.0081[M−H−C8 H8 O]− , 119.0494[M−H−C7 H4 O3 ]− , 137.0230[M+H−C8 H8 O]+

G

647.3789[M−H−C6 H8 O6 ]− , 351.0553[2glucuronic acid−H−H2 O]− , 193.0347[glucuronic acid−H]− , 649.3946[M+H−C6 H8 O6 ]+ , 473.3625[M+H−2C6 H8 O6 ]+ , 455.3519[M+H−2C6 H8 O6 −H2 O]+ 193.0881[M−H−C6 H12 O]− , 99.0819[M−H−C11 H14 O3 ]− , 277.1793[M+H−H2 O]+ , 177.0896[M+H−C6 H14 O2 ]+ , 137.0603 [M+H−C9 H18 O2 ]+

G

645.3630[M−H−C6 H8 O6 ]− , 351.0558[2glucuronic acid−H−H2 O]− , 193.0340[glucuronic acid−H]− , 647.3780[M+H−C6 H8 O6 ]+ , 471.3461[M+H−2C6 H8 O6 ]+ , 453.3365[M+H−2C6 H8 O6 −H2 O]+ 631.3844[M−H−C6 H8 O6 ]− , 351.0554[2glucuronic acid−H−H2 O]− , 193.0349[glucuronic acid−H]− , 633.3991[M−H−C6 H8 O6 ]+ , 457.3670[M+H−2C6 H8 O6 ]+ , 439.3579[M+H−2C6 H8 O6 −H2 O]+ 352.0941[M−H−CH3 ]− , 349.1070[M−H−H2 O]− , 209.0915[[M−H−C8 H6 O4 ]− , 149.9954 [M−H−CH3 −C13 H14 O2 ]− , 354.1097[M+H−CH3 ]+ , 351.1227[M+H−H2 O]+ n.a.

G

366.1097[M−H−CH3 ]− , 339.0863[M−H−C3 H6 ]− , 368.1254[M+H−CH3 ]+

G

338.0784[M−H−CH3 ]− , 201.0910[M−H−C7 H4 O4 ]− , 189.0910[M+H−C8 H6 O4 ]+

G

217.0506[M−H−C9 H10 O2 ]− , 191.0705[M−H−C10 H8 O3 ]− , 173.0605[M−H−C10 H8 O3 −H2 O]− , 149.0606[M−H−C12 H10 O4 ]− , 134.0376[M−H−C10 H8 O3 −CH3 ]− , 193.0859[M+H−C10 H8 O3 ]+ 352.0941[M−H−CH3 ]− , 309.1121[M−H−CH2 O−CO]− , 313.0706[M+H−C4 H8 ]+ , 285.0757[M+H−C4 H6 −CH2 O]+

J

322.1199[M−H−CH3 ]− , 201.0910[M−H−C8 H8 O2 ]− , 149.0597[M−H−C12 H12 O2 ]− , 123.0440[M−H−C14 H14 O2 ]− , 189.0911[M+H−C9 H10 O2 ]+

G

335.0914[M−H−H2 O]− , 323.0914[M−H−CH2 O]− , 269.0444[M−H−C5 H8 O]− , 119.0491[M−H−C12 H10 O5 ]− , 337.1070[M+H−H2 O]+

G

G

G

G

G

Z

G

G

–

G

78

W. Xu et al. / J. Chromatogr. B 986–987 (2015) 69–84

Table 2 (Continued ) No.

tR (min) a

Identification

Formula

103

81.11

Licochalcone A

C21 H22 O4

104

82.44

Glycyrol

C21 H18 O6

105a

83.08

8-Gingerol

C19 H30 O4

106a

85.53

6-Shogaol

C17 H24 O3

107a

98.48

Glycyrrhetinic acid

C30 H46 O4

MS1 (error, ppm) −

337.1441 [M−H] (2.08) 339.1587 [M+H]+ (−0.88) 365.1016 [M−H]− (−0.82) 367.1186 [M+H]+ (2.72) 321.2068 [M−H]− (2.49) 345.2026 [M+Na]+ (−2.90) 275.1645 [M−H]− (1.45) 299.1603 [M+Na]+ (−4.68) 469.3319 [M−H]− (1.49) 471.3450 [M+H]+ (−3.82)

