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Mass spectrometry and NMR spectroscopy: modernhigh-end detectors for high resolution separationtechniques state of the art in natural product HPLC-MS,HPLC-NMR, and CE-MS hyphenations

Christoph Seger, ab Sonja Sturm a and Hermann Stuppner *a

Covering: 2005 to 2013

Current natural product research is unthinkable without the use of high resolution separation techniques

as high performance liquid chromatography or capillary electrophoresis (HPLC or CE respectively)combined with mass spectrometers (MS) or nuclear magnetic resonance (NMR) spectrometers. Thesehyphenated instrumental analysis platforms (CE-MS, HPLC-MS or HPLC-NMR) are valuable tools fornatural product de novo identi cation, as well as the authentication, distribution, and quanti cationof constituents in biogenic raw materials, natural medicines and biological materials obtained frommodel organisms, animals and humans. Moreover, metabolic pro ling and metabolic ngerprintingapplications can be addressed as well as pharmacodynamic and pharmacokinetic issues. This reviewprovides an overview of latest technological developments, discusses the assets and drawbacks of theavailable hyphenation techniques, and describes typical analytical work ows.

1 Introduction2 The role of analytical chemistry in natural product

research3 Qualitative and quantitative analysis3.1 Structure information3.2 Pattern recognition4 Hyphenated analysis platforms4.1 Separation techniques4.2 Hyphenated detectors4.3 Mass spectrometers as HPLC detectors4.4 Mass spectrometers as CE detectors4.5 NMR spectrometers as HPLC detectors4.5.1 HPLC-NMR 4.5.2 Capillary NMR

4.5.3 HPLC-SPE-NMR 5 Applications5.1 HPLC-MS5.2 HPLC-NMR and HPLC-SPE-NMR

6 Conclusion7 References

1 Introduction

Dealing with natural products in modern research is unthink-able without the use of high resolution separation techniques(i.e. high performance liquid chromatography or capillary electrophoresis; HPLC or CE, respectively). They are not only invaluable tools in unraveling the complexity of secondary natural products but in combination with high informationdensity detection systems, such as mass spectrometry (MS) ornuclear magnetic resonance (NMR) spectroscopy, they areextremely bene cial in terms of fostering the identi cation of

analytical entities of interest. Combining these instrumentalanalysis core technologies in hyphenated devices as CE-MS,HPLC-MS or HPLC-NMR is one of the most vivid research

elds in modern analytical chemistry as well as in applicativenatural product related science branches such as phytochem-istry or pharmacognosy. Here typical applications includeregulatory issues established by industrial partners or publicauthorities ( i.e. pesticide contamination screens, adulterationdetection), natural product pharmacokinetics, secondary metabolite biogenesis, biosystematics, and secondary metabo-lite discovery/structure elucidation. Either purely qualitativepro ling approaches as metabolic pro ling, metabolic

a Institute of Pharmacy/Pharmacognosy, CCB - Centrum of Chemistry and Biomedicine,University of Innsbruck, Innrain 80 82, A-6020 Innsbruck, Austria. E-mail: [email protected]; Fax: +43 512 507 58499; Tel: +43 512 507 58401b Institute of Medical and Chemical Laboratory Diagnostics (ZIMCL), University Hospital/Landeskrankenhaus Innsbruck, Anichstrae 35, A-6020 Innsbruck. E-mail:[email protected]; Tel: +43 512 504 81155

Citethis: Nat. Prod. Rep. , 2013, 30 , 970

Received 20th February 2013

DOI: 10.1039/c3np70015a

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970 | Nat. Prod. Rep. , 2013, 30 , 970 987 This journal is The Royal Society of Chemistry 2013

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ngerprinting, and metabolomics or target orientated quanti-tative assays are used to address such tasks. This review provides an overview of latest technological developments,discusses the assets and drawbacks of the available hyphen-ation techniques, and describes typical analytical work ows.

2 The role of analytical chemistry in naturalproduct research

In academia, laboratory scale workup of a crude extract towardspuri ed single constituents is usually accompanied by analyticalassays and pharmacological investigations, e.g. if the strategy of bioassay guided fractionation is chosen. 1 3 Only the combinationof these methods a valid analysis of the fractions by a highresolution separation technique and a reproducible assessment of extract bioactivity might give the investigator a chance to

identify the bioactive principle of the material. To be used for invitro and in vivo testing, the identity and purity of such materialshas to be assessed by characterizing physico-chemical ( e.g.melting point, crystal properties, residual solvent contamina-tions, water content, etc.) and spectroscopical/spectrometrical(e.g. UV/VIS, IR, Raman, NMR, MS/MS) properties. This also holdstrue, of course, if in an industrialized process secondary metab-olites are isolated from natural sources to be used as nature

derived drugs ( e.g. digoxin, digitoxin, cyclosporine, tacrolimus, vinblastine/vincristine etc.).4 In addition, hyphenated instru-mental analysis techniques, mostly HPLC-MS/MS, accompany pharmacological investigations from research setups to clinicaltrials. Since these measurements are performed in bio uids, suchassays are generally considered bio-analytical methods. 5

In industry, but also in academia, a detailed qualitative andquantitative assessment of starting materials is necessary if working with natural products. If applicable, this includesauthentication of the crude drug, exclusion of (fraudulent)adulterations with other plants or xenobiotic drugs, microbialscreening, and targeted analysis of undesired impurities such

as pesticides, herbicides, or mycotoxins. Ideally a chain of control measurements monitors the integrity of the product throughout the production process from the raw materials tothe shelf. Since the used biological materials are derived fromliving systems and do re ect ecological uctuations in theirsecondary metabolite pattern, inter-batch product consistency over the products life time has to be scrupulously monitored toensure the claimed product e ffi cacy. 6

3 Qualitative and quantitative analysis

In analytical chemistry detector readouts can be used in many

diff erent ways

depending on the purpose of the assay. Most o en a quantitative result is sought; here comparison of samplereadouts with readouts from reference samples of known

Sonja Sturm studied Pharma-ceutical Sciences at the Univer-sity of Innsbruck. A er receiving her Ph.D. in Pharmaceutical Biology in 1993, she joined the Kinghorn group at the Universityof Illinois, Chicago as post-

doctoral fellow. Since 1994 sheis an academic sta ff member at the Institute of Pharmacy of theUniversity of Innsbruck. Her main research interests are thedevelopment of qualitative and

quantitative separation methods for plant materials and phyto- pharmaceutical preparations derived thereof utilizing both CE and HPLC instrumentations equipped with MS, or NMR instruments asanalyte detectors. In addition she serves as scienti c committeemember for ESCOP.

Hermann Stuppner studied Pharmaceutical Sciences at theUniversity of Innsbruck. A er receiving his Ph.D. in Pharma-ceutical Biology from theUniversity of Munich he was a postdoctoral fellow at the

Department of Developmental and Cell Biology of the Univer-sity of California, Irvine. Since 2001 he is Full Professor of Pharmacognosy at the Instituteof Pharmacy of the University of

Innsbruck. His main research interests are: isolation and struc-tural elucidation of secondary metabolites from higher plants withanti-in ammatory and antitumor activity, analysis and qualityassessment of (medicinal) plants and phytopharmaceuticals,discovery of pharmacologically active natural products by meansof computer aided models.

Christoph Seger studied Chem-istry at the University of Vienna, Austria. A er focusing on NMRbased structure elucidation of Natural Products (PhD 2001,University of Vienna) and peptides (Max Planck Institute, Martinsried) he nally joined theteam of Hermann Stuppner. Herehis major research focus was theestablishment of HPLC-UV/MS/ NMR based qualitative and quantitative assays. In 2008 he

received his venia docendi in Pharmacognosy from the University of Innsbruck. Currently he is in a leadership position in a HPLC-MS/

MS laboratory at the University Hospital Innsbruck and enjoysanalytical phytochemistry as an independent researcher in the Pharmacognosy group at the University of Innsbruck.

