samples freezing at 80 C instantly after collection is difficult
[22]. The common strategy for urine samples freezing is to super-
induce antibacterial preservatives (e.g., sodium azide (0.05–0.1%
wt/vol)) and storage samples at 4Cor 20 C before being
frozen at 80 C[23, 24]. Methanol is a perfect organic solvent
of quenching solutions, since it is miscible with water and has a low
freezing point (much lower than ethanol or glycerol). The cold
methanol (60%, vol/vol) allows a rapid termination of metabolism
in sub-second time scale for mammalian cells andEscherichia coli
[25]. Quenching in liquid nitrogen coupled with filtration has been
developed as a widely used strategy of preparation for microorgan-
isms, because the risk of cell leakage and damage in this strategy is
considerably lower than that of the methanol-quenching strategy
[26]. The metabolomics study of feces sample is significant to
reveal metabolic interactions between host and gut flora. The
instant preparation of fecal samples is important since living envi-
ronment of gut flora is changed during sample collection, which
may lead to metabolic profile distortion. Fecal samples frozen at
20 C manifested remarkably higher amino acids than that of
crude fecal control samples, suggesting a release of microbial intra-
cellular metabolites [27, 28]. The recommendation for fecal sam-
ples is collected at 4C and processed within 1 h of collection
[28]. After that, samples should be stored under low temperature
(commonly at 80 C). Freeze-thaw cycles should be avoided to
prevent the variation of metabolites. However, urine sample is
insensitive to freeze-thaw cycle [24]. A practicable approach is to
divide the quenched sample into aliquots and utilize one aliquot
sample in each time.3.1.2 Metabolite
Extraction
Metabolite extraction is a key step in the metabolomics analytical
procedure, which directly affects the quality of the final data. To
keep the nature of metabolome, minimal sample preparation is
essential for metabolomics research. The basic step of sample prep-
aration is to remove interferes (e.g., salts and proteins) and make
the extracts compatible with the detecting technique [17]. The
extraction methods are determined by the physicochemical proper-
ties of samples and detective technique for samples. The conven-
tional extraction methods for untargeted metabolomics include
liquid-liquid extraction (LLE), solid-liquid extraction (SLE), solid
phase extraction (SPE), microwave-assisted extraction (MAE),
accelerated solvent extraction (ASE), microdialysis, and ultrafiltra-
tion [29]. During extraction step, it is preferable to spike analytical
standards or isotope-labeled internal standards into samples for
efficiency evaluation of the metabolite recoveries. In general, pro-
tein precipitation is required after metabolite extraction. For this
purpose, organic solvent is added to the extractive complex fol-
lowed by centrifugation or ultrafiltration [30]. Methanol and ace-
tonitrile are the most usual organic solvents of protein precipitation270 Jing Cheng et al.