Determination Of Twenty Free Amino Acids In Flue-cured Tobacco Leaves Using Ultra High Performance Liquid Chromatography-Single Quadruple Mass Spectrometry And Pre-column Derivatization

Free amino acids in flue-cured tobacco leaves were investigated using the ultra high performance liquid chromatography-single quadruple mass spectrometry detection (UPLC-SQD) and pre-column derivatization method. A total of138tobacco leaves were collected from14provinces in2011. Univariate and multivariate statistical analysis were used for data mining.An optimal method was developed. The sample weight was reduced to40mg and ultrasonic time to45min. The validation results showed that the method could meet the analytical requirements. The relative standard deviations (RSDs) of the repeatability for derivatized amino acids were less than10%e xcept Homoserine. The average RSDs of intra-day and inter-day precision are less than10%and13%, respectively. The method showed good linearities and a wide linear range (the linear range of Gly is0.008～12.5mg/L). The limit of detection (LOD) and quantification(LOQ) were1.9-17.8ug/L and6.4～59.4ug/L, respectively. The average recoveries of the method were96.84%～103.48%.The relative standard deviations (RSDs) of the contents of free amino acids in different growing regions ranged from28.50%～94.20%, and those of asparagines and glutamine were over80%. The RSDs of the contents of free amino acids in full aroma tobacco leaves were larger than those in fresh aroma and medium aroma tobacco leaves. The free amino acids of the same aroma type grown in different regions or different aroma types in the same province showed great variation. The contents of free amino acids of full aroma tobacco grown in Southeast region were much lower than those in Huanghuai region. The contents of free amino acids in Hunan province were much lower than the average contents.Principal Component Analysis (PCA) and non-parameters Mann-Whitney U test were used for analysis of tobacco free amino acids fromYunnan Gold Corridor Tobacco Cultivation Region, non-Gold Corridor Tobacco Cultivation Region and Fujian province. Ser%Asp%Tyr showed a difference between Yunnan Gold Corridor Tobacco Cultivation Region and non-Gold Corridor Tobacco Cultivation Region. The contents of free amino acid contained in tobacco of Yunnan province is three times of Fujian province.Besides, the influence of varieties and environment on amino acids content was studied. The results showed that the amino acids content was affected by both varieties and environment.