Fragment ions (m/z)

Source −

−

322.1201[M−H−CH3 ] , 307.0980[M−H−2CH3 ] , 281.0822[M−H−C4 H8 ]− , 243.1026[M−H−C6 H6 O]− , 217.1234[M−H−C7 H4 O2 ]− , 163.1125[M−H−C9 H6 O2 −CO]− , 324.1356[M+H−CH3 ]+ 337.1070[M−H−CO]− , 309.1121[M−H−2CO]− , 352.0941[M+H−CH3 ]+ , 339.1227[M+H−CO]+

G

193.0875[M−H−C8 H16 O]− , 127.1123[M−H−C11 H14 O3 ]− , 305.2105[M+H−H2 O]+ , 177.0899[M+H−C8 H18 O2 ]+ , 137.0590[M+H−C11 H22 O2 ]+

J

139.1123[M−H−C8 H8 O2 ]− , 137.0592[M+H−C9 H16 O]+

J

425.3419[M−H−CO2 ]− , 409.3096[M−H−2CH2 O]− , 355.2639[M−H−CO2 −C5 H10 ]− , 427.3510[M+H−CO2 ]+

G

G

Note: tR : retention time; n.a.: not available; T: Trichosanthis Radix; C: Cinnamomi Ramulus; P: Paeoniae Radix Alba; G: Glycyrrhizae Radix; Z: Zingiberis Rhizoma Recens; J: Jujubae Fructus. a Compared with a reference standard.

3.1.2. Identification of galloyl glucoses A total of 4 galloyl glucoses were deduced and all of them originated from Paeoniae Radix Alba, including three mono-galloyl sucroses and one pentagalloylglucose (Table 2). Compound 48 was unambiguously identified as pentagalloylglucose via the standard reference comparison, a major galloylglucose previously reported existing in Radix Paeoniae [26]. The fragment ions at m/z 169, 295, 447, 465, 617, 769 and 787 were observed in the MS/MS spectrum, indicating the successive neutral losses of gallic acids (170 Da) and galloyl radicals (152 Da). The mass spectrum and proposed fragmentation of pentagalloylglucose were shown in Fig. 3B. Compounds 9, 10 and 11 had the same molecular formula C19 H26 O15 , and isomers of 1 -O-galloylsucrose, 6 -O-galloylsucrose and 6-Ogalloylsucrose matched it, which were reported previously exsiting in Paeoniae Radix lactiflora [26]. Their fragment ions further indicated that the losses of Glc (180 Da) and galloyl (170 Da) were from the precursor of [M−H]− , thus these three compounds were assigned as 1 -O-galloylsucrose (9), 6 -O-galloylsucrose (10) and 6-O-galloylsucrose (11).

3.1.3. Identification of phenolic acids A total of 17 phenolic acids were definitely identified by comparison with references (Table 2). The negative ion mode was much more suitable for the analysis of this kind of compounds. Except for methyl gallate (21), ethyl gallate (32), protocatechuic aldehyde (18) and succinic acid (4), other 13 phenolic acids including gallic acid (8), protocatechuic acid (13), neochlorogenic acid (16), chlorogenic acid (19), p-hydroxybenzoic acid (20), vanillic acid (24), 2-methoxycinnamic acid (25), caffeic acid (27), 4hydroxycinnamic acid (35), ferulic Acid (39), 3-hydroxycinnamic acid (45), 2-hydroxycinnamic acid (54) and cinnamic acid (65) had one carboxylic group in their structures, and their characteristic fragmentation behavior was the loss of CO2 (44 Da) (take cinnamic acid as an example, Fig. 3C). Moreover, the loss of CH3 or C2 H5 was the characteristic fragmentation behavior of methyl gallate (21), vanillic acid (24), ethyl gallate (32), 2-methoxycinnamic acid (25) and ferulic acid (39) with one methyl or ethyl group or moiety in their structures [27]. Succinic acid (4) contained two carboxyl groups, and there were no fragmentation ions in both positive and negative ion modes [28].

