3 Sample Collection and Preparation
Metabolomics usually requires a prior step of sample preparation.
The aims of sample preparation are to extract analytes from a
complicated biological matrix and to improve the detectability of
analytes by chemical derivatization. Sample preparation procedure
mainly includes metabolism quenching, sample storage, metabolite
extraction, and chemical derivatization. When biological samples
are collected from organism, metabolic reaction should be effi-
ciently terminated to achieve a true snapshot of metabolome of
biological samples. Thus quenching is the first step instantly to
deactivate metabolic reaction by inhibiting endogenous enzymes.
After that, most biological samples are stored until enough samples
are collected. The storage factors, such as storage time, tempera-
ture, and freeze-thaw cycle, should be considered comprehensively
since these factors affect metabolic stability significantly. Metabolite
extraction is a key step of metabolomics study, which is coupled
with protein precipitation, super-centrifugation, and liquid-liquid
extraction [14]. Finally, some metabolites should be derivatized
before detection. The whole sample preparation steps should not
induce significant variations in chemical and physical properties as
well as the concentration of metabolites. It is vital to build standard
protocols for specific sample and instrument platform, in which the
re-productivity across batches and laboratories should be consid-
ered thoroughly.3.1 Sample
Preparation for
MS-Based
Metabolomics
3.1.1 Sample Quenching
and Storage
After collection, metabolic reaction in biological samples, such as
biofluids, tissues, and cells, should be deactivated instantly by inhi-
biting endogenous enzymes. In this way, the snapshot of metabo-
lome can be amenable to subsequent detection step. It has been
found that the levels of lactic acid, citric acid, and lipids are changed
in the plasma sample which is not quenched [15]. Some hydrolases
and phosphatase tend to remain active even in methanol solution at
ambient temperatures [16]. It is worth to note that metabolic
reaction may last less than 1 s. Therefore an ideal quenching strat-
egy should allow a rapid metabolism interruption in a sub-second
time scale, which may not induce significant variations of metabo-
lome. The general strategies for sample quenching is freezing cou-
pled with pH regulation by adding acid or alkali [17]. Snap-
freezing by liquid nitrogen ( 196 C) is an effective quenching
approach especially for tissue samples, in which removing contami-
nants from the anesthetic drug in residual blood is of importance
[18–20]. A pilot study of stability of urine metabolome based on
GC-TOF-MS by Dunn et al. [21] indicated that the metabolome of
urine sample stored at 4C for 24 h before being frozen at 80 C
was not significantly changed as compared to that of immediately
frozen at 80 C after collection. It is reported that the urineMetabolomics: A High-Throughput Platform for Metabolite Profile Exploration 269