Mass spectrometry and NMR spectroscopy: modern

high-end detectors for high resolution separation

techniques 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. These
hyphenated instrumental analysis platforms (CE-MS, HPLC-MS or HPLC-NMR) are valuable tools for
natural product de novo identication, as well as the authentication, distribution, and quantication
of constituents in biogenic raw materials, natural medicines and biological materials obtained from
model organisms, animals and humans. Moreover, metabolic proling and metabolic ngerprinting
applications can be addressed as well as pharmacodynamic and pharmacokinetic issues. This review
provides an overview of latest technological developments, discusses the assets and drawbacks of the
available hyphenation techniques, and describes typical analytical workows.
1 Introduction
2 The role of analytical chemistry in natural product
research
3 Qualitative and quantitative analysis
3.1 Structure information
3.2 Pattern recognition
4 Hyphenated analysis platforms
4.1 Separation techniques
4.2 Hyphenated detectors
4.3 Mass spectrometers as HPLC detectors
4.4 Mass spectrometers as CE detectors
4.5 NMR spectrometers as HPLC detectors
4.5.1 HPLC-NMR
4.5.2 Capillary NMR
4.5.3 HPLC-SPE-NMR
5 Applications
5.1 HPLC-MS
5.2 HPLC-NMR and HPLC-SPE-NMR
6 Conclusion
7 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 information
density detection systems, such as mass spectrometry (MS) or
nuclear magnetic resonance (NMR) spectroscopy, they are
extremely benecial in terms of fostering the identication of
analytical entities of interest. Combining these instrumental
analysis 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 applicative
natural product related science branches such as phytochem-
istry or pharmacognosy. Here typical applications include
regulatory issues established by industrial partners or public
authorities (i.e. pesticide contamination screens, adulteration
detection), natural product pharmacokinetics, secondary
metabolite biogenesis, biosystematics, and secondary metabo-
lite discovery/structure elucidation. Either purely qualitative
proling approaches as metabolic proling, metabolic
a
Institute of Pharmacy/Pharmacognosy, CCB - Centrum of Chemistry and Biomedicine,
University of Innsbruck, Innrain 8082, A-6020 Innsbruck, Austria. E-mail: Hermann.
Stuppner@uibk.ac.at; Fax: +43 512 507 58499; Tel: +43 512 507 58401
b
Institute of Medical and Chemical Laboratory Diagnostics (ZIMCL), University
Hospital/Landeskrankenhaus Innsbruck, Anichstrae 35, A-6020 Innsbruck. E-mail:
Christoph.Seger@uki.at; Tel: +43 512 504 81155
Cite this: Nat. Prod. Rep., 2013, 30, 970
Received 20th February 2013
DOI: 10.1039/c3np70015a
www.rsc.org/npr
970 | Nat. Prod. Rep., 2013, 30, 970987 This journal is The Royal Society of Chemistry 2013
NPR
REVIEW
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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 workows.
2 The role of analytical chemistry in natural
product research
In academia, laboratory scale workup of a crude extract towards
puried single constituents is usually accompanied by analytical
assays and pharmacological investigations, e.g. if the strategy of
bioassay guided fractionation is chosen.
13
Only the combination
of these methods a valid analysis of the fractions by a high
resolution 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 in
vitro and in vivo testing, the identity and purity of such materials
has 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 holds
true, 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 clinical
trials. Since these measurements are performed in biouids, such
assays are generally considered bio-analytical methods.
5
In industry, but also in academia, a detailed qualitative and
quantitative assessment of starting materials is necessary if
working with natural products. If applicable, this includes
authentication of the crude drug, exclusion of (fraudulent)
adulterations with other plants or xenobiotic drugs, microbial
screening, 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 to
the shelf. Since the used biological materials are derived from
living systems and do reect ecological uctuations in their
secondary metabolite pattern, inter-batch product consistency
over the products life time has to be scrupulously monitored to
ensure the claimed product ecacy.
6
3 Qualitative and quantitative analysis
In analytical chemistry detector readouts can be used in many
dierent ways depending on the purpose of the assay. Most
oen a quantitative result is sought; here comparison of sample
readouts with readouts from reference samples of known
Sonja Sturm studied Pharma-
ceutical Sciences at the Univer-
sity of Innsbruck. Aer receiving
her Ph.D. in Pharmaceutical
Biology in 1993, she joined the
Kinghorn group at the University
of Illinois, Chicago as post-
doctoral fellow. Since 1994 she
is an academic sta member at
the Institute of Pharmacy of the
University of Innsbruck. Her
main research interests are the
development 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 as
analyte detectors. In addition she serves as scientic committee
member for ESCOP.
Hermann Stuppner studied
Pharmaceutical Sciences at the
University of Innsbruck. Aer
receiving his Ph.D. in Pharma-
ceutical Biology from the
University 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 Institute
of Pharmacy of the University of
Innsbruck. His main research interests are: isolation and struc-
tural elucidation of secondary metabolites from higher plants with
anti-inammatory and antitumor activity, analysis and quality
assessment of (medicinal) plants and phytopharmaceuticals,
discovery of pharmacologically active natural products by means
of computer aided models.
