Analysis of N-acylhomoserine lactones after alkaline hydrolysis and anion-exchange solid-phase extraction by capillary zone electrophoresis-mass spectrometry

A quantitative, specific, and sensitive method for the determination of N‐acylhomoserine lactones (HSLs – a group of bacterial semiochemicals) in the form of their hydrolysis products (N‐acylhomoserines, HSs) is presented. Real samples were analyzed by capillary zone electrophoresis‐mass spectrometry (CZE‐MS) after alkaline lactonolysis and extraction by mixed‐mode anion‐exchange solid‐phase extraction. The presented cleanup significantly speeds up the HSL extraction procedure, strongly reduces sample consumption, and is more selective compared to the commonly used liquid/liquid extraction. Completeness of the hydrolysis reaction was examined by nuclear magnetic resonance spectroscopy. This CZE‐MS method complements recently published capillary separation techniques (nano liquid chromatography‐MS, partial‐filling micellar electrokinetic chromatography‐MS, gas chromatography‐MS) and provides a possibility to differentiate quantitatively between the homoserines (as naturally occurring degradation products) besides the intact homoserine lactones. The method was found to be quantitative down to a concentration of 0.05 µg/mL (limit of quantification), while the limit of detection was determined with 0.01 µg/mL – sufficient for the analysis of culture supernatants.