Volume 45, Number 1 (January - February 2000)

Key words:  HPLC, methylxanthines, tobacco alkaloids

The HPLC method for the separation and determination of alkaloides and their metabolites such as theophylline, theobromine, caffeine, nicotine, 7-methylxanthine, 1,7-dimethylxanthine, cotinine and 1,3-dimethyluric acid has been developed. A forementioned xanthines and tobbaco alkaloides can be analysed in the chromatographic system on octadecylsilane LiChrospher column, 250 x 4 mm  (5 mm), eluent: water-acetonitrile-mathanol-acetic buffer (50+2+29+20 v/v). Acetic acid was used for adjusting pH.The best chromatographic separation of theobromine, theophyline, cotinine and caffeine was obtained at pH = 4.3, while at pH = 4.8 it is possible to determine metabolites in the presence of alkaloides - theobromine and 7-methylxanthine; nicotine and cotinine; caffeine and 1,7-dimethylxanthine in mixtures. Determination of the mixture of metabolites such as: 7-methylxanthine, 1,7-dimethylxanthine and 1,3-dimethyluric acid can be carried out at pH = 7 and at pH = 5.6 in the presence of cotinine. These compounds can be determined by use of DAD detector within the concentration range 1-100 mg ml^-1.