Christoph Seger studied Chem-
istry at the University of Vienna,
Austria. Aer focusing on NMR
based structure elucidation of
Natural Products (PhD 2001,
University of Vienna) and
peptides (Max Planck Institute,
Martinsried) he nally joined the
team of Hermann Stuppner. Here
his major research focus was the
establishment 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 enjoys
analytical 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, 970987 | 971
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content allows quantication of analytes in investigated speci-
mens.
7
If the identity of an analyte has to be deduced, a
maximum of structure related information has to be gained
from the detector readout
8,9
to ensure a proper structure eluci-
dation.
10
If qualitative information is to be retrieved, i.e. if a
certain component or series of components (a ngerprint)
should be present or absent, visual or chemometric pattern
comparisons 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 emphasized
that this data alone, even if tandem mass spectrometers with
high resolution mass selector technologies are employed as
detectors in chromatography, can hardly provide more than the
molecular formula at its best,
12,13
if databases are not used or
the elucidation process can be restricted to a common scaold/
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 scaold (i.e. geometrical isomers,
position isomers), not to speak of its 3D structure.
18
In plant
metabolomics and metabolic proling, which use analytical
platforms with MS based readout devices, the limited possibility
to elucidate the structure of unknown analytes from the
generated MS data has been recognized as major methodolog-
ical bottleneck. In too many case studies, analytes showing up
as discriminators in a sample set (the putative biomarkers) have
been le unidentied.
11
Key players in mass spectrometry based
metabolomics and metabolic proling addressed in several
reviews that a major drawback of metabolomic technologies
yet to be overcome is the vast number of unknown compound
structures,
19
that the identication of metabolites whose MS/
MS spectra are not present in the spectral libraries remains very
challenging and is being addressed through both empirical and
computational mass spectrometry
20
and as a consequence that
increasing the number of metabolite identications within
existing proling platforms is prerequisite for a substantial
improved scope of proling 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 some
science managers within the last few years NMR spectroscopy of
small molecules has morphed to a routine analysis method to
be handled by sta scientists. This might be possible for the
mere 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 workows in the desired holistic compound
class independent manner.
2225
It still holds true that the
development of systems for computer-assisted structure eluci-
dation based on spectroscopic eects has been considered as a
highly challenging problem from the very beginnings of che-
moinformatics. Thus, a lot of work from excellent scientists has
been 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 detailed
knowledge of nuclear spin systems to be expected and strategic
routes to unravel the oen incomplete and/or redundant NMR
data, still discriminates a successful structure elucidator from
less experienced researchers.
27
3.2 Pattern recognition
Whereas in quantitative assays usually only the minimal
amount of information needed to fulll the given task is gath-
ered (i.e. one ion trace from a mass spectrum, one UV trace from
a UV spectrum.), proling and ngerprinting approaches tend
to use all the data available from a detector. In some cases, such
holistic datasets can even be used to derive quantitative or at
least semi-quantitative information on selected analytes (i.e. in
NMR spectroscopy). To investigate this huge amount of data,
to extract meaningful information from the analytical and
biological noise, to nd biomarkers underpinning a given
hypothesis, chemometrical approaches have to be applied. Here
a multitude of statistical methodologies, including data
pretreatment of oen imperfect detector readouts, are available,
which will not be discussed further in this overview.
11,2833
4 Hyphenated analysis platforms
4.1 Separation techniques
Among the dierent analytical separation systems available, a
predominance of high performance liquid chromatography
(HPLC, oen abbreviated as LC) and gas chromatography
(GC) can be observed in natural product analysis. Although GC
still is unmet in its separation capacity, its application is
however limited by the need to vaporize samples prior to anal-
ysis. If not applying GC to volatile matrices (i.e. terpenoid rich
essential oils) extensive sample preparation protocols including
analyte extraction and derivatization are usually necessary and
thermally labile analytes or analytes still involatile aer deriv-
atization cannot be analyzed at all. Electrophoretic separation
techniques such as capillary electrophoresis (CE) or capillary
electrochromatography (CEC) are always valuable alternatives
to HPLC- or GC-based assays in small organic molecule analysis
but hampered in their routine use by the relatively low sensi-
tivities caused by low mass ow and the reduced stability of the
utilized fused silica capillaries.
34
Hence, amongst all separation techniques available, HPLC is
currently the best compromise. As a robust and more or less
universal separation technique combinable with a variety of
reasonably sensitive or selective detector systems it is applied in
almost any natural science laboratory in one way or another.
