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Development of High Performance LiquidChromatography and Mass Spectrometry: a Key Engineof TCM Modernization

Zhengxiang Zhanga, Xue Qiaob, Min Yeb, Manyu Zhanga, Yue Songa and Tao Boa*aAgilent Technologies (China) Co., Ltd., No. 3, Wang Jing Bei Lu, Chao Yang District, Beijing 100102, P.R. ChinabState Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China*Correspondence: Dr. Tao Bo, Agilent Technologies (China) Co., Ltd., Wang Jing Bei Lu, Chao Yang District, Beijing 100102, P.R. China,Email: [email protected]

ABSTRACT

Traditional Chinese Medicine (TCM) has been popular for thousand years in prevention and treatment of chronic diseases synergistically withWestern medicine while producing mild healing effects and lower side effects. Although many TCMs have been proven effective by modernpharmacological studies and clinical trials, their bioactive constituents and the remedial mechanisms are still not well understood. Researchershave made great efforts to explore the real theory of TCM for many years with different strategies. Development of high performance liquidchromatography (HPLC) and mass spectrometry within recent decade can provide scientists with robust technologies for disclosing themysterious mask of TCM. In this paper, important innovations of HPLC and mass spectrometry are reviewed in the application of TCManalysis from single compound identification to metabolomic strategy.Key words: TCM, High performance liquid chromatography, Mass spectrometry, Metabolomics, Active ingredients, Quality control

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

INTRODUCTION

Traditional Chinese Medicine (TCM) has been used inclinical and health care practice for more than 2000 yearsin China[1]. More and more oversea countries and regionshave taken active action to accept TCM as an alternative toWestern medicine. Medicinal plants, animals, and mineralsare major starting materials to make TCM preparations, inwhich medicinal plants are the most dominant. TCMbecomes more and more popular due to the fact that it iswidely available, relatively inexpensive, and produces littleadverse effect[2]. Although many TCMs have been proveneffective by modern pharmacological studies and clinicaltrials, their bioactive constituents and the remedial mechan-isms are still not well understood. There are critical questionsahead of us: what are the in vivo metabolites and theirpharmacokinetic properties after oral administration ofTCMs; what is the toxicity of the TCM, which attractedextensive attention due to its significant impact on TCM safetyand trade; how and why TCM works, what its strengths andweaknesses are; and how to control the quality of TCMpreparations not just from a single active ingredient, etc.

For the purpose of better understanding the complex TCMand controlling their quality, powerful analytical techniquesare essential. Recently, fast development of HPLC and massspectrometry with their hyphenation technology significantlyenhance the ability to dissect TCM with rapid increase inTCM research interests. Especially, ultra-high pressure liquid

chromatography (UHPLC) became the modern standardHPLC platform. UHPLC, with its shorter analysis time andfaster column equilibration, is suited to rapid methoddevelopment ideally[3–5], allowing the high-throughput ana-lysis of complex TCM samples within 10min. Althoughconventional one-dimensional (1D) chromatographicapproaches have been widely used for the analysis of multiplecomponents in TCMs, the complexity of TCM samples oftenexceeds the maximal capacity of any single separation mode.Two-dimensional (2D) LC separation systems, based on twoindependent columns with different separation mechanisms,have proven to be more powerful than 1D technique andhave been used successfully to separate and analyze TCMsamples with excellent performance[6–7]. As a powerfultechnique, supercritical-fluid chromatography (SFC) is uti-lized for chiral and achiral separation. Recently, SFC has beenwidely used to separate chiral mixtures of pharmaceuticalproducts and natural products, including triterpenoids,steroids, and bile acids[8–12].