This journal is The Royal Society of Chemistry 2013 Nat. Prod. Rep. , 2013, 30 , 970 987 | 971

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content allows quanti cation of analytes in investigated speci-mens. 7 If the identity of an analyte has to be deduced, amaximum of structure related information has to be gainedfrom the detector readout 8,9 to ensure a proper structure eluci-dation. 10 If qualitative information is to be retrieved, i.e. if acertain component or series of components ( a ngerprint )should be present or absent, visual or chemometric patterncomparisons are applied to the data. 11

3.1 Structure information

Mass spectrometry is generally said to yield reasonable struc-ture information. However, it has to be strongly emphasizedthat this data alone, even if tandem mass spectrometers withhigh resolution mass selector technologies are employed asdetectors in chromatography, can hardly provide more than themolecular formula at its best, 12,13 if databases are not used orthe elucidation process can be restricted to a common sca ff old/a structurally narrow compound class e.g. peptides, 14,15

DNA/RNA,16 or avonoids. 17 Electron impact (in GC-MS) and

secondary fragmentation mass spectra (as in ESI-MS/MS) may allow a deeper insight into the molecular structure of the ana-lyte under investigation, but also clearly fail in unequivocally ascertaining the molecular sca ff old (i.e. geometrical isomers,position isomers), not to speak of its 3D structure. 18 In plant metabolomics and metabolic pro ling, which use analyticalplatforms with MS based readout devices, the limited possibility to elucidate the structure of unknown analytes from thegenerated MS data has been recognized as major methodolog-ical bottleneck. In too many case studies, analytes showing upas discriminators in a sample set (the putative biomarkers) havebeen le unidenti ed. 11 Key players in mass spectrometry basedmetabolomics and metabolic pro ling addressed in severalreviews that a major drawback of metabolomic technologies yet to be overcome is the vast number of unknown compoundstructures ,19 that the identi cation of metabolites whose MS/MS spectra are not present in the spectral libraries remains very challenging and is being addressed through both empirical andcomputational mass spectrometry 20 and as a consequence that increasing the number of metabolite identi cations withinexisting pro ling platforms is prerequisite for a substantialimproved scope of pro ling studies .21

At the present time, de novo structure elucidation of secondary natural products in solution (in solid phase X-ray analysis can be pursued, if appropriate crystals of the

compound of interest can be obtained) can be only achieved by the exhaustive use of 1D and 2D homo- and heteronuclear NMR measurements assisted by other spectroscopic methods,including high resolution MS. In the understanding of somescience managers within the last few years NMR spectroscopy of small molecules has morphed to a routine analysis method tobe handled by sta ff scientists . This might be possible for themere operation of a modern day NMR spectrometer including 1D/2D NMR spectra acquisition, a task easily manageable by graduate chemists supported by one or more technicians. Swi ,economic NMR spectra interpretation and structure elucida-tion, however, is still a demanding task, which has, in spite of

numerous attempts over recent years, not been replaceable by computer aided work ows in the desired holistic compoundclass independent manner. 22 25 It still holds true that thedevelopment of systems for computer-assisted structure eluci-dation based on spectroscopic e ff ects has been considered as ahighly challenging problem from the very beginnings of che-moinformatics. Thus, a lot of work from excellent scientists hasbeen put into this eld. Nevertheless, none of the systems

developed so far has found broad use in chemical laborato-ries .26 Hence a long time expertise, especially the detailedknowledge of nuclear spin systems to be expected and strategicroutes to unravel the o en incomplete and/or redundant NMR data, still discriminates a successful structure elucidator fromless experienced researchers. 27

3.2 Pattern recognition

Whereas in quantitative assays usually only the minimalamount of information needed to ful ll the given task is gath-ered ( i.e. one ion trace from a mass spectrum, one UV trace froma UV spectrum . ), pro ling and ngerprinting approaches tendto use all the data available from a detector. In some cases, suchholistic datasets can even be used to derive quantitative or at least semi-quantitative information on selected analytes ( i.e. inNMR spectroscopy). To investigate this huge amount of data,to extract meaningful information from the analytical andbiological noise, to nd biomarkers underpinning a givenhypothesis, chemometrical approaches have to be applied. Herea multitude of statistical methodologies, including datapretreatment of o en imperfect detector readouts, are available, which will not be discussed further in this overview. 11,28 33

4 Hyphenated analysis platforms4.1 Separation techniques

Among the di ff erent analytical separation systems available, apredominance of high performance liquid chromatography (HPLC, o en abbreviated as LC ) and gas chromatography (GC) can be observed in natural product analysis. Although GCstill is unmet in its separation capacity, its application ishowever limited by the need to vaporize samples prior to analysis. If not applying GC to volatile matrices ( i.e. terpenoid richessential oils) extensive sample preparation protocols including analyte extraction and derivatization are usually necessary andthermally labile analytes or analytes still involatile a er deriv-

atization cannot be analyzed at all. Electrophoretic separationtechniques such as capillary electrophoresis (CE) or capillary electrochromatography (CEC) are always valuable alternativesto HPLC- or GC-based assays in small organic molecule analysisbut hampered in their routine use by the relatively low sensi-tivities caused by low mass ow and the reduced stability of theutilized fused silica capillaries. 34

Hence, amongst all separation techniques available, HPLC iscurrently the best compromise. As a robust and more or lessuniversal separation technique combinable with a variety of reasonably sensitive or selective detector systems it is applied inalmost any natural science laboratory in one way or another.


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HPLC based chromatography is a thriving and innovativeinstrumental analysis eld, latest developments are devoted toreduce analysis time by the use of highly separation e ffi cient (high resolution) stationary phase materials with sub-2 mmparticle sizes. Equipped with various detection devices, it can beused for more or less any analytical task encountered, sinceapolar analyte classes, such as terpenoids, lipids or depsipep-tides, can be addressed as well as polar metabolites, such as

polyphenol-glycosides, saponins or amino acids. However, it shall not be ignored that, although HPLC analysis itself usually does not require analyte derivatization prior to chromatog-raphy, extensive sample preparation work may precede bothqualitative pro ling/ ngerprinting and quantitative target analytical assays. By including one or more sample preparationsteps like (i) sample homogenization (milling, sieving,

ltration), (ii) sample extraction (liquid liquid extraction, par-titioning, preparative solid phase extraction) or (iii) chromato-graphic clean-up (preparative column chromatography, highspeed counter current chromatography (HSCCC), analyticalsolid phase extraction) in the pre-analytical work ow, unde-

sired matrix in uences are minimized and investigated analytesare enriched. 35,36