3.1.4. Identification of flavonoids A total of 36 flavonoids were identified or tentatively presumed in this study which mainly originated from Glycyrrhizae Radix. The detailed fragmentation information of flavonoids were listed in Table 2. 3.1.4.1. Flavones. 11 compounds were characterized as flavones, 10 of them were definitely identified via standard references comparisons except for compounds 30 (vicenin-2), which was tentatively deduced via comparing its molecular mass and MS/MS spectra with the literature data [28–31]. Majority of them exist in the form of glycosides with sugars attached to the flavonoid aglycones via C–O or C–C bond, except for 3 aglycones. The fragments 0.2X0 and Y0 in both negative and positive ion modes were considered as the characteristic fragment ions to differentiate Cglycoside and O-glycoside flavonoids. Vicenin-2 (30), schaftoside (33) and isoschaftoside (34) have similar fragmentation pathways to produce diagnostic fragments 0.2X0 (loss of C4 H8 O4 , 120 Da) and 0.3X0 (loss of C3 H6 O3 , 90 Da), suggesting these compounds are C-glycosyl flavonoids (take schaftoside as an example, Fig. 3D). Rutin (42), quercetin-7-O-glucopyranoside (43), isoquercitrin (55), astragalin (56) and luteoloside (64) have similar fragmentation pathways to produce diagnostic fragment Y0 (loss of Glc or rutinose group (Glc6 -1 Rha)), suggesting these compounds are O-glycosyl flavonoids. These compounds produced two diagnostic fragment Y0 − or [Y0 −H]− ions in the negative ion mode [32] (take rutin as an example, Fig. 3E). In addition, 3 flavone aglycones (compounds 64, 70 and 71) were clearly identified as kaempferol, quercetin and luteolin, and the characteristic fragmentation behaviors of the flavone aglycones were successive or simultaneous losses of H2 O (18 Da), CH2 O (30 Da), CO (28 Da), CO2 (44 Da), C2 H2 O (42 Da) and C3 O2 (68 Da), and reverse Diels-Alder (RDA) reaction [33] (take quercetin as an example, Fig. 3F) 3.1.4.2. Isoflavones. 6 compounds were characterized as isoflavones in this study. Compounds 28, 61 and 88 were clearly identified via standard references comparison, while compounds 67, 97 and 102 were tentatively identified via comparing their molecular mass and MS/MS spectra with the literature data [29,34]. Compound 28 (C-glycosyl isoflavonoid) was identified as puerarin which had the diagnostic fragment 0.2X0 (loss of C4 H8 O4 ,

W. Xu et al. / J. Chromatogr. B 986–987 (2015) 69–84

79

Fig. 1. Chemical structures of the identified compounds in GLGZD.

120 Da) [20]. Compounds 61 was identified as ononin (O-glycosyl isoflavonoid) which gave [M+HCOO]− ion in the negative ion mode and produced the diagnostic fragment Y0 − and [Y0 −CH3 ]− (Fig. 3G). Compound 88 (formononetin) was aglycone of ononin, which produced [M−CH3 ] ion in the both negative and positive ion mode. Compounds 67 and 97 had one methyl in their structures which characteristic fragmentation behavior also was the loss of CH3 (15 Da), and tentatively identified as biochanin A and licoricone [29,34]. Compound 102 was deduced as gancaonin C via comparing its molecular mass and MS/MS spectra (losses of H2 O, CH2 O and RDA fragmentations) with the literature data [29]. 3.1.4.3. Flavanones. 10 compounds were characterized as flavanones, including 7 O-glycosyl flavanones and 3 flavanone aglycones. Compounds 40, 41, 46, 52 and 63 were clearly identified