The development of mass spectrometry allows scientists togain 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 thecomplex samples such as TCMs, food matrix, blood serum,etc. Recently, high-resolution quadrupole time-of-flight massspectrometry (Q-TOF), one of the most sophisticated andpromising accurate mass instrumentation, with strongqualitative and quantitative capabilities, has been widely

DOI: 10.15806/j.issn.2311-8571.2015.0006

Modern Research on Chinese Materia Medica

April 2015 |Vol. 1 | Issue 2 24 www.wjtcm.org

mailto:[email protected]

http://dx.doi.org/10.15806/j.issn.2311-8571.2015.0006

employed in TCM analysis[13]. It provides accurate mono-isotopic mass measurement and high resolution MS/MSspectrum for target confirmation and unknown identifica-tion. For complex sample analysis, Q-TOF combined withpowerful separation techniques like liquid chromatography(LC), capillary electrophoresis (CE), and gas chromatography(GC) is more popular and reasonable. Separation process cangreatly reduce sample complexity and matrix effect, anddiscriminate structural isomers. ESI, APCI, and APPI areusually equipped in LC/Q-TOF and CE/Q-TOF for com-pounds with changed to from weak to strong polarities.

This paper aims to demonstrate the important applicationsof TCM study based on the recent development in HPLC andmass spectrometry from single compound identification tometabolomic strategy.

BREAKTHROUGH OF LIQUIDCHROMATOGRAPHY TECHNOLOGY: NEWERA OF TCM ANALYSIS

1. UHPLC tandem Q-TOF technology for highthroughput analysis for TCMUHPLC technology has been applied successfully since itscommercial introduction in 2004 to a wide range of samplesand conditions, in combination with spectroscopy and MS[3].

The main advantage of UHPLC is the possibility to achieveultra-fast and/or high resolution separations, with reducedsolvent consumption, using columns packed with sub-2-μmparticles and chromatographic systems compatible with pres-sures from 600 bar or above. Because the benefits of using smallparticles can be extended to chromatographic techniques otherthan RPLC, there is a trend towards using UHPLC technologyin several modes, including chiral liquid chromatography (LC),size exclusion chromatography (SEC), ion exchange chromato-graphy (IEX), hydrophilic interaction chromatography (HILIC),and supercritical-fluid chromatography (SFC). The theory andthe development of UHPLC and UHPLC-MS for applicationswere comprehensively reviewed[3–5], including bioanalysis,TCMs, multi-residue screening, and metabolomics.

Bear bile is a precious traditional Chinese medicine contain-ing abundant bile acids as shown in Figure 1. The content ofthese bile acids varies significantly. A rapid and reliable methodwas established to simultaneously monitor major and minorbile acids, using UHPLC coupled with Q-TOF mass spectro-metry[14]. The samples were separated on a 1.8 µm ZorbaxEclipse- Plus C18 column with acetonitrile and water (contain-ing 4 mM of ammonium acetate) as the mobile phase. A 5 minanalysis allowed the characterization of 21 steroids and thequantitation of two major constituents, taurochenodeoxycholicacid (TCDCA) and tauroursodeoxycholic acid (TUDCA) as

Figure 1. Chemical structures of the internal standard (GLCA) and characterized bile acids from bear bile. (Reprinted with permission from [14]. © 2014Royal Society of Chemistry)

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shown in Figure 2. The simultaneous qualitative and quanti-tative analyses of bear bile powder were achieved, and minorsteroid constituents were identified.

2. 2D-LC for TCM profilingSince Erni and Frei first introduced the 2D-LC technique in1978, the development of 2D-LC techniques has been moving

forward[15]. To date, many 2D-LC combinations based onvarious separation modes, such as normal-phase (NP),reversed-phase (RP), ion exchange (IEX), size exclusionchromatography (SEC) or affinity chromatography (AC)have been employed to generate increased peak capacity,higher resolution and improved selectivity. 2D-LC hassignificantly improved the separation power of LC for

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)

© World Journal of Traditional Chinese Medicine High Performance Liquid Chromatography and Mass Spectrometry


complex samples. Its theory consideration, configuration andapplications in a wide range of analytical fields, such as foodanalysis, life sciences, TCM and bioenergy and polymers wererecently reviewed[6–7].

