[Determination of six secondary metabolites including chlorogenic acid in tobacco using high performance liquid...

A procedure utilizing reversed-phase high performance liquid chromatography coupled with a coulometric array detection system was developed for the characterization of several major secondary metabolites in tobacco. Chromatographic separation was achieved on a Hypersil BDS C-18 column (4.6 mm x 200 mm), with linear gradient elution of 5% -70% (v/v) of acetonitrile in a buffer containing 30 mmol/L NaH2PO4, 0.25 mmol/L dodecyl sulfate sodium salt (pH 3.5) at a flow rate of 1 mL/min and column temperature of 30 degrees C. Ten serial coulometric detectors were set on -20, 140, 210, 310, 400, 450, 490, 730, 800 and 900 mV. The method was reliable and sensitive. The relative standard deviations ranged from 0.71% to 15.31%. The recoveries were between 52.0% and 85.2%. The detection limits were 0.2 - 2 ng, and there were good linear correlations when injection amount was in a range from 20 ng to 500 ng, with the regression coefficient of standard calibration curves from 0.991 0 to 0.999 8. This method is easy to perform and can be applied to determine secondary metabolites in tobacco.