CN113447594A - Method for measuring free nicotine in tobacco - Google Patents
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- CN113447594A CN113447594A CN202110825042.9A CN202110825042A CN113447594A CN 113447594 A CN113447594 A CN 113447594A CN 202110825042 A CN202110825042 A CN 202110825042A CN 113447594 A CN113447594 A CN 113447594A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/62—Detectors specially adapted therefor
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- G01N30/7206—Mass spectrometers interfaced to gas chromatograph
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/04—Preparation or injection of sample to be analysed
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- G01N2030/062—Preparation extracting sample from raw material
Abstract
The invention discloses a method for measuring free nicotine in tobacco, which comprises the following steps: (1) drying tobacco leaves or tobacco shred samples at the temperature of less than or equal to 40 ℃, crushing, and sieving by a standard sieve of 60 meshes; (2) accurately weighing 0.10g of sieved sample powder, adding the sample powder into a 100mL SCHOTT sample bottle, accurately adding 50.0mL of a cyclohexane solution containing anethole, wherein the cyclohexane solution contains 10.0 mu g/mL of anethole, and standing at the temperature of 22 +/-2 ℃ for 24 +/-2 hours to obtain a standing solution to be detected; (3) the standing solution to be tested was filtered through a 0.2 μm nylon 66 syringe filter and analyzed by GC-MS. The detection limit of the analysis method is 5.3 mu g/g, the relative standard deviation is 1.7-2.7% (n is 5), the linear correlation coefficient of the standard solution is 0.9997, and the standard recovery rate is 99.0-106.7%. The determination method is simple to operate and accurate in result, and avoids the transformation influence of the aqueous solution on the original nicotine form distribution in the tobacco.
Description
Technical Field
The invention relates to the accurate determination of free nicotine content in tobacco, in particular to a method for determining free nicotine in tobacco.
Background
Nicotine is the most important characteristic component of tobacco and has a very important effect on the sensory quality of cigarettes. From the view of molecular structure, nicotine is a weak secondary base composed of a pyridine ring and a hydrogenated pyrrole ring, and when the nicotine is contacted with an acidic component, at most two protons can be captured to generate salt, so that the nicotine can exist in 3 forms of a free state, a single proton state and a double proton state. In tobacco, nicotine is usually present in a salt form combined with an inorganic acid or an organic acid, and a small amount is present in the form of free nicotine. Free nicotine has strong lipophilicity, is easy to penetrate through the oral mucosa of a human body and be absorbed, and has strong physiological effect. The protonic nicotine has strong hydrophilicity and is slowly absorbed and metabolized in human body. The presence of nicotine also affects its rate of transfer into the smoke. Compared with the combined nicotine, the free nicotine has strong volatility and high transfer rate to smoke, and has more important influence on the sensory quality of the cigarettes, such as strength, irritation and the like. Therefore, the method for accurately measuring the content of nicotine in different forms in the tobacco, particularly the content of free nicotine, has very important significance for the aspects of evaluation of the quality of tobacco leaves and cigarettes, tar and harm reduction research, formulation work of the cigarettes and the like.
At present, pH value calculation algorithm and solvent extraction method are mainly adopted for measuring the free nicotine in the tobacco.
The pH value calculation algorithm is to measure the total amount of nicotine in different forms in tobacco (smoke particulate matters collected by tobacco leaves, cigarette tobacco or Cambridge filter sheets), prepare a solution with a certain concentration, measure the pH value of the solution and calculate the content of free nicotine by a classical acid-base balance theory. The disadvantages of this method are: the sample to be tested is generally a solid or solid-like heterogeneous system, after the sample is prepared into a solution, because the sample to be tested contains a plurality of acidic or alkaline components, the components jointly form the pH value of the prepared solution, and at the moment, the calculated content of the free nicotine in the solution at the pH value and the nicotine state distribution in the actual sample have a larger difference.
Solvent extraction methods are most widely used and there are two main ways: one method is to extract free nicotine in a sample to be tested with water (deionized water, distilled water, neutral water with a pH value of 7.00), and then extract the free nicotine from the water with a proper organic solvent for determination. When the free nicotine in the sample is extracted by adopting the mode, the free nicotine is dissolved in water and then is converted into concentration distribution under the current pH value no matter what the original form of the nicotine; resulting in incorrect measurement results and low recovery due to the conversion of nicotine (part of free nicotine is converted into proton) when the actual sample detection and recovery experiment is carried out. Another method comprises extracting with organic solvent, purifying with water (deionized water, distilled water, and neutral water with pH of 7.00), and measuring. This also leads to a change in the original morphological distribution of nicotine in the sample, making the test result incorrect.
