What is the background and overview of the dangers of nornicotine
Tobacco is a member of the genus Nicotiana, in the order Tubiflorae, the order Dicotyledoneac, the family Solanaceae, and is mostly an annual herb. It is an important economic crop in my country. The height of the tobacco main stem ranges from a dozen to several hundred centimeters. The single leaves are alternate, with or without petioles, and the leaf shape varies greatly. Cyme-shaped inflorescences, flower colors include yellow, white, purple, red, etc. Tubular or bell-shaped calyx, tube-cylindrical corolla. Five stamens are attached to the base of the corolla, and the ovary has three or four rooms. Each capsule contains approximately 2,000 seeds. Stems, leaves and calyxes all have glandular hairs that secrete mucus. The most important thing is that most Nicotiana plants can produce a unique alkaloid with a content of 0.5%-10%. The composition and content of alkaloids in tobacco are one of the important indicators for measuring the quality of tobacco leaves. The biosynthesis of nicotine and the regulatory mechanism of the conversion between nicotine and nornicotine have always been hot topics in tobacco research. The research will use genetic engineering technology to further elucidate the molecular mechanism of nicotinic metabolism regulation, providing a theoretical basis for tobacco “reducing scorch and harm reduction”.
What are the dangers of nornicotine
Nicotine is the main alkaloid in tobacco (Nicotiana tabacum L.), accounting for 90%-95% of the total alkaloid content. The nornicotine content is usually less than 5% of the alkaloids. However, some burley and flue-cured tobacco varieties convert nicotine into nornicotine during ripening and processing, making nornicotine the most abundant alkaloid. High temperature conditions reduce nicotine to produce myosmine and substituted pyridine compounds, affecting the original flavor of tobacco. In addition, it is harmful to human health. It is the synthetic precursor of the potential carcinogen nitrosonornicotine (NNN). Nornicotine itself can also directly induce abnormal glycosylation of proteins in the plasma of smokers and is closely related to steroid drugs. A covalent anti-potassium bicarbonate reaction occurs, affecting efficacy and toxicity.
What are the dangers of nornicotine and how to measure its content
Nornicotine is one of the main alkaloids in cigarette cut tobacco, which has an important impact on cigarette quality. It is also the precursor of tobacco’s unique nitrosamine – nitrosonornicotine (NNN). The nornicotine molecule contains a chiral center, so nornicotine has two enantiomers: S-(-)-nornicotine (referred to as S-nornicotine) and R-(+)-nornicotine (referred to as R-nornicotine). Depending on the enantiomeric properties, the two enantiomers of nornicotine may have completely different metabolic mechanisms and physiological properties. Therefore, it is necessary to analyze and determine the enantiomeric content of nornicotine in cigarette cut tobacco. At present, the literature reports that the methods for determination of nornicotine enantiomers mainly include gas chromatography-mass spectrometry, multidimensional gas chromatography-mass spectrometry, capillary electrophoresis, etc. However, GC-MS and MDGC-MS require pre-processing. Derivatization is performed during the process, which is time-consuming and labor-intensive. CE only uses retention time for characterization, which has relatively poor qualitative ability and is prone to false positive results. Therefore, it is necessary to develop a method with high sensitivity, good precision, and suitable for accurately quantifying S-nornicotine and R-nornicotine in cigarette cut tobacco in large quantities. Based on the above technical deficiencies, some studies have established a method for determining the nornicotine enantiomeric content in cut tobacco using convergence chromatography-tandem mass spectrometry. This method does not require a derivatization process during the pretreatment process, achieves baseline separation of S-nornicotine and R-nornicotine, and uses secondary mass spectrometry for qualitative and quantitative purposes to avoid false positive results. Therefore, this method It has the advantages of easy operation, high sensitivity and accurate determination. This is achieved through the following technical solution: cigarette cut tobacco samples are dried, crushed and sieved. After the samples are soaked with sodium hydroxide solution, they are extracted with methylene chloride. After the extract is purified by matrix dispersion solid phase extraction, a series chiral column is used. – Convergence chromatography-tandem mass spectrometry analysis, using peak area normalization method to quantitatively detect the ratio of S-nornicotine and R-nornicotine in cigarette cut tobacco. The specific steps are as follows:
1) Preparation of standard solution: Weigh about 100.0 mg of S-(-)-nornicotine standard, dilute it with methanol solution to a 50 mL brown volumetric flask, and prepare it to a concentration of about 2.0 mg/ mL of S-(-)-nornicotine standard stock solution; weigh approximately 100.0 mg of R-(+)-nornicotine standard, dilute it with methanol solution to a 50 mL brown volumetric flask, and prepare to a concentration R-(+)-nornicotine standard stock solution of approximately 2.0 mg/mL; dilute S-(-)-nornicotine standard stock solution and R-(+)-nornicotine standard stock solution to prepare S- Mixed standard solutions with (-)-nornicotine and R-(+)-nornicotine concentrations of 100 μg/mL and 100 μg/mL respectively
2) Mixing of cigarette cut tobacco samples: Place the cigarette cut tobacco samples in an oven at 40°C. After 1 hour, take out the cigarette cut tobacco samples, crush them and pass them through a standard sieve with an aperture of 0.45 mm.
