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Table of Contents
MODERN RESEARCH ON CHINESE MATERIA MEDICA
Year : 2015  |  Volume : 1  |  Issue : 2  |  Page : 24-37

Development of high performance liquid chromatography and mass spectrometry: a Key engine of TCM modernization


1 Agilent Technologies (China) Co., Ltd., No. 3, Wang Jing Bei Lu, Chao Yang District, Beijing 100102, P.R. China
2 State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China

Date of Web Publication21-Sep-2020

Correspondence Address:
Tao Bo
Agilent Technologies (China) Co., Ltd., Wang Jing Bei Lu, Chao Yang District, Beijing 100102
P.R. China
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Source of Support: None, Conflict of Interest: None


DOI: 10.15806/j.issn.2311-8571.2015.0006

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  Abstract 


Traditional Chinese Medicine (TCM) has been popular for thousand years in prevention and treatment of chronic diseases synergistically with Western medicine while producing mild healing effects and lower side effects. Although many TCMs have been proven effective by modern pharmacological studies and clinical trials, their bioactive constituents and the remedial mechanisms are still not well understood. Researchers have made great efforts to explore the real theory of TCM for many years with different strategies. Development of high performance liquid chromatography (HPLC) and mass spectrometry within recent decade can provide scientists with robust technologies for disclosing the mysterious mask of TCM. In this paper, important innovations of HPLC and mass spectrometry are reviewed in the application of TCM analysis from single compound identification to metabolomic strategy.
Abbreviations: TCM, Traditional Chinese Medicine; Q-TOF, Quadrupole time-of-flight mass spectrometry; LC, Liquid chromatography; CE, Capillary electrophoresis; SFC, supercritical-fluid chromatography; QQQ, triple quadrapole

Keywords: TCM, High performance liquid chromatography, Mass spectrometry, Metabolomics, Active ingredients, Quality control


How to cite this article:
Zhang ZX, Qiao X, Ye M, Zhang MY, Song Y, Bo T. Development of high performance liquid chromatography and mass spectrometry: a Key engine of TCM modernization. World J Tradit Chin Med 2015;1:24-37

How to cite this URL:
Zhang ZX, Qiao X, Ye M, Zhang MY, Song Y, Bo T. Development of high performance liquid chromatography and mass spectrometry: a Key engine of TCM modernization. World J Tradit Chin Med [serial online] 2015 [cited 2022 Oct 3];1:24-37. Available from: https://www.wjtcm.net/text.asp?2015/1/2/24/295624




  Introduction Top


Traditional Chinese Medicine (TCM) has been used in clinical and health care practice for more than 2000 years in China[1]. More and more oversea countries and regions have taken active action to accept TCM as an alternative to Western medicine. Medicinal plants, animals, and minerals are major starting materials to make TCM preparations, in which medicinal plants are the most dominant. TCM becomes more and more popular due to the fact that it is widely available, relatively inexpensive, and produces little adverse effect[2]. Although many TCMs have been proven effective by modern pharmacological studies and clinical trials, their bioactive constituents and the remedial mechanisms are still not well understood. There are critical questions ahead of us: what are the in vivo metabolites and their pharmacokinetic properties after oral administration of TCMs; what is the toxicity of the TCM, which attracted extensive attention due to its significant impact on TCM safety and trade; how and why TCM works, what its strengths and weaknesses are; and how to control the quality of TCM preparations not just from a single active ingredient, etc.

For the purpose of better understanding the complex TCM and controlling their quality, powerful analytical techniques are essential. Recently, fast development of HPLC and mass spectrometry with their hyphenation technology significantly enhance the ability to dissect TCM with rapid increase in TCM research interests. Especially, ultra-high pressure liquid chromatography (UHPLC) became the modern standard HPLC platform. UHPLC, with its shorter analysis time and faster column equilibration, is suited to rapid method development ideally[3],[4],[5], allowing the high-throughput analysis of complex TCM samples within 10min. Although conventional one-dimensional (1D) chromatographic approaches have been widely used for the analysis of multiple components in TCMs, the complexity of TCM samples often exceeds the maximal capacity of any single separation mode. Two-dimensional (2D) LC separation systems, based on two independent columns with different separation mechanisms, have proven to be more powerful than 1D technique and have been used successfully to separate and analyze TCM samples with excellent performance[6],[7]. As a powerful technique, supercritical-fluid chromatography (SFC) is utilized for chiral and achiral separation. Recently, SFC has been widely used to separate chiral mixtures of pharmaceutical products and natural products, including triterpenoids, steroids, and bile acids[8],[9],[10],[11],[12].