972 | Nat. Prod. Rep., 2013, 30, 970987 This journal is The Royal Society of Chemistry 2013
NPR Review
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HPLC based chromatography is a thriving and innovative
instrumental analysis eld, latest developments are devoted to
reduce analysis time by the use of highly separation ecient
(high resolution) stationary phase materials with sub-2 mm
particle sizes. Equipped with various detection devices, it can be
used for more or less any analytical task encountered, since
apolar 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 both
qualitative proling/ngerprinting and quantitative target
analytical assays. By including one or more sample preparation
steps like (i) sample homogenization (milling, sieving,
ltration), (ii) sample extraction (liquidliquid extraction, par-
titioning, preparative solid phase extraction) or (iii) chromato-
graphic clean-up (preparative column chromatography, high
speed counter current chromatography (HSCCC), analytical
solid phase extraction) in the pre-analytical workow, unde-
sired matrix inuences are minimized and investigated analytes
are enriched.
35,36
4.2 Hyphenated detectors
In contrast to GC, which in natural product research relies
mostly on the unspecic ame ionization detector (FID) to
monitor analytes if a mass spectrometry hyphenation (GC-MS or
GC-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 oen they are
implemented as diode array detectors (DAD), allowing online
recording of UV/VIS spectra by paralleled acquisition of a broad
range of wavelengths. Since light absorption in the UV/VIS
range requires conjugated double bond systems (chromo-
phores) at least to some extent, this technique is limited to
analytes bearing such substructures. In addition, UV/VIS
detection limits the use of HPLC mobile phases and mobile
phase 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 aerosol
detector (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 oen utilized in natural product
analysis but of unquestioned value as highly specic detectors
in bioanalytical HPLC, uorescent light detectors (FLD) or
analyzers 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 the
physical detection principles summarized above, enzyme or
antibody based assays e.g. frontal anity chromatography
with mass spectrometry as detector (FAC-MS), post HPLC on-
ow reactors, pulsed ultra-ltration mass spectrometry, or
surface plasmon resonance (SPR) based biosensors can be
applied to screen extracts or substance libraries for novel
bioactivities.
40,41
4.3 Mass spectrometers as HPLC detectors
Whereas standalone mass spectrometry has accompanied
structure elucidation eorts in natural product research for
more than half a century,
42,43
only within the last two decades
mass spectrometers have become easily applicable HPLC
detectors. John B. Fenn's invention of the atmospheric pressure
ionization (API) technique ESI (electrospray ionization) in the
mid 1980s can be considered as the major breakthrough in so
ionization techniques.
4446
ESI facilitates the transfer of analyte
molecules from uncharged liquid phase species to gas phase
ions, hence making the hyphenation of mass spectrometers to
liquid chromatography systems technically feasible. First
commercial HPLC-ESI-MS hyphenations became available in
the 1990s, with rst applicative bio-analytical and natural
product analysis publications emerging shortly thereaer.
47
Two more API techniques, atmospheric pressure chemical
ionization (APCI) and the much younger atmospheric pressure
photo 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), hybrid
mass spectrometers, such as the QqTOF instruments with a
quadrupole mass analyzer (Q) followed by a collision cell (q) and
time of ight (TOF) mass analyzer as a readout device, or
Fourier-Transform mass spectrometers (FT-MS) can be
combined with separation devices such as HPLC and CE.
49,50
The dierent mass spectrometer designs utilized in HPLC-
MS/MS hyphenations allow the realization of distinctively
dierent mass spectrometry experiments with optimal appli-
cation ranges. The selected reaction monitoring (SRM) experi-
ment realizable with tandem mass spectrometers of the QqQ
type is unmatched in its selectivity and reproducibility, which
translates to exceptional sensitivity and large linear ranges. Its
major application eld is target analysis investigations with
predened 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 one
or only a few preselected single ion traces per analyte prohib-
iting a deeper insight into the complexity of an investigated
sample. Mass spectrometers with time of ight (ToF) mass
analyzers are in contrast designed to record high resolution
mass data by scanning a preset mass range. The gathered data
usually enables the investigator to reconstruct one or a few
molecular formulas for each chromatographic feature detected
in the ion traces of the HPLC euent, 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 specic fragments usually allow an even deeper insight
into the molecular scaold of an investigated metabolite.
Whereas in QqQ mass selector based SRM experiments ion
currents of one or more predened ion pairs (precursor ion
charged in the ion source and product ion generated by the gas
phase fragmentation process) are recorded at low resolution
(0.51.0 mass units peak widths), ToF mass selector based
experiments are recording preset mass ranges at high resolu-
tion (1/10001/100 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 this
context, 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, SRMexperiments are
usually of unmatched sensitivity. If, however, several analytes
have to be monitored (i.e. in a quality control screen), SRM
experiments which are performed in a strictly serial manner
with observation times in the lower millisecond range, are
quickly reaching total cycling times (the sum of all individual
SRM experiment times), exceeding the sampling rate of about
500 ms (2 Hz) needed to suciently represent a chromato-
graphic peak in the recorded ion traces with about 812 data
points per peak.