Disclosure of Invention
The invention discloses a gas chromatography-mass spectrometry (GC-MS) analysis method for determining free nicotine in tobacco, which adopts cyclohexane to extract free nicotine (without purification), and carries out GC-MS analysis after standing for 24 h. Compared with a pretreatment mode of firstly extracting with water (deionized water, distilled water or neutral water with the pH value of 7.00) and then extracting the free nicotine from the water extract by using an organic solvent, or firstly extracting with the organic solvent and then purifying the organic extract by using water and then measuring, the method has the advantages of simple operation, avoiding the transformation influence of the aqueous solution on the original nicotine form distribution in the tobacco, and avoiding the interference of the existence of the combined nicotine such as the malate, the citrate, the nicotine and the like on the measurement of the free nicotine.
The invention adopts the following technical scheme:
a method for measuring free nicotine in tobacco comprises the following steps:
(1) drying tobacco leaves or tobacco shred samples at the temperature of less than or equal to 40 ℃, crushing, and sieving by a standard sieve of 60 meshes;
(2) accurately weighing 0.10g of sieved sample powder, adding the sample powder into a 100mL SCHOTT sample bottle (German Schottky sample bottle), accurately adding 50.0mL of a cyclohexane solution containing anethole, wherein the cyclohexane solution contains 10.0 mu g/mL of anethole, and standing at 22 +/-2 ℃ for 24 +/-2 hours to obtain a standing solution to be detected;
(3) the standing solution to be tested was filtered through a 0.2 μm nylon 66 syringe filter and analyzed by GC-MS.
The GC-MS analysis was carried out using HP-5 MS UI chromatography column from Agilent.
The column was a 30m 0.250mm 0.25 μm HP-5 MS UI column and the stationary phase was 5% phenyl containing methylpolysiloxane.
The conditions for the GC-MS analysis were as follows: column oven: 45 ℃; sample introduction amount: 1 mu L of the solution; sample inlet temperature: 250 ℃; the split ratio is as follows: 20: 1; carrier gas: he. Flow rate: 1.0 mL/min; purging the spacer: 3 mL/min; temperature rising procedure: the initial temperature is 45 ℃, the temperature is kept for 1min, then the temperature is raised to 280 ℃ at the speed of 20 ℃/min, and the temperature is kept for 2.25 min; solvent retardation: 7.0 min; electron energy: 70 eV; transmission line: 280 ℃; an ion source: 230 ℃; a quadrupole rod: 150 ℃; detecting by adopting a Selected Ion Monitoring mode (SIM), and quantifying by adopting an internal standard method; the mass-to-charge ratio of the quantitative ion of nicotine is 84, and the mass-to-charge ratio of the qualitative ion: 133. 161, 162; the quantitative ion of the anethole as an internal standard is 148, and the qualitative ion: 77. 117, 147.
The preparation method of the standard solution adopted by the internal standard method comprises the following steps:
weighing 0.100g of anethole, adding the anethole into a 50mL volumetric flask, metering the volume with cyclohexane, and shaking up to prepare an internal standard solution with the concentration of 2.00 mg/mL; 30, 60, 90, 120 and 150 mu L of nicotine standard solution with the concentration of 9.6mg/mL are accurately measured, added into a 50mL volumetric flask, and 250 mu L of the internal standard solution is respectively added to prepare the nicotine standard solution with the concentrations of 5.76, 11.52, 17.28, 23.04 and 28.80 mu g/mL.
Compared with the prior art, the invention has at least the following beneficial effects:
the invention provides a GC-MS analysis method for determining free nicotine in tobacco. The method adopts cyclohexane to extract free nicotine in tobacco, and GC-MS analysis is carried out after the standing for 24 hours. Compared with a pretreatment mode of firstly extracting with water (deionized water, distilled water or neutral water with the pH value of 7.00) and then extracting the free nicotine from the water extract by using an organic solvent, or firstly extracting with the organic solvent and then extracting and purifying the organic extract by using water and then measuring, the method has simple operation and avoids the transformation influence of the aqueous solution on the original nicotine form distribution in the tobacco. By verification, when the method is used for detection, the detection of the free nicotine by the bound nicotine such as malic acid nicotine salt, citric acid nicotine salt and the like in the tobacco is not interfered. The standard recovery rate of the experimental method is 99.0-106.7%, the detection limit is 5.3 mu g/g, the relative standard deviation is 1.7-2.7% (n is 5), the linear correlation coefficient of the standard solution is 0.9997, and the linearity is good. Among the 52 tobacco samples tested, the relative deviation of the parallel samples is between 0.1% and 3.2%, and the parallelism of the experimental method is also better.
