Direct enantioselective gradient reversed‐phase ultra‐high performance liquid chromatography tandem mass spectrometry method for 3‐hydroxy alkanoic acids in lipopeptides on an immobilized 1.6 μm amylose‐based chiral stationary phase

3‐Hydroxy fatty acids are important chiral building blocks of lipopeptides and metabolic intermediates of fatty acid oxidation, respectively. The analysis of the stereochemistry of such biomolecules has significant practical impact to elucidate and assign the enzymatic specificity of the biosynthesis machinery. In this work, a new mass spectrometry compatible direct chiral ultra high performance liquid chromatography separation method for 3‐hydroxy fatty acids without derivatization is presented. The application of amylose tris(3,5‐dimethylphenyl carbamate) based polysaccharide chiral stationary phase immobilized on 1.6 μm silica particles (CHIRALPAK IA‐U) allows the enantioseparation of 3‐hydroxy fatty acids under generic electrospray ionization mass spectrometry friendly reversed phase gradient elution conditions. Adequate separation factors were achieved with both acetonitrile and methanol as organic modifiers, covering hydrocarbon chain lengths between C 6 and C 14 . Elution orders were derived from rhamnolipid (R‐95) of which enantiomerically pure or enriched ( R )‐3‐hydroxy fatty acids were recovered after ester hydrolysis. The S ‐configured acids consistently eluted before the respective R‐ enantiomers. The method was successfully applied for the elucidation of the absolute configuration of 3‐hydroxy fatty acids originating from a novel lipopeptide with unknown structure. The work furthermore demonstrates that gradient elution is a viable option also in enantioselective (ultra)high performance liquid chromatography, even for analytes with modest separation factors, although less commonly exploited.