Analysis of Protein Amino Acids in Tobacco Using Microwave Digestion of Plant Material

This paper describes a technique using microwave digestion and gas chromatography-mass spectrometry (GC-MS), which makes possible the analysis of protein amino acids in tobacco. The technique involves first the measurement of free amino acids, a hydrolysis using microwave digestion, and a measurement of total resulting amino acids. The content of protein amino acids is determined from the difference of total and free amino acids. The digestion is performed with aqueous 6 N HCl (with 1% phenol) for two hours in a microwave at 120°C in sealed vials. The GC-MS analysis is performed after the amino acids are derivatized with N-methyl-N-(t-butyldimethylsilyl)trifluoroacetamide (MTBSTFA). The technique provides reliable results with less than 10% relative standard deviation (RSD) for most amino acids. Only the determination of very low level amino acids is affected by larger errors. The method provides results for free amino acids that are in very good agreement with those obtained by high performance liquid chromatography (HPLC), and also results for protein levels in tobacco in agreement with data previously reported in the literature. Results are given for several single grade tobaccos and for tobacco blends from four Kentucky reference cigarettes.