Electrospray Tandem Mass Spectrometry for Analysis of Native Muramic Acid in Whole Bacterial Cell Hydrolysates

Muramic acid is an amino sugar found in eubacterial cell walls and not elsewhere in nature. This study explored the use of electrospray tandem mass spectrometry (ESI MS/MS) in analysis of underivatized muramic acid in bacterial hydrolysates. Fungal hydrolysates were used as negative controls. The only processing used was hydrolysis in sulfuric acid followed by extraction with an organic base (N,N-dioctylmethylamine) to remove the acid prior to ESI MS/MS analysis. Compared with pure muramic acid, bacterial hydrolysates produced more complex ESI mass spectra, such that the protonated molecular ion at m/z 252 was barely detectable. In contrast, product ion spectra of m/z 252 were identical among pure muramic acid, Gram positive bacteria, and Gram negative bacteria. However, no characteristic product ion spectrum was manifested from m/z 252 in fungal samples. This allowed ready, visual differentiation of bacteria and fungi. Multiple reaction monitoring (MRM) following muramic acid fragmentations (m/z 252 → 144 and m/z 252 → 126) increased sensitivity and allowed quantitative differentiation when compared with the MRM of the internal standard N-methyl-D-glucamine (m/z 196 → 44). ESI MS/MS required minimal sample preparation and allowed rapid sample throughput for analysis of muramic acid in whole bacterial cell hydrolysates.