反相高效液相色谱法测定烤烟叶片发育过程中的类胡萝卜素类物质

Determination of Carotenoids in Flue-Cured Tobacco Leaves During its Growth by Reversed-Phase High Performance Liquid Chromatography

建立了采用反相高效液相色谱测定烤烟叶片中类胡萝卜素的方法。烤烟叶片先用含0．1％丁基羟基甲苯（BHT）的90％丙酮水溶液萃取，然后加入0．1g醋酸铅，于4℃下以10000r／min离心5min以去除蛋白质。色谱柱为C18反相柱（3．9mmi．d．×150mm，5μm）。流动相：A，甲醇-异丙醇（体积比为1：1）；B，超纯水。洗脱程序：0～10min，70％A＋30％B；10～17min，100％A；17～30min（90％A＋10％B）。流速：0．5mL／min。进样量：10μL。检测波长：450nm。该方法简化了样品的前处理过程，4种类胡萝卜素物质的加标回收率为91．77％～97．42％，相对标准偏差为3．46％～0．98％。用该方法研究了烤烟发育过程中类胡萝卜素含量的变化规律，获得了与文献较为一致的结果。

A reversed-phase high performance liquid chromatographic （RP-HPLC） method for the determination of carotenoids in flue-cured tobacco leaves was developed. Carotenoids were extracted from flue-cured tobacco leaves by acetone-water （90 ： 10, v/v） solution containing 0.1% butylated hydroxytoluene （BHT）. Plant proteins were eliminated by adding 0.1 g Pb（Ac）2 and by centrifugation （10000 r/min） for 5 min at 4℃. Lutein, β-carotene, neoxanthin, violaxanthin and other plant pigments were separated on a reversed-phase C18 column （3.9 mm i.d.×150 mm, 5μm）, with a mobile phase of （A） methanol-isopropyl alcohol （1 ： 1, v/v） and （B） water using a gradient elution at a flow rate of 0.5 mL/min. The optimum elution gradient was as follows： 0 - 10 min, 70% A ＋ 30% B; 10 - 17 min, 100% A; 17 - 30 min, 90% A ＋ 10% B. The recoveries of carotenoids in flue-cured tobacco leaves were 91.77% - 97.42%, and relative standard deviations were 3.45% - 0.98%. This method was applied to determine carotenoids in flue-cured tobacco leaves during its growth with satisfactory results.