Anal Bioanal Chem (2007) 387: 525–527
DOI 10.1007/s00216-006-0687-8
TRENDS
Zhengzheng Pan . Daniel Raftery
Comparing and combining NMR spectroscopy and mass
spectrometry in metabolomics
Published online: 12 August 2006
# Springer-Verlag 2006
Introduction
The rapidly expanding field of metabolomics has been
driven in recent years by advances in the analytical
methods of high-resolution nuclear magnetic resonance
(NMR) spectroscopy and mass spectrometry (MS), and
their combination with multivariate statistics [1, 2].
Widespread application of metabolomics has been
achieved in the areas of system biology, drug discovery,
pharmaceutical research, early disease detection, toxicol-
ogy, food and nutrition science and others [3–6]. The
potential scientific payoff is also driving the development
of new methodologies that promise to fuel a further
expansion of the field. In addition, because of the
complementary analytical features of NMR and MS,
opportunities for leveraging both methods are being
considered which will create more comprehensive meta-
bolic profiling. While only a few studies to date have
combined both analytical techniques with statistical anal-
ysis [7], this approach has exciting potential for the field.
Current state of metabolomics
The high sensitivity (typically pg level) of MS detection
makes it an important method for measuring metabolites in
complex biosamples. While electrochemical methods
provide excellent sensitivity, MS methods are more
popular because they allow for reliable metabolite identi-
fication. Since the 1970s, chromatographic methods have
been used to separate complex mixtures of metabolites for
improved analysis and identification. While GC (gas
chromatography) and later GC–MS methods have been
used for quantitative metabolic profiling, GC–MS is
Z. Pan . D. Raftery (*)
Department of Chemistry, Purdue University,
560 Oval Drive,
West Lafayette, IN 47907, USA
e-mail: raftery@purdue.edu
largely limited to volatile compounds. The developments
in LC (liquid chromatography)–MS and CE (capillary
electrophoresis)–MS have significantly broadened the
applicability of MS-based metabolomics [8], although the
need to separate or purify the sample before it is directed
into the mass analyzer can make the analysis time-
consuming. In addition, the selectivity of GC/LC–MS for
classes of analytes provides both benefits and complica-
tions. Tandem MS and so-called accurate mass (time of
flight) methods are most often used to validate the
identities of unknown analytes. Fourier transform (FT)
MS provides an alternative approach with extremely high
resolution and a mass accuracy better than 1 ppm [9].
However, the high cost of the instrument has limited its use
to date. Current challenges for MS-based metabolomics
include the development of more robust methods for
chromatographic separation, data reduction methods to
deal with the enormous file sizes, and of course the
reduction of matrix effects, including ion suppression, that
can cause widely varying signal intensities.
Compared with MS, NMR spectroscopy yields rela-
tively low-sensitivity measurements, with limits of detec-
tion on the order of 10 μM or a few nmol at high fields
using new cryoprobes. Nevertheless, NMR-based meta-
bolic profiling can be performed successfully because
NMR is highly quantitative and reproducible. These
properties are especially important when metabolomic
data are analyzed by multivariate statistical methods [10],
and they compensate for the limited sensitivity of NMR.
Furthermore, NMR sensitivity does not depend on metab-
olite pKa or hydrophobicity, which makes NMR an
excellent choice for broad-based analyses (see Fig. 1),
and suitable for samples in different conditions. Recent
clinical examples of this approach include promising
studies involving the detection of ovarian cancer and
inborn errors of metabolism [11, 12]. Progress has been
made to improve the resolution and sensitivity of NMR
spectroscopy by means of the J-resolved experiment [13]
and other 2-D methods. Using advanced high-throughput
NMR methodology, up to 500 samples can be measured
within one day with the assistance of flow-injection probes
526
Fig. 1 A schematic comparison of NMR spectroscopy and mass
spectrometry as a function of different metabolite characteristics
and automated liquid handlers. The detection limit can also
be decreased to tens of nanograms through the use of
microcoil probes [14]. However, the complexity inherent in
biosamples generates a large number of peaks in a small
chemical shift range (∼10 ppm) in the 1H-NMR spectrum.
Therefore, peaks generated by different species have more
chance of overlapping with each other in the spectrum,
especially in the aliphatic region. As a result, potentially
important compounds present at smaller concentrations are
often overshadowed by larger peaks, and are thus less
likely to be analyzed.