TCMs contain a large number of minor constituents,which could contribute to their therapeutic effects andprovide valuable lead compounds for drug discovery. How-ever, to explore minor constituents from complicated herbalextracts is usually laborious and time-consuming. In order todiscover minor novel herbal constituents efficiently, wecombined heart-cutting and comprehensive two-dimensionalliquid chromatography (HC-2DLC) to remove major com-ponents from herbal extracts, and then characterized theminor ones by mass spectrometry as shown in Figure 3 and4. This strategy was employed to analyze Pueraria lobata andPueraria thomsonii, the roots of which are used as theChinese herbal medicine Ge-Gen[16]. Five major compoundsin Ge-Gen extract were removed by on-line heart-cutting,and the minor compounds were separated on an RP × RP2DLC system (1D, Acquity CSH C18, 2.1×100 mm, 1.7 µm;2D, Poroshell Phenyl-Hexyl, 3.0×50 mm, 2.7 µm). Asynchronized gradient elution program was used to improvechromatographic resolution of the second dimension. Byusing this 2D-LC system, a total of 271 and 254 peaks wereseparated in P. lobata and P. thomsonii within 35 min,respectively. The practical and effective peak capacity was1593 and 677, respectively, and the orthogonality was around70%. Structures of 12 selected compounds were tentativelycharacterized by mass spectrometry, and 9 of them werediscovered from Ge-Gen for the first time. Contents of theseminor compounds in Ge-Gen were preliminarily determinedto be 0.01–0.1% (w/w). The HC-2DLC/MS system is apowerful and convenient tool to explore minor novelchemical constituents from complex herbal extracts.

3. SFC for TCM analysisAs a powerful technique, supercritical-fluid chromatography(SFC) is for chiral and achiral separation. Due to lowviscosity and high diffusivity of supercritical fluid CO2, SFCis operated at a higher flow rate with lower back pressure,and is generally faster and more efficient than HPLC[17]. SFChas gained interest and acceptance in a wide variety ofapplications. This is due to unique selectivity, high separationspeed, green chemistry and low operating costs. As there isno universal stationary phase available for SFC separations,screening of different columns is required in order to achieveoptimal separation. Rapid column equilibration and conve-nient mobile phase removal also make SFC attractive forpreparative scale separation. Recently, SFC has been widelyused to separate chiral mixtures of pharmaceutical productsand natural products, including triterpenoids, steroids, andbile acids[8–11].

Ergostanes are major bioactive constituents of the medicinalmushroom Antrodia camphorate. These tetracyclic triterpe-noids usually occur as 25R/S epimeric pairs as shown in Figure5(A), which render their chromatographic separation difficult.We used analytical supercritical-fluid chromatography (SFC)to separate seven pairs of 25R/S-ergostanes from A. campho-rate[18]. The (R)- and (S)-forms for each of the seven pairscould 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 by10%MeOH in CO2 at 2 mL/min with a back pressure of 120bar and a column temperature of 40 ◦C. Particularly, thischiral-SFC method could rapidly and efficiently separate low-polarity epimers like antcin A and antcin B, which were verydifficult for RP-HPLC. A 3-min preparative-scale method wasestablished to purify (25S)- and (25R)-antcin. However, OJ-Hcolumn suffered from peak overlapping of different pairs ofergostanes. We found that Princeton 2-ethylpyridine column(2-EP, 4.6×250 mm, 3 µm, achiral) could effectively separate

Figure 3. Construction of the heart-cutting and comprehensive two-dimensional liquid chromatography system (HC-2DLC). (Reprinted with permissionfrom [16]. © 2014 Elsevier)


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different pairs, although the resolutions for 25-R/S forms ofeach epimeric pair were not as good as OJ-H column.Meanwhile, all the (25S)-forms showed stronger retentionsthan the corresponding (25R)-epimers on the 2-EP column.These results demonstrated different selectivity of chiral- andachiral-SFC in separating 25R/S-ergostane epimers. Asidefrom high separation efficiency, SFC also showed advantageover HPLC in short analysis time and low consumption oforganic solvents. Finally, both OJ-H and 2-EP columns wereused on analytical SFC to separate 25R/S-ergostanesin anextract of A. camphorata.

