Abstract:

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×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 ℃. 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 0.9910 to 0.9998. This method is easy to perform and can be applied to determine secondary metabolites in tobacco.

Key words: coulometric array detection, secondary metabolites , tobacco, reversed-phase high performance liquid chromatography(RP-HPLC)