|Year : 2017 | Volume
| Issue : 49 | Page : 175-179
Rapid analysis of components in Coptis chinensis Franch by ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry
Peng-peng Tian1, Xiao-xu Zhang1, Hong-ping Wang2, Pu-ling Li3, Yu-xin Liu4, Shao-jing Li5
1 Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Dongzhimen, Beijing; Laboratory of Cell Pharmacology, College of Pharmaceutical Sciences, Hebei University, Baoding, China
2 Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Dongzhimen, Beijing, China
3 Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Dongzhimen, Beijing; Henan University of Traditional Chinese Medicine, Zhengzhou, China
4 Laboratory of Cell Pharmacology, College of Pharmaceutical Sciences, Hebei University, Baoding; Drug Quality Control Key Laboratory of Hebei Province, Hebei University, Baoding, China
5 Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Dongzhimen, China
|Date of Submission||11-Jan-2016|
|Date of Acceptance||12-May-2016|
|Date of Web Publication||06-Jan-2017|
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences,Dongzhimen, Beijing
College of Pharmaceutical Sciences, Hebei University, Baoding
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Coptis chinensis Franch is a traditional Chinese medical herb.Objective: In this article, ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was used to rapidly, qualitatively, and comprehensively identify the components in Coptis chinensis Franch. Materials and Methods: Chromatographic separation was achieved on an Agilent Zorbax RRHD Eclipse Plus C18 column. The mobile phase consisted of 0.1% formic acid water (A) and 0.1% formic acid acetonitrile (B) with a gradient program. Qualitative analysis was performed on an Agilent 6540 quadrupole time-of-flight mass spectrometer, which was equipped with a Dual AJS ESI source operating in negative mode. Results: A total of 30 alkaloid and non-alkaloid components of Coptis chinensis Franch were identified in only 14 min.Conclusion: This study helped to provide a basis for the quality control of Coptis chinensis Franch.
Abbreviations used: Q-TOF-MS: quadrupole time-of-flight mass spectrometry, UPLC: ultra-performance liquid chromatography, pos: positive, neg: negative.
Keywords: Alkaloids, Coptis chinensis Franch, non-alkaloids, Q-TOF, UPLC
|How to cite this article:|
Tian Pp, Zhang Xx, Wang Hp, Li Pl, Liu Yx, Li Sj. Rapid analysis of components in Coptis chinensis Franch by ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry. Phcog Mag 2017;13:175-9
|How to cite this URL:|
Tian Pp, Zhang Xx, Wang Hp, Li Pl, Liu Yx, Li Sj. Rapid analysis of components in Coptis chinensis Franch by ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry. Phcog Mag [serial online] 2017 [cited 2022 May 21];13:175-9. Available from: http://www.phcog.com/text.asp?2017/13/49/175/197635
- Qualitative analysis method of chlorogenic alkaloids and non-alkaloids in Coptis chinensis Franch is developed by Ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) method.
- Established UPLC-Q-TOF-MS/MS analysis method is validated with rapidness and accuracy.
- The developed method was successfully applied for qualitative analysis of Coptis chinensis Franch sample collected from cultivation place in China.
| Introduction|| |
The traditional Chinese medicine, Coptis chinensis, is the dry root and stem of Coptis chinensis Franch, and is widely used in clinic. It is also called 'Weilian'. Coptis chinensis is a common detoxification agent in traditional Chinese medicine, which can purge fire and clear heat, with a very bitter taste. Earlier assessment has verified the antibacterial and anti-inflammatory functions of the active components of Coptis chinensis. Many studies,,, have also evaluated the pharmacodynamics effects of the active components on high blood sugar, high cholesterol, arrhythmia, cerebral ischemia, and heart failure. Liu et al., studied the main active components in Coptis chinensis using high-performance liquid chromatography. However, the analysis was incomplete, and non-alkaloids were seldom reported.
Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) has been widely used in the field of analytical chemistry and in the quality control of traditional Chinese medicine,,, because of its high resolution, high sensitivity, and high resolution. UPLC-Q-TOF-MS/MS can extrapolate the molecular formula and chemical structural composition of compounds according to the molecular weight and fragment ions in the secondary MS of the compound. In this study, UPLC-Q-TOF-MS/MS was used to identify the alkaloids and non-alkaloids in Coptis chinensis Franch.
| Materials and Methods|| |
Chemicals and instruments
Methanol, formic acid, and acetonitrile (all LC-MS grade) were purchased from Thermo Fisher (United States). Other reagents were of analytical grade. Agilent 1290 UPLC, which was equipped with a binary pump, an online degasser, a column oven, an autosampler, and a diode array detector, was purchased from Agilent Technologies Inc. The Agilent 6540 TOF resolution mass spectrometer, which was equipped with a Dual AJS ESI ion source and a Masshunter Data Acquisition Online Workstation and Qualitative Analysis Offline Analysis Software, was purchased from Agilent Technologies Inc. A KQ-250B ultrasonic cleaner was purchased from Kunshan Ultrasonic Instrument Co., Ltd. An N-1100 rotary evaporator was purchased from Shanghai Ailang Instrument Co., Ltd. A BP211D Balance was purchased from Sartorius Scientific Instrument Co., Ltd. A DFT-50 type grinder was purchased from Wenling Linda Machinery Co., Ltd.
Coptis chinensis was purchased from Chongqing Wanglong Berberine Ltd. and identified to be the root and stem of Coptis chinensis Franch by Dr. Wei Sun of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences.
Coptis chinensis Franch was crushed into powder, which was filtered with a 40-mesh screen. Then, 1.0 g of powder, which was mixed with 10 mL of methanol (70%), was extracted ultrasonically 30 min before the collection of the filtrate. The remaining powder was treated with methanol (70%) twice according to the above-mentioned method. Three mixed filtrates were concentrated by evaporation using a rotary evaporator until the methanol was completely evaporated. The 160 × sample preparation was finished after the mixture of methanol (70%), and the evaporated extract was standardized to be 5 mL. Then, the 160 × sample was diluted and filtered with a 0.22 μm microporous membrane before the injection.
For the analysis, a 1290 series UPLC system coupled to a 6540 quadrupole TOF MS was used. The 6540 Q-TOF system was equipped with an Agilent JetStream ESI interface and was operated by Masshunter Workstation B.04.01 software. Precursor and production selection, and the optimization of collision energies, were performed with flow injection of single-analyte solutions using Masshunter Optimizer software. The analytical column was a ZORBAX RRHD Eclipse Plus C18 (100 × 3 mm, 1.8 μm) column from Agilent Technologies. Chromatographic separation was performed at 45°C with a flow rate of 800 μL/min. Eluent A was composed of water/formic acid (99.9:0.1, v/v), and eluent B was composed of acetonitrile/formic acid (99.9:0.1, v/v). The total time of the chromatographic run was 14 min, which comprised the following: 0–4 min, 5–15% B; 4–5 min, 15–17% B; 5–12 min, 17–24% B; and 12–14 min, 24–95% B.
The general source settings in the positive (pos.) and negative (neg.) ionization modes were as follows: gas temperature, 300°C; gas flow, 5 L/min; nebulizer, 35 psi; sheath gas temperature, 350°C; sheath gas flow, 11 L/min; capillary voltage, 4000 V (pos.) and 3500 V (neg.); nozzle voltage, 1500 V; capillary outlet voltage, 175 V; collision energy, 30 V; and reference mass, m/z 121.0509, 922.0098 (pos.), 119.0363, 1033.9881 (neg.).
| Results|| |
UPLC-Q-TOF Total Ion current chromatogram of the Extract of Coptis chinensis Franch
As shown in [Figure 1], the components of the sample solution were collected and analyzed qualitatively in both positive and negative modes within 14 min, and they were well separated.
