Sample Preparation in Global Metabolomics of Biological Fluids and Tissues - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 63

using LC-MS to maximize metabolite cov-
erage. 86 Ellinger et al. recently addressed the
need for automation by designing a promising
semiautomated batch-extraction system capable
of tissue disruption/homogenization, extrac-
tion, and

However, to date in vivo microdialysis has not
been applied to global metabolite studies in
combination with LC-MS, presumably due to
the dif

culties in obtaining a comprehensive
metabolite pro le especially for highly bound

and hydrophobic species and severe ionization
suppression that is observed for commonly
employed buffers in microdialysis.
A second method that can be applied for
in vivo sampling of a metabolome is in vivo
solid-phase microextraction (SPME). This tech-
nique successfully addresses some of the limita-
tions of microdialysis in the context of
untargeted studies by providing direct compati-
bility with LC-MS and balanced extraction of
both hydrophilic and hydrophobic metabolites.
In addition to extensive applications of in vivo
SPME for sampling of volatile and semivolatile
metabolome in combination with GC-MS anal-
ysis, including biomarker discovery appli-
cations such as breath and skin analysis, 98 e 100
the usefulness of this technique was also recently
shown for direct in vivo sampling of circulating
mouse blood metabolome in awake animals. 27
Brie

filtration with comparable precision
(12 e 14% mean RSD) to manual extractions
(14 e 18% mean RSD) in combination with NMR
analysis. 93

NEW TRENDS IN SAMPLE
PREPARATION FOR GLOBAL
METABOLOMICS

In Vivo Sampling: Microdialysis

and Solid-Phase Microextraction
In vivo sampling and sample preparation are
particularly attractive for global metabolomics,
because the process of sampling and taking the
sample out of its biological milieu is likely to
disturb the metabolite pro le by exposure to

oxygen, solvents, and pH changes and can acti-
vate various biological processes. Microdialysis
is the gold standard technique for the sampling
of small molecular weight metabolites directly
in vivo from both blood and tissue samples. Its
utility has been established for tissue metabolo-
mics without the need for biopsy in combination
with NMR, 94,95 GC-MS, 96 and HPLC/electro-
chemical detection. 97 Anesthesia was found to
signi

y, in SPME, sorbent particles are immobi-
lized directly on the outside of metal wire, and
this thin coated wire can easily be housed inside
of a hypodermic needle, as shown in Figure 5 A
(in contrast to SPE, in which the sorbent is
housed inside of a cartridge). Second, the ratio
of sorbent to sample used in SPME is low in
comparison to SPE, so exhaustive extraction of
analytes will not occur. Instead, equilibrium is
established between the sample solution and
sorbent phase if the two phases are left in contact
for suf

les of the liver
and had a profound effect on biological data
interpretation of oxidative stress induced by
doxorubicin dosing, showing distinct advantages
of awake in vivo animal procedures. 94 The in vivo
sampling approach also allowed repeated
sampling of the same animals to examine diurnal
and interanimal variation in liver metabolome. 94
Wibom et al. showed the utility of the technique
to intracranially sample extracellular

cantly affect metabolite pro

ciently long extraction times. The needle-
based SPME device is inserted directly into the
circulating bloodstream (or tissue) of an animal
for a short time and used to extract the analytes
of interest. 27,101 e 104 The main steps of the tech-
nique are shown in Figure 5 and include a 2
min in vivo extraction, a 30 sec rinse of the device
with water, and solvent desorption steps. The
coating for such applications is specially
designed to be biocompatible in order to avoid

fluid from
glioblastoma patients (151 total metabolites) and
found distinct metabolic differences between
tumor and tumor-adjacent regions of the brain. 96