4.2 Hyphenated detectors

In contrast to GC, which in natural product research reliesmostly on the unspeci c ame ionization detector (FID) tomonitor analytes if a mass spectrometry hyphenation (GC-MS orGC-MS/MS) is not realized; analyte detection in HPLC and CE/CEC can be facilitated in many ways. Besides mass spectrometry and NMR spectroscopy, to be discussed further below, spec-troscopic detectors recording data in the range of ultraviolet and visible light (UV/VIS) are predominant. Most o en they areimplemented as diode array detectors (DAD), allowing onlinerecording of UV/VIS spectra by paralleled acquisition of a broadrange of wavelengths. Since light absorption in the UV/VISrange requires conjugated double bond systems (chromo-phores) at least to some extent, this technique is limited toanalytes bearing such substructures. In addition, UV/VISdetection limits the use of HPLC mobile phases and mobilephase additives to ones not absorbing light in the detection wavelength ranges. Other metabolites e.g. saponins, most terpenoids, carbohydrates, sugar alcohols, and many more,have to be derivatized with chromophore-bearing ligands ( e.g.ninhydrin derivatization for amino acids analysis) or have to be

detected by alternative techniques. Here the evaporative light scattering detector (ELSD) 37 and the corona charged aerosoldetector (CAD) 38 have emerged over the past few years as valu-able alternatives in combination with HPLC separations,despite being unselective and showing non linear concentra-tion/signal relationships. Less o en utilized in natural product analysis but of unquestioned value as highly speci c detectorsin bioanalytical HPLC, uorescent light detectors (FLD) oranalyzers relying on electrochemical properties ( e.g. ampero-metric detection) are successfully applied in targeted assays 39

but have limited use in screening setups. In addition to thephysical detection principles summarized above, enzyme or

antibody based assays e.g. frontal a ffi nity chromatography with mass spectrometry as detector (FAC-MS), post HPLC on-

ow reactors, pulsed ultra- ltration mass spectrometry, orsurface plasmon resonance (SPR) based biosensors can beapplied to screen extracts or substance libraries for novelbioactivities. 40,41

4.3 Mass spectrometers as HPLC detectors Whereas standalone mass spectrometry has accompaniedstructure elucidation e ff orts in natural product research formore than half a century, 42,43 only within the last two decadesmass spectrometers have become easily applicable HPLCdetectors. John B. Fenn's invention of the atmospheric pressureionization (API) technique ESI (electrospray ionization) in themid 1980s can be considered as the major breakthrough in so ionization techniques. 44 46 ESI facilitates the transfer of analytemolecules from uncharged liquid phase species to gas phaseions, hence making the hyphenation of mass spectrometers toliquid chromatography systems technically feasible. First

commercial HPLC-ESI-MS hyphenations became available inthe 1990s, with rst applicative bio-analytical and naturalproduct analysis publications emerging shortly therea er. 47

Two more API techniques, atmospheric pressure chemicalionization (APCI) and the much younger atmospheric pressurephoto ionization (APPI) allowed extending the range of analyz-able molecules to apolar species hardly ionizable by ESI. 48

Several types of mass spectrometers, i.e. tandem mass spec-trometers (QqQ), ion trap mass spectrometers (IT-MS), hybridmass spectrometers, such as the QqTOF instruments with aquadrupole mass analyzer (Q) followed by a collision cell (q) andtime of ight (TOF) mass analyzer as a readout device, orFourier-Transform mass spectrometers (FT-MS) can becombined with separation devices such as HPLC and CE. 49,50

The di ff erent mass spectrometer designs utilized in HPLC-MS/MS hyphenations allow the realization of distinctively diff erent mass spectrometry experiments with optimal appli-cation ranges. The selected reaction monitoring (SRM) experi-ment realizable with tandem mass spectrometers of the QqQtype is unmatched in its selectivity and reproducibility, whichtranslates to exceptional sensitivity and large linear ranges. Itsmajor application eld is target analysis investigations withprede ned analytes panels to be covered. Major drawbacks of the SRM experiment are (i) the low inherent mass resolution of quadrupoles hampering the user in obtaining meaningful

molecular formulas from the data and (ii) the recording of oneor only a few preselected single ion traces per analyte prohib-iting a deeper insight into the complexity of an investigatedsample. Mass spectrometers with time of ight (ToF) massanalyzers are in contrast designed to record high resolutionmass data by scanning a preset mass range. The gathered datausually enables the investigator to reconstruct one or a few molecular formulas for each chromatographic feature detectedin the ion traces of the HPLC e ffl uent, thereby strongly sup-porting the structure elucidation process of unknown analytes.In combination with ion fragmentations in a collision cell(QqToF) or within the ionization process, exact masses of


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analyte speci c fragments usually allow an even deeper insight into the molecular sca ff old of an investigated metabolite. Whereas in QqQ mass selector based SRM experiments ioncurrents of one or more prede ned ion pairs (precursor ioncharged in the ion source and product ion generated by the gasphase fragmentation process) are recorded at low resolution(0.5 1.0 mass units peak widths), ToF mass selector basedexperiments are recording preset mass ranges at high resolu-

tion (1/1000 1/100 mass units peak widths).In any hyphenation of mass spectrometers to separation

devices, MS experiments have to be recorded on ight .Consequently, the data acquisition rate of the mass spectrom-eter has to be adequate to record the chromatographic peaks with uncompromised reproducibility and accuracy. In thiscontext, QqQ and ToF mass analyzers show distinctive advan-tages and disadvantages. By narrowing the observation range of a mass spectrometer down to one ion pair, SRM experiments areusually of unmatched sensitivity. If, however, several analyteshave to be monitored ( i.e. in a quality control screen), SRMexperiments which are performed in a strictly serial manner

with observation times in the lower millisecond range, arequickly reaching total cycling times (the sum of all individualSRM experiment times), exceeding the sampling rate of about 500 ms (2 Hz) needed to su ffi ciently represent a chromato-graphic peak in the recorded ion traces with about 8 12 datapoints per peak. 51 With state of the art QqQ mass spectrometers,three to four dozen SRM experiments can be summed up toone LC-MS/MS assay. If more analytes have to be covered,data dependent SRM recording strategies relying on scouting experiments have to be utilized. 52

If narrow chromatographic peaks, i.e. produced by theapplication of chromatographic devices with optimized peak capacities by utilizing modern sub-2 mm stationary phaseparticles and high mobile phase ow rates, 53 have to be moni-tored, the application of SRM experiments comes to its limita-tions, since short cycle times of less than 50 ms to 100 ms (10Hz 20 Hz) have to be realized for appropriate chromatographicpeak coverage. Here modern ToF mass analyzer based detectorsystems are used as alternatives. Due to the registration of repetitive scans, the recording time of a single data point issigni cantly shorter than in the SRM experiment. Conse-quently, the sensitivity of ToF based experiments is limitedcompared to tandem MS instruments operated in the SRMexperiment setup. 54 On the other hand ToF mass spectrometerscans allow post-analysis data mining; the complete high

resolution data set is available for detailed investigations.Hence, ToF based chromatography readouts provide utmost exibility in the assessment of the gathered data both quali-

tative and quantitative analyses are possible. 55 It should,however, not be overlooked that ToF scanning experiments canbe of impaired selectivity if complex matrices ( i.e. crude bio-

uids as plasma) have to be investigated. 56 Here QqToFinstruments allowing selected ions to be monitored in the rst mass selector and to read out gas phase reaction fragment ionsin the second (ToF) mass selector have to be utilized. 55

Besides mass selector designs based on ion travel times(ToF) or on ion de ections in electrical elds (QqQ), the

frequency of ions oscillating in an ion trap can be used to derivetheir mass over charge ( m/ z ) ratio. Such devices can producemass spectra of unmatched resolution by observing the ionclouds selected and transferred to the readout devices withmass selectors of conventional design. Similar to NMR spec-troscopy, a Fourier transformation of the complex frequency pattern generated by the oscillating ions cloud is producing themass spectrum. Since mass resolution in Fourier transform

mass spectrometry 57 (FT-MS) using magnetic eld ion traps (FT-ICR-MS) or electrostatic ion traps (such as the orbitrap ), isproportional to the observation time, recording of high reso-lution mass scans typically needs up to 2 3 s an eternity

compared to the lower millisecond time regimen of QqQ or ToFinstruments. Consequently, such devices cannot be used torecord narrow chromatographic peaks with sampling ratessuffi ciently short to reconstruct chromatographic peaks withhigh accuracy and precision. This pivotal drawback is circum- vented in the instrumental design of one vendor by splitting theions stream generated by the ion source between a conventionallow resolution linear ion trap mass selector recording ion traces

with scan speeds allowing proper recording of chromatographicpeaks and high resolution mass spectra derived from thereadout of the FT-MS unit orbitrap itself.

