Precolumn derivatization LC-MS/MS method to simultaneous quantitation of 10 monosaccharides in rat plasma

Monosaccharides are essential for maintaining the normal physiological functions of living organisms. Under disease states, metabolic disorders in vivo will inevitably affect the levels of monosaccharides, which brings the possibility of monosaccharides as a biomarker of some diseases. In this study, a method was developed and validated for simultaneously determining 10 monosaccharides (glucose, galactose, mannose, rhamnose, fucose, xylose, iduronic acid, glucuronic acid, N-acetylgalactosamine and N-acetylglucosamine) in SD rat plasma using liquid chromatography-tandem mass spectrometry. The method employed 1-phenyl-3-methyl-5-pyrazolone (PMP) as a derivatization reagent, considerably improved the chromatographic retention and ionization efficiency of monosaccharides. After protein precipitation of plasma samples, monosaccharides and isotope internal standards were derivatized and liquid-liquid extraction was performed to remove excess PMP. To achieve the baseline separation of several isomers, the resulting derivatives were chromatographed on a Bridged ethyl hybrid (BEH) Phenyl column using gradient elution with a total run time of 8 min. The method was linear within the range of 0.0100-5.00 & mu;g/mL for rhamnose, 0.0500-25.0 & mu;g/mL for fucose, xylose, iduronic acid, glucuronic acid, N-acetylgalactosamine and N-acetylglucosamine, 1.00-500 & mu;g/mL for galactose, 10.0-5000 & mu;g/mL for mannose, and 50.0-25,000 & mu;g/mL for glucose. And the accuracy and precision verification of surrogate matrix samples and plasma samples met the required criteria. The method has been used successfully to study the effect of hepatic insufficiency on monosaccharide levels in rats. It was found that the concentration of glucuronic acid in SD rat plasma was abnormally increased in rats with liver injury.

Automated summary

A method for simultaneous determination of 10 monosaccharides in rat plasma using LC-MS/MS was developed and validated. The method employed PMP as a derivatization reagent, improving the chromatographic retention and ionization efficiency of monosaccharides. The method was linear and met the accuracy and precision criteria. It was used to study the effect of hepatic insufficiency on monosaccharide levels in rats.