groups) of compounds, and they are transformed into a structurally
altered complexes, which have some features improving effective-
ness of the subsequent chromatographic retention, separation,
detection, and identification [36].
Currently, there are two common used derivation methods,
i.e., silylation derivatization and alkylation derivatization. As for
silylation derivatization, most GC-MS based metabolomics studies
apply a two-step process, including the first oximation and
subsequent trimethylsilylation (TMS). The aim of oximation is to
prevent side derivatization of compounds containing carbonyl
group (such as pyruvic acid,α-ketoglutaric acid, and reducing
sugars), by reacting with methoxyamine hydrochloride [37].N-
Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% tri-
methylchlorosilane (TMCS) andN,O-bis(Trimethylsilyl)trifluoroa-
cetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) are two
commonly used TMS reagents in GC-MS metabolomics
[37–39]. The sequential oximation and TMS derivatization can
improve effectiveness of metabolic analysis of compounds contain-
ing nitrogen (amino acids, amines, purine, pyrimidine, and nucleo-
sides), carboxylic acids (fatty acids, the intermediates of citric acid
cycle, hydroxyl acids), polyols, carbohydrates, sugar acids, choles-
terol and free bile acids, and phenolics. These compounds cover the
main metabolites in central carbon metabolism, which are very
important in the study of biological science. In general, the silyla-
tion derivatization requires a non-aqueous environment, while the
alkylation derivatization can be performed in an aqueous environ-
ment. For the alkyl chloroformate derivatization, proposed by
Husek, its chemical reaction system is consists of alkyl chlorofor-
mate, water, ethanol, and pyridine [40, 41]. During the derivatiza-
tion reaction, alkoxycarbonyl esters of amino and hydroxyl groups
are stable, but the alkoxycarbonyl ester of carboxylic group is
unstable, and it subsequently transformed into stable alkyl esters
by an exchange reaction with ethanol [27]. Alkyl chloroformate
derivatization allows simultaneous esterification of carboxylic
group, amino group, and hydroxyl group linked to a benzene
ring or the side chain, which remarkably improve peak intensity
and chromatographic separation [42]. The alkyl chloroformate
derivatization has higher reproducibility and stability than the
TMS derivatization [27, 43]. However, carbohydrates and polyols
existed in biofluids can be detected by the TMS derivatization but
not the alkyl chloroformate derivatization. While alkyl chlorofor-
mate derivatization can help detect short-chain fatty acids, amino
acids, carboxylic acids, hydroxyl acids, phenolic acids, benzoyl and
phenyl derivatives, and indoles [27, 42].
Generally, metabolomics based on LC-MS just apply a simple
protein precipitation step in sample preparation process, allowing
hundreds to thousands of metabolites to be routinely detected and272 Jing Cheng et al.