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

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis Pub Date : 2025-09-15 DOI:10.1016/j.jpba.2025.117146

Nikita V. Basov , Ekaterina A. Butikova , Maria A. Sotnikova , Ivan A. Razumov , Yulia S. Sotnikova , Yuriy V. Patrushev , Artem D. Rogachev , Nariman F. Salakhutdinov , Andrey G. Pokrovsky

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引用次数: 0

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.

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LC-MS/MS代谢组学筛选细胞培养样品制备特点

代谢组学分析已成为生命科学的重要工具，提供了对细胞代谢的见解。然而，制备用于代谢组学筛选的细胞培养物仍然具有挑战性，特别是含有可变细胞数量的样品。需要标准化和可重复的协议来确保可靠的数据，同时保持与高效液相色谱-串联质谱（HPLC-MS/MS）的兼容性。以黑色素瘤细胞系SK-MEL-28（人）和B16（小鼠）为模型，建立并优化了一种简便的样品制备方案，用于HPLC-MS/MS代谢组学筛选。该研究的重点是优化关键步骤，包括细胞裂解、代谢物提取和精确半定量分析的规范化策略。采用亲水相互作用液相色谱法（HILIC）和反相色谱法（RP）评价细胞计数对代谢组学覆盖率和检测灵敏度的影响。该方案能够有效地检测几种代谢物类别的样品中含有少至10,000个细胞。发现用于可靠分析的最佳细胞计数为400,000 - 500,000个细胞，确保在该方法的分析覆盖范围内进行一致和可重复的检测。我们的发现强调了细胞大小和数量在代谢组学研究中的重要性，因为更大的细胞提供了更好的代谢组学覆盖。此外，代谢物表现出不同的检测限，突出了根据代谢物特征调整样品制备策略的必要性。该方案为通过HPLC-MS/MS对粘附黑色素瘤细胞培养物进行代谢组学筛选提供了一种可靠且可重复的方法，并可适用于非粘附细胞和其他细胞类型。该方法平衡了灵敏度、重现性和可行性，为药物代谢组学、癌症研究及相关领域的细胞代谢组学研究提供了标准化的解决方案。

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来源期刊

Journal of pharmaceutical and biomedical analysis 医学-分析化学

CiteScore

6.70

自引率

5.90%

发文量

588

审稿时长

37 days

期刊介绍： This journal is an international medium directed towards the needs of academic, clinical, government and industrial analysis by publishing original research reports and critical reviews on pharmaceutical and biomedical analysis. It covers the interdisciplinary aspects of analysis in the pharmaceutical, biomedical and clinical sciences, including developments in analytical methodology, instrumentation, computation and interpretation. Submissions on novel applications focusing on drug purity and stability studies, pharmacokinetics, therapeutic monitoring, metabolic profiling; drug-related aspects of analytical biochemistry and forensic toxicology; quality assurance in the pharmaceutical industry are also welcome. Studies from areas of well established and poorly selective methods, such as UV-VIS spectrophotometry (including derivative and multi-wavelength measurements), basic electroanalytical (potentiometric, polarographic and voltammetric) methods, fluorimetry, flow-injection analysis, etc. are accepted for publication in exceptional cases only, if a unique and substantial advantage over presently known systems is demonstrated. The same applies to the assay of simple drug formulations by any kind of methods and the determination of drugs in biological samples based merely on spiked samples. Drug purity/stability studies should contain information on the structure elucidation of the impurities/degradants.