AbstractIn this work, four different metabolite purification approaches are investigated prior to metabolomics of human HT29 colon cancer cells. Namely, methanol deproteinization, ultrafiltration and two SPE methods using C18 and polymer‐based cartridges were studied. The extracts were characterized via a metabolomic approach based on the application of CE TOF MS (CE‐MS). CE‐MS analysis time was less than 20 min per sample and allowed the simultaneous and reproducible analysis of more than 80 metabolites in a single run with a minimum consumption of sample and reagents. Metabolome analysis revealed in some cases important differences among the studied metabolite purification procedures. No significant differences were observed in the metabolite profile using C18 and polymer‐based cartridges, or between ultrafiltration and methanol deproteinization. However, important differences were observed in the metabolomic profiles obtained from SPE and methanol deproteinization samples. These results demonstrate the crucial role of the metabolite purification strategy in metabolomics since it can bias (and in some cases mislead) the conclusions achieved by the metabolomic study.