Drawings
FIG. 1 is a GC-MS analysis chromatogram of a standard solution; in the figure, reference numeral 1 represents anethole, and reference numeral 2 represents nicotine.
FIG. 2 is a GC-MS analysis chromatogram of a sample solution; in the figure, reference numeral 1 represents anethole, and reference numeral 2 represents nicotine.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Materials, reagents and apparatus used in the examples
Materials: flue-cured tobacco and cigar tobacco leaves used by the tobacco industry Limited liability company in Sichuan and commercial flue-cured tobacco and cigar in 2020 and 2021 years.
Reagent: 9.6mg/mL nicotine standard solution (solvent: isopropanol) and 100. mu.g/mL nicotine standard solution (solvent: water) were prepared using 99.9% nicotine (CAS #:54-11-5) (Tianjin Altar technologies, Inc.); nicotine (CAS #:54-11-5, 99.9%, national tobacco quality supervision and inspection center); anethole (CAS #:4180-23-8, 98.5%, Beijing Bailingwei science and technology Co., Ltd.); cyclohexane (CAS #:110-82-7, HPLC grade, Saimer Feishell science Co., Ltd.); anhydrous sodium sulfate (CAS #:7757-82-6, ≥ 99.0%), citric acid monohydrate (CAS #:5949-29-1, 99.5%), glacial acetic acid (CAS #:64-19-7, ≥ 99.5%) (analytically pure) and DL-malic acid ((CAS #:6915-15-7, ≥ 99.0%) were purchased from national pharmaceutical group chemical Co., Ltd.
The instrument comprises the following steps: model 7890B/5977B GC (Agilent, USA); model Milli-Q ultra pure water apparatus (Millipore, USA); 8510E-DTH type ultrasonic cleaner (Branson Ultrasonics, USA); an HY-8 type adjustable-speed oscillator (Guohua electric Co., Ltd.); an electronic balance of type AX504 (sensory 0.0001g, Mettler Toledo, Switzerland); helium (greater than or equal to 99.999%, V/V, Szechwan Meissel gas products Co., Ltd.); syringe filters (0.2 μm, Nylon 66, jin teng laboratory instruments ltd, Tianjin); SCHOTT sample bottles (sealed ring, 100mL, DURAN Group, Germany).
Method adopted by various embodiments
1.1 Standard solution preparation
0.100g (accurate to 0.1mg) of anethole is weighed, added into a 50mL volumetric flask, and shaken up after the constant volume is realized by cyclohexane to prepare an internal standard solution with the concentration of 2.00 mg/mL. 30, 60, 90, 120 and 150 mu L of nicotine standard solution with the concentration of 9.6mg/mL are accurately measured, added into a 50mL volumetric flask, and 250 mu L of the internal standard solution is respectively added to prepare the nicotine standard solution with the concentrations of 5.76, 11.52, 17.28, 23.04 and 28.80 mu g/mL.
1.2 sample pretreatment
Drying the tobacco leaf (or tobacco shred) sample at temperature not higher than 40 deg.C, pulverizing, and sieving with 60 mesh standard sieve (aperture 0.3 mm). 0.10g (to the nearest 0.1mg) of the sieved sample powder was accurately weighed, added to a 100mL SCHOTT sample bottle, and 50.0mL of a cyclohexane solution (containing 10.0. mu.g/mL anethole) was accurately added, and then allowed to stand at (22. + -. 2) ° C for (24. + -. 2) hours, filtered through a 0.2. mu. mNylon 66 syringe filter, and subjected to GC-MS analysis.
1.3GC-MS analysis
The separation was carried out using an HP-5 MS UI column (30m 0.250mm 0.25 μm, 5% phenyl-containing methylpolysiloxane as the stationary phase) from Agilent. Column oven: 45 ℃; sample introduction amount: 1 mu L of the solution; sample inlet temperature: 250 ℃; the split ratio is as follows: 20: 1; carrier gas: he. Flow rate: 1.0 mL/min; purging the spacer: 3 mL/min; temperature rising procedure: the initial temperature is 45 ℃, the temperature is kept for 1min, then the temperature is raised to 280 ℃ at the speed of 20 ℃/min, and the temperature is kept for 2.25 min; solvent retardation: 7.0 min; electron energy: 70 eV; transmission line: 280 ℃; an ion source: 230 ℃; a quadrupole rod: at 150 ℃. Detection is performed by using a Selected Ion Monitoring mode (SIM), and quantification is performed by using an internal standard method. The mass-to-charge ratio of the quantitative ion of nicotine is 84, and the mass-to-charge ratio of the qualitative ion: 133. 161, 162; the quantitative ion of the anethole as an internal standard is 148, and the qualitative ion: 77. 117, 147.