HIGH-RESOLUTION MASS SPECTROMETRYAND RELATED NOVEL TECHNOLOGY:POWERFUL TOOL FOR DEMYSTIFYINGTRADITIONAL CHINESE MEDICINE

As the most attractive high-resolution mass spectrometry,Q-TOF has proven as powerful tool in TCM analysis due toits strong qualitative and quantitative capabilities in asingle platform. Recently, we have been invited to write areview about Q-TOF technology in the TCM study, inwhich Q-TOF based achievements about TCM analysisincluding profiling of active ingredients and their metabo-lites, screening of harmful components, and applications ofcutting edge metabolomic strategies are comprehensivelyreviewed[12]. The novel applications employing Q-TOF and

metabolomics strategy we just achieved are demonstratedas follows.

1. Quality control of TCM based on Q-TOF andmetabolomics strategyQuality control is vital for ensuring safety and efficacy ofTCMs. Usually, TCMs are used as whole plant and/orcombination of several herbs, and multiple constituents areresponsible for the therapeutic effects. Therefore, qualitycontrol of TCM is very difficult. To date, the valid methodfor quantitatively evaluating the quality of TCM is poor.Recent applications of key analytical techniques in qualityassurance and authentication of herbs and their extracts werereviewed[19–20], which highlight the emerging role of chemicalfingerprinting of TCMs and the latest regulatory requirementsimposed on TCM utilizing chromatographic fingerprinting.

Batch to batch reproducibility is very important for TCMinjection manufacturing. Development of a fast QC solutionis significant to reduce test cost and improve manufacturingthroughput for pharmaceutical factories. A statistical analysisbased strategy has been successfully established and appliedto QC analysis for Shuxuetong Injection by high resolutionQ-TOF mass spectrometry in our laboratory[21]. Bothunsupervised and supervised pattern recognition algorithmswere utilized to perform clustering analysis and buildstatistical model of Shuxuetong Injection. In this work, wedesigned a workflow to assess manufacture reproducibilityand to filter out disqualified product as shown in Figure 6(A).

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)



Shuxuetong Injections of different batches were analyzeddirectly without further sample preparation. Accurate massfingerprint profile of each sample was obtained by usingUHPLC/Q-TOF system. Chromatograms with reproducibleretention time, signal response, and accurate mass wereachieved. Molecular feature extraction algorithm wasemployed to automatically find compounds in each chroma-togram. All the rest of the data mining process wasperformed on Agilent metabolomic research platform, i.e.Mass Profiler Professional software. Principal ComponentAnalysis (PCA) plot can clearly show intra-batch reproduci-bility. A Partial Least Squares Discrimination (PLSD) modelwas built and validated on the basis of known samples as

shown in Figure 6(B). Components that result in statisticallysignificant differences between qualified and disqualifiedproducts were identified by Agilent Molecular StructureCorrelator (MSC) software based on MS/MS data as seenfrom Figure 7. The established strategy can also be applied todistinguish products from different manufacturers.

2. Natural product identification, geographic origindeduction, and manufacturing process discriminationby high resolution mass spectrometryThe active ingredients in TCM vary a lot with respect todifferent origin, planting conditions and extract process, whichplays essential roles in the TCM quality and therapy effects.

Figure 5. (A) Chemical structures of ergostanes isolated from Antrodia camphorate; (B) Simultaneous separation of seven pairs of 25R/S-ergostaneepimers by SFC. (a) Chiralcel OJ-H column (chiral), Overlaid chromatograms of each epimeric pair; (b1)Princeton 2-ethylpyridine column (achiral), mixture ofall 14 standards; (b2) 2-ethylpyridine column, mixture of (S)-form and (R)-form ergostanes, respectively. (Reprinted with permission from [18]. © 2014Elsevier)


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Tea is the second most widely consumed beverage in theworld and grown in about 30 countries worldwide. Manyhealth benefits including prevention of cancer and heartdiseases result from the consumption of tea. It is found thattea’s constituents are significantly affected by geographicorigin and manufacturing processes. For food safety purpose,it is important to distinguish the fake or low-grade teaproduct from the first-rate ones and to better understand thefate of tea after entering the human body. In our laboratory, ahigh resolution mass spectrometry combined with metabo-lomic software tool and dedicated database was employed to

identify the components from tea extract and identify teas ofdifferent kinds and origins[22].

