[31, 32]. In addition, the lipids in the supernatant may interfere
with mass spectrometry due to ion suppression. Therefore, the
combination of protein precipitation and lipid removal by chloro-
form is recommended to improve metabolite coverage and repro-
ducibility [33]. The extraction methods of different samples are
various because of their different natural characterization. Urine
samples usually contain relative less protein than plasma or tissues.
Thus, the sample preparation is performed through a simple centri-
fugation commonly. For the urine of renal disease, however, the
injury kidney function usually causes the excessive excretion of
urine proteins, and thus protein precipitation by organic solvent is
an essential step [34]. Compared to biofluids (e.g., plasma and
urine), the sample preparation of tissues is labor-intensive and
time-consuming, because an additional step of homogenization
(physical disruption of tissue sample) is prerequisite. The break-
down of the frozen tissue is performed through grinding in liquid
nitrogen with mortar and pestle, in which a specifically designed
tissue lyzer is utilized to homogenize multiple tissue samples simul-
taneously [19]. For specific tissues such as the lung and heart, a
prior step of enzyme digestion before bead-beater treatment can
effectively improve homogenization [35]. Methanol/water in dif-
ferent proportions (50–100%) is the common solvent for homoge-
nization. After that, proteins in tissue homogenates are precipitated
by a one-step solvent method (adding methanol/water mixture)
[18] or a two-step solvent method (adding methanol/water (1:1,
vol/vol) mixture followed by dichloromethane/methanol (3:1,
vol/vol) mixture) [19]. The feces is a kind of complicated sample
including undigested food residues, metabolites, and gut microbes.
To avoid the disturbance from the intracellular contents of gut
microbes especially amino acids, super-centrifugation
(171,500gat 4C for 2 h) of feces samples within 24 h after
collection is recommended [27, 28]. Short-chain fatty acids and
bile acids are two kinds of targeted metabolites closely correlating
to the interactions between host and gut microbes. Interestingly,
our study found that these metabolites were almost not affected by
the intracellular contents of gut microbes when the feces samples
were stored at low temperature (e.g., 80 C) [ 27].3.1.3 Chemical
Derivatization for
MS-Based Metabolomics
Due to the complexity of metabolome and the diversity of physico-
chemical property of metabolites, it is practically impossible to
simultaneously detect all compounds in a biological sample
through MS-based detection methods. To detect more metabo-
lites, derivatization is a prerequisite step in GC/MS based on
methods for rendering highly polar compounds with sufficiently
volatile and increasing ionization efficiency of these compounds. In
derivatization, a chemical reagent is applied to react with one or
more active groups (such as carboxylic, amide, amino, and hydroxylMetabolomics: A High-Throughput Platform for Metabolite Profile Exploration 271