|Figure 1: UHPLC-Q-TOF total ion current chromatogram of the extract of Coptis chinensis Franch. A: Negative mode; B: Positive mode|
Click here to view
Identification of alkaloids in the extract of coptis chinensis Franch by UPLC-Q-TOF
The main alkaloids identified in the extract of Coptis chinensis Franch included three apomorphine alkaloids, three tetrahydroprotoberberines alkaloids, and 17 protoberberine alkaloids, as shown in [Figure 2]. Most of the side chains of the alkaloids were connected with -O-CH2-O-, -OCH3, -OH, and -CH3. There is a quasi-molecule ion peak of M]+ or [M+H]+ in the positive-mode ESI mass spectrum of most alkaloids. The collision-induced dissociation (CID) qualified the cleavage of the side chain and the opening and closing of the cyclic structure in the MS2 spectra.
|Figure 2: Chemical structures of the most reported alkaloids in the extract of Coptis chinensis Franch. A: Apomorphine alkaloids; B: Tetrahydroprotoberberines alkaloids; C: Protoberberine alkaloids|
Click here to view
In the MS2 spectra of peaks 5, 8, and 11, the [M-C2H7-N]+], [M-C2H7-CH3], and [M-C2H7N-CH3-CH3OH]+ ions were observed, which was consistent with the structural assignment of the apomorphine alkaloid, suggesting that peaks 5, 8, and 11 may be this type of alkaloid.
Peak 5 shows three strong absorption bands (225-235, 270-280, and 315-335 nm) in the ultraviolet (UV) spectrum. As shown in [Table 1], ions of m/z 342.1703 ([M]+) were observed, and their inferred molecular formula was C20H24NO4. In the MS2 spectra, m/z 297.11 ([M-C2H7N]+), 282.0886 ([M-C2H7N-CH3]+), 265.0854 ([M-C2H7N-CH3OH]+), and 237.0900 ([M-C2H7N-CH3OH-CO]+) were observed after the energy collisions to infer that peak 11, peak 5 [Figure 3]a, and peak 8 were Menisperine, Magnoflorine, and Norisocorydine, respectively.
|Table 1: Identification and analysis of the extract of Coptis chinensis Franch|
Click here to view
|Figure 3: MS2 spectra of typical alkaloids. A: Magnoflorine; B: Stecepharine; C: Palmatine|
Click here to view
RDA reaction, is a relatively common mass craking reaction. The six-membered ring containing endo-double bond is decomposed into a conjugated diene, alkene or alkyne under high temperature conditions. It is a concerted reaction. RDA reaction, is a relatively common mass craking reaction. The six-membered ring containing endo-double bond is decomposed into a conjugated diene, alkene or alkyne under high temperature conditions. It is a concerted reaction. RDA reaction occurs after the collision energy occurs in the tetrahydroprotoberberines alkaloids, which can cause the end chain, such as -CH3, to fracture. Thus, it was inferred that peaks 13, 14, and 20 belonged to this type of alkaloid.
Peak 14 was one of the characteristic alkaloid absorption peaks in the UV graph. As shown in [Table 1], ions of m/z 372.1806 ([M]+) were observed in MS1. Hence, its inferred molecular formula was C21H26NO5. Simultaneously, ions of [M-C8H22O2]+, [M-C8H22O2-CH3]+, and [M-C8H22O2-CH3-H2O]+ were also observed in MS2, so peak 14 was inferred to be Stecepharine [Figure 3]b, N-methlylcorydalmine, or its isomers.
As the important components in alkaloids, the fracture of side chains such as -O-CH2-O-, -OCH3, -OH, and -CH3 in protoberberine alkaloids can produce the 28 Da (CO), 15 Da (CH3), and 18 Da (H2O) fragment ions. Thus, 17 components, such as peaks 3, 4, 12, 15, and 16, could be tentatively identified as protoberberine alkaloids based on the reported literature.,,,,
The m/z of peak 27 was 352.1561 [Table 1], and peak 27 could be identified as a type of alkaloid according to the UV chromatogram and ESI(+) MS. Thus, the inferred molecular formula of peak 27 was C20H18NO5. In the MS2 experiment, the predominant ions appeared at m/z 337.1287 [M-CH3]+, 336.1233 [M-CH3-H]+, 308.1280 [M-CH3-H-CO]+, 322.1071[M-2CH3]+, and 294.1121 [M-2CH3-CO]+, which was consistent with the reported literature. Hence, we inferred that peak 27 was Palmatine [Figure 3]c shows its MS2 chromatogram. The other 16 peaks were also observed and identified using this method.
