Actinobacteria are one of the most promising producers of medically and industrially relevant secondary metabolites. However, screening of such compounds in actinobacteria growth demands simple, fast, and efficient extraction procedures that enable detection and precise quantification of biologically active compounds. In this regard, solid phase microextraction (SPME) emerges as an ideal extraction technique for screening of secondary metabolites in bacteria culture due to its non-exhaustive, minimally invasive, and non-destructive nature: its integrated sample preparation workflow; balanced coverage feature; metabolism quenching capabilities; and superior cleanup, as well as its versatility in configuration, which enables automation and high throughput applications. The current work provides a comparison of micro-scale and direct immersion SPME (DI-SPME) for screening of secondary metabolites, describes the optimization of the developed DI-SPME method, and introduces the developed technique for mapping of target secondary metabolites as well as its direct coupling to mass spectrometry for such applications. The optimized DI-SPME method provided higher amounts of extracted ions and intensity signals, yielding superior extraction and desorption efficiency as compared with micro-scale extraction. Studied compounds presented stability on the coating for 24 h at room temperature. The DI-SPME mapping approach revealed that lysolipin I and the lienomycin analog are distributed along the center and edges of the colony, respectively. Direct coupling of SPME to MS provided a similar ions profile as SPME-LC-MS while enabling a significant decrease in analysis time, demonstrating its suitability for such applications. DI-SPME is herein presented as an alternative to micro-scale extraction for screening of secondary metabolites in actinobacteria solid medium, as well as a feasible alternative to DESI-IMS for mapping of biologic radial distribution of secondary metabolites and cell life cycle studies. Lastly, the direct coupling of DI-SPME to MS is presented as a fast, powerful technique for high throughput analysis of secondary metabolites in this medium.

中文翻译：

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固相微萃取是筛选放线菌中次生代谢产物的有力替代方法。

放线菌是医学上和工业上相关的次级代谢产物最有前途的生产者之一。然而，在放线菌生长中对此类化合物的筛选需要简单，快速和有效的提取程序，以实现对生物活性化合物的检测和精确定量。在这方面，固相微萃取（SPME）由于其非穷举，微创和无损性质而成为筛选细菌培养中次生代谢物的理想提取技术。均衡的覆盖功能；代谢淬灭能力；以及出色的清理功能以及其配置的多功能性，可实现自动化和高吞吐量的应用程序。当前的工作提供了用于次级代谢物筛选的微型和直接浸入式SPME（DI-SPME）的比较，描述了开发的DI-SPME方法的优化，并介绍了用于绘制目标次级代谢物的图谱以及它可以直接与质谱联用，用于此类应用。优化的DI-SPME方法提供了更高数量的离子提取和强度信号，与微量萃取相比，具有更高的萃取和解吸效率。所研究的化合物在室温下可在涂层上保持24小时的稳定性。DI-SPME作图方法表明，溶血素I和lienomycin类似物分别沿菌落的中心和边缘分布。SPME与MS的直接偶联提供了与SPME-LC-MS相似的离子分布，同时大大缩短了分析时间，证明了其在此类应用中的适用性。本文介绍了DI-SPME，作为微型提取法在放线菌固体培养基中筛选次级代谢物的替代方法，以及DESI-IMS的可行替代物，用于绘制次级代谢物的生物学径向分布图和细胞生命周期研究。最后，DI-SPME与MS的直接偶联是一种快速，强大的技术，可用于该培养基中次级代谢物的高通量分析。本文介绍了DI-SPME，它是用于筛选放线菌固体培养基中次级代谢产物的微型提取方法的替代方法，以及用于绘制次级代谢产物的生物学径向分布图和细胞生命周期研究的DESI-IMS的可行替代方法。最后，DI-SPME与MS的直接偶联是一种快速，强大的技术，可用于该培养基中次级代谢物的高通量分析。本文介绍了DI-SPME，它是用于筛选放线菌固体培养基中次级代谢产物的微型提取方法的替代方法，以及用于绘制次级代谢产物的生物学径向分布图和细胞生命周期研究的DESI-IMS的可行替代方法。最后，DI-SPME与MS的直接偶联是一种快速，强大的技术，可用于该培养基中次级代谢物的高通量分析。