samples should be collected by lithium heparin tubes, because
EDTA could result in extra resonances. They are usually diluted
with saline-buffer solution containing 10% D 2 O. Similar to MS,
proteins in serum or in plasma should be removed before NMR
measurements by either organic solvent precipitation or by ultrafil-
tration using a 10 kDa molecular weight as cutoff [51]. Urine
samples should be supplemented with NaN 3 (in a total concentra-
tion of sodium azide of min 0.05%, wt/vol) after sample collection
and stored at 40 C before analysis [52]. The supernatant should
be mixed with potassium phosphate buffer (containing 10 mM
sodium trimethylsilyl [2,2,3,3-D 4 ] propionate TMSP, in 100%
D 2 O). For the urine of subjects with injury kidney function, addi-
tional steps of protein precipitation by organic solvent or ultrafil-
tration should be conducted. For tissues and cells samples, it is
necessary to quench metabolic reactions by flash-freezing in liquid
nitrogen after collection. Tissues can be analyzed directly when
NMR machine is equipped with HR MAS probe, which allows
the sample to be spun with a high speed (more than 2 kHz) at
the magic angle 54.7[53]. However, HR MAS NMR spectra
might be overcrowded by the overlapping lipid signals. Thus, it is
of necessity to perform an extraction procedure prior to NMR
detection. Inorganic acids (e.g., perchloric acid) can be applied to
extract polar metabolites, but the risks of metabolite degradation
and the ionic strength increasement of the final extracts are raised.
The solvent of methanol/water (2:1, vol/vol) can be used to
extract low molecular weight metabolites, while acetonitrile/
water (2:1, v/v) is used for lipids and macromolecules. Solvents
containing methanol, chloroform, and water can be utilized for
extraction of polar and lipophilic metabolites [54]. For cell samples,
it is recommended to disrupt these samples by repeated freeze-thaw
procedure. Then the supernatant is collected after centrifugation in
2000 gat 4C for 3 min [55]. The approaches of protein
precipitation and metabolite extraction are the same as those of
tissue samples. For fecal samples, multiple solvents, such as phos-
phate buffer, methanol, and alkalized solutions, can be utilized for
metabolite extraction. The recommended extraction solvent is
phosphate buffer (0.1 M, pH 7.4), which present maximum signal-
to-noise ratio for subsequent analysis [55, 56].4 Multiple Detecting Platforms for Data Acquisition
The ambition of metabolomics is to detect and quantify all small
molecular metabolites in a biological sample, which is a huge
challenge in practice because of the diverse physicochemical proper-
ties of metabolites (e.g., polarity, concentration, stability). Multiple
analytical techniques are required to detect metabolites as many as
possible. Currently, most metabolomics studies are performed274 Jing Cheng et al.