Behrens Beate, Engelen Jeannine, Tiso Till, Blank Lars Mathias, Hayen Heiko
Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstr. 30, 48149, Münster, Germany.
Department of Food Chemistry, Faculty of Mathematics and Natural Sciences, University of Wuppertal, Gaussstr. 20, 42119, Wuppertal, Germany.
Anal Bioanal Chem. 2016 Apr;408(10):2505-14. doi: 10.1007/s00216-016-9353-y. Epub 2016 Feb 15.
Rhamnolipids are surface-active agents with a broad application potential that are produced in complex mixtures by bacteria of the genus Pseudomonas. Analysis from fermentation broth is often characterized by laborious sample preparation and requires hyphenated analytical techniques like liquid chromatography coupled to mass spectrometry (LC-MS) to obtain detailed information about sample composition. In this study, an analytical procedure based on chromatographic method development and characterization of rhamnolipid sample material by LC-MS as well as a comparison of two sample preparation methods, i.e., liquid-liquid extraction and solid-phase extraction, is presented. Efficient separation was achieved under reversed-phase conditions using a mixed propylphenyl and octadecylsilyl-modified silica gel stationary phase. LC-MS/MS analysis of a supernatant from Pseudomonas putida strain KT2440 pVLT33_rhlABC grown on glucose as sole carbon source and purified by solid-phase extraction revealed a total of 20 congeners of di-rhamnolipids, mono-rhamnolipids, and their biosynthetic precursors 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) with different carbon chain lengths from C8 to C14, including three rhamnolipids with uncommon C9 and C11 fatty acid residues. LC-MS and the orcinol assay were used to evaluate the developed solid-phase extraction method in comparison with the established liquid-liquid extraction. Solid-phase extraction exhibited higher yields and reproducibility as well as lower experimental effort.
鼠李糖脂是一类具有广泛应用潜力的表面活性剂，由假单胞菌属细菌在复杂混合物中产生。发酵液分析通常需要繁琐的样品制备，并且需要联用分析技术，如液相色谱-质谱联用（LC-MS），以获取有关样品组成的详细信息。在本研究中，提出了一种基于色谱方法开发以及通过LC-MS对鼠李糖脂样品材料进行表征的分析程序，同时比较了两种样品制备方法，即液液萃取和固相萃取。使用混合丙基苯基和十八烷基硅烷基改性硅胶固定相在反相条件下实现了高效分离。对以葡萄糖作为唯一碳源生长并通过固相萃取纯化的恶臭假单胞菌KT2440 pVLT33_rhlABC菌株的上清液进行LC-MS/MS分析，结果显示共有20种不同碳链长度（从C8到C14）的二鼠李糖脂、单鼠李糖脂及其生物合成前体3-(3-羟基链烷酰氧基)链烷酸（HAAs）同系物，包括三种具有不常见C9和C11脂肪酸残基的鼠李糖脂。与既定的液液萃取相比，使用LC-MS和间苯二酚测定法对所开发的固相萃取方法进行了评估。固相萃取表现出更高的产率和重现性以及更低的实验工作量。