Examples 1 to 52 the free nicotine content was measured in 52 tobacco samples by the above-mentioned method, and the moisture content and the dry content were calculated by the YC/T31-1996 method, and the results are shown in Table 1.
Table 1 actual measurements of free nicotine content in 52 samples
As can be seen from Table 1, the free nicotine content of the flue-cured tobacco leaf, the cigar leaf, the finished cigarette product and the finished cigar product in the measured samples is respectively 2.70-8.65, 2.15-12.66, 3.96-5.27 and 7.28-14.78 mg/g. The relative deviation of the experimental method is between 0.1% and 3.2%, and the parallelism is good during actual detection.
Second, method verification
2.1 Standard Curve and detection Limit
A series of standard solutions were prepared according to the method of "1.1" and subjected to GC-MS analysis to obtain a linear regression equation and related parameters (Table 2).
TABLE 2 Standard Curve and detection Limit of the Experimental methods
As can be seen from Table 2, the linear correlation coefficient R of the experimental method in the concentration range of 5.76-28.80 mug/mL is 0.9997, and the linearity is good. The Detection Limit (LOD) of the experimental method is 5.3 mu g/g (S/N is 3) by adopting a stepwise dilution method for determination, and the determination requirement of the content of free nicotine in tobacco can be completely met.
The chromatogram of the standard solution and the actual sample in GC-MS analysis is shown in FIGS. 1-2. As can be seen from fig. 1 and 2, the Total Ion Current (TIC) in SIM mode of both the standard solution and the sample GC-MS are clean, and no component interfering with detection exists. The peak shapes of nicotine and the internal standard anethole are also better, and the baseline separation is complete.
2.2 precision and recovery from spiked samples
5 parts of the sieved sample powder are weighed in parallel, each part is 0.10g (accurate to 0.1mg), then sample pretreatment and GC-MS detection are carried out according to the methods shown in 1.2 and 1.3, and the detection results are shown in Table 3.
TABLE 3 precision and recovery from spiking of the experimental method
As can be seen from Table 3, the Relative Standard Deviation (RSD) of the experimental method is between 1.7% and 2.7%, indicating that the precision of the experimental method is better. The accuracy of the method is verified by measuring the standard addition recovery rate of the sample, the standard addition recovery rates of low, medium and high 3 concentrations are 99.0%, 106.7% and 105.7% respectively, and the accuracy of the method is shown to be good.
Third, control test
Comparative example 1
The free nicotine is extracted from the sample to be tested by using fresh deionized water, and then the free nicotine is extracted from the water by using a proper organic solvent for testing. The principle of the method is as follows: free nicotine and some protic nicotine, which has a better solubility in water, are first extracted into water. When both the water phase and the organic phase exist, the protonic nicotine is still remained in the water phase because of being easily dissolved in water, and the free nicotine is selectively extracted out because of being more easily dissolved in the organic phase, thereby achieving the purpose of purification. As a result, the recovery rate of the experimental method was found to be low (less than 80%). After consulting the literature and carrying out theoretical calculation, the method has the following findings: the pH value of the tobacco water solution is lower, and is generally between 4.8 and 6.5; at this pH, the free nicotine content should be between 0.08% and 4.09% (table 4); when deionized water (or distilled water, neutral water with a pH value of 7.00) is adopted to extract free nicotine in a sample, the free nicotine is converted into concentration distribution under the current pH value after being dissolved in water no matter what the original form of the nicotine is; in the actual sample detection and recovery rate experiment, part of free nicotine is converted into proton due to the conversion of nicotine, so that the measurement result is incorrect and the recovery rate is low.
TABLE 4 Nicotine content distribution in aqueous solution in 3 forms
Note: the calculation used pKb 1-3.15 and pKb 2-7.87.
