Trace Detection of Underivatized Muramic Acid in Environmental Dust Samples by Microcolumn Liquid Chromatography−Electrospray Tandem Mass Spectrometry

Abstract

Muramic acid (MA) is a universal chemical marker for bacterial cell wall polymers (peptidoglycan) present in complex environmental, industrial, and clinical matrices. Trace analysis of MA has been limited to a handful of laboratories because of the lengthy nature of derivatization procedures necessary for GC/MS and GC/MS/MS. The focus of the current report concerns environmental analysis (organic dust) using LC/MS/MS of native MA. Dust was heated in sulfuric acid to release MA from cell wall polymers. Acid was removed by extraction with an organic base. The samples were passed through a hydrophobic C-18 extraction column to remove nonpolar contaminants. This was followed by binding of MA to a strong cation-exchange extraction column to eliminate neutral and anionic components of the sample. MA was then eluted with hydrochloric acid, which was removed by evaporation. Analysis was done by microscale LC ES/MS/MS using a custom-made Lichrosphere-diol capillary column. 13C-Labeled MA was used as an internal standard. Total sample preparation takes approximately 12 h. The simplicity of the sample preparation may eventually allow MA analysis to be used in a host of applications, including air monitoring for biocontamination. This is the first report, to our knowledge, of microscale LC ES/MS/MS analysis for MA (or, indeed, any other sugar monomer) in a complex matrix.

Mass Spectrometry: Identification and Biodetection, Lessons Learned and Future Developments

“…Furthermore, for complex matrices, GC-MS-MS have proved to have much greater sensitivity than LC-MS-MS for trace detection of monomers. [15][16][17] This helps explain why LC-MS-MS has not been widely adopted for trace analysis in the microbiology field.…”

Characterisation of bacteria by matrix-assisted laser desorption/ionisation and electrospray mass spectrometry

“…This study pioneers the MALDI MS investigation of regulation of secondary metabolites and demonstrates the feasibility of localising compounds in subcellular fractions by MS. Flow injection ES MS and ES MS/MS was used to distinguish a wide variety of gram-negative and gram-positive bacteria (including B. anthracis) from fungi through the detection of muramic acid in crude cell lysates [111]. With the same objective, the compound has also recently been analysed in dust [112]. Muramic acid is an amino sugar that is exclusively found in eubacteria (but not in all eubacteria) and that can be liberated from the cell wall peptidoglycan by simple acid hydrolysis.…”

Molecular indicators (biomarkers) of past life

“…Typical instrument sensitivities are in the low ng (10 -9 g) range. High-resolution MS or tandem MS (MS/ MS) (Shahgholi et al, 1997;Kozar et al, 2001) coupled with GC are other options for special cases, although they have slightly lower sensitivity in some applications. Unknown biomarker structures are determined, after purification of the compound, by nuclear magnetic resonance (NMR) spectroscopy or by X-ray crystallography (Greiner et al, 1977;Hussler et al, 1984;Trendel et al, 1990;Hauke et al, 1992a, b).…”