A specific database including 333 compounds with com-pound name, molecular structure, formula, accurate mass, CASNo, and partial high resolution MS/MS spectra was successfullyestablished on the basis of literature reference. More thantwenty kinds of components, i.e., catechins, flavonols, flavones,flavonol glycosides, flavone glycosides, proanthocyanidins andbisflavanols, theasinensins, theaflavins, thearubigins, hydrolyz-able tannins, phenolic acids and derivatives, purine alkaloids,amino acids, carotenoids, chlorophylls, carbohydrates, vitamins,

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.



organic acids, acrylamide, and lipids were embodied. Unknownidentification and confirmation based on MS, MS/MS, andretention time matching were performed with the use of theabove database and Agilent Molecular Structure Correlatorsoftware. Agilent metabolomic research platform, i.e. MassProfiler Professional software, was utilized for statistical analysisand class prediction model building. Many structural isomers,like (+)-catechin vs. (�)-epicatechin, theobromine vs. theo-phylline were well resolved and identified by current method. APLSD model was established and successfully applied todistinguish tea from different geographic origin and fromdifferent manufacturing processes as seen from Figure 8. In

addition, biotransformation pathways of important biomar-kers contributing to differentiation were also discussed. Thisresearch is meaningful to tea quality control and fake teascreening.

3. CE-MS for screening the active componentsin TCMCapillary electrophoresis (CE) is a family of electromigrationtechniques that employ small diameter capillaries to performhigh efficiency separation of both large and small moleculeswhether charged or uncharged. CE and CE-MS technologieshave been widely used for analyzing the TCMs. The

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


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applications of CE methods to phytochemical analysis andquality control of herbal drugs have been reviewed[23–25].

Amino acids in Chinese Traditional Medicines (TCMs) areimportant bio-active components, which have attracted muchattention. In this study, we developed an advanced CE-Q-TOFtandem technique for characterizing sixteen amino acids inherbal medicines[26]. This technique proved to be rapid andhighly sensitive, involving data processing (Mass MoleculeFeature Extraction, MFE), accurate mass database search, andstructural confirmation based on MS/MS fragmentation.

MS and MS/MS data were acquired on an Agilent Q-TOFsystem in positive mode with Jet Stream ESI. Separation wasperformed on the Agilent 7100 HPCE (High PerformanceCapillary Electrophoresis) using 1 M formic acid as runningbuffer and 5mMacetate ammonium in 50%methanol as sheath

liquid. The separation voltage was 30 kV by employing 50 µmID and 80 cm length silica capillary. The mass spectrometer wasoperated in MS mode at 2 spectra/second and MS/MS mode at3 spectra/second for profiling. MS parameters: 100 V Fragmen-tor; 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 analysisof sixteen amino acids in positive ion mode using anelectrolyte with a pH value below the analyte’s isoelectricpoint (<2.77). An accurate mass TCM database (containing10,500 active compounds) was established for rapid screen-ing of the amino acids in the TCM samples, after the raw MSdata was processed by the MFE algorithm to remove thebackground ions producing a compound list. The conditionsof CE were systematically optimized with respect to running

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#.



buffer system and sheath liquid components to obtain thehigh-efficient and fast separation. The mass spectrometerparameters were optimized for the best sensitivity withregard to the Jet Stream ESI parameters, fragmentor voltageand acquisition rate. Especially, leucine and iso-leucine canbe well separated at the optimized conditions in the RadixAstragali injection samples as seen from Figure 9. Reprodu-cibility of migration time and peak area was excellent withRSD 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 structuralconfirmation and fragmentation mechanism study of tar-geted amino acids in real samples. The study demonstratesthat the established CE-Q-TOF method offers high-through-put, good sensitivity and high selectivity for characterizingthe amino acids in TCMs. In addition, this technique can beapplied to profiling profile amino acids in other complexmatrices 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 inTraditional Chinese Medicines Using UHPLC-QQQMass spectrometry with triggered MRMDue to the complexity of TCM matrices, false positiveidentification during the quantitation (including activeingredients and illegally chemical additives) in TCM is amajor concern when employing LC-QQQ technique. Onlytwo MRMs cannot ensure reliable confirmation for mostcases, especially for trace analysis in the complex foodmatrices. As recently developed technique, triggered MRM(tMRM) can effectively avoid false positive occurrence by theacquisition of additional confirmatory ions and performingfurther reference library matching[28]. Due to the use ofoptimized collision energy and dwell time for each MRM,tMRM is very sensitive with excellent reproducible spectraeven at trace concentrations.