The third prominent mass spectrometer construction typeused as a detector in hyphenated techniques is the low massresolution ion trap. Two di ff erent designs can be distinguished the more common spherical ion trap or the linear ion trap.Both trap designs allow performing gas phase collision experi-ments on molecular ions con ned in the trap leading to product ion distributions in accordance with the thermodynamic andkinetic laws of gas phase ion chemistry, thus being qualitatively and quantitatively reproducible and at least to some extent

predictable. Consequently, mass spectra derived from suchinstruments can be used in structure elucidation of unknowncompounds or combined with database orientated datamining approaches to identify analytes in a general unknownscreening approach. Compared to conventional QqQ designs orQqTOF designs, which can also produce fragment spectra fromanalytes reacting in the collision cell ( q ) of the instrument where on the ight gas phase reactions are carried out, theadvantage of ion trap instruments lies on the one hand inhigher concentration of ions con ned in the trap and on theother hand the possibility to con ne ionized reaction productsagain and expose them to another gas phase reaction the MS/MS/MS experiment. Hence ion trap instruments can (at least

theoretically) produce MSn

mass spectra of ions, which under- went n 1 gas phase reactions. Tandem mass spectrometers incontrast are strictly restricted to one gas phase reaction result-ing in a MS 2 (MS/MS) mass spectrum.

4.4 Mass spectrometers as CE detectors

The utilization of mass spectrometers as detectors for electro-phoretical separation devices is less common than in liquidchromatography. Due to low ow rates (nL min 1 range) andthe necessity to ensure a closed electrical circuit to maintain ahigh voltage across the separation capillary, interfacing is less


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straightforward than in LC-MS. The rst successful hyphen-ation of CE to MS via an ESI interface was reported in 1987 by Olivares et al. 58 More than two decades later, ESI has remainedthe principal ionization technique 59 61 although generally alltypes of mass spectrometers bearing an API ion source can behyphenated to CE instruments their speci c advantages anddisadvantages in the CE-MS coupling are, however, identical tothe HPLC-MS coupling.

Three types of interfaces for CE-ESI-MS hyphenations havebeen developed up to now, sheathless, sheath-liquid, and liquid junction interfaces. Currently only the sheath-liquid interfacesare commercially available, here make-up liquid and nebulizing gas are added coaxially to the CE e ffl uent, the interface isgrounded and the inlet of the MS is put onto high voltage. Oneof the major drawbacks of this robust and user friendly hyphenation is the dilution of the CE e ffl uent by the sheathliquid, impairing the analytical sensitivity of the device.

In CE selectivity can be easily adjusted by varying the back-ground electrolytes, by addition of chiral modi ers, or by theaddition of neutral micelle formers for the separation of neutral

analytes. Since ESI is susceptible to ion suppression eff

ectscaused by the most frequent employed background electrolytes,such as high concentrations salts, non-volatile salts andorganics constituents, or surfactants, such additives have to beavoided. Consequently, the application range of CE-MS is incomparison to CE-UV dramatically narrowed. A promising approach to overcome this limitation seems to be the imple-mentation of APCI 62 or APPI63 as ionization techniques, bothhighly compatible with nonvolatile background electrolytes.Several applications demonstrated the potential of these inter-faces for CE-MS hyphenations, however, designed for higher

ow rates the adaptation for the extremely low ow rates in CEare still not optimized and are under development. The numberof publications in CE-ESI-MS is limited, besides proof of prin-ciple contributions, most natural product applications aredevoted to the qualitative analysis of alkaloids 64 72 (Fig. 1) and toprimary metabolite pro ling (Table 1). 73 75

4.5 NMR spectrometers as HPLC detectors

In analogy to mass spectrometry, NMR spectroscopy has itsunquestioned merits in the structure elucidation of organicanalytes. For decades it has been considered as one of the majorcornerstones to establish molecular structures by analyzing connectivity networks on the atomic level. 76 79 Due to the signal

richness of its spectra, NMR, however, works best if the inves-tigated analyte is present in high purity whenever mixtures arepresent, the number of overlapping signals rises dramatically.Consequently, especially if secondary metabolites with complex spin patterns are investigated, analyte puri cation prior to NMR based structure analysis is a well established work- ow modelin phytochemistry. Due to the metabolic complexity of naturalmaterial derived extracts, this task is burdensome anddemanding in terms of workforce and costs. Therefore, the on-line combination of analyte separation by HPLC and NMR spectroscopy (HPLC-NMR or LC-NMR) was envisioned from thelate 1970s onwards 80 82 and realized in commercially available

setups shortly therea er. 83,84 From these pioneering works on,HPLC-NMR and related ow probe NMR technologies evolvedto well established analytical platforms around the turn of thecentury. 85 87

4.5.1 HPLC-NMR. In HPLC-NMR ow probes equipped with conventional Helmholtz (saddle) type RF coils matching analytical HPLC dimensions are used. Whereas in conventionalNMR spectrometers a NMR sample tube is placed into thecentre of these coils, in HPLC-NMR this tube is replaced by a

ow cell connected to the LC module with a capillary. Themobile phase eluting from the HPLC column is entering the

ow cell and NMR spectra are recorded permanently. Whilst thechromatographic peak of an analyte is crossing the ow cell,NMR signals of its proton sca ff old are recorded. Typically, toincrease the signal to noise ratio, several NMR experiments ( i.e.

16 scans, recording time less than one minute) are accumulatedfor one NMR spectrum. As in HPLC-MS, the acquisition time of NMR spectra has to be matched to the average peak widthsproduced by the chromatographic system to allow recording of one or more NMR spectra for a chromatographic peak.

The major advantages of on- ow HPLC-NMR lie in the on-line real-time access to structure information-rich NMR data of chromatographic peaks. If a HPLC-DAD-MS/NMR hyphenationis utilized, 88 such a setup can generate all the structural infor-mation usually needed for structural characterization of smallorganic analytes. The major drawbacks of HPLC-NMR are theinherently low sensitivity of NMR spectroscopy, the time

Fig. 1 Non aqueous CE-ESI-MS electropherograms (base peaks, m / z 200 500)of crude Corydalis sp. extracts of four European Corydalis species. Identi cation ofthe analytes was possible by comparison of reference compounds which wereeither purchased, isolated and identi ed by o ff -line NMR or by HPLC-SPE-NMR.174

Reprinted with permission from Sturm et al. 72


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( L R )

[ 6 6 ]

E s c h s c h o l z i a c a l i f o r n i c a ,

A l k a l o i d s

E x t r a c t N F 1 0 0 m M

, F A ( p H

3 )

,

1 0 % M e O H

5 m M F A i n A C N

Q

E S I +

S C ( L R )

[ 6 7 ]

B e r b e r i s v u l g a r i s

,

N F 7 0 m

M , F

A ( p H

4 )

,

4 0 % A C N

J a t e o r h i z a p a l m a t a ,

N F 7 0 m

M , F

A ( p H

4 )

,

4 0 % A C N

C h e l i d o n i u m m a j u s

N F 7 0 m

M , F

A ( p H

4 )

,

2 0 % A C N

S o l a n u m s p . (

p o t a t o e s )

A l k a l o i d s


M , 1 . 2

M A A i n

A C N / M e O H ( 9 0 : 1 0 )

,

1 % A A i n W A / M e O H ( 1 : 1 )

S I T

E S I +

S C ( L R )

, M S n

( L R )

[ 6 8 ]

S t e m o n a s p .

