Determination Of Various Tobacco Alkaloids By High Performance Liquid Chromatography And Its Application

Many chemical components in tobacco are important factors affecting the quality of tobacco leaves,among which alkaloids have a great influence on the key sensory qualities of tobacco,such as aroma temperament,aroma quantity,taste and irritation,and are extremely important chemical components.For the determination of alkaloid content is a key link in the process of identifying the tobacco leaf quality,in this paper,the calculation formula of ultraviolet spectrophotometry to detect the contents of nicotine is optimized,and to establish the HPLC method to determine the content of four kinds of tobacco alkaloids simultaneously,for different areas,different types and different blade location of the four kinds of alkaloid content in tobacco samples of testing and analysis.The experimental results are as follows:(1)To improve the traditional calculation formula of ultraviolet spectrophotometry measuring nicotine content in application scope and improve the calculation accuracy of flue-cured tobacco as raw material,the screening of a variety of extraction solvent,and fitting base on the results of HPLC,fitting a new formula of nicotine,with four different types of tobacco,flue-cured tobacco,cigars,dark sun-red tobacco and burley tobacco)compared with the original formula to calculate the results for the object.The results show that the linear range of nicotine absorbance by UV spectrophotometer is 0.047-2.088,which is wider than 0.167-0.671 when the original formula was proposed.Methanol aqueous solution was better than hydrochloric acid,sodium hydroxide and ammonia aqueous solution as extraction solvent.Taking flue-cured tobacco as the object,the ratio between the calculated results of the new formula and the test results of liquid chromatography was 0.75-1.25,accounting for 76.47%,which was significantly improved compared with the original formula of 55.88%.It was not as accurate as for cigars,dark sun-red tobacco and bur ley tobacco.(2)In order to quickly and accurately determine the content of alkaloids in tobacco leaves,the extraction conditions were optimized,and a HPLC method was established for the determination of four alkaloids in tobacco,including nicotine,anatabine,anabasine and nornicotine.The pretreatment was 100 mg/20 mL solid-liquid ratio,5%methanol was extracted at 25℃ for 30 min,and the chromatography was performed on A C18 column(250 mm×4.6 mm,5 μm)with mobile phase A(90%water+10%methanol,pH 6.8,0.15%sodium acetate)and mobile phase B(90%methanol+10%water,pH 3.8).The standard curves of the four alkaloids showed good linear relationship(R2>0.99)after elution by gradient.The detection limit of nicotine was 0.00065 μg·mL-1,the average recovery was 100.79%,RSD was 3.11%.The detection limit of anatabine was 0.0884 μg·mL-1,the average recovery was 98.53%,RSD was 3.75%.The detection limit of anabasine was 0.0348 μg·mL-1,and the average recovery was 98.70%with RSD of 3.63%.The detection limit of nornicotine was 0.1509 μg·mL1,the average recovery was 101.33%,RSD was 4.61%.The method is simple,rapid,sensitive and reproducible,and can meet the requirements of four alkaloids in tobacco samples.A total of 11 different samples of 4 different types of tobacco leaves were compared and tested according to industry standards to verify the reliability of the method.(3)In order to find out the difference of alkaloid content in different tobacco leaves and discuss the influence of different factors on alkaloid content in tobacco leaves,so as to provide theoretical basis for flue-cured tobacco production and processing of tobacco raw materials,four alkaloid contents were detected in tobacco samples from different regions,different types and different leaf locations.Nicotine content:Henan>Sichuan>Jiangxi>Yunnan,burley tobacco>dark sun-red tobacco>cigar tobacco>fluecured tobacco;Anatabine content Henan>Jiangxi>Yunnan>Sichuan,cigars>fluecured tobacco;Anabasine content:Henan>Yunnan>Sichuan>Jiangxi,dark sun-red tobacco>cigars>flue-cured tobacco>burley tobacco;Nornicotine content reduction Yunnan>Sichuan>Jiangxi>Henan,burley tobacco>cigars>dark sun-cured tobacco.The contents of four alkaloids in flue-cured tobacco and cigars were as follows:leaf tip>leaf base,leaf margi>main vein on both sides.