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Although the analysis of large biomolecules is the prime application of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS), there is also increasing interest in lipid analysis. Since lipids possess relatively small molecular weights, matrix signals should be as small as possible to avoid overlap with lipid peaks. Although 2,5-dihydroxybenzoic acid (DHB) is an established MALDI matrix, the question whether just this isomer is ideal for lipid analysis was not yet addressed. UV absorptions of all six DHB isomers were determined and their laser desorption spectra recorded. In addition, all isomers were used as matrices to record positive and negative ion mass spectra of selected phospholipids (phosphatidylcholine and -serine): In the order 2,5-, 2,6-, 2,3- and 2,4-DHB, the quality of the positive ion lipid spectra decreases. This correlates well with the decreasing acidity of the applied DHB isomers. The 3,4- and 3,5- isomers give only very weak positive ion signals especially of acidic lipids. In contrast, the most suitable matrices in the negative ion mode are 2,5-, 2,4- and 3,5-DHB. 2,6-DHB does not provide any signal in the negative ion mode due to its marked acidity. Finally, differences in the crystallization behavior of the pure matrix and the matrix/lipid co-crystals were also monitored by atomic force microscopy (AFM): 2,5-DHB gave the smallest crystals and the skinniest layer. It is concluded that basically all DHB isomers can be used as MALDI matrices but the 2,5-isomer represents the most versatile compound.

In this study the efficacy of two different methods for extracting lipopeptides produced by five Bacillus strains-ethyl acetate extraction, and acid precipitation followed by methanol extraction—was investigated using mass spectrometry. High performance thin layer chromatography (HPTLC) was also used for the simultaneous separation of complex mixtures of lipopeptide extracts and for the determination of antimicrobial activity of their components. The mass spectra clearly showed well-resolved groups of peaks corresponding to different lipopeptide families (kurstakins, iturins, surfactins, and fengycins). The ethyl acetate extracts produced the most favorable results. The extracts of SS-12.6, SS-13.1, and SS-38.4 showed the highest inhibition zones. An iturin analog is responsible for the inhibition of Xanthomonas arboricola and Pseudomonas syringae phytopathogenic strains. HPTLC bioautography effectively identified the active compounds from a mixture of lipopeptide extracts, proving in situ its potential for use in direct detection and determination of antimicrobials. In the test of potential synergism among individual extracts used in different mixtures, stronger antimicrobial effects were not observed. Biochemical and phylogenetic analysis clustered isolates SS-12.6, SS-13.1, SS-27.2, and SS-38.4 together with Bacillus amyloliquefaciens, while SS-10.7 was more closely related to Bacillus pumilus.

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