51
With state of the art QqQmass spectrometers,
three to four dozen SRM experiments can be summed up to
one 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 the
application of chromatographic devices with optimized peak
capacities by utilizing modern sub-2 mm stationary phase
particles 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 (10
Hz20 Hz) have to be realized for appropriate chromatographic
peak coverage. Here modern ToF mass analyzer based detector
systems are used as alternatives. Due to the registration of
repetitive scans, the recording time of a single data point is
signicantly shorter than in the SRM experiment. Conse-
quently, the sensitivity of ToF based experiments is limited
compared to tandem MS instruments operated in the SRM
experiment setup.
54
On the other hand ToF mass spectrometer
scans 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 can
be of impaired selectivity if complex matrices (i.e. crude bio-
uids as plasma) have to be investigated.
56
Here QqToF
instruments allowing selected ions to be monitored in the rst
mass selector and to read out gas phase reaction fragment ions
in the second (ToF) mass selector have to be utilized.
55
Besides mass selector designs based on ion travel times
(ToF) or on ion deections in electrical elds (QqQ), the
frequency of ions oscillating in an ion trap can be used to derive
their mass over charge (m/z) ratio. Such devices can produce
mass spectra of unmatched resolution by observing the ion
clouds selected and transferred to the readout devices with
mass 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 the
mass 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), is
proportional to the observation time, recording of high reso-
lution mass scans typically needs up to 23 s an eternity
compared to the lower millisecond time regimen of QqQ or ToF
instruments. Consequently, such devices cannot be used to
record narrow chromatographic peaks with sampling rates
suciently short to reconstruct chromatographic peaks with
high accuracy and precision. This pivotal drawback is circum-
vented in the instrumental design of one vendor by splitting the
ions stream generated by the ion source between a conventional
low resolution linear ion trap mass selector recording ion traces
with scan speeds allowing proper recording of chromatographic
peaks and high resolution mass spectra derived from the
readout of the FT-MS unit orbitrap itself.
The third prominent mass spectrometer construction type
used as a detector in hyphenated techniques is the low mass
resolution ion trap. Two dierent 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 conned in the trap leading to product
ion distributions in accordance with the thermodynamic and
kinetic laws of gas phase ion chemistry, thus being qualitatively
and quantitatively reproducible and at least to some extent
predictable. Consequently, mass spectra derived from such
instruments can be used in structure elucidation of unknown
compounds or combined with database orientated data
mining approaches to identify analytes in a general unknown
screening approach. Compared to conventional QqQ designs or
QqTOF designs, which can also produce fragment spectra from
analytes reacting in the collision cell (q) of the instrument
where on the ight gas phase reactions are carried out, the
advantage of ion trap instruments lies on the one hand in
higher concentration of ions conned in the trap and on the
other hand the possibility to conne ionized reaction products
again and expose them to another gas phase reaction the MS/
MS/MS experiment. Hence ion trap instruments can (at least
theoretically) produce MS
n
mass spectra of ions, which under-
went n 1 gas phase reactions. Tandem mass spectrometers in
contrast 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 liquid
chromatography. Due to low ow rates (nL min
1
range) and
the necessity to ensure a closed electrical circuit to maintain a
high voltage across the separation capillary, interfacing is less
974 | Nat. Prod. Rep., 2013, 30, 970987 This journal is The Royal Society of Chemistry 2013
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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 remained
the principal ionization technique
5961
although generally all
types of mass spectrometers bearing an API ion source can be
hyphenated to CE instruments their specic advantages and
disadvantages in the CE-MS coupling are, however, identical to
the HPLC-MS coupling.
Three types of interfaces for CE-ESI-MS hyphenations have
been developed up to now, sheathless, sheath-liquid, and liquid
junction interfaces. Currently only the sheath-liquid interfaces
are commercially available, here make-up liquid and nebulizing
gas are added coaxially to the CE euent, the interface is
grounded and the inlet of the MS is put onto high voltage. One
of the major drawbacks of this robust and user friendly
hyphenation is the dilution of the CE euent by the sheath
liquid, impairing the analytical sensitivity of the device.
In CE selectivity can be easily adjusted by varying the back-
ground electrolytes, by addition of chiral modiers, or by the
addition of neutral micelle formers for the separation of neutral
analytes. Since ESI is susceptible to ion suppression eects
caused by the most frequent employed background electrolytes,
such as high concentrations salts, non-volatile salts and
organics constituents, or surfactants, such additives have to be
avoided. Consequently, the application range of CE-MS is in
comparison to CE-UV dramatically narrowed. A promising
approach to overcome this limitation seems to be the imple-
mentation of APCI
62
or APPI
63
as ionization techniques, both
highly 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 CE
are still not optimized and are under development. The number
of publications in CE-ESI-MS is limited, besides proof of prin-
ciple contributions, most natural product applications are
devoted to the qualitative analysis of alkaloids
6472
(Fig. 1) and to
primary metabolite proling (Table 1).
