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In order to preserve the in vivo metabolite levels of cells, a quenching protocol must be quickly executed to avoid degradation of labile metabolites either chemically or biologically. In the case of mammalian cell cultures cultivated in complex media, a wash step previous to quenching is necessary to avoid contamination of the cell pellet with extracellular metabolites, which could distort the real intracellular concentration of metabolites. This is typically achieved either by one or multiple centrifugation/wash steps which delay the time until quenching (even harsh centrifugation requires several minutes for processing until the cells are quenched) or filtration. In this article, we describe and evaluate a two-step optimized protocol based on fast filtration by use of a vacuum pump for quenching and subsequent extraction of intracellular metabolites from CHO (Chinese hamster ovary) suspension cells, which uses commercially available components. The method allows transfer of washed cells into liquid nitrogen within 10-15s of sampling and recovers the entire extraction solution volume. It also has the advantage to remove residual filter filaments in the final sample, thus preventing damage to separation columns during subsequent MS analysis. Relative to other methods currently used in the literature, the resulting energy charge of intracellular adenosine nucleotides was increased to 0.94 compared to 0.90 with cold PBS quenching or 0.82 with cold methanol/AMBIC quenching., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)