The development of mass spectrometry allows scientists to gain deeper insight into the TCMs from a molecular level. Triple quadrapole (QQQ) with multiple reaction monitoring (MRM) is the gold criteria of trace quantitation in the complex samples such as TCMs, food matrix, blood serum, etc. Recently, high-resolution quadrupole time-of-flight mass spectrometry (Q-TOF), one of the most sophisticated and promising accurate mass instrumentation, with strong qualitative and quantitative capabilities, has been widely employed in TCM analysis[13]. It provides accurate monoisotopic mass measurement and high resolution MS/MS spectrum for target confirmation and unknown identification. For complex sample analysis, Q-TOF combined with powerful separation techniques like liquid chromatography (LC), capillary electrophoresis (CE), and gas chromatography (GC) is more popular and reasonable. Separation process can greatly reduce sample complexity and matrix effect, and discriminate structural isomers. ESI, APCI, and APPI are usually equipped in LC/Q-TOF and CE/Q-TOF for compounds with changed to from weak to strong polarities.

This paper aims to demonstrate the important applications of TCM study based on the recent development in HPLC and mass spectrometry from single compound identification to metabolomic strategy.


  Breakthrough of Liquid Chromatography Technology: New Era of TCM Analysis Top


1. UHPLC tandem Q-TOF technology for high throughput analysis for TCM

UHPLC technology has been applied successfully since its commercial introduction in 2004 to a wide range of samples and conditions, in combination with spectroscopy and MS[3]. The main advantage of UHPLC is the possibility to achieve ultra-fast and/or high resolution separations, with reduced solvent consumption, using columns packed with sub-2-μm particles and chromatographic systems compatible with pressures from 600 bar or above. Because the benefits of using small particles can be extended to chromatographic techniques other than RPLC, there is a trend towards using UHPLC technology in several modes, including chiral liquid chromatography (LC), size exclusion chromatography (SEC), ion exchange chromatography (IEX), hydrophilic interaction chromatography (HILIC), and supercritical-fluid chromatography (SFC). The theory and the development of UHPLC and UHPLC-MS for applications were comprehensively reviewed[3],[4],[5], including bioanalysis, TCMs, multi-residue screening, and metabolomics.

Bear bile is a precious traditional Chinese medicine containing abundant bile acids as shown in [Figure 1]. The content of these bile acids varies significantly. A rapid and reliable method was established to simultaneously monitor major and minor bile acids, using UHPLC coupled with Q-TOF mass spectrometry[14]. The samples were separated on a 1.8 μm Zorbax Eclipse- Plus C18 column with acetonitrile and water (containing 4 mM of ammonium acetate) as the mobile phase. A 5 min analysis allowed the characterization of 21 steroids and the quantitation of two major constituents, taurochenodeoxycholic acid (TCDCA) and tauroursodeoxycholic acid (TUDCA) as shown in [Figure 2]. The simultaneous qualitative and quantitative analyses of bear bile powder were achieved, and minor steroid constituents were identified.
Figure 1: Chemical structures of the internal standard (GLCA) and characterized bile acids from bear bile. (Reprinted with permission from [14]. © 2014 Royal Society of Chemistry

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Figure 2: Total ion current of bear bile powder sample and extracted ion chromatograms of 21 characterized bile acids. Ion extraction width, 100 mDa; IS, internal standard. Compounds marked with * were identified by reference compounds. (Reprinted with permission from [14]. © 2014 Royal Society of Chemistry)

