Determination of Chlorogenic Acid and Its Isomers in Tobacco Leaves by High Performance Liquid Chromatography

A portion of dried and powdered tobacco leaf sample (0.100 0 g) was extracted ultrasonically with 20 mL of CH₃OH-H₂O (1+1) mixture for 20 min. The extract obtained was filtered through 0.45 μm filtering membrane and the filtrate was used for HPLC analysis. Diamonsil Plus C₁₈ column was used as stationary phase and mixtures of (A) methanol and (B) CH₃COOH (1+99) solution in various proportions were used as mobile phase in the programmed gradient elution, to have chlorogenic acid and its isomers (neochlorogenic acid and crypto-chlorogenic acid) clearly separated. It was shown that linearity range for chlorogenic acid was found between 1.01-101.22 mg·L^-1, while those for its isomers (i.e., neochlorogenic acid and cryptochlorogenic acid) were found same between 0.25-25.00 mg·L^-1, and that values of detection limits (3S/N) for the 3 compounds were 0.013, 0.025, 0.028 mg·L^-1 respectively. Test for recovery was made by standard addition method, giving values of recovery in the range from 92.0% to 104%, and RSDs (n=5) in the range from 1.1% to 4.0%.