反相高效液相色谱法测定烟草中的类胡萝卜素及其异构体

采用反相高效液相色谱−二极管阵列检测器（RP−HPLC−DAD）分离和测定烟草中类胡萝卜素及其异构体的组成和含量。烟草样品经过含有0.1%丁基羟基甲苯（BHT）的冰丙酮溶液萃取，浓缩后，经Zorbax SB C18色谱柱分离。流动相组成:A，乙腈−水（体积比为88:12）；B，乙酸乙酯。梯度洗脱程序:0 − 25min，100%A；25 − 50 min，B由0%线性增加为60%；50 − 55 min， 40%A+ 60% B；55 − 60 min，A由40%线性增加为100%。 检测波长:450 nm。进样量:10 µL。流速:1.0 mL/min。该方法简化了样品的前处理，共分离出烟草中11种类胡萝卜素及其异构体。类胡萝卜素物质的加标回收率为87.7−94.6%，；相对标准偏差为3.01−4.29%。同时研究了新鲜烟叶和烘烤后烟叶中类胡萝卜素的分布和含量，结果显示:烟叶中类胡萝卜素的组成及含量与烟叶品种、部位以及调制有关。 

Determination of carotenoids and isomers in tobacco leaves by RP-HPLC

A reversed−phase high performance liquid chromatographic (RP−HPLC) with the photodiode array detection (DAD) was developed for the determination of compositions and contents of carotenoids and their isomers in tobacco leaves. Carotenoids were extracted from tobacco leaves by cold acetone solution containing 0.1% butylated hydroxytoluene (BHT), and then it was concentrated and separated on a Zorbax SB C18 column. A mobile phase of acetonitrile-H2O (88:12, v/v) (A) and ethyl acetate (100%) (B) with the following gradient elution was used: 100% A in the beginning, maintained for 25 min, decreased to 40% A in 50 min, maintained for 5 min and returned to 100% A in 60 min. A total of 11 carotenoids and some isomers in tobacco leaves were resolved with the flow-rate at 1.0 mL/min，injection at 10 µL and detection at 445 nm. The recoveries of carotenoids in tobacco leaves were 87.7−94.6%, and relative standard deviations were 3.01−4.29%. This method was successfully applied for the quantification of carotenoids in green, fresh flue-cured tobacco leaves. The results show that it presented many differences not only in carotenoids contents but also in carotenoids profiles in various tobacco leaves.