A l k a l o i d s


M , A

A 1 M

, 1 0 %

M e O H i n A C N

2 - p r o p a n o l : W A ( 1 : 1 )

S I T

E S I +

S C ( L R )

, M S n

( L R )

[ 6 9 ]

C o p t i s s p .

A l k a l o i d s


M , N

H 4

O H i n

M e O H / A C N ( 8 0 : 2 0 )

( p H

6 . 8

) , N A 5 0 m M

,

N H

4 O H i n M e O H / A C N

( 5 0 : 5 0 ) ( p H

6 . 8 )

0 . 5 % A A i n W A / M e O H ( 1 : 1 ) T O F

E S I +

S C ( H R )

[ 7 0 ]

F u m a r i a o ffi c i n a l i s

A l k a l o i d s


M , A

A 2 . 2 M

,

1 0 % M e O H i n A C N

2 - p r o p a n o l : W A ( 1 : 1 )

S I T

E S I +

S C ( L R )

, M S n

( L R )

[ 7 1 ]

C o r y d a l i s s p .

A l k a l o i d s


M , A

A 1 M

, 1 0 %

M e O H i n A C N

2 - p r o p a n o l : W A ( 1 : 1 )

S I T

E S I +

S C ( L R )

, M S n

( L R )

[ 7 2 ]

C y s t o s e i r a a b i e s m a r i n a

A m i n o a c i d s , o r g a n i c a c i d s

R a t

u r i n e

1 0 % M e O H

, F A 0 . 8 M

i n W A

F A 1 m M i n M e O H / W A ( 1 : 1 ) T O F

E S I +

S C ( H R )

[ 7 3 ]

T o k i - S

h a k u y a k u - S a n

A m i n o a c i d s , s h i k i m a t e -

d e r i v e d m e t a b o l i t e s

E x t r a c t F A 1 M i n W A

( p H

1 . 8

)

h e x a k i s - ( 2

, 2 - d

i u o r o t h o x y ) -

p h o s p h a z e n e 0 . 1 m M i n M e O H /

W A ( 1 : 1 )

T O F

E S I +

S C ( H R )

[ 7 6 ]

a A C N a c e t o n i t r i l e

, M e O H m e t h a n o l , W A w a t e r , A

A a c e t i c a c i d

, F A f o r m i c a c i d

, N A a m m o n i u m a c e t a t e , N F a m m o n i u m f o r m a t e . b

S I T s p h e r i c a l i o n t r a p , Q

s i n g l e q u a d r u p o l e i n s t r u m e n t , T O F t i m e

o f i g h t i n s t r u m e n t , q T O F q u a d r u p o l e

t i m e o f

i g h t h y b r i d i n s t r u m e n t . c E S I e l e c t r o s p r a y i o n i z a t i o n .

d S C s c a n m o d e , M S n f r a g m e n t a t i o n e x p e r i m e n t . ( H R ) h i g h r e s o l u t i o n M S d e t e c t i o n , ( L R )

l o w r e s o l u t i o n M S d e t e c t i o n .


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constraints of the on- ow data acquisition, and the NMR shi referencing problem occurring whenever solvent conditions arechanging i.e. if a LC solvent gradient is applied. 89 Conse-quently, HPLC-NMR was evolved by improvement on both theHPLC and the NMR side. Within the past twenty years setupschanged from on- ow experiment designs to stopped ow HPLC-NMR and further to the temporal segregation of chro-matography and NMR using loop collection and loop storage

devices for peak parking. 76 Modern commercial HPLC-NMR installations allowing these operation options are availablefrom all major NMR instrument producers. Whereas on ow experiments have nearly vanished from the literature within thelast few years due to their insensitivity, stopped ow and loopcollection experiments are still widely applied in naturalproduct chemistry 90,91 and the pharmaceutical industry. 87,92 94

Phytochemical applications range from secondary plant metabolite identi cation in plants 95 or cell cultures, 96 to bio-degradation studies. 97

4.5.2 Capillary NMR. Following the introduction of capil-lary HPLC for the analysis of mass limited samples, the

hyphenation to NMR became feasible with the development of capillaryNMRprobes withsolenoidal RF coils. 98,99 Thisapproachresulted in the design and commercial availability of microcoilprobes with active sample volumes (capillary volume within theRF sender/receiver coil) down to 1.5 ml. Although capillary NMR can be successfully hyphenated to matching micro HPLCequipment 100 or can be combined with sample robots in indus-trialstyle highthroughputsetups, 101 it is currentlymostly used inan o ff -line mode with driedsamples taken up in a fewmicrolitersofNMRsolvent andintroduced into theprobemanuallyor by theaid of a syringe pump. 102 Whenever an analyte is not concentra-tion-limited, meaning that it can be dissolved in a microliteramount of NMR solvent, microcoil NMR is superior to conven-tional NMR machines due to the inherently higher sensitivity of smaller-diameter coils. 98 A sensitivity gain factor of up to ve canbe realized compared to conventional NMR setups using sampletubes. Consequently, recording heteronuclear 2D NMR spectrabecomes feasible for analyte amounts not addressable withregular NMR equipment. 103 CapillaryNMR has beensuccessfully used to characterize mass limited natural product samples within decent NMR acquisition times. Overnight recording of 13 C/1 H shi correlations (HSQC andHMBC) spectra for100 mg of a 500 Da analyte ( 40 mM solution) was reported. 103,104

4.5.3 HPLC-SPE-NMR. Developing the concept of HPLC-NMR further, the high performance liquid chromatography

solid phase extraction nuclear magnetic resonance (HPLC-SPE-NMR) hyphenation emerged about 15 years ago when Gri ffi thsand Horton described a post HPLC column trapping setupusing a guard column as an analyte enrichment device. Theirsetup already allowed on-line trapping of an analyte peak eluting from the HPLC onto a pre-equilibrated guard columncartridge, removing the HPLC mobile phase by washing thisstationary phase with a solvent of as little elutropic strength asD2 O or H 2 O, and nally transferring the analytes from the solidphase to the NMR ow probe with a NMR solvent of su ffi cient elution power ( e.g. CD3 OD, CD3 CN). Since it was found that observed signal to noise (S/N) gains were inversely proportional

to the chromatographic peak widths (peak volumes), theauthors reasoned that concentrating up the analyte to anelution volume close to the ow cell volume of the LC-NMR probe head results in an optimal sensitivity enhancement. 105

This work ow and the argument to match HPLC peak volumes with NMR ow cell volumes are major keystones forthe successful application of HPLC-SPE-NMR. About a decadeago a highly automated and re ned HPLC-SPE-NMR setup

allowing full control over HPLC-UV or HPLC-MS triggeredtrapping events, solid phase extraction (SPE) cartridge handling in a 96 well plate format, and analyte elution from the SPE toeither a NMR spectrometer or another collecting device (autosampler, NMR tubes etc.) was introduced by one of the majorNMR manufacturers.

