Quantitative analysis of per-O-acetylated carbohydrates
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Free neutral oligosaccharides, and sometimes mono- and disaccharides, are traditionally separated either using ion chromatographic (IC) or HILIC methods following detection by pulsed amperometric or refractive index detectors. In LC-MS analysis neutral carbohydrates can be detected using electrospray (ESI) or atmospheric pressure chemical ionization techniques (APCI). Mono and disaccharides were detected in negative APCI mode as [M+Hal]- adducts (Hal= Cl, I) after post-column addition of chloroform or methyl iodide and in positive ESI mode as cation adducts [M+Me]+ (Me=Li+, Na+, Cs+).
The disadvantages of LC-MS analysis of free neutral carbohydrates are low efficiency of atmospheric pressure ionization of neutral carbohydrates, post-column addition of immiscible solvents, or non-volatile inorganic salts.
In this study, mono-, di- and oligosaccharides were converted into per-acetylated derivatives following reversed-phase LC-HRMS analysis. Peracetylated derivatives were detected as ammonium adducts in positive ESI mode.
Instrumental: Orbitrap Discovery hybrid FT mass spectrometer coupled with Accela UPLC (Thermo Scientific); positive ESI; m/z 150-1500. Chromatographic separations were carried out using the Gemini Hexyl-Phenyl column (2×150 mm, 3µ particle size, Phenomenex) using a linear gradient of ACN/water (with 10 mM AmAc).
Carbohydrates: glucose, glucose-13C6 (as IS), mannose, galactose, rhamnose, ribose, fructose, myoinositol, sucrose, lactose, trehalose, maltose, sorbitol, mannitol, galactinol, raffinose, stachyose.
Peracetylated carbohydrates were prepared with acetic anhydride using iodine as a catalyst.
Positive ESI mass spectrum of per-acetylated sorbitol:
a) (above) using ACN/water (with 0.1% acetic acid) and,
b) (below) ACN/water (with 10 mM ammonium acetate).
LC-MS EIC chromatograms of per-O-acetylated mixture of carbohydrates (fructose, glucose, myoinositol, rhamnose, sorbitol, mannitol, ribose, galactinol, raffinose, stachyose)
Demonstration of sensitivity of the method: extracted ion accurate mass chromatograms of per-acetylated mixture of carbohydrates (amount of each compound injected on-column was 0.5 pg).
Calibration curves of glucose, fructose, stachyose, and raffinose obtained using glucose-13C6 as an internal standard.
Structures of carbohydrates detected in plants in this study.
Results and conclusions
The method was successfully applied for the analysis of carbohydrates in plant samples.
This work was published in the Journal of Plant Physiology, "Effect of CMV infection and high temperatures on the enzymes involved in raffinose family oligosaccharide biosynthesis in melon plants", 2012, 169(10), 965-70.
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