High performance liquid chromatography in the analysis of methylxanthines in the presence of tobacco alkaloids

The HPLC method for the separation and determination of alkaloides and thei r metabolites such as theophylline, theobromine, caffeine, nicotine, 7-meth ylxanthine, 1,7-dimethylxanthine, cotinine and 1,3-dimethyluric acid has be en developed. Aforementioned xanthines and tobacco alkaloides can be analys ed in the chromatographic system on octadecylsilane LiChrospher column, 250 x 4 mm (5 mu m), eluent: water-acetonitrile-methanol-acetic buffer (50+2+2 9+20 v/v). Acetic acid was used for adjusting pH. The best chromatographic separation of theobromine, theophylline, 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 cotin ine; caffeine and 1.7-dimethylxanthine in mixtures. Determination of the mi xture 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 presenc e of cotinine. These compounds can be determined by the use of DAD detector within the concentration range 1-100 mu g/ml.