高效液相色谱同时检测不同类型烟叶中的多种生物碱含量

林雨晟 金洪石 张皓楠 金江华 郭伟 李玉娥 赵铭钦 刘鹏飞

摘 要：为快捷准确地测定烟叶中生物碱的含量，本研究通过优化提取条件，建立了测定烟草中烟碱、新烟草碱、假木贼碱和降烟碱的高效液相色谱检测方法。优化后的检测步骤为：干烟叶和5%甲醇的料液比为100 mg/20 mL，在25 ℃下振荡提取30 min，色谱条件：C18柱（250 mm×4.6 mm，5 μm），流动相A（90%水+10%甲醇，pH 6.8，0.15%醋酸钠），流动相B（90%甲醇+10%水，pH 3.8）。烟碱在0.1～50 μg/mL、其余3种在0.005～2 μg/mL的范围内，4种生物碱均具有较好的线性关系（R2>0.99）;烟碱检出限为0.000 65 μg/mL，平均回收率100.79%，RSD 3.11%;新烟草碱检出限0.088 4 μg/mL，平均回收率98.53%，RSD 3.75%;假木贼碱检出限0.034 8 μg/mL，平均回收率98.70%，RSD 3.63%;降烟碱检出限0.150 9 μg/mL，平均回收率101.33%，RSD 4.61%。對4种不同类型烟叶共11个不同样品进行检测验证，检测结果可靠。该方法前处理简单快捷，灵敏度高，重复性较好，可以满足烟草样品中4种生物碱的检测要求。

关键词：烟草;烟碱;新烟草碱;假木贼碱;降烟碱;液相色谱法

Abstract： In order to quickly and accurately determine the content of alkaloids in tobacco leaves， the extraction conditions were optimized， and a method for the determination of nicotine， anatabine， anabasine and nornicotine contents in tobacco by HPLC was established. The optimized detection steps are as follows： 100 mg/20 mL solid-liquid ratio， 5% methanol was extracted by oscillation at 25 ℃ for 30 min， the chromatographic conditions were C18 column （250 mm×4.6 mm， 5 μm）， mobile phase A （90% water +10% methanol， pH 6.8， 0.15% sodium acetate）， mobile phase B （90% methanol +10% water， pH 3.8）. Nicotine was in the range of 0.1-50 μg/mL and the other three alkaloids were in the range of 0.005-2 μg/mL， with the standard curves of the four alkaloids all havig good linear relationship （R2 > 0.99）. The limits of detection （LOD） of nicotine were 0.000 65 μg/mL， and the average recoveries were 100.79%， RSD 3.11%; the LOD of anatabine were 0.088 4 μg/mL， and the average recoveries were 98.53%， RSD 3.75%; the LOD of anabasine were 0.034 8 μg/mL， and the average recoveries were 98.70%， RSD3.63%; the LOD of nornicotine were 0.150 9 μg/mL， and the average recoveries were 101.33%， RSD 4.61%. A total of 11 different samples of 4 different types of tobacco leaves were tested， and the results were reliable. The method is simple and fast， sensitive and repeatable， and can meet the requirements of detecting four alkaloids in tobacco samples.

Keywords： tobacco; nicotine; anatabine; anabasine; nornicotine; HPLC

生物碱是一类存在于植物体中的特殊的含氮有机物，是植物体随外界环境变化合成的次生代谢产物，一般具有特殊的生理活性，如抗炎、镇痛和成瘾性[1]。烟草中含有超过50种生物碱，含量较高的有烟碱（尼古丁）、新烟草碱、假木贼碱、降烟碱（去甲基烟碱）等[2]。其中烟碱含量最高，一般占总生物碱组分的90%以上，其他各种生物碱所占的比例一般不超过10%[3]。不同类型烟叶的生物碱含量差异主要是烟碱和降烟碱的含量差异，而假木贼碱和新烟草碱在各类型烟叶中的含量均较低，且较少出现转移或降解[4]。

生物碱的组成和含量是影响烟叶质量的重要因素，直接影响着烟草制品的劲头、刺激性、吃味和安全性[5]。烟碱……