Rapid Analysis of the Main Components of the Total
Glycosides of Ranunculus japonicus by UPLC/Q-TOF-MS
Wen Ruia, Hongyuan Chenb, Yuzhi Tanc, Yanmei Zhonga and Yifan Fenga,*aCentral laboratory, GuangDong Pharmaceutical University, Guangzhou, P.R.China 510006 bDepartment of Microbiology Immunology, GuangDong Pharmaceutical University, Guangzhou, P.R.China 510006
cDepartment of Pharmaceutical Sciences, GuangDong Pharmaceutical University, Guangzhou, P.R.China 510006
Received: January 12th, 2010; Accepted: March 9th, 2010
A rapid method for the analysis of the main components of the total glycosides of Ranunculus japonicus (TGOR) was developed using ultra-performance liquid chromatography with quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS). The separation analysis was performed on a Waters Acquity UPLC system and the accurate mass of molecules and their fragment ions were determined by Q-TOF MS. Twenty compounds, including lactone glycosides, flavonoid glycosides and flavonoid aglycones, were identified and tentatively deduced on the basis of their elemental compositions, MS/MS data and relevant literature. The results demonstrated that lactone glycosides and flavonoids were the main constituents of TGOR. Furthermore, an effective and rapid pattern was established allowing for the comprehensive and systematic characterization of the complex samples.
Keywords: total glycosides, Ranunculus japonicus, UPLC/Q-TOF, MS/MS, lactone glycosides, flavonoid glycosides.
Ranunculus japonicus Thunb. is a Chinese herbal
medicine traditionally used in the treatment of malaria, jaundice, migraine, stomach ache, arthralgia, crane-like arthropathy, ulcer, toothache, and eye inflammation [1]. Pharmacological studies have showed that the total glycosides of R. japonicus (TGOR) possess anti-inflammatory and analgesic activities [2]. In addition, TGOR has also been found to inhibit angiotensin II (AngII) from inducing cardiac myocyte hypertrophy in rats [3]. Phytochemical studies of Ranunculus species have led to the isolation of lactones, flavonoids, saponins, organic acids, steroids, and alkaloids [4]. However, there are few relevant reports about the chemical constituents of R. japonicas [4a,4b]. Three lactones, ranunculinin, isoranunculinin, and dihydroranunculinin, have been previously isolated and identified using conventional column chromatographic and spectroscopic procedures [4b]. Therefore, the development of an effective method to identify the main components in TGOR is very important. Compared with conventional methods, ultra-performance liquid chromatography with quadruple-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) is rapid, sensitive in separation, accurate in mass measurement and tandem
mass spectrometry (MS/MS), and can be used for the analysis of complex components [5]. This paper describes an effective method to analyze TGOR using UPLC/Q-TOF MS, and attempts to reveal more useful information about the drugs bioactivity.
The main components of TGOR are glycosides, which are highly polar compounds. Therefore, aqueous formic acid in the mobile phase, and a high-gradient slope were applied to the chromatographic method. As a result, each glycoside in TGOR was separated in 30 minutes and the efficiency in the ionization of compounds was increased for Q-TOF-MS. The chromatogram of TGOR by UPLC/Q-TOF-MS is shown in Figure 1. Also, it was found that the glycosides of TGOR were broken easily when the cone voltage was set over 30 V. Due to this, the cone voltage was evaluated at 15, 20, 25 and 30 V, respectively. As a result, the cone voltage at 15V in the positive mode and 20V in the negative mode were applied for minimal fragmentation and better sensitivity. The collision energy (CE) is the most effective parameter for MS/MS of TGOR. The value of CE was evaluated from 15eV to 60eV in order to obtain
No. 5
783 - 788
Figure 1: The base peak ion (BPI) chromatograms of TGOR obtained by UPLC/Q-TOF-MS in (a) positive ESI mode and (b) negative ESI mode. Peak
numbers are consistent with those in Tables 1 and 2.
adequate characteristic fragment ions for most of the compounds of TGOR.
The base peak ion (BPI) chromatograms of TGOR obtained by UPLC/Q-TOF-MS in both positive and negative ion mode are presented in Figures 1 a and b. It was found that the feature ions were quasi-molecular [M+H]+, adduct [M+Na]+ and quasi-molecular [M−H] + ions.