The sensitivity of this commercially available HPLC-SPE-NMR instrumentation is more or less comparable to theintrinsically more sensitive capillary NMR if applied in routinenatural product analysis setups. 103,104 As a rule of thumb for atypical secondary metabolite with a molecular weight of 500Da about 2 mg analyte per ml NMR solvent have to be transferred

to the active HPLC-SPE-NMR probe volume ( i.e. 120 m

g analyteina60 ml ow probe with a 30 ml active cell volume mounted in a600 MHz NMR spectrometer equipped with a room temperatureprobehead) to obtain a set of high quality homo- and hetero-nuclear 2D NMR spectra ( e.g. a DQF-COSY, TOCSY, HSQC, andHMBC) overnight. 87 If only 1 H-NMR spectra are required ( i.e. formetabolic ngerprinting purposes) about 10 nmol substance(i.e. 5 mg at500 Da) are neededto obtain a su ffi ciently good NMR spectrum ( i.e. visibility of all signals including CH 2 multiplets toallow the interpretation of 1 H 1 H coupling) within about onehour measurement time. 87,106,107

From an analytical point of view, major advantages anddisadvantages of HPLC-SPE-NMR compared to HPLC-NMR andcapNMR have to be addressed. The possibility to refocus achromatographic peak a er leaving the HPLC column by trap-ping it on the stationary phase of the SPE column in a narrow precipitation band and releasing it from this phase by ushing with a small volume of deuterated NMR solvent allows match-ing the peak volume to the active volume of the NMR ow cell.Consequently, in the design phase of a HPLC-SPE-NMR assay the optimization of SPE trapping and elution conditions isnecessary e.g. the amount and composition of the post LCadded makeup ow used to reduce the organic modi erconcentration is a critical parameter to be carefully tuned. 108

In the majority of HPLC-SPE-NMR applications, deuterated

acetonitrile (CD 3 CN) and deuterated methanol (CD 3 OD) areused as NMR solvents and lipophilic stationary phases (dive-nylbenzene type polymer or RP-C18 silica) are utilized as SPEmaterials. 87 Herein lies one of the major drawbacks of HPLC-SPE-NMR.109 Any analyte which cannot be precipitated on theSPE stationary phase a er already eluting from the HPLC unit islost for the subsequent transfer to the NMR spectrometer. Insuch cases, i.e. very polar metabolites, other approaches, suchas the use of alternative SPE materials or the application of capNMR, have to be pursued.

Compared to HPLC-NMR were mobile phase water in theHPLC system has to be replaced by deuterated water (D 2 O) to


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allow a proper solvent suppression in the NMR detector, theSPE mediated solvent change from conventional HPLC solvent systems (bulk mobile phase with additives) to a small volume of pure NMR solvent means a signi cant cost reduction. In addi-tion, using deuterated NMR solvents reduces the need tosuppress residual non-deuterated solvent signals which alwaysmeans loss of spectral information in the vicinity of attenuatedsignals. Finally, recording spectra in solvents of identical

polarity enables the comparison of the derived data a key toeffi cient structure elucidation unachievable in HPLC-NMR. 93

The possibility of multiple SPE trapping by accumulating the analyte peak e ffl uent of several HPLC runs onto the identicalSPE cartridge prior to starting the elution/transfer process to theNMR spectrometer enables the user to use analytical HPLCequipment but to bring semi-preparative HPLC amounts of analyte (200 300 mg) to the NMR detector. With such sampleamounts, no solvent suppression is needed and 2D NMR spectra can be recorded within decent time frames.

5 Applications

Within the following paragraphs, typical application examplesfor the hyphenated instrumental analysis setup described aboveare given. The application focus of each technology mirrors itsspeci c advantages. Whereas LC-NMR and LC-SPE-NMR hyphenations are predominantly used to elucidate the structureof analytes, HPLC-MS/MS instruments are either utilized inscreening/ ngerprinting applications (ToF mass analyzers) orare thebackbone of targetanalysis (quadrupole mass analyzers),e.g. in pharmacokinetics/pharmacodynamics (PK/PD) investi-gationsof isolatednatural productsor inherbalmedicinequality control settings (Table 2).

5.1 HPLC-MS

Within the past years a paradigm change has taken place innatural product analysis. The classical use of o ff -line highresolution MS data acquisition to obtain a molecular formulafor an investigated analyte and to characterize the analyte by analyzing the obtained mass spectra was replaced by hyphen-ated HPLC-UV/MS routine platforms used to monitor theisolation and puri cation process of an analyte as well as aiding in its structural characterization. 110 In parallel to developmentsin medicinal chemistry, pharmacology, and clinical chem-istry, 111 115 natural products bioanalysis in model organisms

and humans abandoned HPLC-UV platforms and now reliesalmost exclusively on HPLC-MS/MS technologies. Especially intraditional Chinese Medicine (TCM) where due to the concept of synergistic e ff ects a multitude of herbal remedies are combinedinto one medicine, multi-parameter quantitative assays seem tobe needed to investigate drug pharmacokinetics as well as tomonitor TCM production and product quality. 116 Typical HPLC-MS/MS publications in this eld describe the quantitativeassessment of one or more isolated secondary metabolites inplant matrices or bio uids. If investigating herbal prepara-tions 117 or crude drug batches 118 120 in a quantitative manner,tandem quadrupole (QqQ) based HPLC-MS/MS assays are most

o en utilized. 121,122 Analyte diversity covers the whole range of secondary metabolites, but adulterants are also addressed. 123

Analyte numbers range from few to up to thirty and the assaysare usually based on commercially available referencecompounds of high purity if such materials are not thoroughly characterized ( i.e. purity and content check by qNMR 124,125 ), theaccuracy of an assay cannot be guaranteed. Due to the more orless general unavailability of stable isotope labelled reference

materials either no or analogue internal standards are used. Validation protocols follow ICH, FDA, or similar guidelines,usually data on assay limitations and assay reproducibility as well on interference analyses (if applicable 126 ) is presented. Inapplications more devoted to the qualitative assessment of biological materials, ToF mass selectors and QqToF hybridinstruments dominate. 127 Although such instruments can alsobe used in quantitative assays, 128 ngerprinting and metabolicpro ling are the true strongholds of such instrumentations withhigh resolution MS and MS/MS data combined with diode array detector (DAD) derived UV/VIS spectra, o en allowing tentativeanalyte assignments at least on the metabolite class level. 129 131

In addition, by using sophisticated MS and MS/MS spectra datamining tools, ToF based analysis of bio uids enables thecomprehensive assessment of phase I and phase II metabolitesof administered natural products in vivo132 and in vitro.133

Quantitative assays monitoring dedicated natural productsin bio uids can be considered bioanalytical methods. Methodspresented in this context rely on reference materials isolatedfrom natural materials. As stated above, their quality is crucialfor the accuracy of the quantitative data derived from the HPLC-MS/MS data. As all analytical pitfalls of bioanalytical methodapplications apply, monitoring ion suppression e ff ects is asmandatory as a thorough assay validation. 7,134 Applicationexamples from TCM and other phytomedicines deal withall kind of secondary metabolite classes, e.g. alkaloids, areas well investigated as avonoids, coumarins or terpenoidcompounds. 135 140 Most of the applications utilize tandemquadrupole (QqQ) instruments, only few reports are describing the use of ToF mass analyzers. 135,141 Besides TCM plant derivedanalytes, 142,143 secondary metabolite pharmacokinetics of key phytopharmaceuticals are addressed. The metabolic fate of Hypericum perforatum pharmacological key components 144,145 isinvestigated as well as Ginko biloba terpenoids, 146 and Echinaceaangustifolia alkamides. 147 Besides plant products of phyto-pharmaceutical relevance, food plant products ( e.g. orange juice

avonoids 148 151 ) are frequently investigated. Of special interest

in this context is a recent publication by Mattivi and co- workers, 152 who presented a SRM based quantitative assay devoted to the quanti cation of 135 phenolic metabolites fromdiff erent structure classes in fruits and beverages an under-taking still not pursued in a phytopharmaceutical relevant research context (Fig. 2).

