Revisiting monosaccharide analysis – quantitation of a comprehensive set of monosaccharides using dynamic multiple reaction monitoring

Abstract

A rapid method for the quantitation of sixteen neutral and acidic monosaccharides, from both animal and plant sources was developed using ultra-high performance liquid chromatography triple quadrupole mass spectrometry (UHPLC/QqQ-MS) in dynamic multiple reaction monitoring (dMRM) mode. Monosaccharides including three pentoses (ribose, xylose, arabinose), two deoxyhexoses (rhamnose, fucose), five hexoses (fructose, mannose, allose, glucose, galactose), two hexuronic acids (glucuronic acid, galacturonic acid), and two N-acetyl-hexosamines (GlcNAc, GalNAc), were derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP), while underivatized sialic acids, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), were simultaneously analyzed with a 10-minute run. With the optimized UHPLC conditions, baseline separations of the isomers were achieved. The sensitivity and calibration ranges of this method were determined. The limits of detection were between femtomoles and attomoles with linear ranges spanning four to six orders of magnitude and CVs ≤ 7.2%. Spiking experiments performed on a pooled fecal sample demonstrated the high accuracy of this method even when applied to samples with complicated matrices. The validated method was applied to fecal samples from an infant transitioning from breast milk to weaning foods. Major milk monosaccharides including galactose, fucose, glucose, GlcNAc, and Neu5Ac were found to be the most abundant components in the feces of milk-fed infants. PMP-derivatives of other monosaccharides including apiose, lyxose, altrose, talose, gulose, glucosamine, galactosamine, mannosamine, and N-acetylmannosamine (ManNAc) were also tested and could be added to the quantitation method depending on the need. The speed and sensitivity of the method makes it readily adaptable to rapid throughput analysis of monosaccharides in biological samples.

A nonenzymatic method for cleaving polysaccharides to yield oligosaccharides for structural analysis

“…However, it could not identify monosaccharide constituents, linkages, or the anomeric character of the linkage (alpha versus beta). The nearly 200 collected fractions were further analyzed by rapid throughput monosaccharide and linkage analysis to provide nearly complete structures of each oligosaccharide component 54 – 57 .

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“…Each fraction was analyzed for monosaccharide compositions using a recently described rapid throughput automated workflow (Fig. 8b ) 54 , 57 . The linkage information was similarly obtained using a separate recently reported workflow for comprehensive linkage analysis 55 , 56 .…”

Campylobacter Abundance in Breastfed Infants and Identification of a New Species in the Global Enterics Multicenter Study

“…Since C. jejuni lacks fucosidases, it grows only on fucose-modified gut oligosaccharides in the presence of other gut microbes, such as Bacteroides vulgatus, suggesting a scavenging lifestyle within the intestine (35). Many commensal bacteria in the gut, including Bifidobacterium and Bacteroides, possess fucosidases and are predominant in breastfed infants (18)(19)(20), suggesting that breastfeeding may benefit fuc ϩ C. jejuni strains, particularly since fecal levels of free L-fucose can reach milligram amounts in breastfed infants (22).…”

A seven-membered cell wall related transglycosylase gene family in Aspergillus niger is relevant for cell wall integrity in cell wall mutants with reduced α-glucan or galactomannan

“…The LC-system was coupled to an amaZon speed ESI-MSn detector (Bruker Daltonik, Bremen, Germany). PMP derivatized monosaccharides were separated based on a method previously described ( Xu et al, 2018 ), using the following 14 min gradient elution profile: 0–1 min isocratic 95% A (95% H 2 O, 5% acetonitrile, 25 mM ammonium acetate, pH 7.8); 1–9 min linear from 5% to 25% B (5% H 2 O, 95% acetonitrile); 9–9.5 min linear from 25% B to 99% B; 9.5–12 min isocratic 99% B; column re-equilibration: 12–12.5 min linear from 99% B to 95% A; 12.5–14 min isocratic 100% A. 2 µl of sample were injected into the system and samples were measured in UltraScan mode and negative ion mode (Auto MS 2 ) with the following parameters: capillary voltage 4000 V; plate offset voltage = 500 V; isolation width = 4 m z-1; nebulizer pressure = 1.03 bar.…”

“…Dry gas = 8 L min −1 ; dry temperature = 200 °C ICC target = 100000; max. accumulation time 50 ms; CID fragmentation; For each derivatized monosaccharide (monosaccharide-PMP 2 ), one of the most abundant fragment ions of the precursor ion [M−H] − was used for quantification as previously described ( Xu et al, 2018 ) (see Table 2 ). To minimize fragmentation of non-target molecules, we included only precursor masses in the following m / z ranges ( Fig.…”