Fast and sensitive detection of amino sugar, neutral sugar and uronic acid biomarkers using 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization and reversed phase liquid chromatography coupled to Orbitrap mass spectrometry

Soil organic matter is composed to a large extent of microbial necromass, including fragmented cell wall residues and other cytoplasmic components from dead fungi and bacteria. These components accumulate in soil over long periods of time and have been used as biomarkers to trace microbial residues. Amino sugars are key components of microbial cell walls and can be found in polymeric forms as fungal chitin and bacterial peptidoglycan in soils. Among the most abundant amino sugars in soil are glucosamine, galactosamine, mannosamine and muramic acid. Glucosamine is used as a biomarker for both fungal and bacterial necromass, while muramic acid is exclusively found in bacterial peptidoglycan. Neutral sugars can also be used as biomarkers, where pentose:hexose ratios are used to determine the contribution of plant biomass relative to microbial necromass to soil organic matter (SOM). Potentially uronic acids can also be used as plant versus microbial biomarkers. Acid hydrolysis breaks apart polymers such as peptidoglycan, chitin, and plant matter into monomers, which can later be quantified to estimate the contribution of bacterial, fungal and plant necromass to stabilized SOM. Due to their structural similarity and complexity, high-throughput identification and quantification of these compounds has remained a challenge. Derivatization using 1-phenyl-3-methyl-5-pyrazolone (PMP) has been used to characterize carbohydrates because of its simple and rapid reaction mechanism and enhanced ionization efficiency with ESI-MS. Our aim was to develop a highly sensitive method to quantify sugar-containing compounds in a single rapid assay using pre-column PMP derivatization. Separation and quantification of the PMP-derivatives was carried out using reversed-phase ultra-high performance liquid chromatography (RP-UHPLC) coupled to high resolution-high accuracy Orbitrap mass spectrometry (MS). Our method allowed the simultaneous separation and quantification of >20 compounds, including hexosamines, muramic acid, N-acetylhexosamines, hexuronic acids, pentoses, hexoses, deoxyhexoses, cellobiose, chitobiose, and chitotriose. All PMP derivatives were separated within 20 minutes. This method provides high resolution and high sensitivity for the quantification of diverse sugar-related compounds in one single assay, which demonstrates its potential in measuring complex and heterogeneous mixtures. Likewise, this method can also be applied in isotope tracing studies, where the turnover and stabilization of ¹⁵N and ¹³C labeled compounds in necromass are traced through the different soil pools using UPLC-Orbitrap mass spectrometry.