as liquirtin apioside, liquiritin, taxifolin, naringin and liquiritigenin by comparison with reference standards. As for O-glycosyl flavanones including glucoliquiritin apioside (26), naringenin 7-O(2-␤-d-apiofuranosyl)-␤-d-glucopyranoside (36), neoisoliquiritin (37), liquirtin apioside (40), liquiritin (41), liquiritigenin-7, 4diglucoside (50) and naringin (52), they produced diagnostic fragment Y0 − and further yielded fragment ions via RDA reaction (m/z 135 [A1,3 ]− , 119 [B1,3 ]− , and 153 [A1,3 +H2 O]− ) (take liquiritin as an example, Fig. 3H). Liquiritigenin (63), the aglycone of liquirtin (41), yielded diagnostic fragment ions via RDA reaction at m/z 135 [A1,3 ]− , 119 [B1,3 ]− , and 153 [A1,3 +H2 O]− in the negative ion mode [28,29,35–37]. Flavanone aglycone taxifolin (16) yielded diagnostic fragment ions via successive or simultaneous losses of H2 O (18 Da), CO (28 Da), CO2 (44 Da), CH2 O2 (46 Da), and RDA reaction at m/z 151 [A1,3 ]− and 177 [B1,4 ]− , while dihydroapigenin (81) yielded

80

W. Xu et al. / J. Chromatogr. B 986–987 (2015) 69–84

Fig. 2. HPLC–QTOF-MS/MS total ion chromatogram of GLGZD from negative ion mode (A) and positive ion mode (B).

diagnostic fragment ions via the loss of ring B and RDA reaction at m/z 165 [A1,2 ]− and 151 [A1,3 ]− . 3.1.4.4. Chalcones. 7 compounds were characterized as chalcones, 4 of them (compounds 57, 59, 90 and 103) were unambiguously identified via the reference compounds comparison while the other 3 compounds (58, 60 and 62) were tentatively identified via comparing their molecular mass and MS/MS spectra with the literature data [28,29,35–37]. Chalcones with C-2 -OH could transform to corresponding flavanone isomers, and then produced the same typical fragment ions via Y0 and RDA reaction [28,35]. For example, isoliquiritin (59) could transform to the corresponding flavanone isomer liquirtin (41), they both produced the same typical fragment ions Y0 at m/z 255 and RDA reaction at m/z 135 [A1,3 ]− , 119 [B1,3 ]− and 153 [A1,3 +H2 O]− in the negative ion mode (Fig. 3I). Compound 60 also had the same fragment pathway which was deduced as neoisoliquiritin via comparison with the literature data [29,35]. Compound 58 and isoliquiritin apioside (57) showed the same molecule formula and fragment ions at m/z 417 [M−H−Api]− (apiose, Api), 255 [M−H−Api−Glc]− , 135 [A1,3 ]− , 119 [B1,3 ]− and 153 [A1,3 + H2 O]− , so it was tentatively identified as neolicuraside [35,36]. Compounds 62, 95 and 103 had one methyl in their structures which characteristic fragmentation behavior was the loss of CH3 (15 Da), and yielded fragment ions via RDA reaction (A1,3 , B1,3 ), So they were deduced as licochalcone B, glycyrrhisoflavanone and licochalcone A, respectively [28,29,35,37]. In addition, compound 23 was identified as catechin via comparing with reference standard, which was only flavan-3-ols type flavonoid found in GLGZD [25,26]. 3.1.5. Identification of gingerols Compounds 92, 99, 105 and 106 were clearly identified as 6gingerol, curcumin, 8-gingerol and 6-shogaol by comparing with their reference compounds, which were originated from Zingiberis Rhizoma Recens [38,39]. These compounds had one carbonyl connected with a methylene (O C–CH2 –) in their structures. Their characteristic fragmentation behaviors often took place in ˛ or ˇposition of carbonyl group [40] (take 6-gingerol as an example, Fig. 3J). 3.1.6. Identification of triterpene saponins Triterpene saponins were recognized as the major active ingredients in Glycyrrhizae Radix [41]. In this work, a total of 20 triterpene saponins were identified and showed similar MS/MS behaviors. In the negative ion mode, all saponin compounds

