Amperometric detection of muramic acid in high-performance liquid chromatography with a post-column reaction

Highly sensitive detection of muramic acid has been developed by using an amperiometric detector in high-performance liquid chromatography. The reducing ability of muramic acid was coupled with the redox reaction of bis(1,10-phenanthroline)copper(II), CBP, as mediator. CBP, reduced by muramic acid in a post-column reaction, was reoxidized with the amperometric detector, resulting in a highly sensitive and selective detection. A doublet peak was obtained, due to the alpha- and beta-anomers of muramic acid. This was merged into a single peak by adding phosphoric acid to the sample solution. Addition of phosphoric acid to serum, spiked with muramic acid, also clarified the chromatogram. The detection limit was 0.05 ppm (4 pmol of muramic acid injected) in serum samples.