7375
4.5 NMR spectrometers as HPLC detectors
In analogy to mass spectrometry, NMR spectroscopy has its
unquestioned merits in the structure elucidation of organic
analytes. For decades it has been considered as one of the major
cornerstones to establish molecular structures by analyzing
connectivity networks on the atomic level.
7679
Due to the signal
richness of its spectra, NMR, however, works best if the inves-
tigated analyte is present in high purity whenever mixtures are
present, the number of overlapping signals rises dramatically.
Consequently, especially if secondary metabolites with complex
spin patterns are investigated, analyte purication prior to NMR
based structure analysis is a well established work-ow model
in phytochemistry. Due to the metabolic complexity of natural
material derived extracts, this task is burdensome and
demanding 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 the
late 1970s onwards
8082
and realized in commercially available
setups shortly thereaer.
83,84
From these pioneering works on,
HPLC-NMR and related ow probe NMR technologies evolved
to well established analytical platforms around the turn of the
century.
8587
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 conventional
NMR spectrometers a NMR sample tube is placed into the
centre of these coils, in HPLC-NMR this tube is replaced by a
ow cell connected to the LC module with a capillary. The
mobile phase eluting from the HPLC column is entering the
ow cell and NMR spectra are recorded permanently. Whilst the
chromatographic peak of an analyte is crossing the ow cell,
NMR signals of its proton scaold are recorded. Typically, to
increase the signal to noise ratio, several NMR experiments (i.e.
16 scans, recording time less than one minute) are accumulated
for one NMR spectrum. As in HPLC-MS, the acquisition time of
NMR spectra has to be matched to the average peak widths
produced 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 hyphenation
is utilized,
88
such a setup can generate all the structural infor-
mation usually needed for structural characterization of small
organic analytes. The major drawbacks of HPLC-NMR are the
inherently low sensitivity of NMR spectroscopy, the time
Fig. 1 Non aqueous CE-ESI-MS electropherograms (base peaks, m/z 200500)
of crude Corydalis sp. extracts of four European Corydalis species. Identication of
the analytes was possible by comparison of reference compounds which were
either purchased, isolated and identied by o-line NMR or by HPLC-SPE-NMR.
174
Reprinted with permission from Sturm et al.
72
This journal is The Royal Society of Chemistry 2013 Nat. Prod. Rep., 2013, 30, 970987 | 975
Review NPR
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976 | Nat. Prod. Rep., 2013, 30, 970987 This journal is The Royal Society of Chemistry 2013
NPR Review
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constraints of the on-ow data acquisition, and the NMR shi
referencing problem occurring whenever solvent conditions are
changing i.e. if a LC solvent gradient is applied.
89
Conse-
quently, HPLC-NMR was evolved by improvement on both the
HPLC and the NMR side. Within the past twenty years setups
changed 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 available
from all major NMR instrument producers. Whereas on ow
experiments have nearly vanished from the literature within the
last few years due to their insensitivity, stopped ow and loop
collection experiments are still widely applied in natural
product chemistry
90,91
and the pharmaceutical industry.
87,9294
Phytochemical applications range from secondary plant
metabolite identication 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
capillary NMRprobes withsolenoidal RFcoils.
98,99
This approach
resulted in the design and commercial availability of microcoil
probes with active sample volumes (capillary volume within the
RF sender/receiver coil) down to 1.5 ml. Although capillary NMR
can be successfully hyphenated to matching micro HPLC
equipment
100
or can be combined with sample robots in indus-
trial style highthroughput setups,
101
it is currently mostly usedin
ano-line mode withdriedsamples takenupina fewmicroliters
of NMRsolvent andintroducedintothe probe manually or by the
aid of a syringe pump.
102
Whenever an analyte is not concentra-
tion-limited, meaning that it can be dissolved in a microliter
amount 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 can
be realized compared to conventional NMR setups using sample
tubes. Consequently, recording heteronuclear 2D NMR spectra
becomes feasible for analyte amounts not addressable with
regular NMRequipment.
103
Capillary NMRhas beensuccessfully
used to characterize mass limited natural product samples
within decent NMR acquisition times. Overnight recording of
13
C/
1
Hshicorrelations (HSQCand HMBC) spectra for 100 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 Griths
and Horton described a post HPLC column trapping setup
using a guard column as an analyte enrichment device. Their
setup already allowed on-line trapping of an analyte peak
eluting from the HPLC onto a pre-equilibrated guard column
cartridge, removing the HPLC mobile phase by washing this
stationary phase with a solvent of as little elutropic strength as
D
2
O or H
2
O, and nally transferring the analytes from the solid
phase to the NMR ow probe with a NMR solvent of sucient
elution power (e.g. CD
3
OD, CD
3
CN). Since it was found that
observed signal to noise (S/N) gains were inversely proportional
to the chromatographic peak widths (peak volumes), the
authors reasoned that concentrating up the analyte to an
elution volume close to the ow cell volume of the LC-NMR
probe head results in an optimal sensitivity enhancement.