Statistical pattern recognition methods, when combined
with either MS or NMR, create enormous opportunities for
metabolomics research beyond simple data reduction
methods. Among a variety of statistical methods employed,
principal component analysis (PCA) has proven to be
suitable for differentiating sample groups on the basis of
metabolite composition. As an unsupervised method, PCA
requires no knowledge of the nature of samples prior to the
data analysis, and any significant difference between the
groups of samples will be detected, whether or not this
difference is important to the study. Other methods are
described as “supervised” because classes of samples are
identified before the mathematical analysis begins. These
include partial least squares descriminant analysis (PLS-
DA) and soft independent modeling of class analogies
(SIMCA). These methods have been used for the classi-
fication of spectroscopic results, and are especially useful
for detecting markers which differentiate the pre-identified
classes of samples, such as diseased and undiseased. In
addition, variable stability scaling (VAST) and orthogonal
signal correction (OSC) methods have been reported to
improve NMR-based metabolomics sample classification
[15].
New methods
While the current technologies used in metabolomics are
already fueling a growing number of applications, new
approaches are certain to accelerate this trend. For MS,
new atmospheric pressure ionization methods dramatically
simplify the sample treatment and introduction process. For
example, desorption electrospray ionization (DESI) MS
makes it possible to measure biosamples without pre-
separation or sample pretreatment [16]. DESI utilizes
charged droplets to ionize chemical species from a surface
located outside the mass analyzer. A somewhat similar
approach is used by the DART (direct analysis in real time)
method [17]. These direct measurements significantly
shorten the experimental time compared to GC– or LC–
MS analysis. DESI spectra can be used as inputs for
statistical analysis for metabolomics studies, and initial
studies have been carried out to provide fast and
comprehensive measurements of urine samples from
tumor-bearing mice [18] and the detection of inborn errors
of metabolism [19]. However, two potential concerns for
this approach are the overshadowing of small signals by
larger ones (similarly encountered in NMR) and ion
suppression, which could limit their utility for broad-based
metabolomics.
New NMR methods have focused on improving the
resolution in complex spectra. A relatively simple approach
is to fractionate the hydrophilic and hydrophobic compo-
nents, which improves the resolution somewhat [20]. As an
alternative approach, the use of the selective TOCSY (total
correlation spectroscopy) experiment [21] allows one to
focus on certain target molecules with much improved
resolution and sensitivity. NMR detection in this case is
focused on J-coupling in the system, i.e., hydrogen atoms
linked by covalent bonds in a single molecule. Using
selective TOCSY, important metabolites, which may
appear as low-concentration species, can be emphasized
so as to achieve a better discriminatory ability when
performing metabolomics analyses [22]. Selective TOCSY
can be multiplexed to improve throughput using Hadamard
methods [23].
An exciting recent development is the combination of
NMR and MS data, which allows improved identification
of unknown analytes and creates an opportunity to expand
the scope of metabolomics research. A statistical correla-
tion tool has been developed by Nicholson and coworkers
to interrelate the metabolites from NMR and UPLC-MS
[7]. Different metabolites, including some possible bio-
markers, measured with NMR and UPLC-MS individually
were linked by their mutual correlation values and then
illustrated by a corresponding contour plot. This correla-
tion approach has been applied to the study of inborn errors
of metabolism using the unique combination of NMR and
DESI-MS [19]. Correlation of several known biomarkers
identified by NMR spectroscopy with a group of
metabolites seen in the DESI–MS results allowed a new
set of metabolites to be studied and rationalized in terms of

their relationship to the disease and their impact on related
metabolic pathways.
Outlook
A key goal of metabolomics is the selection of a set of
reliable biomarker metabolites that can be used to identify
and follow changes in systems biology. To this end, more
sensitive, reliable, faster, cheaper, and easier analytical
tools to perform metabolomics research are highly
desirable, and thus provide an impetus for instrument
development. New statistical tools, such as advanced
multivariate analyses and correlation methods, will likely
be developed and applied to different types of samples,
including various biofluids and even tissues, etc. In the
longer run it may also be possible to correlate metabo-
lomics more extensively with proteomics and genomics,
although this has been an overwhelmingly difficult
problem to date. Ultimately, the real challenge will be to
place discovered biomarkers in their biochemical contexts
in terms of pathways and processes. For the present,
however, advanced and correlated NMR- and MS-based
analyses will definitely move the field of metabolomics
into a new era.
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