Comparative example 2
Extracting with organic solvent, purifying the organic extractive solution with water, and detecting. In analysis, free nicotine and a part of nicotine salt combined with organic acid are first extracted into organic solvent, and then certain amount of distilled water (or deionized water, neutral water with pH 7.00) is added to remove protonic nicotine in organic solvent. The method is based on the following steps: when both phases are present, the nicotine salt is removed by entering the aqueous phase, since it is more soluble in the aqueous phase; the free nicotine is more soluble in the organic phase and thus remains in the organic phase and is measured. In the experiment, the blank spiking recovery rate of the method is still low. The recovery rate test method comprises the following steps: preparing a nicotine standard solution (with the number of A) with the concentration of 5.76 mu g/mL by adopting a nicotine standard solution with the concentration of 9.6mg/mL and cyclohexane as a solvent; preparing a nicotine standard solution (No. B) with the concentration of 5.87 mu g/mL by using a pure nicotine product (CAS #:54-11-5, the content: 99.9%) and cyclohexane as a solvent; respectively weighing 25mL of A, B solution, adding 25mL of newly prepared deionized water, shaking for 1 hour, separating an organic phase by using a 250mL separating funnel, and then respectively adding 0.2g of anhydrous sodium sulfate for dehydration and drying to obtain a solution to be detected C, D; the four solutions A-D were filtered through a 0.2 μm Nylon 66 syringe filter and analyzed by GC-MS, the results of which are shown in Table 5.
TABLE 5 blank spiking recovery for the experimental procedure
As can be seen from Table 5, the blank standard recovery rates of A, B after the two nicotine standard solutions were treated with water were 20.6% -27.6%, and both were very low. The reason for this should be: the content of free nicotine in deionized water (or distilled water, neutral water with pH 7.00) is very low (see Table 4), not more than 5%; when the pure nicotine (free nicotine) added into cyclohexane exists in two phases, part of the pure nicotine is dissolved into water, most of the free nicotine dissolved in the water is changed into a proton state, and the reaction is further converted to the proton state; finally, most of the free nicotine dissolved in cyclohexane is also transferred to water and removed as protic nicotine.
It can be seen from the above two comparative examples that the determination of the free nicotine in tobacco after extraction or purification with water as solvent results in the change of the original form distribution of nicotine in the sample, and the detection result is inaccurate.
Fourthly, verifying the detection conditions
4.1 Effect of coexisting bound Nicotine in tobacco on the assay
Malic acid and citric acid are two organic acids with the highest content in tobacco, and acetic acid is also contained in tobacco leaf, which is highThe salts formed by combining these acids with nicotine (nicotine in the combined form) are present in tobacco in high amounts. If the detection of the method is carried out, the bound nicotine interferes the detection, and the salt formed by the acid and the nicotine has great influence on the detection result, so the 3 acids are selected as representatives to verify the experimental method. 4 parts of nicotine standard solution (prepared by adopting free nicotine with the purity of 99.9 percent; solvent: water) with the concentration of 100 mug/mL is precisely measured, and each part is 0.5 mL; adding malic acid 0.1g (1#), citric acid monohydrate 0.1g (2 #), respectively#) Glacial acetic acid 0.1mL (3)#) Fresh deionized water 0.1mL (4)#)Properly shaking and standing for 24 hours; adding 50mL of cyclohexane into each sample, and shaking for 1 h; the organic phase was separated into 25mL portions by a separatory funnel, dehydrated and dried by adding 0.25g of anhydrous sodium sulfate, filtered through a 0.2 μm Nylon 66 syringe filter, and analyzed by GC-MS. As a result, it was found that#-3#No nicotine was detected in the sample solution, 4#The detection concentrations of nicotine in the sample solution are 0.857 and 0.851 mug/mL (average value is 0.854 mug/mL), which indicates that the detection of the nicotine in the combined state coexisting in the sample by the method has little interference on the determination. In addition, since cyclohexane is an inert solvent for the aprotic donors, it also has little effect on the original morphological distribution of nicotine in the sample.
4.2 comparison of 3 extraction methods of shaking, ultrasound and standing
The extraction effect of free nicotine in tobacco by three extraction modes of shaking, ultrasonic oscillation and standing for 24h is examined. The experimental method comprises the following steps: accurately weighing 0.10g (accurate to 0.1mg) of sieved sample powder, adding the powder into a 100mL SCHOTT sample bottle, accurately adding 50.0mL cyclohexane solution containing an internal standard (10.0 mu g/mL anethole), and then carrying out corresponding extraction and GC-MS detection according to shaking, ultrasound and standing time listed in Table 6, wherein the results are shown in Table 6.
