Lipids are biomolecules with a broad variety of chemical structures, which renders them essential not only for various biological functions but also interestingly for biotechnological applications. Rhamnolipids are microbial glycolipids with surface-active properties and are widely used biosurfactants. They are composed of one or two l-rhamnoses and up to three hydroxy fatty acids. Their biosynthetic precursors are 3-hydroxy(alkanoyloxy)alkanoic acids (HAAs). The latter are also present in cell supernatants as complex mixtures and are extensively studied for their potential to replace synthetically derived surfactants. The carbon chain lengths of HAAs determine their physical properties, such as their abilities to foam and emulsify, and their critical micelle concentration. Despite growing biotechnological interest, methods for structural elucidation are limited and often rely on hydrolysis and analysis of free hydroxy fatty acids losing the connectivity information. Therefore, a high-performance liquid chromatography-mass spectrometry method was developed for comprehensive structural characterization of intact HAAs. Information is provided on chain length and number of double bonds in each hydroxy fatty acid and their linkage by tandem mass spectrometry (MS/MS). Post-column photochemical derivatization by online Paternὸ–Büchi reaction and MS/MS fragmentation experiments generated diagnostic fragments allowing structural characterization down to the double bond position level. Furthermore, the presented experiments demonstrate a powerful approach for structure elucidation of complex lipids by tailored fragmentation.

脂质是具有多种化学结构的生物分子，这不仅使脂质对于各种生物学功能至关重要，而且对于生物技术应用也很有趣。鼠李糖脂是具有表面活性的微生物糖脂，是广泛使用的生物表面活性剂。它们由一两个l组成-鼠李糖酶和最多三个羟基脂肪酸。它们的生物合成前体是3-羟基（烷酰氧基）链烷酸（HAAs）。后者也以复杂混合物形式存在于细胞上清液中，并对其替代合成表面活性剂的潜力进行了广泛研究。HAA的碳链长度决定了它们的物理性质，例如它们的起泡和乳化能力以及临界胶束浓度。尽管对生物技术的兴趣日益增长，但用于阐明结构的方法仍然受到限制，并且通常依赖水解和游离羟基脂肪酸的分析，从而失去了连通性信息。因此，开发了一种用于完整HAAs的全面结构表征的高效液相色谱-质谱法。通过串联质谱（MS / MS）提供有关每个羟基脂肪酸中双键的链长和双键数量及其连接的信息。通过在线Paternὸ–Büchi反应进行柱后光化学衍生化和MS / MS片段化实验生成了诊断片段，从而可将结构特征降至双键位置水平。此外，提出的实验证明了通过有针对性的片段化方法对复杂脂质进行结构解析的有效方法。