produced [M−H]− ion, and the [M−H]− ion was further fragmented into ions at m/z 193 [glucuronic acid]− and 351 [2 glucuronic acid−H]− in MS/MS spectra (take glycyrrhizic acid as an example, Fig. 3K) [36]. In the positive ion mode, saponin compounds produced [M+H]+ or [M+Na]+ ion, and generated base peak ion at m/z [M+H−2GluA−H2 O]+ in MS/MS spectra. All these ions were important features for the identification of licorice saponin compounds [41]. Compoud 107 was the aglycone of compound 86, and both of them were unambiguously identified as glycyrrhetinic acid and glycyrrhizic acid via comparison with their reference compounds. The remaining compounds were tentatively assigned as uralsaponin C (66), 22-hydroxyl-glycyrrhizin or isomer (68 and 77), 22-hydroxyllicorice saponin G2 (69), licorice saponin H2 (72), licorice saponine A or isomer (73 and 75), 24-hydroxyl-licorice E2 (78), licorice saponine G2 or isomer (82 and 84), licorice saponine E2 (83), licorice saponine B2 (87), uralsaponin B or isomer (89 and 93), licorice saponine J2 (91) and 22-dehydroxyl-uralsaponin C (94) via comparing their exact molecular masses and MS/MS spectra with the literature data [28,29,36,41]. In addition, 2 triterpene saponins (compounds 80 and 85) originated from Jujubae Fructus were unambiguously identified as jujuboside A and jujuboside B via the reference compounds comparison, which were only detected in the negative ion mode. Their characteristic fragmentation behavior were the losses of Ara (130 Da) and Ara-Glc (294 Da) (take jujuboside A as an example, Fig. 3L). 3.1.7. Identification of others Gentiobiose (2), 3 -hydroxy-4 -O-metthylglabridin (98), glycycoumarin (100), 4’-O-methylglabridine (101) and glycyrol (104) were tentatively identified by comparing their exact molecular masses and MS/MS spectra with the literature data [29,35,37] except compounds 1 and 38 which were definitely identified as citrulline and scopoletin via comparison with the reference standards. 3.2. UPLC–QqQ MS quantitative analysis of GLGZD samples 3.2.1. Selection and confirmation of marker constituents The qualitative results indicated that monoterpene glycosides, galloyl glucoses, phenolic acids, flavonoids, gingerols and triterpene saponins were the major constituents in GLGZD. Among them, monoterpene glycosides from Radix paeniae Alba had protective effects on cerebral ischemic rats [42,43] and albiflorin and paeoniflorin, two monoterpene glycoside compounds, have been used as

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phytochemical markers of Radix Paeoniae Alba in Chinese Pharmacopoeia (2010 version) for their various bioactivities [44]. Galloyl glucoses such as pentagalloylglucose expresses multiple pharmaceutical including antioxidant, anti-inflammatory, cytoprotection

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and neuroprotective effect [45]. Phenolic acids offen showed good antioxidant activity, such as Cinnamic acid, a free radical scavenger, showed significant anti-inflammatory and anti-oxidation effects in a transient MCAO model, and also has been used as a phytochemical

Fig. 3. The ESI–QTOF-MS spectra and the proposed fragmentation pathway of paeoniflorin (A), pentagalloylglucose (B), cinnamic acid (C), schaftoside (D), rutin (E), quercetin (F), ononin (G), liquiritin (H), isoliquiritin (I), 6-gingerol (J), glycyrrhizic acid (K), and jujuboside A (L).

