School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China.
Department of Chemistry, Yanbian University, Park Road 977, Yanji 133002, Jilin Province, China.
J Chromatogr A. 2022 May 10;1670:462954. doi: 10.1016/j.chroma.2022.462954. Epub 2022 Mar 10.
Amine/phenol submetabolome has shown critical role in clinical cancer screening and therapy. In suit derivatization has widely applied in the analysis of amine/phenol submetabolome by LC/MS for simplifying the pretreatment procedures, improving the separation and sensitivity. However, the complexity of biological matrix and trace amount of metabolites in plasma that lead to the limited detection coverage, poor repeatability and low extraction efficiency are still issues for in suit derivatization. Herein, we proposed an isotope labelled in suit derivatization-extraction integrated system for targeted analysis of all the metabolites in amine/phenol submetabolome with high efficiency and repeatability by LC-MS. The processes of in suit derivatization, alkalization and extraction were performed simultaneously in the nanopores highly dispersed between the carbon nanofibers based on the nanoconfinement effect. Isotope labelling derivatization (ILD) reagents benzoyl chloride (BzCl) and BzCl-d were used to enhance the accuracy of identification and relative quantification. The detection sensitivity was increased up to 5.91-fold and detection coverage was enhanced more than 25% compared with conventional derivatization method. After systematical validation, the established methodology was applied to profile the amine/phenol submetabolome of human plasma and 1498 metabolites were screened out, among which, 1004 (67.02%) were positively or putatively identified. Furthermore, 106 amine/phenol metabolites exhibited significant difference between lung cancer patients and healthy controls by using multiple data processing methods. Taken together, the isotope labelled in suit derivatization-extraction integrated system was a useful approach for the analysis of amine/phenol submetabolome in plasma with broad metabolome coverage, simple pretreatment steps, high detection sensitivity and accuracy, and could be a potential tool for clinical biomarker discovery of disease.
胺/酚亚代谢组在临床癌症筛查和治疗中具有重要作用。衍生化在 LC/MS 分析胺/酚亚代谢组中得到了广泛应用，可简化预处理步骤，提高分离度和灵敏度。然而，生物基质的复杂性和血浆中痕量代谢物导致检测覆盖率有限、重复性差和提取效率低等问题仍然存在。在此，我们提出了一种同位素标记的原位衍生-萃取一体化系统，用于通过 LC-MS 高效、重复地分析胺/酚亚代谢组中的所有代谢物。基于纳米限域效应，在基于碳纳米纤维的纳米孔中同时进行原位衍生化、碱化和萃取过程。同位素标记衍生化 (ILD) 试剂苯甲酰氯 (BzCl) 和 BzCl-d 用于提高鉴定和相对定量的准确性。与传统衍生化方法相比，检测灵敏度提高了 5.91 倍，检测覆盖率提高了 25%以上。经过系统验证，该方法被应用于人血浆的胺/酚亚代谢组分析，筛选出 1498 种代谢物，其中 1004 种（67.02%）被正向或推测鉴定。此外，通过使用多种数据处理方法，发现 106 种胺/酚代谢物在肺癌患者和健康对照者之间存在显著差异。总之，同位素标记的原位衍生-萃取一体化系统是一种用于分析血浆中胺/酚亚代谢组的有用方法，具有广泛的代谢组覆盖范围、简单的预处理步骤、高检测灵敏度和准确性，可作为疾病临床生物标志物发现的潜在工具。
