Department of Pathology and Immunology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, United States; Texas Children's Microbiome Center, Department of Pathology, Texas Children's Hospital, 1102 Bates Ave, Houston, TX 77030, United States.
Food Science and Technology Department, the University of Nebraska-Lincoln, 1901 N 21(st) Street, Lincoln, NE 68588, United States.
J Microbiol Methods. 2020 Oct;177:106020. doi: 10.1016/j.mimet.2020.106020. Epub 2020 Aug 11.
Recently, an opportunity to perform a broad ruggedness assessment of our liquid chromatography-tandem mass spectrometry (LC-MS/MS) system presented itself during the analytical planning phase of a large-scale human fecal microbiome study. The specific aim of this project was to study the microbial-mediated metabolism of a targeted set of bile acids/salts by mixed bacterial communities cultured from the feces of 12 healthy volunteers when grown in a custom growth medium and following exposure to different clinically-relevant antibiotics. The magnitude of this study offered a rare opportunity to significantly stress procedures and LC-MS/MS system components comprised in our bile acid/salt targeted metabolomics method. With this second specific aim in mind, we modified the sample analysis plan to include a series of figure-of-merit (FoM)-based tests that are commonly used in regulated bioanalytical labs to assess LC and MS system ruggedness for a specific assay - these FoM-based testing parameters were monitored continuously over the course of sample analysis and the results are presented in this report. In total, the assessment included 1206 sequential injections (180 calibration standards, 136 blank-internal standard samples, and 890 diluted medium samples) that took place over 8-days. Completion of the 8-days of non-stop sample analysis revealed no critical hardware or software failures, and the analysis of the FoM-based tests indicated no observable degradation of system performance over the number of samples and time tested. The FoM-based test metrics presented may be used as a template to assess the ruggedness of any LC-MS/MS-based targeted metabolomics workflow.
最近，在一项大规模人类粪便微生物组研究的分析规划阶段，我们有机会对我们的液相色谱-串联质谱（LC-MS/MS）系统进行广泛的稳健性评估。该项目的具体目的是研究混合细菌群落从 12 名健康志愿者的粪便中培养出来后，在定制生长培养基中生长并暴露于不同临床相关抗生素时，对一组靶向胆汁酸/盐的微生物介导代谢。这项研究的规模为我们的胆汁酸/盐靶向代谢组学方法中的程序和 LC-MS/MS 系统组件提供了一个难得的压力测试机会。考虑到这第二个具体目标，我们修改了样品分析计划，包括一系列基于性能指标（FoM）的测试，这些测试通常用于监管生物分析实验室，以评估特定测定的 LC 和 MS 系统稳健性 - 这些基于 FoM 的测试参数在样品分析过程中进行连续监测，并在本报告中呈现结果。总共进行了 1206 次连续进样（180 个校准标准品、136 个空白-内标样品和 890 个稀释培养基样品），这些样品分析在 8 天内完成。完成 8 天不停歇的样品分析没有发现关键的硬件或软件故障，并且基于 FoM 的测试分析表明，在测试的样品数量和时间范围内，系统性能没有观察到可降解。提出的基于 FoM 的测试指标可以用作评估任何基于 LC-MS/MS 的靶向代谢组学工作流程的稳健性的模板。
