A liquid chromatography–tandem mass spectrometry assay for Marfey’s derivatives of l-Ala, d-Ala, and d-Ala-d-Ala: Application to the in vivo confirmation of alanine racemase as the target of cycloserine in Escherichia coli

Bacterial cell wall biosynthesis is the target of several antibacterial agents and is also of interest as a target for future antibacterial agent development. Given the now widespread availability of liquid chromatography–tandem mass spectrometry (LC–MS/MS) instruments, the development of LC–MS/MS assays for cell wall biosynthesis intermediates would fill a needed gap in the analytical methodology available for antibacterial agent discovery and characterization. An LC–MS/MS assay for several early cell wall intermediates—l-Ala, d-Ala, and d-Ala-d-Ala—has been developed. This method relies on derivatization of bacterial extracts with Marfey’s reagent. Marfey’s reagent adducts of l-Ala and d-Ala were cleanly separated chromatographically, allowing Marfey’s adducts of d-Ala and l-Ala to be separated prior to mass spectrometry (MS) detection and quantitation. The Marfey’s adduct of d-Ala-d-Ala was also readily detectable using this same approach. This method shows good linearity (R2 > 0.99), with a lower limit of quantitation of 1 pmol. This assay was demonstrated for characterization of the in vivo effect of cycloserine on Escherichia coli. Cycloserine resulted in a dramatic lowering of both d-Ala and d-Ala-d-Ala levels. Ampicillin had little effect on levels of these three metabolites, consistent with the actions of ampicillin on the later stages of cell wall biosynthesis. These observations indicate that cycloserine inhibits alanine racemase production of d-Ala in E. coli and demonstrates the utility of this assay in directly assessing d-Ala branch targeted antibacterial agents.