relatively quantitated. However, there are still numerous metabo-
lites that can’t be detected by the standard LC-MS techniques, due
to the weak retention on the chromatographic column, lower
responses of mass detection, wide concentration range, poor ioni-
zation, low abundance, matrix effects, and potential degradation of
compounds. Chemical derivatization of compounds can help to
improve the detection effectiveness of LC-MS methods, in which
a charged or chargeable functional group is introduced into com-
pounds. An ideal derivatization reaction should be conducted in a
mild condition with high yields and less side reactions, and the
resulting derivatives should have good chemical stability in the
subsequent analytical procedures. The stable-isotope-labeled deri-
vatives, presumed to have identical physicochemical properties to
those of the corresponding unlabeled compounds, are utilized as
internal standard (IS) to correct for the variability arising from
sample preparations and instrumental analyses. Generally, the(^13) C- or (^15) N-labeled ISs are more preferable than deuterium (d-)
labeled ISs because they avoid chromatographic isotope effect that
could lead to low accuracy in quantitative LC-MS assays. For
example, Guo and his colleagues synthesized a new reagent of
(^13) C
2 -dansyl chloride to derivatize standard compounds as ISs,
and then 93 metabolites containing amino or phenol hydroxy
group were absolutely quantified from human urine samples by
the RPLC-ESI-MS method [44]. This approach was afterward
extended for metabolomics studies of body fluids [45–48]. Another
widely available derivatization reagent is^13 C-labeled benzoyl chlo-
ride (BzCl), which is used for primary and secondary amines,
phenols, thiols, and ribose hydroxyls. Compared to^13 C 2 -dansyl
chloride,^13 C-labeled benzoyl chloride has the following advan-
tages in derivatization: (1) the derivative reaction of BzCl is a fast
(seconds) and mild (room temperature) reaction at a wider pH
range; (2) the reaction does not need to scavenge reagents; (3) ben-
zoyl chloride and the benzoylated products are insensitive to light;
and (4)^13 C-labeled benzoyl chloride is commercially available at
relatively low cost. It was reported by using the^13 C-labeled benzoyl
chloride as derivatization reagents, up to 70 neurochemicals,
including catecholamines, indoleamines, amino acids, polyamines,
trace amines, antioxidants, energy compounds, and their metabolic
products were quantified from cerebrospinal fluid, plasma, tissue,
and hemolymph with a 10 nM detection limits [49, 50].
3.2 Sample
Preparation for
NMR-Based
Metabolomics
Compared with MS-based metabolomics, the sample preparation
in NMR-based metabolomics is simpler. The alteration in pH value
and salt concentration affect the chemical shift of metabolites,
which lead to spurious clustering of compounds in metabolomics
studies. Therefore, it is necessary to adjust the pH value of each
sample through a phosphate buffer (pH 7.4). Various preparation
methods are required for different samples. Serum or plasma
Metabolomics: A High-Throughput Platform for Metabolite Profile Exploration 273