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Synthesis and application of Fmoc-hydrazine for the quantitative determination of saccharides by reversed-phase high-performance liquid chromatography in the low and subpicomole range

Analytical Biochemistry
Publication Date
DOI: 10.1016/0003-2697(91)90312-h
  • Biology


Abstract A new derivatization reagent, Fmoc-hydrazine, has been synthesized from the reaction of Fmoc-chloroformate with hydrazine as a precolumn fluorometric labeling reagent for reducing sugars such as glucose, galactose, mannose, fructose, fucose, ribose, xylose, arabinose, lactose, and maltose. The optimization of derivatization conditions was examined in detail. Using a reversed-phase high-performance C-8 column and a mobile phase consisting of acetonitrile-aqueous acetic acid, seven sugar derivatives were separated under either isocratic or gradient conditions within 20 min. The Fmoc-hydrazine and sugar Fmoc-hydrazone derivatives exhibit excellent stability. The extent of the hydrazone formation was 77 and 82% for mannose and fucose as assessed by Dionex high-performance anion-exchange chromatography with pulsed amperometric detection. Linear calibration graphs were established in the range from 0.5 to 2 pmol and 12 to 110 pmol for individual sugar derivatives. The determination limits were 0.05 – 0.09 pmol for mannose, galactose, and ribose; 0.1 pmol for maltose, xylose, and glucose; 0.2 pmol for fucose and lactose; 0.3 pmol for arabinose; and 0.4 pmol for fructose. The component monosaccharides of ultramicroquantities of two glycoproteins (e.g., from 7 ng fetuin and ovalbumin) were determined in the subpicomole range.

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