The purpose of the study was to develop a procedure forthe detection of 214 common synthetic adulterants in dietary

supplements and TCM, using ultrahigh UHPLC-QQQ withthe innovation scan mode, triggered multiple reactionmonitoring (tMRM). The 214 drugs belonging to thirtythree pharmacological classes were grouped in suites,comprising aphrodisiacs, slimming drugs, anti-diabeticdrugs, anti-epileptic drugs, hormones, anabolic drugs, psy-chotropic drugs and antibiotics, etc., as shown in Figure 10(A). Validation was achieved according to the criteriapublished 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 1.8µm particles was applied to achieve excellent separation withsymmetric peak shapes. Dietary supplements and TCM wereanalyzed 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 or0.05 ng/ml for all target pesticides with internal standard

Figure 10. (A) The workflow of monitoring 214 adulterants in the TCM; (B) Triggered MRM for monitoring 214 adulterants.



(Triphenylphosphate as the ISTD with the concentration of2.0 ng/ml in spiked tea samples). Moreover, good precisionand linearity were obtained for quantitative determination intea 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 developedtMRM method offered highly selective and accurate detec-tion of multi-targets screening (MTS) applications for TCMquality control.

5. Ion Mobility-QTOF Mass Spectrometry: Bigpotential to TCM AnalysisThe field of ion mobility-mass spectrometry (IM-MS QTOF)has grown with significant momentum in recent years inboth fundamental advances and pioneering applications[29–31].A search of the terms “ion mobility” and “mass spectrometry”returns more than 2,000 papers, with over half of these beingpublished in the past 4 years. This increased interest has beenmotivated in large part by improved technologies which have

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 singlebore capillary. The front ion funnel improves the sensitivity by efficiently transferring gas phase ions into the trapping funnel while pumping away excessgas 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 20V/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 forresolving two isobaric trisaccharides.


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enabled contemporary IM-MS QTOF to be amendable to avariety of samples in biology and medicine with highsensitivity, resolving power, and sample throughput. As shownin Figure 11(A), the new generation of IM-QTOF LC/MSsystem enables very precise and accurate collision cross section(CCS) measurements without class dependent calibrationstandards, which operates under uniform low field conditions(allowing drift time information for ions to be directlyconverted to collision cross section information). The innova-tive ion funnel technology dramatically increases the ionsampling into the mass spectrometer and results in higherquality MS/MS spectra at trace levels[32].

The IM-QTOF mass spectrometry can deliver an addeddimension of separation, provide direct measurement ofaccurate collision cross sections, preserve structural charac-teristics of molecular conformations and expand coveragemaps for the complex TCM samples. As seen from Figure 11(B), a 1:1 mixture of melezitose and raffinose was infusedusing a syringe pump. These two carbohydrates can bebaseline separated using the ion mobility drift cell anddetected using the Q-TOF mass analyzer as sodium adducts,which cannot be resolved by using the traditional massspectrometry. The ion mobility resolving power for thisseparation is 60. As seen from Figure 12, the fast separationof four natural products in the ion mobility drift cell of IM-QTOF mass spectrometry with their CSS calculation within1 min was achieved in Flowing Injection Mode, provides arobust approach for ultra-high throughput screening of theactive ingredients in TCMs with extra 3D identificationrelating to CCS measurement. The IM-QTOF technologyespecially contributes to the discrimination of isomers andactive ingredients with structural similarity in TCMs, whichis very attractive for TCM metabolomics and the activecomponent discovery in herbal medicines.