The Q-TOF MS provided the elemental compositions of both molecular and some characteristic fragment ions. The elemental compositions of 20 peaks were calculated by the Masslynx software, and the results are shown in Table 1. The structures of the detected compounds were deduced by TOF-MS, MS/MS fragment ions, comparison with standard compounds, and literature data [6]. The results indicate that the main components of TGOR are lactone glycosides, flavonoid glycosides, and aglycones; the lactone glycosides include ranunculin and its derivates, flavonoid glycosides, including fifteen C-glycosides and O-glycosides of which the major mother nuclei flavones are luteolin, apigenin and tricin.
It was demonstrated that ions [M−H−60]−, [M−H−90]− and [M−H−120]− were characteristic of C-glycoside flavonoids [5e]. An example is presented as follows. The MS/MS peak at 10.43 min in the positive ion mode was confirmed as vitexin by its retention time and MS compared with the standard compound. The predominant ions appeared at m/z 313 and 283, which corresponded to the ring cleavage of glycosyl, followed by a loss of 30 Da (CH2O) [7]. The fragment ions at m/z
415, 397 and 379 via the loss of one, two and three molecules of H2O are characteristic fragmentation
pathways. The product ion [M+H−162]+ corresponds to
the loss of one molecule of glucose. Pathways of
cleavage are shown in Figure 2. Analogous MS/MS data appeared in compounds 6-12, 14 and 16.
Compounds 8 and 10 are isomers. The difference in these two compounds was observed in negative mode in their MS/MS, which gave ions for [M−H−90]− at m/z 357, [M−H−120]− at m/z 327 and [M−H−150]− at
m/z 297, consistent with the characteristic ions of a C-glycoside. The relative intensity of ions in the
ESI-MS/MS is shown in Table 2. A more reliable means to differentiate the 8-C- and 6-C-glucosides of orientin and isorientin is based on the product ion at m/z 327 obtained in the MS/MS experiment, since certain ions [M−H−120]- are observed at higher abundance in the spectra of 6-C-glucosides than those in the spectra of 8-C-glucosides [8]. According to the elemental compositions, MS/MS and literature data, compounds 8 and 10 were identified as orientin and isorientin, respectively [4k, 6b].
The MS/MS spectra of flavonoid O-flavonoid glycosides such as compounds 3, 5, 13, 15 and 17-18 showed the predominant neutral loss of 162u, 132u and 176u, corresponding to hexose, pentose and glucuronic acid residues respectively. Compound 1 and compound 15 yielded ions at both m/z 493 [M+H]+ and m/z 491 [M−H]−. From the MS fragments and literature data, compound 13 was plausibly characterized as
tricin-7-O-β-D-glucoside [4e,6d], and compound 15 was
tentatively deduced as tricin with a hexose.
Using an accurate method to measure elemental composition and applying the relevant MS fragmentation rules reported before, three lactone glycosides, fifteen flavonoid glycosides, and two flavonoid aglycones were either identified or tentatively deduced from TGOR. Among them, only ranunculin and tricin have been previously reported for Ranunculus
Table 1: MS and MS/MS data of (+) ESI-MS and (−) ESI-MS, and identification of the constituents of TGOR.