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Fig. 3. (Continued )

marker of Cinnamomi Ramulus [46]. Vanillic acid, a phenolic acids from Trichosanthis Radix, had antiplatelet aggregation effect that expressed potentials neuroprotective [47]. Flavonoids as an important kind of active compounds in GLGZD, their activity including anti-inflammatory, anti-apoptotic, antioxidant, neuroprotective effect etc. [48]. Liquiritin, a flavonoid compound, was found to protect against injury resulting from focal cerebral ischemia via its anti-oxidative and anti-apoptotic properties [49,50]. Gingerols, pungent agents of Zingiberis Rhizoma Recens such as 6-gingerol, was found to have neuroprotective effect against ischemic brain injury in BV-2 and primary microglial cell cultures [51]. In addition, Triterpene saponins, another major compounds in GLGZD. Glycyrrhizic acid, a phytochemical marker of Glycyrrhizae Radix,

and its related compounds were demonstrated to possess robust neuroprotection in the postischemic brain by inhibiting high mobility group box 1 phosphorylation and secretion [52,53]. Jujuboside A, a triterpene saponin constituent from Jujubae Fructus, upregulated inhibitory amino acid levels of neurotransmitters and reduced apoptosis in hippocampus Cerebral Ischemia in rats [54]. Therefore, 24 compounds either with high contents or strong bioactivities, including three monoterpene glycosides (oxypaeoniflorin, paeoniflorin and albiflorin), seven phenolic acids (gallic acid, protocatechuic acid, 4-hydroxybenzoic acid, methyl gallate, vanillic acid, 3-hydroxycinnamic acid, 2-hydroxycinnamic acid and cinnamic acid), six flavonoids (catechin, liquiritin, astragalin, isoliquiritin, liquiritigenin and licochalcone A), three gingerols (6-gingerol,

Fig. 4. The MRM chromatograms of 24 markers and 3 internal standards: (1) gallic acid; (2) protocatechuic acid; (3) oxypaeoniflorin; (4) catechin; (5) 4-hydroxybenzoic acid; (6) methyl gallate; (7) vanillic acid; (8) albiflorin; (9) paeoniflorin; (10) pentagalloylglucose; (11) liquiritin; (12) astragalin; (13) 3-hydroxycinnamic acid; (14) isoliquiritin; (15) 2-hydroxycinnamic acid; (16) liquiritigenin; (17) jujuboside a; (18) cinnamic acid; (19) glycyrrhizic acid; (20) 6-gingerol; (21) licochalcone a; (22) 8-gingerol; (23) 6-shogaol; (24) glycyrrhetinic acid; (IS1) swertiamarin; (IS2) nicotiflorin; (IS3) methylparaben.

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8-gingerol and 6-shogaol), three triterpene saponins (glycyrrhizic acid, glycyrrhetinic acid and jujuboside A) and one galloyl glucose (pentagalloylglucose) were selected as quantification markers in this study (Fig. 4). 3.2.2. Optimization of chromatographic systems and sample preparation In order to improve the resolution and sensitivity, the UPLC conditions were optimized, including type of column, mobile phase system, flow rate and column temperature. After compared with different brands of columns including Waters CORTECS C18 (2.10 mm × 100 mm, 1.6 ␮m, USA), Phenomenex kintecx (2.10 mm × 100 mm, 1.7 ␮m, USA) and Waters HSS T3 C18 (2.10 mm × 100 mm, 1.8 ␮m, USA), Waters CORTECS C18 finally was chosen for its successful separation of multi-component mixtures. In addition, different kinds of mobile phase, such as acetonitrile and methanol with a variety of modifiers (including 0.1% formic acid, 0.2% acetic acid and 5 mM ammonium acetate) were tested. The mixture of acetonitrile and 0.1% formic acid water solutions was the suitable mobile phase. Meanwhile, column temperature (25, 35 and 45 ◦ C) and flow rates (0.20, 0.25 and 0.30 mL/min) were studied. Finally, column temperature at 45 ◦ C and flow rate seting as 0.25 mL/min were used. For the MS conditions, the desired abundance of each transition and the collision energy values were optimized with manual tuning mode (Table 1). In order to achieve an efficient extraction, the parameters of different extraction method (reflux; ultrasonic and soxhlet extraction method), different extraction solvent systems (methanol-water solution (30, 50, 70 and 100%), different sample-solvent ratios (1:25, 1:50 and 1:100, w/v) and different extraction times (15, 30 and 45 min) were investigated. The optimal sample preparation was found to be the ultrasonic extraction of 0.50 g sample powder with 25 mL of 50% methanol-water solution in an ultrasonic water bath for 30 min. 3.2.3. Method validation 3.2.3.1. Linearity, LODs and LOQs. The internal standard method was employed to calculate the contents of 24 chemical markers in GLGZD. The stock solution was diluted with chromatographic grade methanol to seven different concentrations for the construction of calibration curves. Triplicate experiments were conducted for each

