Uncovering the fragmentation and separation characteristics of sophorolipid biosurfactants with LC-MS-ESI.

The application of liquid chromatography and mass spectrometry (MS) is a challenging area of research for structural identification of sophorolipids, owing to the large number of possible variations in structure and limited knowledge on the separation and fragmentation characteristics of the variants. The aims of this work was to provide a comprehensive analysis of the expected characteristics and fragmentation patterns of a wide range of sophorolipid biosurfactant congeners, providing a methodology and process alongside freely available data to inform and enable future research of commercial or novel sophorolipids. Samples of acidic and lactonic sophorolipid standards were tested using reverse-phase ultra-high performance liquid chromatography and identified using electrospray ionization MS. 37 sophorolipid variants were identified and compared for their elution order and fragmentation pattern under MS/MS. The retention time of sophorolipids was increased by the presence of lactonization, unsaturation, chain length, and acetylation as hydrophobic interactions with the C18 stationary phase increased. A key finding that acidic forms can elute later than lactonic variants was obtained when the fatty acid length and unsaturation and acetylation are altered, in contradiction to previous literature statements. Fragmentation pathways were determined for lactonic and acidic variants under negative [M-H]- and positive [M+NH4]+ ionization, and unique patterns/pathways were identified to help determine the structural components present. The first publicly available database of chromatograms and MS2 spectra has been made available to aid in the identification of sophorolipid components and provide a reliable dataset to accelerate future research into novel sophorolipids and shorten the time to innovation. ONE-SENTENCE SUMMARY: This article describes the process and challenges in identifying different structures of eco-friendly biosurfactants, providing a novel database to compare results.