Whenever quantitative information is not in the focus of anassay, but the identi cation of analytes giving rise to chro-matographic peaks is sought in a screening approach, highresolution mass spectra are utilized in HPLC-DAD/MS hyphen-ations. Combining molecular formula short lists with UV spectra and retention time ( polarity) information allows at


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T a

b l e 2

( C o n t d

. )

J i t a i t a b l e t

P h e n o l i c a c i d s

R a t p l a s m a

U

R P - C

1 8

W A , A C N ( g )

Q q Q

E S I

S R M ( L R )

[ 1 4 3 ]

( 1 0 0

3 ) , 1 . 8

F A 0 . 1 %

A n g e l i c a p u b e s c e n s

C o u m a r i n s

R a t p l a s m a

U

R P - C

1 8

W A , A C N ( g )

Q q Q

E S I +

S R M ( L R )

[ 1 3 8 ]

( 1 0 0

4 . 6 )

, 1 . 8

N A 1 m M

I s a t i s i n d i g o t i c a

A l k a l o i d s

R a t p l a s m a

U

R P - C

1 8

W A , A C N ( g )

Q q Q

E S I +

S R M ( L R )

[ 1 3 9 ]

I s a t i s t i n c t o r i a

( 1 0 0

2 . 1 )

, 1 . 8

F A 0 . 1 %

P i p e r l o n g u m

A l k a l o i d s

R a t p l a s m a

H

R P - C

1 8

W A , A C N ( g )

Q q Q - L

I T

E S I +

S R M ( L R )

[ 1 4 0 ]

( 2 0

2 . 1 )

, 3

F A 0 . 1 %

G l y c y r r h i z a u r a l e n s i s

F l a v o n o i d s

, t r i t e r p e n e s a p o n i n s

R a t p l a s m a

H

R P - C

1 8

W A , A C N ( g )

Q q Q

E S I

S R M ( L R )

[ 1 4 2 ]

( 2 0

3 . 9 )

, 5

F A 0 . 1 %

C i t r u s a u r a n t i u m

F l a v o n o i d s

R a t p l a s m a

H

R P - C

1 8

W A , A C N ( g )

Q q Q

E S I

S R M ( L R )

[ 1 4 8 ]

( 1 5 0

2 . 1 )

, 5

F A 0 . 1 %

H y p e r i c u m p e r f o r a t u m

F l a v o n o i d s

H u m a n p l a s m a

H

R P - C

1 8

W A , A C N ( i )

Q q Q

E S I

S R M ( L R )

[ 1 4 4 ]

( 1 5 0

2 . 1 )

, 3 . 5

N F 5 m M

G i n k g o b i l o b a

T e r p e n o i d s


H

R P - C

1 8

W A , M e O H ( g )

S I T

E S I

S I M ( L R )

[ 1 4 6 ]

( 1 5 0

2 . 1 )

, 3 . 5

A A 0 . 1 %

E c h i n a c e a a n g u s t i f o l i a

A l k a m i d e s


H

R P - C

1 8

W A , A C N ( g )

S I T

E S I +

S R M ( L R )

[ 1 4 7 ]

( 5 5

2 ) , 3

F A 0 . 1 %

O r a n g e j u i c e

F l a v o n o i d s


H

R P - C

1 8

W A , A C N ( g )

Q q Q

E S I

S R M ( L R )

[ 1 4 9 ]

( 1 5 0

2 . 1 )

, 3 . 5

N A 2 m M


F l a v o n o i d s

H u m a n p l a s m a , h u m a n u r i n e

H

R P - C

1 2

W A , A C N ( g )

S I T

E S I

S I M ( L R )

[ 1 5 0 ]

( 2 5 0

2 ) , 4

F A 1 %


F l a v o n o i d s

H u m a n u r i n e

H

R P - C

1 8

W A , A C N ( g )

S I T

E S I

S I M ( L R )

[ 1 5 1 ]

( 1 5 0

4 . 6 )

, 3 . 5

F A 0 . 0 1 %

a H

c o n v e n t i o n a l H P L C , U

h i g h r e s o l u t i o n H P L C .

b F i r s t l i n e : M a t e r i a l , S e c o n d l i n e : d i m e n s i o n ( l e n g t h

d i a m e t e r i n m

m ) , p a r t i c l e s i z e ( m m ) . c F i r s t l i n e : p o l a r s o l v e n t / a p o l a r s o l v e n t : A C N

a c e t o n i t r i l e

, M e O H m e t h a n o l , W A w a t e r , ( g ) g r a d i e n t m o d e , ( i ) i s o c r a t i c m o d e . S

e c o n d l i n e : a d d i t i v e s , A

A a c e t i c a c i d , F A f o r m i c a c i d

, T F A t r i u o r o a c e t i c a c i d

, N A a m m o n i u m a c e t a t e , N B

a m m o n i u m b i c a r b o n a t e , N F a m m o n i u m f o r m a t e .

d S I T s p h e r i c a l i o n t r a p , L I

T l i n e a r i o n t r a p ,

Q s i n g l e q u a d r u p o l e i n s t r u m e n t , Q q Q t a n d e m q u a d r u p o l e i n s t r u m e n t , T O F t i m e o f

i g h t

i n s t r u m e n t , q T O F q u a d r u p o l e

t i m e o f

i g h t h y b r i d i n s t r u m e n t .

e E S I e l e c t r o s p r a y i o n i z a t i o n , A P C I a t m o s p h e r i c p r e s s u r e c h e m i c a l i o n i z a t i o n , A P P I a t m o s p h e r i c p r e s s u r e p h o t o i o n i z a t i o n , +

p o s i t i v e m o d e ,

n e g a t i v e m o d e ,

p o s i t i v e a n d n e g a t i v e m o d e .

f S C s c a n m o d e , S I M s e l e c t e d i o n m o n i t o r i n g ,

S R M s e l e c t e d r e a c t i o n m o d e , M S n f r a g m e n t a t i o n e x p e r i m e n t . ( H R ) h i g h

r e s o l u t i o n M S d e t e c t i o n , ( L R ) l o w r e s o l u t i o n M S d e t e c t i o n .


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least tentative assignments of chromatographic features tocompound classes. 153 158 If more detailed structural informationis needed, usually NMR data is desired to be obtained either ina HPLC-NMR/HPLC-SPE-NMR setting or by the preparativeisolation of the analyte under investigation.

5.2 HPLC-NMR and HPLC-SPE-NMR

In natural product chemistry, HPLC-SPE-NMR is the best established HPLC/NMR hyphenation technology. Typicalapplications are either using crude plant extract HPLC separa-tions to transfer chromatographic peaks to the SPE unit 159,160 ordo work with extracts obtained from one or more semi-prepar-ative enrichment steps as liquid liquid extractions, solidphase extractions or HSCCC/column chromatography basedseparation procedures. 107,161 Additional spectroscopic data (UV

spectra, MS/MS spectra) is either obtained by splitting the HPLCeffl uent between the SPE trapping unit and a mass spectrometer(HPLC-MS/SPE-NMR hyphenation) or by transferring a chro-matographic system established on a HPLC-DAD-MS/MSinstrument to a HPLC-SPE-NMR hyphenation. In contrast toHPLC-NMR solely HPLC columns with analytical dimensionsare needed, semi-preparative equipment with high chromato-graphic solvent consumption is not necessary. 162 HPLC-SPE-NMR orientated research is always directed towards the struc-tural characterization of analytes; important metabolite classesaddressed in the past years are for example aromatic alka-loids, 163 diarylheptanoids, 160 avonoids, 164 iso avonoids, 165

lignans, 166 terpenoids, 159,167 steroids, 168 iridoids, 169 and sapo-nins. 170 As in offl ine NMR based structure elucidation, thenumber and nature of 1D- and 2D-NMR experiments ( i.e.diff erent type of shi correlations) needed for an unequivocaldeduction of the molecular sca ff old of an analyte depends onthe complexity of the investigated metabolite and on the avail-ability of additional spectroscopic data as high resolution massspectra allowing the deduction of a molecular formula and asconsequence the determination of double bond equivalents. 77,78