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concentration. The ratio of peak areas (Ai /As ) of each analyte to its corresponding internal standard was plotted against the concentrations (C). All the calibration curves indicated good linearity with determination coefficients (r) from 0.9959 to 0.9999. The LODs and LOQs were evaluated at a signal-to-noise ratio (S/N) of 3 and 10, respectively. The values of LODs and LOQs in this experiment were 0.03–30.6 and 0.12–70.9 ng/mL, respectively. (Table S-1). 3.2.3.2. Precision, repeatability, stability and recovery. To evaluate the precision of the present method, the intra- and inter-day precision were calculated by analyzing the working solution under optimized conditions, with respective RSD values less than 3.64% and 4.85%. In addition, the acceptable RSD values of repeatability (n = 6) and stability in 24 h (n = 6) were 1.45–3.87% and 2.11–4.84%, respectively (Table S-2). The recovery was validated by adding a known amount of stock standard solutions at different concentration levels (high, middle and low, n = 3) into a selected sample. The mixtures were analyzed in triplicate with optimized method. The recovery of this method varied from 94.94% to 103.66%, with the RSD values ranging from 1.46% to 5.12% (Table S-3). 3.2.4. Analysis of chemical makers of GLGZD samples The validated UPLC–QqQ MS analytical method was subsequently applied for the quantification of 24 representative compounds in 10 batches of GLGZD samples (Fig. 4). The analysis time was shortened to 7.5 min. Each marker compound was calculated by their respective calibration curve, and the quantification results from three parallel determinations were shown in Table 3. As a result, all 24 compounds were detected in the 10 GLGZD samples. Triterpene saponins and monoterpene glycosides were the predominant constituents. However, their contents varied widely in different samples. Glycyrrhizic acid, the maker component in Glycyrrhizae Radix, ranged from 2.23 to 4.51 mg/g, which could be affected mainly by different sources of plant material [55]. Paeoniflorin and albiflorin, two major marker components in Radix Paeoniae Alba, ranged from 1.37 to 4.63 mg/g and 1.45 to 3.68 mg/g, respectively. It was reported that these two compounds were affected by processing time or temperature in the manufacturing procedure which could cause their degradation [56]. It suggested that the quality control study of GLGZD should be focused on quality control of raw material and optimization of processing parameters.

Table 3 The contents of 24 compounds in 10 batches GLGZD (mg/g). Compounds

Lot.1

Lot.2

Lot.3

Lot.4

Lot.5

Lot.6

Lot.7

Lot.8

Lot.9

Lot.10

Gallic acid Protocatechuic acid Oxypaeoniflorin Catechin 4-Hydroxybenzoic Acid Methyl Gallate Vanillic Acid Albiflorin Paeoniflorin Pentagalloylglucose Liquiritin Astragalin 3-Hydroxycinnamic Acid Isoliquiritin 2-Hydroxycinnamic acid Liquiritigenin Jujuboside A Cinnamic acid Glycyrrhizic acid 6-Gingerol Licochalcone A 8-Gingerol 6-Shogaol Glycyrrhetinic acid

0.2668 0.0141 0.0223 0.1722 0.0236 0.0306 0.0203 4.2135 3.3029 0.2414 0.2947 0.0151 0.0105 0.1419 0.0251 0.1231 0.0104 0.0786 2.6935 0.0356 0.0194 0.0176 0.0082 0.0127

