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 acid oxidation, respectively. The analysis the stereochemistry such biomolecules has significant practical impact to elucidate assign enzymatic specificity biosynthesis machinery. In this work, a new mass spectrometry compatible direct ultra high performance liquid chromatography separation method for 3-hydroxy without derivatization is presented. application amylose tris(3,5-dimethylphenyl carbamate) based polysaccharide stationary phase immobilized on 1.6 μm silica particles (CHIRALPAK IA-U) allows enantioseparation under generic electrospray ionization friendly reversed gradient elution conditions. Adequate factors were achieved with both acetonitrile methanol as organic modifiers, covering hydrocarbon chain lengths between C6 C14. Elution orders derived from rhamnolipid (R-95) which enantiomerically pure or enriched (R)-3-hydroxy recovered after ester hydrolysis. S-configured consistently eluted before respective R-enantiomers. was successfully applied elucidation absolute configuration originating novel lipopeptide unknown structure. work furthermore demonstrates that viable option also in enantioselective (ultra)high chromatography, even analytes modest factors, although less commonly exploited.