Related Experiment Video
Updated: Jan 17, 2026
Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry
Published on: June 9, 2019
Features of sample preparation of cell culture samples for metabolomic screening by LC-MS/MS
Nikita V Basov1, Ekaterina A Butikova2, Maria A Sotnikova3
1N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia; Novosibirsk State University. Institute of Medicine and Medical Technologies, Novosibirsk, Russia; Novosibirsk State University. Department of Natural Sciences, Novosibirsk, Russia.
Abstract:
Metabolomic analysis has become an essential tool in the life sciences, providing insights into cellular metabolism. However, preparing cell cultures for metabolomic screening remains challenging, especially with samples containing variable cell numbers. Standardized and reproducible protocols are required to ensure reliable data while maintaining compatibility with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Using melanoma cell lines SK-MEL-28 (human) and B16 (mouse) as models, we developed and optimized a convenient sample preparation protocol for metabolomic screening by HPLC-MS/MS. The study is focused on optimizing key steps, including cell lysis, metabolite extraction, and normalization strategies for accurate semiquantitative analysis. The effects of cell count on metabolomic coverage and detection sensitivity were evaluated using hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) chromatography. The protocol enables efficient detection of several metabolite classes from samples containing as few as 10,000 cells. The optimal cell count for reliable analysis was found to be 400,000 - 500,000 cells, ensuring consistent and reproducible detection within the method's analytical coverage. Our findings emphasize the importance of cell size and number in metabolomic studies, as larger cells provide improved metabolomic coverage. Moreover, metabolites exhibited varying detection limits, highlighting the need to adjust sample preparation strategies according to metabolite characteristics. The proposed protocol offers a robust and reproducible approach for the metabolomic screening of adherent melanoma cell cultures by HPLC-MS/MS and can be adapted for non-adherent and other cell types. Balancing sensitivity, reproducibility, and feasibility, this method provides a standardized solution for cell metabolomic studies in pharmacometabolomics, cancer research, and related fields.