105
This workow and the argument to match HPLC peak
volumes with NMR ow cell volumes are major keystones for
the successful application of HPLC-SPE-NMR. About a decade
ago a highly automated and rened HPLC-SPE-NMR setup
allowing full control over HPLC-UV or HPLC-MS triggered
trapping events, solid phase extraction (SPE) cartridge handling
in a 96 well plate format, and analyte elution from the SPE to
either a NMR spectrometer or another collecting device (auto
sampler, NMR tubes etc.) was introduced by one of the major
NMR manufacturers.
The sensitivity of this commercially available HPLC-SPE-
NMR instrumentation is more or less comparable to the
intrinsically more sensitive capillary NMR if applied in routine
natural product analysis setups.
103,104
As a rule of thumb for a
typical secondary metabolite with a molecular weight of 500
Da about 2 mg analyte per ml NMR solvent have to be transferred
to the active HPLC-SPE-NMR probe volume (i.e. 120 mg analyte
in a 60 ml owprobe with a 30 ml active cell volume mounted in a
600 MHz NMR spectrometer equipped with a room temperature
probehead) to obtain a set of high quality homo- and hetero-
nuclear 2D NMR spectra (e.g. a DQF-COSY, TOCSY, HSQC, and
HMBC) overnight.
87
If only
1
H-NMR spectra are required (i.e. for
metabolic ngerprinting purposes) about 10 nmol substance
(i.e. 5 mg at 500 Da) are needed to obtain a suciently good NMR
spectrum(i.e. visibility of all signals including CH
2
multiplets to
allow the interpretation of
1
H
1
H coupling) within about one
hour measurement time.
87,106,107
From an analytical point of view, major advantages and
disadvantages of HPLC-SPE-NMR compared to HPLC-NMR and
capNMR have to be addressed. The possibility to refocus a
chromatographic peak aer 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 is
necessary e.g. the amount and composition of the post LC
added makeup ow used to reduce the organic modier
concentration 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) are
used as NMR solvents and lipophilic stationary phases (dive-
nylbenzene type polymer or RP-C18 silica) are utilized as SPE
materials.
87
Herein lies one of the major drawbacks of HPLC-
SPE-NMR.
109
Any analyte which cannot be precipitated on the
SPE stationary phase aer already eluting from the HPLC unit is
lost for the subsequent transfer to the NMR spectrometer. In
such cases, i.e. very polar metabolites, other approaches, such
as the use of alternative SPE materials or the application of
capNMR, have to be pursued.
Compared to HPLC-NMR were mobile phase water in the
HPLC system has to be replaced by deuterated water (D
2
O) to
This journal is The Royal Society of Chemistry 2013 Nat. Prod. Rep., 2013, 30, 970987 | 977
Review NPR
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allow a proper solvent suppression in the NMR detector, the
SPE mediated solvent change from conventional HPLC solvent
systems (bulk mobile phase with additives) to a small volume of
pure NMR solvent means a signicant cost reduction. In addi-
tion, using deuterated NMR solvents reduces the need to
suppress residual non-deuterated solvent signals which always
means loss of spectral information in the vicinity of attenuated
signals. Finally, recording spectra in solvents of identical
polarity enables the comparison of the derived data a key to
ecient structure elucidation unachievable in HPLC-NMR.
93
The possibility of multiple SPE trapping by accumulating
the analyte peak euent of several HPLC runs onto the identical
SPE cartridge prior to starting the elution/transfer process to the
NMR spectrometer enables the user to use analytical HPLC
equipment but to bring semi-preparative HPLC amounts of
analyte (200300 mg) to the NMR detector. With such sample
amounts, no solvent suppression is needed and 2D NMR
spectra can be recorded within decent time frames.
5 Applications
Within the following paragraphs, typical application examples
for the hyphenated instrumental analysis setup described above
are given. The application focus of each technology mirrors its
specic advantages. Whereas LC-NMR and LC-SPE-NMR
hyphenations are predominantly used to elucidate the structure
of analytes, HPLC-MS/MS instruments are either utilized in
screening/ngerprinting applications (ToF mass analyzers) or
are the backbone of target analysis (quadrupole mass analyzers),
e.g. in pharmacokinetics/pharmacodynamics (PK/PD) investi-
gations of isolatednatural products or inherbal medicine quality
control settings (Table 2).
5.1 HPLC-MS
Within the past years a paradigm change has taken place in
natural product analysis. The classical use of o-line high
resolution MS data acquisition to obtain a molecular formula
for 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 the
isolation and purication process of an analyte as well as aiding
in its structural characterization.
110
In parallel to developments
in medicinal chemistry, pharmacology, and clinical chem-
istry,
111115
natural products bioanalysis in model organisms
and humans abandoned HPLC-UV platforms and now relies
almost exclusively on HPLC-MS/MS technologies. Especially in
traditional Chinese Medicine (TCM) where due to the concept of
synergistic eects a multitude of herbal remedies are combined
into one medicine, multi-parameter quantitative assays seem to
be needed to investigate drug pharmacokinetics as well as to
monitor TCM production and product quality.