0.3753 0.0226 0.0374 0.1664 0.0311 0.0254 0.0186 1.9277 3.5218 0.3929 0.3322 0.0145 0.0142 0.1427 0.0241 0.1375 0.0097 0.0589 4.1612 0.0471 0.0139 0.0145 0.0064 0.0175

0.3572 0.0195 0.0332 0.1691 0.0341 0.0328 0.0173 2.7882 3.5092 0.2585 0.2946 0.0129 0.0175 0.1232 0.0269 0.1040 0.0102 0.0390 3.5679 0.0547 0.0156 0.0150 0.0053 0.0140

0.2325 0.0215 0.0350 0.1271 0.0211 0.0275 0.0262 2.3403 2.5583 0.3470 0.3260 0.0208 0.0209 0.1040 0.0160 0.1758 0.0138 0.0600 4.5134 0.0247 0.0186 0.0074 0.0088 0.0258

0.3279 0.0227 0.0370 0.1612 0.0303 0.0201 0.0206 3.2198 2.9453 0.3800 0.3401 0.0131 0.0071 0.1140 0.0140 0.1124 0.0098 0.0716 4.1385 0.0482 0.0232 0.0141 0.0176 0.0160

0.1823 0.0245 0.0291 0.1884 0.0247 0.0286 0.0120 2.8039 2.4306 0.3136 0.1956 0.0162 0.0109 0.1190 0.0096 0.1362 0.0066 0.0421 3.7570 0.0270 0.0141 0.0127 0.0072 0.0122

0.3172 0.0265 0.0421 0.1979 0.0270 0.0258 0.0178 1.6817 3.2342 0.3126 0.2896 0.0317 0.0151 0.1332 0.0200 0.1504 0.0145 0.0832 3.3752 0.0425 0.0178 0.0081 0.0071 0.0125

0.2114 0.0170 0.0440 0.1587 0.0158 0.0233 0.0257 2.5668 1.3720 0.2953 0.2743 0.0131 0.0189 0.1091 0.0200 0.2044 0.0102 0.1041 2.9903 0.0589 0.0210 0.0107 0.0075 0.0124

0.3064 0.0291 0.0261 0.1930 0.0247 0.0322 0.0229 1.4547 3.0897 0.2783 0.2583 0.0115 0.0129 0.1041 0.0230 0.1799 0.0106 0.1151 2.6089 0.0559 0.0136 0.0164 0.0084 0.0189

0.2014 0.0113 0.0380 0.1640 0.0229 0.0222 0.0159 3.6767 4.6278 0.2612 0.2432 0.0131 0.0076 0.1001 0.0260 0.1951 0.0094 0.0961 2.2282 0.0499 0.0286 0.0267 0.0041 0.0168

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4. Conclusion For the first time, the chemical profile of GLGZD was thoroughly and systematically investigated by HPLC–QTOF-MS. 106 compounds were unambiguously or tentatively identified, the characteristic behaviors of monoterpene glycosides, galloyl glucoses, phenolic acids, flavonoids, gingerols and triterpene saponins were summarized. Based on the qualitative analysis, a rapid method was established for quantitative analysis of 24 representative compounds in GLGZD by UPLC–QqQ MS, which has been demonstrated to be effective for the analysis of 10 batches of GLGZD. This study would facilitate the quality control of GLGZD for safe and efficacious use. It could also provide a basis for further study in vivo of GLGZD. Acknowledgements This work was done in the Collaborative Innovation Center for Rehabilitation Technology and TCM Rehabilitation Research Center of SATCM. It was supported by the Important Subject of Fujian Province Science and Technology Hall of China (2012Y0041), the Important Subject of Fujian province Education Hall of China (JA12176), the National Natural Science Foundation of China (81373940), the Specialized Research Fund for the Doctoral Program of Higher Education (20133519120001) and the Fujian University of Traditional Chinese Medicine Foundation (X2013014 and X2013015). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb. 2015.02.002. Referemces [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]

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