Peaks 10 and 18 were identified as types of alkaloids according to the UV chromatogram. The m/z values of peaks 10 and 18 were 308.0918 and 368.1510, respectively, which was also consistent with the literature., Thus, peaks 10 and 18 were identified as Lycoranine B and lincangenine/stephabine, respectively.
Identification of non-alkaloids in the extract of coptis chinensis franch by UPLC-Q-TOF
Most of the non-alkaloids that were identified in the extract of Coptis chinensis Franch belonged to feruloylquinic acid in positive mode. According to the accurately measured m/z of peaks 2, 7 and 9, they had almost the same m/z (367.10) as the fragment ion of [M–H]–]. Thus, it was inferred that their molecular formula was C17H20O9. In addition, both fragment ions with m/z 191.0551 [M-Feruloyl H] and 173.0449 [M-Feruloyl H-H2O] were observed in the three peaks, from which we can infer that peaks 2, 7, and 9 may be isomers. Peaks 2, 7, and 9 were inferred to be 5-O-feruloylquinic acid, 3-O-feruloylquinic acid, and 4-O-feruloylquinic acid, respectively, because of the difference in retention time, which is caused by different polarities, according to previous reports., The chemical structure of them is shown in [Figure 4].
|Figure 4: Chemical structures of three non-alkaloids identified in the extract of Coptis chinensis Franch. A: 3-O-Feruloylquinic acid; B: 4-O-Feruloylquinic acid; C: 5-O-Feruloylquinic acid|
Click here to view
| Conclusion|| |
UPLC-Q-TOF-MS/MS was used to identify the alkaloids and non-alkaloids in the extract of Coptis chinensis Franch. The m/z value was accurately measured, and the probable molecular composition and formula were analyzed according to the fragment ions of the extract. In total, 30 components were identified (including 25 alkaloids in positive mode and five non-alkaloids in negative mode) in 14 min according to the fragment ions in MS2 and accurate m/z in MS1 of the extract. The method developed in this article may provide a reference for the identification and analysis of compound used traditional Chinese medicine, and other samples, because of the rapid and comprehensive qualitative analysis of the extract of Coptis chinensis Franch. Two unknown components were not reported, and it remains to be determined if they are new components in the extract of Coptis chinensis Franch.
Financial support and sponsorship
This work was supported by grants from the National Natural Science Foundation of China (No. 81303261 and No. 81274133), the Major Scientific and Technological Special Project for Significant New Drugs Creation (No. 2012ZX09103-201-055) and the Fundamental Research Funds for the Central Public Welfare Research Institutes of China (ZZ2014005, ZZ2014060).
Conflicts of interest
There are no conflicts of interest
| References|| |
Yang YH, Gan CL. Isolation and identification of chemical constituents of Coptidis Rhizoma
. Heilongjiang Med J 2009; 22:480-1.
Ding NN, Tong SS, Ding LX, Xu XM, Yu JN. Research progress in analysis methods and pharmacological effects of berberine. Chinese J Pharm Anal 2012; 3: 1296-300.
Wu JF, Shi YJ, Liu TP. Protective effects of berberine on cerebral ischemia in mice and rats. Chinese J Pharm Toxicol 1995; 9:100-3.
Zhang YB, Wang J, Wang Y, Li S, Wang LY, Zhou GQ. Effect of berberine on expression and apoptosis of Bcl-2 and Bax in cerebral ischemia reperfusion in rats. J Chinese Pr Diagn Ther 2011; 25: 126-9.