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2. 2D-LC for TCM profiling

Since Erni and Frei first introduced the 2D-LC technique in 1978, the development of 2D-LC techniques has been moving forward[15]. To date, many 2D-LC combinations based on various separation modes, such as normal-phase (NP), reversed-phase (RP), ion exchange (IEX), size exclusion chromatography (SEC) or affinity chromatography (AC) have been employed to generate increased peak capacity, higher resolution and improved selectivity. 2D-LC has significantly improved the separation power of LC for complex samples. Its theory consideration, configuration and applications in a wide range of analytical fields, such as food analysis, life sciences, TCM and bioenergy and polymers were recently reviewed[6],[7].

TCMs contain a large number of minor constituents, which could contribute to their therapeutic effects and provide valuable lead compounds for drug discovery. However, to explore minor constituents from complicated herbal extracts is usually laborious and time-consuming. In order to discover minor novel herbal constituents efficiently, we combined heart-cutting and comprehensive two-dimensional liquid chromatography (HC-2DLC) to remove major components from herbal extracts, and then characterized the minor ones by mass spectrometry as shown in [Figure 3] and [Figure 4]. This strategy was employed to analyze Pueraria lobata and Pueraria thomsonii, the roots of which are used as the Chinese herbal medicine Ge-Gen[16]. Five major compounds in Ge-Gen extract were removed by on-line heart-cutting, and the minor compounds were separated on an RP × RP 2DLC system (1D, Acquity CSH C18, 2.1×100 mm, 1.7 μm; 2D, Poroshell Phenyl-Hexyl, 3.0×50 mm, 2.7 μm). A synchronized gradient elution program was used to improve chromatographic resolution of the second dimension. By using this 2D-LC system, a total of 271 and 254 peaks were separated in P. lobata and P. thomsonii within 35 min, respectively. The practical and effective peak capacity was 1593 and 677, respectively, and the orthogonality was around 70%. Structures of 12 selected compounds were tentatively characterized by mass spectrometry, and 9 of them were discovered from Ge-Gen for the first time. Contents of these minor compounds in Ge-Gen were preliminarily determined to be 0.01–0.1% (w/w). The HC-2DLC/MS system is a powerful and convenient tool to explore minor novel chemical constituents from complex herbal extracts.
Figure 3: Construction of the heart-cutting and comprehensive two-dimensional liquid chromatography system (HC-2DLC). (Reprinted with permission from [16]. © 2014 Elsevier)

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Figure 4: Contour plots for P. lobata by using conventional comprehensive 2DLC (A) and heart-cutting and comprehensive 2DLC; (B) Dark areas in plot (B) indicate heart-cutting sections. (Reprinted with permission from [16]. © 2014 Elsevier)

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3. SFC for TCM analysis

As a powerful technique, supercritical-fluid chromatography (SFC) is for chiral and achiral separation. Due to low viscosity and high diffusivity of supercritical fluid CO2, SFC is operated at a higher flow rate with lower back pressure, and is generally faster and more efficient than HPLC[17]. SFC has gained interest and acceptance in a wide variety of applications. This is due to unique selectivity, high separation speed, green chemistry and low operating costs. As there is no universal stationary phase available for SFC separations, screening of different columns is required in order to achieve optimal separation. Rapid column equilibration and convenient mobile phase removal also make SFC attractive for preparative scale separation. Recently, SFC has been widely used to separate chiral mixtures of pharmaceutical products and natural products, including triterpenoids, steroids, and bile acids[8],[9],[10],[11].