CONCLUSION

Versatile data acquisition modes combined with various datamining techniques and dedicated databases and librariesmake QQQ and Q-TOF suitable for the quantitation andcharacterization of complex sample. As the high-resolutionmass spectrometry, Q-TOF has proven as a powerful tool inTCM analysis due to its strong qualitative and quantitativecapabilities on single platform. Q-TOF based metabolomicstrategies have accepted widespread interests from TCMresearchers and achieved more and more successful applica-tions focusing on quality control, therapeutic effect assess-ment, and toxicity investigation. Overall, Q-TOF has madegreat contributions to the development of both TCM theoryand practice. With the combination with the liquid chroma-tography development including UHPLC, 2D LC and SFC,LC-MS technology can play key roles in the TCM relatedresearch and manufacture process.

ACKNOWLEDGEMENTS

We gratefully acknowledge the technique direction fromRong An (Greater China Application Support Manager fromAgilent Technologies) for this review.

REFERENCES

1. Tang F, Zhang QL, Nie Z, Chen B, Yao SZ. Sample preparation foranalyzing traditional Chinese medicines. Trends in Analytical Chem-istry 2009, 28:1253–1262.

2. Steinmann D., Ganzera M. Recent advances on HPLC/MS in medicinalplant analysis. Journal Pharmceutical and Biomedical Analysis 2011,55:744–757.

3. Dong MW, Zhang K. Ultra-high-pressure liquid chromatography(UHPLC) in method development. Trends in Analytical Chemistry2014, 63:21–30.

Figure 12. The fast separation of four natural products in the ion mobility drift cell of IM-QTOF mass spectrometry by flowing injection Mode.



4. Walter TH, Andrews RW. Recent innovations in UHPLC columns andinstrumentation. Trends in Analytical Chemistry 2014, 63:14–20.

5. Fekete S, Schappler J, Veuthey JL, Guillarme D. Current and futuretrends in UHPLC. Trends in Analytical Chemistry 2014, 63:2–13.

6. Cao JL, Wei JC, Chen MW, Su HX, Wan JB, Wang YT, Li P.Application of two-dimensional chromatography in the analysis ofChinese herbal medicines. Journal of Chromatography A 2014,1371:1–14.

7. Sarrut M, Cretier G, Heinisch S. Theoretical and practical interest inUHPLC technology for 2D-LC. Trends in Analytical Chemistry 2014,63:104–112.

8. Williams KL, Sander LC. Enantiomer separations on chiral stationaryphasesin supercritical fluid chromatography. Journal of Chromato-graphy A 1997,785:149–158.

9. Miller L. Preparative enantioseparations using supercritical fluidchromatog-raphy. Journal of Chromatography A 2012, 1250:250–255.

10. Lesellier E, Destandau E, Grigoras C, Fougère L, Elfakir C. Fastseparation oftriterpenoids by supercritical fluid chromatography/evaporative light scattering detector. Journal of Chromatography A2012,1268:157–165.

11. Wang Z, Zhang H, Liu O, Donovan B, Development of an orthogonalmethod for mometasone furoate impurity analysis using supercriticalfluid chromatography, Journal of Chromatography A 2011, 1218:2311–2319.

12. Zhang ZX, Bo T, Bai Y, Ye M, An R, Cheng FF, Liu HW. Quadrupoletime-of-flight mass spectrometry: powerful tool for demystifyingtraditional Chinese medicine. Trends in Analytical Chemistry, inrevision.

13. Huang HL, Liu M, Chen P. Recent advances in ultra-high performanceliquid chromatography for the analysis of Traditional ChineseMedicine. Analytical Letters 2014, 47: 1835–1851.

14. Qiao X, Song W, Lin XH, Wang Q, Bo T, Guo DA, Liu JX, Ye M. Rapidchemical analysis of bear bile: 5 minute separation and quantitationof bile acids using UHPLC–qTOF-MS. Analytical Mehtods 2014,6:596–601.

15. Dugo P, Cacciola F, Kumm T, Dugo G, Mondello L. Comprehensivemulti di-mensional liquid chromatography: theory and applications,Journal of Chromatography A, 2008,1184:353–368.

16. Qiao X, Song W, Jia S, Li YJ, Wang Y, Li R, An R, Guo DA, Ye M.Separation and detection of minor constituents in herbal medicinesusing a combination of heart-cutting and comprehensivetwo-dimen-sional liquid chromatography. Journal of Chromatography A 2014,1362:157–167.