If for example molecular formula and compound class arealready known, secondary metabolites of average complexity,i.e. some alkaloids (Fig. 3), 163 avonoids, 171,172 iridoid glyco-sides, 107 or polyacetylenes 173 can be identi ed by easily acces-sible 1 H NMR spectra. If however and this is the morecommon case complex and hardly investigated secondary metabolites have to be investigated, a full set of homo- and

heteronuclear 2D NMR spectra77

have to be recorded.174 177

Dueto the inherent lower sensitivity of 1 H/ 13 C heteronuclear NMR correlation spectra (HSQC, HMBC) recording, the analyteconcentration in the ow cell of the NMR spectrometer has tobe reasonably higher than for 1 H NMR spectra. Since the singleinjection volume onto the HPLC unit is limited, alternatively orin addition to preanalytical sample enrichment procedures ( i.e.pre-fractionation to prepare enriched extracts with a maximalconcentration of the analytes), repeated trapping onto anidentical SPE cartridge by repeating the HPLC analysis of theinvestigated extract leads to a higher concentration of the ana-lyte on the SPE cartridge and under optimized conditions also

Fig.2 HPLC-DAD (A)and HPLC-MS/MS (B)chromatogramsof a polyphenol pro le of a grape extract. Whereas with DADan incomplete separation of themetaboliteshinders theirquanti cation, the SRM (selected reaction monitoring) ion traces in the HPLC-MS/MSchromatogramdisplays clearbaseline separationof co-eluting peaksby the selectivity introduced by the SRM experiment. It should however not be overlooked, that in the HPLC-MS/MS approach solely analytes prede ned in the designphase of the assay can be addressed everything else is missed. Reprinted with permission from Vrhovsek et al. 152


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in the NMR ow cell the prerequisite to obtain 13 C NMR shi information 169,178 or even 13 C NMR spectra 179 in a decent analysis time. If only minute sample amounts are available, i.e. inthe analysis of endophytic fungi 180 or in biosynthesis anal- ysis181,182 it becomes strikingly evident, that HPLC-SPE-NMR is atrue micro-method working on the analytical sample scale,making an experiment scale up for preparative isolation of analytes unnecessary. In an approach similar to impurity identi cation assays in pharmaceutical industry, HPLC-SPE-NMR was used as additional analytical device to identify theHPLC-MS/MS detected adulterants in the herbal medicinepreparation Gold Nine So Capsules spiked with anti-hyper-tensive drugs. 183

If for instrumental setup reasons ( i.e. the high end NMR spectrometer available cannot be equipped with a HPLC-SPE

front-end), alternative approaches can be pursued. The HPLC-SPE instrument can be operated as stand-alone separationdevice, the SPE e ffl uent is not transferred to a NMR spectrom-eter, but to other collection devices. In one bench-mark exper-iment, analytes were trapped with a HPLC-SPE unit inCopenhagen, shipped to Geneva and transferred to a NMR spectrometer equipped with a capNMR probehead. Similarconcentration pro les as in HPLC-SPE-NMR were achieved. Dueto the ltering e ff ect of the SPE column, the sample was freefrom interfering mobile phase additives or contaminants fromsample containers. 184 In an alternative approach, SPE trappedanalytes can be transferred to conventional or low volume NMR

sample tubes again, the SPE serves as a puri cation step andthe preparative workup of an extract to obtain the analyte of interest is avoided. 185

Recent applications of capNMR, strictly speaking not HPLC-NMR hyphenations but to be listed here for the sake of completeness, include the structural characterization of Arabi-dopsis thaliana wound-induced jasmonate and oxylipin deriva-tives detected as sample set discriminators in a HPLC-MSbased metabolic pro ling orientated research approach, 186,187

and the identi cation of mycotoxins induced in the confront-ing zone between two wood decaying fungi raised in an in vitrosetting. 188

More classical recent secondary metabolites capNMR inves-tigations include the structural characterization of sesquiter-pene alkaloids from Greenwayodendron suaveolens 189 or

stilbenoids from diff

erent orchid species.190

If in capNMR thesyringe pump mediated manual sample delivery is replaced by automated liquid handling devices, large sample arrays can behandled in the sophisticated microdroplet 191 or segmented

ow analysis 192 NMR setups, which also found their applica-tion in secondary plant metabolite investigations. 193 By hyphenating capNMR probes to capillary LC devices true LC-capNMR hyphenations can be realized. In this realm, theonline structural characterization of mass limited samples isstill in the focal point of research, 194,195 parallelization, furtherminiaturization and hyphenation to electrophoretical separa-tion devices or to gas chromatography are pursued hot topics. 196

Fig. 3 HPLC-SPE-NMR derived information rich 1 H-NMR spectra of a series of crinane-type alkaloids. Spectra recording under identical solvent conditions (CD 3 OD)allows the detailed comparison of the congeners and the deduction of substitution patterns. Reprinted with permission from Chen et al. 163


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6 Conclusion

The dramatic technological development of the past decade,including the introduction of stable and reliable QqQ tandemmass spectrometers to be used as workhorses in quantitativeHPLC-MS/MS analyses, the invention of novel high resolutionFourier transform mass spectrometers ( i.e. the orbitrap ) andsteady improvements in highresolution ToF massspectrometers

both to be utilized in qualitative HPLC-MS/MS assays, as well asthe maturation of HPLC-NMR by the development of the HPLC-SPE-NMR platform, led to a paradigm shi in bioanalysis andphytoanalysis. In research settings i.e. devoted to unravel thecomplexity of secondary natural product patterns, to investigatethe biosynthesis of natural products, or aiming to isolate conge-ners of novel natural product structure classes, this novelinstrumentation (HPLC-NMR, HPLC-SPE-NMR, HPLC-MS/MS with high resolution mass spectrometers) makes it feasible toelucidate structures online, i.e. without the need to isolate theanalyte under investigation from its complex matrix. Further-more, quantitative NMR spectroscopy and high resolution massspectrometry assays can be used to ensure, that the isolated orpurchased hit candidate materials applied in the testingsetupsis indeed authentic and of the stated purity. Unfortunately, suchself evident pre-analytical measures are rarely seen in naturalproduct publications 6 in academia in vitro and in vivo pharma-cological experiments without intensive characterization of thesubstances under investigation are more rule than exception. Whenever quantitative structure activity relationships (QSAR)arebeing investigated,HPLC-MS/MS assaysenabletheresearcherto trace the research objects, the putative hit candidate and itscongeners, quantitatively in the matrices of the research setting (i.e. cell cultures, bio uids and tissues of model organisms orhumans) to yield a valid data basis for model calculations.

Unfortunately thorough pharmacokinetic (PK) investigations of natural products are still rare, quantitative monitoring of suchanalytes in bio uids or tissues is besides TCM applications

still not widespread in natural product chemistry. Too little isknown, whether or not lead compounds of applied phyto-pharmaceutical truly reach their target tissue, how fast they aremetabolized and if under traditional passed down applicationregimens stable steady state analyte levels can be reached.Consequently, the in vivo mode of action of phytopharmaceut-icals (pharmacodynamics, PD) is o en still concealed, rationaldrug development and PK/PD analysis as well as rational drug/natural product interaction 197,198 investigations are hindered.

Here the presented modern hyphenated analysis platforms

HPLC-MS/MS, CE-MS/MS, and HPLC-SPE-NMR will certainly enable thephytochemistand thenatural productpharmacologist to gain a deeper and more precise insight into secondary naturalproduct biogenesis, distribution, and pharmacology.

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