Ergostanes are major bioactive constituents of the medicinal mushroom Antrodia camphorate. These tetracyclic triterpenoids usually occur as 25R/S epimeric pairs as shown in [Figure 5](A), which render their chromatographic separation difficult. We used analytical supercritical-fluid chromatography (SFC) to separate seven pairs of 25R/S-ergostanes from A. camphorate[18]. The (R)- and (S)-forms for each of the seven pairs could be well resolved (Rs>1.3) on a Chiralcel OJ-H column (4.6×250 mm, 5 μm, chiral) as shown in [Figure 5](B), eluted by 10%MeOH in CO2 at 2 mL/min with a back pressure of 120 bar and a column temperature of 40 °C. Particularly, this chiral-SFC method could rapidly and efficiently separate low-polarity epimers like antcin A and antcin B, which were very difficult for RP-HPLC. A 3-min preparative-scale method was established to purify (25S)- and (25R)-antcin. However, OJ-H column suffered from peak overlapping of different pairs of ergostanes. We found that Princeton 2-ethylpyridine column (2-EP, 4.6×250 mm, 3 μm, achiral) could effectively separate different pairs, although the resolutions for 25-R/S forms of each epimeric pair were not as good as OJ-H column. Meanwhile, all the (25S)-forms showed stronger retentions than the corresponding (25R)-epimers on the 2-EP column. These results demonstrated different selectivity of chiral- and achiral-SFC in separating 25R/S-ergostane epimers. Aside from high separation efficiency, SFC also showed advantage over HPLC in short analysis time and low consumption of organic solvents. Finally, both OJ-H and 2-EP columns were used on analytical SFC to separate 25R/S-ergostanesin an extract of A. camphorata.
Figure 5: (A) Chemical structures of ergostanes isolated from Antrodia camphorate; (B) Simultaneous separation of seven pairs of 25R/S-ergostane epimers by SFC. (a) Chiralcel OJ-H column (chiral), Overlaid chromatograms of each epimeric pair; (b1)Princeton 2-ethylpyridine column (achiral), mixture of all 14 standards; (b2) 2-ethylpyridine column, mixture of (S)-form and (R)-form ergostanes, respectively. (Reprinted with permission from [18]. © 2014 Elsevier)

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  High-Resolution Mass Spectrometry and Related Novel Technology: Powerful Tool For Demystifying Traditional Chinese Medicine Top


As the most attractive high-resolution mass spectrometry, Q-TOF has proven as powerful tool in TCM analysis due to its strong qualitative and quantitative capabilities in a single platform. Recently, we have been invited to write a review about Q-TOF technology in the TCM study, in which Q-TOF based achievements about TCM analysis including profiling of active ingredients and their metabolites, screening of harmful components, and applications of cutting edge metabolomic strategies are comprehensively reviewed[12]. The novel applications employing Q-TOF and metabolomics strategy we just achieved are demonstrated as follows.

1. Quality control of TCM based on Q-TOF and metabolomics strategy

Quality control is vital for ensuring safety and efficacy of TCMs. Usually, TCMs are used as whole plant and/or combination of several herbs, and multiple constituents are responsible for the therapeutic effects. Therefore, quality control of TCM is very difficult. To date, the valid method for quantitatively evaluating the quality of TCM is poor. Recent applications of key analytical techniques in quality assurance and authentication of herbs and their extracts were reviewed[19],[20], which highlight the emerging role of chemical fingerprinting of TCMs and the latest regulatory requirements imposed on TCM utilizing chromatographic fingerprinting.

Batch to batch reproducibility is very important for TCM injection manufacturing. Development of a fast QC solution is significant to reduce test cost and improve manufacturing throughput for pharmaceutical factories. A statistical analysis based strategy has been successfully established and applied to QC analysis for Shuxuetong Injection by high resolution Q-TOF mass spectrometry in our laboratory[21]. Both unsupervised and supervised pattern recognition algorithms were utilized to perform clustering analysis and build statistical model of Shuxuetong Injection. In this work, we designed a workflow to assess manufacture reproducibility and to filter out disqualified product as shown in [Figure 6](A). Shuxuetong Injections of different batches were analyzed directly without further sample preparation. Accurate mass fingerprint profile of each sample was obtained by using UHPLC/Q-TOF system. Chromatograms with reproducible retention time, signal response, and accurate mass were achieved. Molecular feature extraction algorithm was employed to automatically find compounds in each chromatogram. All the rest of the data mining process was performed on Agilent metabolomic research platform, i.e. Mass Profiler Professional software. Principal Component Analysis (PCA) plot can clearly show intra-batch reproducibility. A Partial Least Squares Discrimination (PLSD) model was built and validated on the basis of known samples as shown in [Figure 6](B). Components that result in statistically significant differences between qualified and disqualified products were identified by Agilent Molecular Structure Correlator (MSC) software based on MS/MS data as seen from [Figure 7]. The established strategy can also be applied to distinguish products from different manufacturers.
Figure 6: (A) Workflow to assess manufacture reproducibility and filter out disqualified products; (B) PCA (Principal Component Analysis) Scores Plot: Significant difference between qualified and disqualified samples