116
Typical HPLC-
MS/MS publications in this eld describe the quantitative
assessment of one or more isolated secondary metabolites in
plant matrices or biouids. If investigating herbal prepara-
tions
117
or crude drug batches
118120
in a quantitative manner,
tandem quadrupole (QqQ) based HPLC-MS/MS assays are most
oen 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 assays
are usually based on commercially available reference
compounds of high purity if such materials are not thoroughly
characterized (i.e. purity and content check by qNMR
124,125
), the
accuracy of an assay cannot be guaranteed. Due to the more or
less 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. In
applications more devoted to the qualitative assessment of
biological materials, ToF mass selectors and QqToF hybrid
instruments dominate.
127
Although such instruments can also
be used in quantitative assays,
128
ngerprinting and metabolic
proling are the true strongholds of such instrumentations with
high resolution MS and MS/MS data combined with diode array
detector (DAD) derived UV/VIS spectra, oen allowing tentative
analyte assignments at least on the metabolite class level.
129131
In addition, by using sophisticated MS and MS/MS spectra data
mining tools, ToF based analysis of biouids enables the
comprehensive assessment of phase I and phase II metabolites
of administered natural products in vivo
132
and in vitro.
133
Quantitative assays monitoring dedicated natural products
in biouids can be considered bioanalytical methods. Methods
presented in this context rely on reference materials isolated
from natural materials. As stated above, their quality is crucial
for the accuracy of the quantitative data derived from the HPLC-
MS/MS data. As all analytical pitfalls of bioanalytical method
applications apply, monitoring ion suppression eects is as
mandatory as a thorough assay validation.
7,134
Application
examples from TCM and other phytomedicines deal with
all kind of secondary metabolite classes, e.g. alkaloids, are
as well investigated as avonoids, coumarins or terpenoid
compounds.
135140
Most of the applications utilize tandem
quadrupole (QqQ) instruments, only few reports are describing
the use of ToF mass analyzers.
135,141
Besides TCM plant derived
analytes,
142,143
secondary metabolite pharmacokinetics of key
phytopharmaceuticals are addressed. The metabolic fate of
Hypericum perforatum pharmacological key components
144,145
is
investigated as well as Ginko biloba terpenoids,
146
and Echinacea
angustifolia alkamides.
147
Besides plant products of phyto-
pharmaceutical relevance, food plant products (e.g. orange juice
avonoids
148151
) 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 quantication of 135 phenolic metabolites from
dierent 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 an
assay, but the identication of analytes giving rise to chro-
matographic peaks is sought in a screening approach, high
resolution 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
978 | Nat. Prod. Rep., 2013, 30, 970987 This journal is The Royal Society of Chemistry 2013
NPR Review
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This journal is The Royal Society of Chemistry 2013 Nat. Prod. Rep., 2013, 30, 970987 | 979
Review NPR
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980 | Nat. Prod. Rep., 2013, 30, 970987 This journal is The Royal Society of Chemistry 2013
NPR Review
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least tentative assignments of chromatographic features to
compound classes.
153158
If more detailed structural information
is needed, usually NMR data is desired to be obtained either in
a HPLC-NMR/HPLC-SPE-NMR setting or by the preparative
isolation 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. Typical
applications are either using crude plant extract HPLC separa-
tions to transfer chromatographic peaks to the SPE unit
159,160
or
do work with extracts obtained from one or more semi-prepar-
ative enrichment steps as liquidliquid extractions, solid
phase extractions or HSCCC/column chromatography based
separation procedures.
107,161
Additional spectroscopic data (UV
spectra, MS/MS spectra) is either obtained by splitting the HPLC
euent 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/MS
instrument to a HPLC-SPE-NMR hyphenation. In contrast to
HPLC-NMR solely HPLC columns with analytical dimensions
are 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 classes
addressed in the past years are for example aromatic alka-
loids,
163
diarylheptanoids,
160
avonoids,
164
isoavonoids,
165
lignans,
166
terpenoids,
159,167
steroids,
168
iridoids,
169
and sapo-
nins.
170
As in oine NMR based structure elucidation, the
number and nature of 1D- and 2D-NMR experiments (i.e.
dierent type of shi correlations) needed for an unequivocal
deduction of the molecular scaold of an analyte depends on
the complexity of the investigated metabolite and on the avail-
ability of additional spectroscopic data as high resolution mass
spectra allowing the deduction of a molecular formula and as
consequence the determination of double bond equivalents.
77,78
If for example molecular formula and compound class are
already 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 identied by easily acces-
sible
1
H NMR spectra. If however and this is the more
common case complex and hardly investigated secondary
metabolites have to be investigated, a full set of homo- and
heteronuclear 2D NMR spectra
77
have to be recorded.