Peak no.
tR
(min) Selected ion
Measured mass (m/z)
Calc. mass
(m/z) ppm Formula ESI-MS/MS (m/z Identification 1 3.15 [M+H]+ 571.1906 571.1874 5.6 C 22H35O17 571[M+H]+,355[M+H-hexose-3H 2O]+ ,277[M+H-hexose-pentose]+ ,259[M+H-hexose-pentose-H2O]+,115[M+H-2hexose-pentose]+ [M+Na]+ 593.1717 593.1694 3.9 C 22H34O17Na [M-H]- 569.1699 569.1718 -3.3 C 22H33O17 569[M-H]-,551[M-H-H2O]-,389[M-H-hexose-H2O] -,275[M-H-hexose-pentose]-,113[M-H-2hexose- pentose] -Ranunculin derivative 2 3.85 [M-H]- 275.0784 275.0767 6.2 C 11H15O8 275[M-H]-,113[M-H-glc]-,71[M-H-glc-C2H2O]- Ranunculin 3 6.35 [M+H]+ 759.2011 759.1984 3.6 C 32H39O21 759[M+H]+,465[M+H-hexose-pentose]+ ,303[M+H-2hexose-pentose]+ [M+Na]+ 781.1817 781.1803 1.8 C 32H38O21Na [M-H]- 757.183 757.1827 0.4 C 32H37O21 757[M-H]-,595[M-H-hexose]- ,463[M-H-hexose-pentose] -flavonoid O-glycoside 4 6.60 [M+H]+ 329.1224 329.1236 -3.6 C 15H21O8 329[M+H] +,121[M+H-hexose-CH 2O2]+ ,85[M+H-hexose-C5H6O]+ [M+Na]+ 351.1069 351.1056 3.7 C 15H20O8Na [M-H]- 327.1071 327.1080 -2.8 C 15H19O8 327[M-H]-,165[M-H-hexose]- ,147[M-H-hexose-H2O]-,89[M-H-hexose-H2O-C2H2O2] -lactone glycoside 5 7.02 [M+H]+ 921.2504 921.2512 -0.9 C 38H49O26 921[M+H]+ ,465[M+H-2hexose-pentose]+,303[M+H-3hexose-pentose]+ [M+Na]+ 943.2280 943.2332 -5.5 C 38H48O26Na [M-H]- 919.2363 919.2356 0.8 C 38H47O26 919[M-H]-,757[M-H-hexose]-,595[M-H-2hexose] -,463[M-H-2hexose-pentose] -flavonoid O-glycoside 6 8.05 [M+H]+ 611.1642 611.1612 4.9 C 27H31O16 611[M+H] +,449[M+H-hexose]+ ,287[M+H-2hexose]+ [M+Na]+ 633.1470 633.1432 6.0 C 27H30O16Na [M-H]- 609.1465 609.1456 1.5 C 27H29O16 609[M-H]-,489[M-H-C4H8O4]-,447[M-H-hexose] -,327[M-H-hexose-C4H8O4]-,285[M-H-2hexose] -flavonoid C-glycoside 7 8.85 [M+H]+ 581.1531 581.1506 4.3 C 26H29O15 581[M+H]+,527[M+H-3H 2O]+,497[M+H-3H2 O-2CH2]+,449[M+H-Xyl]+,443[M+H-C4H8O4 -H2O]+,425[M+H-C4H8O4-2H2O]+ ,395[M+H-Xyl-3H2O]+ [M+Na]+ 603.1351 603.1326 4.1 C 26H28O15Na [M-H]- 579.1352 579.135 0.3 C 26H27O15 579[M-H] -,489[M-H-C 3H6O3]-,459[M-H-C4H8O4] -,429[M-H-C4H8O4-CH2O]-,399[M-H-glc-H2O] -Adonivernite 8 9.40 [M+H]+ 449.1085 449.1084 0.2 C 21H21O11 449[M+H]+,413[M+H-2H 2O]+,353[M+H-2H2 O-C2H4O2]+,329[M+H-C4H8O4]+,299[M+H-C4H8O4 -CH2O]+ [M-H]- 447.0937 447.0927 2.2 C 21H19O11 447[M-H] -,357[M-H-C 3H6O3]-,327[M-H-C4H8O4] -,297[M-H-C4H8O4-CH2O]-,285[M-H-glc] -Orientin 9 9.55 [M+H]+ 565.1584 565.1557 4.8 C 26H29O14 565[M+H]+,511[M+H-3H 2O]+,481[M+H-3H2 O-2CH2]+,433[M+H-arabinose]+,427[M+H-C4H8O4 -H2O]+,409[M+H-C4H8O4-2H2O]+,379[M+H- arabinose -3H2O]+ [M+Na]+ 587.1373 587.1377 -0.7 C 26H28O14Na [M-H]- 563.1379 563.1401 -3.9 C 26H27O14 563[M-H] -,503[M-H-C 2H4O2]-,473[M-H-C3H6O3] -,443[M-H-C4H8O4]-,383[M-H-glc-H2O] -6-C-β-D -Glucosyl-8-C-α-L -arabinosylapigenin 10 9.95 [M+H]+ 449.1106 449.1084 4.9 C 21H21O11 [M-H]- 447.0946 447.0927 4.2 C 21H19O11 447[M-H] -,357[[M-H-C 3H6O3]-,327[M-H-C4H8O4] -,297[M-H-C4H8O4-CH2O]-,285[M-H-glc] -Isorientin 11 10.43 [M+H]+ 433.1128 433.1135 -1.6 C 21H21O10 433[M+H]+,415[M+H-H 2O]+,397[M+H-2H2O]+, 379[M+H-3H2O]+,351[M+H-3H2O-CO]+, 323[M+H-2H2O-C3H6O2]+ ,313[M+H-C4H8O4]+,283[M+H-C4H8O4-CH2O]+ ,271[M+H-glc]+ [M-H]- 431.0977 431.0978 -0.2 