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Figure 7: (A) Components that result in statistically significant difference between qualified and disqualified products; (B) The identification of components that result in statistically significant difference between qualified and disqualified products by Molecular Structure Correlator (MSC) software for MS/MS elucidation.

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2. Natural product identification, geographic origin deduction, and manufacturing process discrimination by high resolution mass spectrometry

The active ingredients in TCM vary a lot with respect to different origin, planting conditions and extract process, which plays essential roles in the TCM quality and therapy effects.

Tea is the second most widely consumed beverage in the world and grown in about 30 countries worldwide. Many health benefits including prevention of cancer and heart diseases result from the consumption of tea. It is found that tea’s constituents are significantly affected by geographic origin and manufacturing processes. For food safety purpose, it is important to distinguish the fake or low-grade tea product from the first-rate ones and to better understand the fate of tea after entering the human body. In our laboratory, a high resolution mass spectrometry combined with metabolomic software tool and dedicated database was employed to identify the components from tea extract and identify teas of different kinds and origins[22].

A specific database including 333 compounds with compound name, molecular structure, formula, accurate mass, CAS No, and partial high resolution MS/MS spectra was successfully established on the basis of literature reference. More than twenty kinds of components, i.e., catechins, flavonols, flavones, flavonol glycosides, flavone glycosides, proanthocyanidins and bisflavanols, theasinensins, theaflavins, thearubigins, hydrolyzable tannins, phenolic acids and derivatives, purine alkaloids, amino acids, carotenoids, chlorophylls, carbohydrates, vitamins, organic acids, acrylamide, and lipids were embodied. Unknown identification and confirmation based on MS, MS/MS, and retention time matching were performed with the use of the above database and Agilent Molecular Structure Correlator software. Agilent metabolomic research platform, i.e. Mass Profiler Professional software, was utilized for statistical analysis and class prediction model building. Many structural isomers, like (+)-catechin vs. (–)-epicatechin, theobromine vs. theophylline were well resolved and identified by current method. A PLSD model was established and successfully applied to distinguish tea from different geographic origin and from different manufacturing processes as seen from [Figure 8]. In addition, biotransformation pathways of important biomarkers contributing to differentiation were also discussed. This research is meaningful to tea quality control and fake tea screening.
Figure 8: (A) PCA scores plot for six kinds of tea from statistical result of positive ion mode data; (B) Concentration variation of theophylline (left) and (+)-catechin (right) in six different tea 1#–6#.

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3. CE-MS for screening the active components in TCM

Capillary electrophoresis (CE) is a family of electromigration techniques that employ small diameter capillaries to perform high efficiency separation of both large and small molecules whether charged or uncharged. CE and CE-MS technologies have been widely used for analyzing the TCMs. The applications of CE methods to phytochemical analysis and quality control of herbal drugs have been reviewed[23],[24],[25].

Amino acids in Chinese Traditional Medicines (TCMs) are important bio-active components, which have attracted much attention. In this study, we developed an advanced CE-Q-TOF tandem technique for characterizing sixteen amino acids in herbal medicines[26]. This technique proved to be rapid and highly sensitive, involving data processing (Mass Molecule Feature Extraction, MFE), accurate mass database search, and structural confirmation based on MS/MS fragmentation.