3) Extraction of cigarette cut tobacco samples: Five solvents, n-hexane, methyl tert-butyl ether, ethyl acetate, dichloromethane, and chloroform, were selected to compare the extraction efficiency. The results showed that methylene chloride had the highest extraction efficiency for nornicotine, followed by methyl tert-butyl ether and ethyl acetate, and n-hexane and chloroform had the worst extraction efficiency. Therefore, methylene chloride was selected as the final extraction method. Oxypyridine as solvent. The optimized extraction step is: weigh 0.3 g of the crushed sample into a 15 mL stoppered centrifuge tube, add 2.0 mL of 5% sodium hydroxide solution (mass ratio), vortex for 1 minute to mix, and then let stand for 10 minutes to infiltrate. Sample, take 10 mL dichloromethane and centrifugetube and shake on a vortex mixer at 0 rpm for 30 min.
4) Purification of the extract: Take 1.5 mL of supernatant in a 2 mL purification centrifuge tube (containing 150 mg anhydrous magnesium sulfate, 50 mg PSA adsorbent, 25 mg GCB adsorbent, collectively referred to as matrix dispersion solid phase extraction material), shake on a vortex mixer at 0 rpm for 2 min, and centrifuge at 10000 rpm for 1 min. Pass the supernatant through a 0.45 μm organic phase filter membrane and then proceed to liquid phase analysis.
5) Tandem chiral column-convergence chromatography-tandem mass spectrometry conditions: Analytical column 1 is a Trefoil CEL1 column (column length 150 mm, inner diameter 3.0 mm, stationary phase particle size 2.5 µm), analytical column 2 is Chiralcel OD -H liquid chromatography column (column length 250 mm, inner diameter 4.6 mm, stationary phase particle size 5 µm); mobile phase A is: CO2, mobile phase B: methanol containing 0.05% isopropylamine; flow rate is 1.0 mL/min; Gradient elution conditions were: 0.0 min, 97% A; 0.5 min, 97% A; 8.0 min, 92% A; 12.0 min, 92% A; 30.0 min, 90% A. The column temperature is 40 ℃; the injection chamber temperature is 10 ℃; the injection volume is 2 μL; the back pressure is 1600 psi; the mobile phase of the ISM compensation flow path is methanol containing 0.1% formic acid, and the flow rate is 0.3 mL/min. The total analysis time is 30 minutes. Mass spectrometry conditions: Ion source: electrospray source, scanning mode is positive ion scan, ion source temperature is 300 ℃, electrospray voltage is 5000 V, atomization gas pressure is 40 psi; detection method: multiple reaction monitoring (MRM);. The proportion of S-nornicotine and R-nornicotine to total nornicotine was quantified by normalizing the peak area.
6) Detection limit and quantification limit: Under optimized conditions, the concentration corresponding to the signal-to-noise ratio of the target substance is 3 as the detection limit, and the concentration corresponding to the signal-to-noise ratio of the target substance as 10 is used as the detection limit. . The detection limit and quantification limit of S-nornicotine are 0.12 μg/g and 0.40 μg/g respectively, and the detection limit and quantification limit of R-nornicotine are 0.12 μg/g and 0.40 μg/g respectively.
7) Precision: Take the same cigarette cut tobacco sample and conduct 5 intra-day and inter-day parallel measurements to examine the precision of the above method. The results show that the intra-day and inter-day measurement results of S-nornicotine in the cigarette cut tobacco sample The coefficients of variation are 1.08% and 2.45% respectively, with good precision.
