Quantification of monosaccharides through multiple‐reaction monitoring liquid chromatography/mass spectrometry using an aminopropyl column

Abstract

A simple, sensitive, and reproducible quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was designed for the simultaneous quantification of monosaccharides derived from glycoprotein and blood serum using a multiple-reaction monitoring (MRM) approach. Sialic acids and neutral monosaccharides were efficiently separated using an amino-bonded silica phase column. Neutral monosaccharide molecules were detected as their aldol acetate anion adducts [M + CH(3)CO(2)](-) using electrospray ionization in negative ion MRM mode, while sialic acids were detected as deprotonated ions [M-H](-). The new method did not require a reduction step, and exhibited very high sensitivity to carbohydrates with limits of detection of 1 pg for the sugars studied. The linearity of the described approach spanned over three orders of magnitude (pg to ng). The method was validated for monosaccharides originating from N-linked glycans attached to glycoproteins and glycoproteins found in human blood serum. The method effectively quantified monosaccharides originating from as little as 1 microg of glycoprotein and 5 microL of blood serum. The method was robust, reproducible, and highly sensitive. It did not require reduction, derivatization or postcolumn addition of reagents.

Antibiotic-induced shifts in the mouse gut microbiome and metabolome increase susceptibility to Clostridium difficile infection

“…Extraction of carbohydrates (mannitol and sorbitol) was done using a combination of a modified Bligh-Dyer procedure to remove lipids followed by protein precipitation by adding methanol to aqueous phases 44 . LC-MS instruments used in this work included the Agilent 1200 HPLC, a Quadrupole Time of Flight (Q-TOF) MS/MS (6530) and a triple Quadrupole (QqQ) MS/MS (6450) system.…”

Separation of monosaccharides hydrolyzed from glycoproteins without the need for derivatization

“…In all other cases, pre-spiked samples were dried in a speed vac (ThermoSavant SPD1010) and subjected separately to each of three unique acid hydrolyses. Hydrolysis was performed in a similar manner to previously reported studies by Mechref's group [16,17]. In all cases, gas-phase hydrolysis was achieved within a capped glass vial housing the glass autosampler insert.…”

“…Although there are previous literature reports that describe acid hydrolysis of glycoproteins into their constituent monosaccharide units with subsequent analysis using LC-MS [12][13][14][15][16][17] or capillary electrophoresis [18], each of these studies requires derivitization of monosaccharides for detection, likely incorporating measurement bias in proportion to the labeling inefficiency. The current industry standard for analysis of non-derivatized, hydrolyzed sugars -anion exchange chromatography with pulsed amperometric detection [19] (HPAEC-PAD) -is capable of chromatographic resolution of monosaccharides and can be a very sensitive detection technique, but is inherently incompatible with direct mass spectrometry detection, and therefore incompatible with isotope dilution techniques.…”

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

“…30,31 For monosaccharides, Hammad et al used an aminopropyl column to separate the acetate adducts of label-free reducing monosaccharides with subsequent detection using MRM. 32,33 Due to the high similarity of monosaccharide structures, isomers such as glucose and galactose were not well separated. Recently, Han et al 34 developed a MRM-based method to quantify neutral mono- and disaccharides derivatized by 3-nitrophenylhydrazine employing 13 C-labeled internal standards in negative ion mode.…”