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.
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.
Zhang
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RE
Institute of Chemistry, Academia Sinica, Beijing, People's Republic of China.
Cao
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Hearn
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MW
D000085:Acetates / Q000737:chemistry; D019342:Acetic Acid; D002138:Calibration; D002241:Carbohydrates / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005449:Fluorenes / Q000138:chemical synthesis*; D005453:Fluorescence; D006023:Glycoproteins / Q000737:chemistry; D006834:Hydrazines / Q000138:chemical synthesis*; D006868:Hydrolysis; D007202:Indicators and Reagents / Q000138:chemical synthesis*; D008832:Microchemistry; D015394:Molecular Structure; D012680:Sensitivity and Specificity; D013696:Temperature
D000085:Acetates; D002241:Carbohydrates; D005449:Fluorenes; D006023:Glycoproteins; D006834:Hydrazines; D007202:Indicators and Reagents; C070658:9-fluorenylmethyl chloroformate hydrazine; D019342:Acetic Acid