17. Xu X, Roman JM, Veenstra TD, Anda JV, Ziegler RG, Issaq HJ. Analy-sis of fifteen estrogen metabolites using packed column supercriticalfluid chromatography–mass spectrometry. Analytical Chemistry 2006,78:1553–1558.

18. Qiao X, An R, Huang Y, Jia S, Li L, Tzengc YM, Guo DA, Ye M.Separation of 25R/S-ergostane triterpenoids in the medicinal mush-room Antrodia camphorata using analytical supercritical-fluid chro-matography. Journal of Chromatography A 2014, 1358:252–260.

19. Jing J, Ren WC, Chen SB, Wei M, Parekh HS. Advances in analyticaltechnologies to evaluate the quality of traditional Chinese medicines.Trends in Analytical Chemistry 2013, 44: 39–45.

20. Li SP, Zhao JB, Yang B. Strategies for quality control of Chinesemedicines. Journal of Pharmaceutical and Biomedical Analysis 2011,55:802–809.

21. Zhang ZX, Bo T, Chen W, Zhang ZX. Quality Control for ShuxuetongInjection by High Resolution Mass Spectrometry Based StatisticalAnalysis. Minneapolis: 61rd ASMS Conference on Mass Spectrometry& Allied Topics, 2013, Oral presentation.

22. Zhang ZX, Bo T, Chen W, Zhang ZX. Tea’s Constituents Identification,Geographic Origin Deduction, and Manufacture Process Discrimina-tion by High Resolution Mass Spectrometry. Vancouver: 60rd ASMSConference on Mass Spectrometry & Allied Topics, 2012, Posterpresentation.

23. Gotti R. Capillary electrophoresis of phytochemical substances inherbal drugs and medicinal plants. Journal of Pharmaceutical andBiomedical Analysis 2011, 55:775–801.

24. Rabanes HR, Guidote AM, Quirino JP, Capillary electrophoresis ofnatural products: Highlights of the last five years (2006–2010).Electrophoresis 2012, 33:180–195.

25. Ganzera M. Quality control of herbal medicines by capillary electro-phoresis: Potential, requirements and applications. Electrophoresis2008, 29:3489–3503.

26. Bo T, Zhang ZX. Advanced CE-QTOF tandem technique for char-acterizing sixteen amino acids in herbal medicines. Vancouver: 60rdASMS Conference on Mass Spectrometry & Allied Topics 2012,Poster presentation.

27. Li JZ, Bo T, Wu CL , Lu YF, Chen W, Zhang ZX. Quantitation ofantiviral drugs in chicken samples by ultra-high performance liquidchromatography tandem triple quadrupole mass spectrometry withtriggered MRM for high-accurate confirmation. Baltimore: 62rdASMS Conference on Mass Spectrometry & Allied Topics 2014,Poster presentation.

28. Zhang MY, Song Y, Sun JL, Wang SZ, Lee WY, Chen SA. Determina-tion of Synthetic Adulterants in Traditional Chinese Medicines UsingUHPLC-QQQ with triggered MRM. St. Louis: 63rd ASMS Conferenceon Mass Spectrometry & Allied Topics 2014, Poster presentation.

29. John WP, Roberto CG, David H. Russel l. Elucidation of ConformerPreferences for a Hydrophobic Antimicrobial Peptide by VesicleCapture-Freeze-Drying: A Preparatory Method Coupled to IonMobility-Mass Spectrometry. Analytical Chemistry 2015, 87:578–583.

30. Chan ECY, Lee N, Chun WY, Lin TG, Pharmaceutical metaboliteprofiling using quadrupole/ion mobility spectrometry/time-of-flightmass spectrometry. Rapid Communication of Mass Spectrometry2009,23:384–394.

31. O'Donnell RM., Sun Xiaobo, Harrington PB. Pharmaceutical applica-tions of ion mobility spectrometry. Trends in Analytical Chemistry2008, 27:44–52.

32. May JC, McLean JA. Ion Mobility-Mass Spectrometry: Time-DispersiveInstrumentation. Analytical Chemistry 2015, 87:1422–1436.


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