174177
Due
to the inherent lower sensitivity of
1
H/
13
C heteronuclear NMR
correlation spectra (HSQC, HMBC) recording, the analyte
concentration in the ow cell of the NMR spectrometer has to
be reasonably higher than for
1
H NMR spectra. Since the single
injection volume onto the HPLC unit is limited, alternatively or
in addition to preanalytical sample enrichment procedures (i.e.
pre-fractionation to prepare enriched extracts with a maximal
concentration of the analytes), repeated trapping onto an
identical SPE cartridge by repeating the HPLC analysis of the
investigated 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) chromatograms of a polyphenol prole of a grape extract. Whereas with DAD an incomplete separation of the metabolites
hinders their quantication, the SRM(selected reaction monitoring) ion traces in the HPLC-MS/MS chromatogramdisplays clear baseline separation of co-eluting peaks
by the selectivity introduced by the SRM experiment. It should however not be overlooked, that in the HPLC-MS/MS approach solely analytes predened in the design
phase 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 anal-
ysis time. If only minute sample amounts are available, i.e. in
the analysis of endophytic fungi
180
or in biosynthesis anal-
ysis
181,182
it becomes strikingly evident, that HPLC-SPE-NMR is a
true 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
identication assays in pharmaceutical industry, HPLC-SPE-
NMR was used as additional analytical device to identify the
HPLC-MS/MS detected adulterants in the herbal medicine
preparation 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 separation
device, the SPE euent 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 in
Copenhagen, shipped to Geneva and transferred to a NMR
spectrometer equipped with a capNMR probehead. Similar
concentration proles as in HPLC-SPE-NMR were achieved. Due
to the ltering eect of the SPE column, the sample was free
from interfering mobile phase additives or contaminants from
sample containers.
184
In an alternative approach, SPE trapped
analytes can be transferred to conventional or low volume NMR
sample tubes again, the SPE serves as a purication step and
the 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-MS
based metabolic proling orientated research approach,
186,187
and the identication of mycotoxins induced in the confront-
ing zone between two wood decaying fungi raised in an in vitro
setting.
188
More classical recent secondary metabolites capNMR inves-
tigations include the structural characterization of sesquiter-
pene alkaloids from Greenwayodendron suaveolens
189
or
stilbenoids from dierent orchid species.
190
If in capNMR the
syringe pump mediated manual sample delivery is replaced by
automated liquid handling devices, large sample arrays can be
handled 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, the
online structural characterization of mass limited samples is
still in the focal point of research,
194,195
parallelization, further
miniaturization 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
982 | Nat. Prod. Rep., 2013, 30, 970987 This journal is The Royal Society of Chemistry 2013
NPR Review
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6 Conclusion
The dramatic technological development of the past decade,
including the introduction of stable and reliable QqQ tandem
mass spectrometers to be used as workhorses in quantitative
HPLC-MS/MS analyses, the invention of novel high resolution
Fourier transform mass spectrometers (i.e. the orbitrap) and
steady improvements in high resolution ToF mass spectrometers
both to be utilized in qualitative HPLC-MS/MS assays, as well as
the maturation of HPLC-NMR by the development of the HPLC-
SPE-NMR platform, led to a paradigm shi in bioanalysis and
phytoanalysis. In research settings i.e. devoted to unravel the
complexity of secondary natural product patterns, to investigate
the biosynthesis of natural products, or aiming to isolate conge-
ners of novel natural product structure classes, this novel
instrumentation (HPLC-NMR, HPLC-SPE-NMR, HPLC-MS/MS
with high resolution mass spectrometers) makes it feasible to
elucidate structures online, i.e. without the need to isolate the
analyte under investigation from its complex matrix. Further-
more, quantitative NMR spectroscopy and high resolution mass
spectrometry assays can be used to ensure, that the isolated or
purchased hit candidate materials applied inthe testing setups
is indeed authentic and of the stated purity. Unfortunately, such
self evident pre-analytical measures are rarely seen in natural
product publications
6
in academia in vitro and in vivo pharma-
cological experiments without intensive characterization of the
substances under investigation are more rule than exception.
Whenever quantitative structureactivity relationships (QSAR)
are beinginvestigated, HPLC-MS/MSassays enable theresearcher
to trace the research objects, the putative hit candidate and its
congeners, quantitatively in the matrices of the research setting
(i.e. cell cultures, biouids and tissues of model organisms or
humans) to yield a valid data basis for model calculations.
Unfortunately thorough pharmacokinetic (PK) investigations of
natural products are still rare, quantitative monitoring of such
analytes in biouids or tissues is besides TCM applications
still not widespread in natural product chemistry. Too little is
known, whether or not lead compounds of applied phyto-
pharmaceutical truly reach their target tissue, how fast they are
metabolized and if under traditional passed down application
regimens stable steady state analyte levels can be reached.
Consequently, the in vivo mode of action of phytopharmaceut-
icals (pharmacodynamics, PD) is oen still concealed, rational
drug 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 the phytochemist andthe natural product pharmacologist
to gain a deeper and more precise insight into secondary natural
product biogenesis, distribution, and pharmacology.
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