DURHAM, NEW HAMPSHIRE

University of New Hampshire
 
 

An improved method for the high-performance liquid chromatographic separation of quercetin and isoquercitrin.

--Michael Dowe, Carol Macomber and Anita S. Klein

Published methods (Gerats et al., Biochem. Genet. 22:1161, 1984) for the assay of UDP-glucose 3-O-flavonol glucosyl transferase, the Bronze-1 gene product, require the separation of quercetin and isoquercetrin by high-performance liquid chromatography. While obtaining rapid turnaround times (5-7 min per sample), these procedures require high solvent flow rates and generate column pressures in excess of the manufacturer's specifications. These conditions result in decreased column life and relatively high expenses for solvent components.

A new chromatographic method has been developed which employs a shorter cartridge column and a two pump system. This method allows a turnaround time of 6.5 min per sample with low column pressure and relatively low solvent flow rates. This method is comparable in sensitivity and resolution to published separation methods. The lower limit of detection was 50 picomoles of isoquercetrin.

Sample Preparation: Samples were prepared according to the method of Gerats et al.

Column: Pecosphere 33mm C18 cartridge column and column holder were obtained from Perkin Elmer, Norwalk, CT.

Instrumentation: A Beckman (Fullerton, CA) Model 110A dual pump HPLC system was used with an Altex (Danbury, CT) Model 420 controller. Sample loop size was 100ul. Absorbance was monitored at 355.5nm with an Altex model 155 UV/vis variable wavelength detector. Peak areas were calculated with a Hewlett-Packard (Avondale, PA) Model 3390A integrator.

Chromatographic Conditions: The mobile phase consisted of either solvent A, water:methanol:acetic acid (55:35:10) or solvent B, water:acetic acid (4:1). Samples were eluted with solvent A at a flow rate of 0.5ml/min for 2.5 min. The column was then stripped with solvent B at a flow rate of 2ml/min for one minute. The column was then re-equilibrated with solvent A at a flow rate of 2ml/min for three minutes. Under these conditions, the retention times were 1.7 min. for isoquercetrin and 2.9 min for quercetin.

Chemicals: HPLC grade methanol and reagent A.C.S. grade acetic acid were obtained from Fisher, Pittsburgh, PA.


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