MS and MS/MS data were acquired on an Agilent Q-TOF system in positive mode with Jet Stream ESI. Separation was performed on the Agilent 7100 HPCE (High Performance Capillary Electrophoresis) using 1 M formic acid as running buffer and 5 mM acetate ammonium in 50% methanol as sheath liquid. The separation voltage was 30 kV by employing 50 μm ID and 80 cm length silica capillary. The mass spectrometer was operated in MS mode at 2 spectra/second and MS/MS mode at 3 spectra/second for profiling. MS parameters: 100 V Fragmentor; 280 °C drying gas temperature; 10 L/min drying gas flow; 5 L/min sheath gas; 10 psi nebulizer pressure.

The established method allows the simultaneous analysis of sixteen amino acids in positive ion mode using an electrolyte with a pH value below the analyte’s isoelectric point (<2.77). An accurate mass TCM database (containing 10,500 active compounds) was established for rapid screening of the amino acids in the TCM samples, after the raw MS data was processed by the MFE algorithm to remove the background ions producing a compound list. The conditions of CE were systematically optimized with respect to running buffer system and sheath liquid components to obtain the high-efficient and fast separation. The mass spectrometer parameters were optimized for the best sensitivity with regard to the Jet Stream ESI parameters, fragmentor voltage and acquisition rate. Especially, leucine and iso-leucine can be well separated at the optimized conditions in the Radix Astragali injection samples as seen from [Figure 9]. Reproducibility of migration time and peak area was excellent with RSD of less than 5.0% (n = 3). The detection limits are in the range of 0.5-10 μg/ml for all analytes. Furthermore, high- resolution MS/MS mode was used for reliably structural confirmation and fragmentation mechanism study of targeted amino acids in real samples. The study demonstrates that the established CE-Q-TOF method offers high-throughput, good sensitivity and high selectivity for characterizing the amino acids in TCMs. In addition, this technique can be applied to profiling profile amino acids in other complex matrices such as food and serum.
Figure 9: (A) CE-QTOF for amino acids; (B) MS and MS/MS identification of proline with good repeatability.

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4. Determination of synthetic adulterants in Traditional Chinese Medicines Using UHPLC-QQQ Mass spectrometry with triggered MRM

Due to the complexity of TCM matrices, false positive identification during the quantitation (including active ingredients and illegally chemical additives) in TCM is a major concern when employing LC-QQQ technique. Only two MRMs cannot ensure reliable confirmation for most cases, especially for trace analysis in the complex food matrices. As recently developed technique, triggered MRM (tMRM) can effectively avoid false positive occurrence by the acquisition of additional confirmatory ions and performing further reference library matching[28]. Due to the use of optimized collision energy and dwell time for each MRM, tMRM is very sensitive with excellent reproducible spectra even at trace concentrations.

The purpose of the study was to develop a procedure for the detection of 214 common synthetic adulterants in dietary supplements and TCM, using ultrahigh UHPLC-QQQ with the innovation scan mode, triggered multiple reaction monitoring (tMRM). The 214 drugs belonging to thirty three pharmacological classes were grouped in suites, comprising aphrodisiacs, slimming drugs, anti-diabetic drugs, anti-epileptic drugs, hormones, anabolic drugs, psy-chotropic drugs and antibiotics, etc., as shown in [Figure 10] (A). Validation was achieved according to the criteria published in several World Health Organization (WHO) and European (EU) issued guidelines and acts. In this study, an Agilent Zorbax Eclipse-plus C18 column packed with 1.8 μm particles was applied to achieve excellent separation with symmetric peak shapes. Dietary supplements and TCM were analyzed by liquid-liquid extraction (LLE) prior to auto-injection for direct quantification as seen from [Figure 10](B). Good recoveries were obtained in the range of 60-120% with the limit of quantitation (LOQ, S/N>10) as 0.02 or 0.05 ng/ml for all target pesticides with internal standard (Triphenylphosphate as the ISTD with the concentration of 2.0 ng/ml in spiked tea samples). Moreover, good precision and linearity were obtained for quantitative determination in tea extracts by the proposed UHPLC-MS/MS method with “Dynamic MRM”. The precision was in the ranges of 0.98-9.5% and the R2 value was better than 0.99. The developed tMRM method offered highly selective and accurate detection of multi-targets screening (MTS) applications for TCM quality control.
Figure 10: (A) The workflow of monitoring 214 adulterants in the TCM; (B) Triggered MRM for monitoring 214 adulterants.

