Study on the determination of polyphenols in tobacco by HPLC coupled with ESI-MS after solid-phase extraction.

A high-performance liquid chromatography method coupled with electrospray ionization-mass spectrometry for the determination of polyphenols in tobacco is studied. The polyphenols are extracted from a tobacco sample by being refluxed in a boiling water bath with 80% methanol and purified by solid-phase extraction with a C18 cartridge. The chlorogenic acid, rutin, scopoletin, caffeic acid, scopolin, and other polyphenols are satisfactorily separated on a Nova-Pak C18 chromatographic column (3.9 x 150 mm) with methanol and 0.05 mol/L potassium dihydrogen phosphate buffer solution gradient elution as mobile phase at a flow rate of 0.5 mL/min. Each of the polyphenols is monitored by photodiode array detector at its maximum wavelength: chlorogenic acid, 326.1 nm; rutin, 354.8 nm; scopoletin, 344.0 nm; caffeic acid, 323.7 nm; and scopolin, 365.2 nm. The limits of detection are: 100 ng/mL for chlorogenic acid, 125 ng/mL for rutin, 60 ng/mL for scopoletin, 50 ng/mL for caffeic acid, and 100 ng/mL for scopolin. The key polyphenols in tobacco are identified by comparing the retention time, the UV-spectrum, and the mass spectra with those of the standards. The recovery of tobacco polyphenols is 94-105%, and the relative standard deviations are 1.28-1.49%. This method is successfully applied to qualitatively and quantitatively analyze the polyphenols in tobacco with good results.