C 21H19O10 431[M-H] -,341[[M-H-C 3H6O3]-,311[M-H-C4H8O4]-, 283[M-H-C5H8O5] -Vitexin 12 10.83 [M+H]+ 535.1475 535.1452 4.3 C 25H27O13 535[M+H] +,481[M+H-3H 2O]+ ,463[M+H-C3H4O2]+,445[M+H-C3H6O3]+ [M+Na]+ 557.1273 557.1271 0.4 C 25H26O13Na [M-H]- 533.1284 533.1295 -2.1 C 25H25O13 533[M-H]-,515[M-H-H2O]-,473[M-H-C2H4O2] -,443[M-H-C3H6O3]-,383[M-H-C4H8O4-CH2O]-, 353[M-H-2C3H6O3] -flavonoid C-glycoside 13 11.77 [M+H]+ 493.1364 493.1346 3.7 C 23H25O12 493[M+H]+, 331[M+H-glc]+ [M-H]- 491.1203 491.119 2.6 C 23H23O12 491[M-H]-, 329[M-H-glc] -Tricin-7-O-β-D -glucoside
Table 1 (contd.) 14 12.73 [M+H]+ 491.1202 491.119 2.4 C 23H23O12 491[M+H] +,455[M+H-2H 2O]+,353[M+H-C4H8O4 -H2O]+,329[M+H-glc]+ [M+Na]+ 513.098 513.1009 -5.7 C 23H22O12Na [M-H]- 489.1027 489.1033 -1.2 C 23H21O12 489[M-H]-,429[M-H-C 2H4O2]-,327[M-H-glc] -flavonoid C-glycoside 15 12.84 [M+H]+ 493.137 493.1346 4.9 C 23H25O12 493[M+H]+, 331[M+H- hexose]+ [M-H]- 491.1166 491.119 -4.9 C 23H23O12 491[M-H] -,476[M-H-CH 3]-,461[M-H-2CH3] -,329[M-H- hexose]-,313[M-H-glc-O] -Tricin+hexose 16 13.93 [M+H]+ 475.1262 475.124 4.6 C 23H23O11 475[M+H]+,457[M+H-H 2O]+,397[M+H-H2 O-C2H4O2]+,379[M+H-2H2O-C2H4O2]+,313[M+H- hexose]+,283[M+H- hexose -CH 2O]+ [M-H]- 473.1105 473.1084 4.4 C 23H21O11 473[M-H] -,413[M-H-C 2H4O2]-,311[M-H-hexose] -,281[M-H- hexose -CH2O] -flavonoid C-glycoside 17 14.56 [M+H]+ 447.0929 447.0927 0.4 C 21H19O11 447[M+H]+,271[M+H-C6H8O6]+ [M+Na]+ 469.0754 469.0747 1.5 C 21H18O11Na [M-H]- 445.0745 445.0771 -5.8 C 21H17O11 445[M-H]-,269[M-H-C6H8O6] -Apigenin+ glucuronic acid 18 17.01 [M+H]+ 461.11 461.1084 3.5 C 22H21O11 461[M+H] +,285[M+H-C 6H8O6]+ ,270[M+H-C6H8O6-CH3]+ [M+Na]+ 483.0921 483.0903 3.7 C 22H20O11Na [M-H]- 459.0948 459.0927 4.6 C 22H19O11 459[M-H] -,283[M-H-C 6H8O6]-,268[M-H-C6H8O6 -CH3] -flavonoid O-glycoside 19 17.62 [M+H]+ 331.0819 331.0818 0.3 C 17H15O7 331[M+H] +,315[M+H-O]+,270[M+H-C 2H5O2]+, 242[M+H-C4H9O2]+ [M-H]- 329.0641 329.0661 -6.1 C 17H13O7 329[M-H] -,299[M-H-2CH 3]-,271[M-H-C2H2O2] -,243[M-H-C4H6O2] -Tricin 20 27.91 [M+H]+ 279.1584 279.1596 -4.3 C 16H23O4 279, 149, 121, 54 [M+Na]+ 301.1412 301.1416 -1.3 C 16H22O4Na Uncertain OH O O H OH O OH OH OH OH O OH O O H OH O OH O O H OH CHO O OH O O H OH O OH O O H OH O OH OH O OH OH O O H OH O O OH OH OH O O H OH O O OH -glc -C4H8O4 -CH2O -H2O -H2O OH O O H OH O O -H2O -CO OH O HO OH O OH -C3H6O2 + + + + + + + + m/z 433 m/z 313 m/z 415 m/z 397 m/z 379 m/z 351 m/z 323 m/z 283 m/z 271
Figure 2: The cleavage pathways of vitexin in positive ion mode. Table 2: Relative intensity of ions in ESI-MS/MS of compounds 10 and
8. Peak [M−H−90] − (%) [M−H−120]− (%) [M−H−150]− (%) [M−H−glc]− (%) 8 100 67 20 9 10 16 100 20 12
orientin, isorientin, vitexin, 6-C-β-D-glucosyl-8-C-α-L -arabinosylapigenin, and tricin-7-O-β-D-glucopyranoside from this plant. In this study, a simple, rapid and sensitive method was established for screening the main components in TGOR by UPLC/Q-TOF-MS, and several new glycosides were detected in the present
study. This experiment demonstrates that UPLC/ Q-TOF-MS is a powerful and rapid technique for analyzing and discovering the complex constituents of Chinese herbal medicines.
