Abstract: Microbial metabolomics refers to the qualitative and quantitative analysis of all low molecular weight metabolites present in and around the microbial cells.It has been widely applied in combination with other biological fields of studies for fermentation process control,metabolites analysis and microbial species upgrading.In cultivating microorganisms,the intracellular metabolites can turnover in an extremely rapid pace.Consequently,instantaneous fixation of the metabolism is essential for an accurate sampling.And,the development of a highly efficient and reliable quenching protocol is in order for most metabolomical studies.In this study,5 quenching solvent mixtures were evaluated by comparing their intracellular metabolite retention rates measured by LC-MS using the cold methanol extraction method.The results indicated that cold-methanol/water = 3/2(V/V) containing the buffer agent,HEPES,was the most efficient and reproducible quenching agent for Brevibacillus brevis cell culture.Using this method of quenching,more than 3 000 intracellular metabolites were detected in the positive ion ESI mode,which could detect more metabolites than the negative ion ESI mode.