TY - JOUR

T1 - Features of sample preparation of cell culture samples for metabolomic screening by LC-MS/MS

AU - Басов, Никита Вячеславович

AU - Бутикова, Екатерина Алексеевна

AU - Сотникова, Мария

AU - Разумов, Иван Алексеевич

AU - Сотникова, Юлия Сергеевна

AU - Патрушев, Юрий Валерьевич

AU - Рогачев, Артем Дмитриевич

AU - Салахутдинов, Нариман Фаридович

AU - Покровский, Андрей Георгиевич

N1 - HPLC–MS/MS analysis was conducted with the support of a state project No. FSUS–2025–0012. Cultivation and cell counting were supported by budget project number FWNR–2022–0023. The authors also thank the state project No. 075–00365–25–00 of N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry of the Siberian Branch of the Russian Academy of Sciences for providing resources. The preparation of monolithic columns for HPLC has been conducted with the support of a state project No. FWUR–2024–0032. The authors acknowledge support from the state assignment of RICEL – a branch of IC&G SB RAS, project No. FWNR–2025–0016.

PY - 2025/9/15

Y1 - 2025/9/15

N2 - 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.

AB - 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.

KW - Cell culture metabolomics

KW - Cell number optimization

KW - HILIC

KW - HPLC-MS/MS standardization

KW - RP LC

KW - Targeted metabolomics

UR - https://www.scopus.com/pages/publications/105016313330

UR - https://www.mendeley.com/catalogue/46a63d18-a500-33d3-b826-74ebef33210f/

U2 - 10.1016/j.jpba.2025.117146

DO - 10.1016/j.jpba.2025.117146

M3 - Article

C2 - 40972480

VL - 267

SP - 117146

JO - Journal of Pharmaceutical and Biomedical Analysis

JF - Journal of Pharmaceutical and Biomedical Analysis

SN - 0731-7085

M1 - 117146

ER -