Experimental
Materials and chemicals: Acetonitrile (HPLC grade)
was purchased from Merck (Germany), and formic acid from DIMA (USA); water was purified by an arium 611UV system (Sartorius, Germany). Other solvents and chemicals were bought from Guangzhou Chemical Agent Factory (Guangzhou, China). Vitexin was purchased from SIGMA (USA).
Instruments and methods: A Waters Acquity Ultra
Performance LC system (Waters Co., USA) was used equipped with a binary solvent manager, and tunable UV detector (TUV). The chromatographic conditions were as follows: Waters UPLC BEH C18 column (1.7 µm, 50 mm × 2.1 mm); mobile phase: a gradient elution system of 0.1% aqueous formic acid solution (A) and acetonitrile (B), 0–4 min, 98% A–95% A, 4–20 min, 95% A–70% A, 20–25 min, 70% A–50% A, 25–30 min, 50% A–0% A; flow rate: 0.4 mL/min; column temperature: 30oC; injection volume: 5 µL.
A Waters Micromass Q-TOF micro system (Waters Co., UK) was used equipped with a LockSpray and ESI interface operating in both positive and negative ion
modes. The source parameters were electrospray capillary voltage 3.5 kV for positive ionization mode and 3.2 kV for negative ionization mode; source temperature 110oC and desolvation temperature 350oC. The cone voltage was set at 15V for positive ionization mode and 20V for negative ionization mode. Nitrogen and argon were used for cone and collision gases, respectively. The cone and desolvation gas flows were 60 L/h and 600 L/h, respectively. The TOF mass spectrometer was calibrated routinely in the positive electrospray ionization (ESI+) mode using sodium formate solution. Data were collected from m/z 90 to 1000 in continuum mode, using independent reference lock-mass ions via the LockSpray interface to ensure mass accuracy and reproducibility. The solution of leucine enkephalin (Sigma Chemical Co.) was used as lock-mass, with an [M+H]+ ion at m/z 556.2771 and
[M−H]− ion at m/z 554.2615. The MS/MS experiments
were performed using a variable collision energy (15– 60 eV), which was optimized for each individual compound. The LockSpray frequency was set at 10s. The Acquity UPLC/Q-TOF micro system was operated using MassLynx 4.1 software (Waters Co., USA). The accurate mass and composition for the precursor and fragment ions were calculated by MassLynx 4.1 software.
Plant material and sample preparation: Ranunculus
japonicus Thunb. was collected from Jiujiang (Jiangxi
Province, China). The herb was authenticated by lecturer Jizhu Liu, Guangdong Pharmaceutical University.
The air-dried and cut R. japonicus was extracted under reflux with 85% ethanol, thrice. The solutions were filtered, pooled, and evaporated under vacuo to obtain an EtOH extract. This was suspended in distilled water, and successively partitioned in a 1:1 ratio with ethyl acetate. The water fraction was absorbed onto a D-101 macroporous resin column and eluted with water and ethanol. The ethanol fraction formed the total glucosides of R. japonicus (TGOR). TGOR was dissolved in methanol:water (1:1, v/v). Vitexin was dissolved in methanol and filtered through 0.22 μm membranes for analysis.
Acknowledgments - This work is supported by the Research Foundation of Science and Technology Plan Project in Guangdong Province, P.R. China, under Grant No. 2004B30101002.
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