Wei YZ, Yao ZB, Yuan QF, Chen YC. Protective effect of berberine on rat hippocampus against cerebral ischemia. Chinese J Neuroanat 1995; 11: 315-21.
Lv X, Wang J, Jiang WQ, Zhang HY. Research progress of Coptidis Rhizoma
. J Pharm Res 2004; 22: 33-5.
Liu F, Zhang H, Qing LS. Study on HPLC digital fingerprint of Coptidis Rhizoma and content determination of seven alkaloids. China J Chinese Materia Medica 2013; 21: 3713-9.
Feng M, Huang M, Li XG, He HM, Zhang BS. Research of effective ingredients and amino acid content in Coptidis Rhizoma
and byproducts. J Chinese Med Mater 2006; 28: 753-4.
Xin Y, Pi ZF, Song FR, Liu ZQ, Liu SY. Comparison of intracorporal absorption of hypaconitine in Heishunpian decoction and its compound recipe decoction by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Chinese J Chromatogr 2011; 29: 389-93.
Zhang PP, Zhang FC, Wang CH, Jiang Y, Lu YJ. Simultaneous determination of four trace aconitum alkaloids in urine using ultra performance liquid chromatography-mass spectrometry..Chinese J Chromatogr 2013; 31: 211-17.
Fan JJ, Wen HM, Shan CX, Zhou HG, Chen HB, Wu MH. Chemical constituents in Akebiae Fructus
based on UFLC-Q-TOF/MS. Chinese Tradit and Herb Drugs 2013; 44: 3282-88.
Zhang XX, Wang HP, Yang Y, Du MB, Mao S, Chen C. Rapid analysis of ginsenosides in dried fresh Ginseng by ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry. China Med Her ISTIC 2015; 12: 130-6.
Chen JH, Wang FM, Liu J, Lee FS, Wang XR, Yang HH, Analysis of alkaloids in Coptis chinensis
Franch by accelerated solvent extraction combined with ultra-performance liquid chromatographic analysis with photodiode array and tandem mass spectrometry detections. Anal Chim Acta 2008; 613: 184-95.
Chen JH, Zhao HQ, Wang XR, Lee FS, Yang HH, Zheng L. Analysis of major alkaloids in Rhizoma coptidis by capillary electrophoresis-electrospray-time of flight mass spectrometry with different background electrolytes. Electrophoresis 2008; 29: 2135-47.
Ren LL, Xue XY, Liang XM. Characterization of protoberberine alkaloids in Coptidis Rhizoma (Huanglian) by HPLC with ESI-MS/MS. J Sep Sci 2013; 36: 1389-96.
Wang DD, Liang J, Yang WZ, Hou JJ, Yang M, Da J. HPLC/qTOF-MS-oriented characteristic components data set and chemometric analysis for the holistic quality control of complex TCM preparations: Niuhuang Shangqing pill as an example.J Pharmaceut Biomed 2014;89: 130-41.
Jiang X, Huang LF, Wu LB, Wang ZH, Chen SL. UPLC-QTOF/MS analysis of alkaloids in traditional processed Coptis chinensis
Franch.Evid Based Complement Alternat Med 2012;2012:942-384.
Liu QX, Qiu SY, Yu H, Ke YX, Jin Y, Liang XM. Selective separation of structure-related alkaloids in Rhizoma coptidis with "click" binaphthyl stationary phase and their structural elucidation with liquid chromatography-mass spectrometry. Analyst 2011;136:4357-65.
Yin XY, Fu JJ, Yang HS, Liu HN, Luo YM. Analysis of Flavonoids and Phenolic Acids of Aqueous Extracts in Leaves of Apocynum venetum
L. Inform Technol Agr Eng.Springer Berlin Heidelberg 2012;134:925-33.
Ge AH, Bai Y, Li J, Liu J, He J, Liu EW. An activity-integrated strategy involving ultra-high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry and fraction collector for rapid screening and characterization of the α-glucosidase inhibitors in Coptis chinensis Franch.(Huanglian).J Pharmaceut Biomed 2014;100:79-87.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]