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5. Ion Mobility-QTOF Mass Spectrometry: Big potential to TCM Analysis

The field of ion mobility-mass spectrometry (IM-MS QTOF) has grown with significant momentum in recent years in both fundamental advances and pioneering applications[29],[30],[31]. A search of the terms “ion mobility” and “mass spectrometry” returns more than 2,000 papers, with over half of these being published in the past 4 years. This increased interest has been motivated in large part by improved technologies which have enabled contemporary IM-MS QTOF to be amendable to a variety of samples in biology and medicine with high sensitivity, resolving power, and sample throughput. As shown in [Figure 11](A), the new generation of IM-QTOF LC/MS system enables very precise and accurate collision cross section (CCS) measurements without class dependent calibration standards, which operates under uniform low field conditions (allowing drift time information for ions to be directly converted to collision cross section information). The innovative ion funnel technology dramatically increases the ion sampling into the mass spectrometer and results in higher quality MS/MS spectra at trace levels[32].
Figure 11: (A) Schematic diagram of IM-QTOF mass spectrometry; Ions generated in the source region are carried into the front ion funnel through a single bore capillary. The front ion funnel improves the sensitivity by efficiently transferring gas phase ions into the trapping funnel while pumping away excess gas and neutral molecules. The trapping funnel accumulates and releases ions into the drift tube. The drift cell is ~80 cm long and generally operated at 20 V/cm drift field. Ions exiting the drift tube enter the rear ion funnel that efficiently refocuses and transfers ions to the mass analyzer. (B) IM-QTOF for resolving two isobaric trisaccharides.

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The IM-QTOF mass spectrometry can deliver an added dimension of separation, provide direct measurement of accurate collision cross sections, preserve structural characteristics of molecular conformations and expand coverage maps for the complex TCM samples. As seen from [Figure 11] (B), a 1:1 mixture of melezitose and raffinose was infused using a syringe pump. These two carbohydrates can be baseline separated using the ion mobility drift cell and detected using the Q-TOF mass analyzer as sodium adducts, which cannot be resolved by using the traditional mass spectrometry. The ion mobility resolving power for this separation is 60. As seen from [Figure 12], the fast separation of four natural products in the ion mobility drift cell of IM-QTOF mass spectrometry with their CSS calculation within 1 min was achieved in Flowing Injection Mode, provides a robust approach for ultra-high throughput screening of the active ingredients in TCMs with extra 3D identification relating to CCS measurement. The IM-QTOF technology especially contributes to the discrimination of isomers and active ingredients with structural similarity in TCMs, which is very attractive for TCM metabolomics and the active component discovery in herbal medicines.
Figure 12: The fast separation of four natural products in the ion mobility drift cell of IM-QTOF mass spectrometry by flowing injection Mode.

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  Conclusion Top


Versatile data acquisition modes combined with various data mining techniques and dedicated databases and libraries make QQQ and Q-TOF suitable for the quantitation and characterization of complex sample. As the high-resolution mass spectrometry, Q-TOF has proven as a powerful tool in TCM analysis due to its strong qualitative and quantitative capabilities on single platform. Q-TOF based metabolomic strategies have accepted widespread interests from TCM researchers and achieved more and more successful applications focusing on quality control, therapeutic effect assessment, and toxicity investigation. Overall, Q-TOF has made great contributions to the development of both TCM theory and practice. With the combination with the liquid chromatography development including UHPLC, 2D LC and SFC, LC-MS technology can play key roles in the TCM related research and manufacture process.


  Acknowledgements Top


We gratefully acknowledge the technique direction from Rong An (Greater China Application Support Manager from Agilent Technologies) for this review.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12]


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