TABLE 6 content determination by shaking, sonication and standing 3 extraction methods
Note: firstly, the rotating speed is about 150 r/min; ② after standing for 22 h, 24h and 26h, the average content detected is 5.75 mg/g, 5.70 mg/g and 5.86mg/g respectively.
As can be seen from Table 6, free nicotine in tobacco can not be completely extracted by shaking for 3h and ultrasonic oscillation for 2h, which may be because cyclohexane is an organic solvent with very weak polarity and has weak penetrability to plant cells, and free nicotine is difficult to completely dissolve out of tobacco cells in a short time. In the ultrasonic oscillation extraction method, the temperature of the extract solution is increased by long-term operation, and the measurement accuracy and reproducibility are deteriorated. In addition, the cavitation effect during ultrasonic oscillation can also generate strong instantaneous high temperature and high pressure at the local part of the liquid to be detected, so that the risk of converting the combined nicotine into the free nicotine is high. The extraction method of standing for 24h has long operation time, can completely extract free nicotine, is convenient to operate, has good parallelism during detection, and is not easy to cause degradation of bound nicotine.
4.3 sample inlet temperature investigation
To obtain good GC-MS analysis, the sample is first introduced quantitatively into the gas chromatography system and allowed to vaporize efficiently, for which purpose the inlet temperature of the gas chromatograph is examined. 0.1057 g and 0.1069g of flue-cured tobacco and cigar tobacco samples are weighed respectively and added into a 100mL SCHOTT sample bottle, then sample pretreatment and GC-MS detection are carried out according to 1.2 sample pretreatment and 1.3GC-MS analysis, and the results are shown in Table 7.
TABLE 7 sample temperature investigation
As can be seen from Table 7, when the temperature of the injection port is 200-270 ℃, the peak area of nicotine increases first and then slightly decreases with the increase of the gasification temperature; the response of nicotine signals is strongest at 240-260 ℃; the injection inlet temperature is preferably 250 ℃ by comprehensively considering factors such as precision and the like.
Although the invention has been described herein with reference to illustrative embodiments thereof, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure. More specifically, various variations and modifications may be made to the component parts and/or arrangements of the subject combination arrangement within the scope of the disclosure herein. In addition to variations and modifications in the component parts and/or arrangements, other uses will also be apparent to those skilled in the art.
Claims (5)
1. The method for measuring the free nicotine in the tobacco is characterized by comprising the following steps of:
(1) drying tobacco leaves or tobacco shred samples at the temperature of less than or equal to 40 ℃, crushing, and sieving by a standard sieve of 60 meshes;
(2) accurately weighing 0.10g of sieved sample powder, adding the sample powder into a 100mL SCHOTT sample bottle, accurately adding 50.0mL of cyclohexane solution containing 10.0 mu g/mL of anethole, and standing at 22 +/-2 ℃ for 24 +/-2 hours to obtain a standing solution to be detected;
(3) the standing solution to be tested was filtered through a 0.2 μm nylon 66 syringe filter and analyzed by GC-MS.
2. The method according to claim 1, wherein the GC-MS analysis is performed by using HP-5 MS UI chromatographic column manufactured by Agilent.
3. The method of claim 2, wherein the column is a 30m 0.250mm 0.25 μm HP-5 MS UI column, and the stationary phase is 5% phenyl-containing methylpolysiloxane.
4. The method of claim 1, wherein the GC-MS analysis is performed under the following conditions: column oven: 45 ℃; sample introduction amount: 1 mu L of the solution; sample inlet temperature: 250 ℃; the split ratio is as follows: 20: 1; carrier gas: he. Flow rate: 1.0 mL/min; purging the spacer: 3 mL/min; temperature rising procedure: the initial temperature is 45 ℃, the temperature is kept for 1min, then the temperature is raised to 280 ℃ at the speed of 20 ℃/min, and the temperature is kept for 2.25 min; solvent retardation: 7.0 min; electron energy: 70 eV; transmission line: 280 ℃; an ion source: 230 ℃; a quadrupole rod: 150 ℃; detecting by adopting a Selected Ion Monitoring mode (SIM), and quantifying by adopting an internal standard method; the mass-to-charge ratio of the quantitative ion of nicotine is 84, and the mass-to-charge ratio of the qualitative ion: 133. 161, 162; the quantitative ion of the anethole as an internal standard is 148, and the qualitative ion: 77. 117, 147.
5. The method of claim 4, wherein the standard solution formulation method used in the internal standard method is:
weighing 0.100g of anethole, adding the anethole into a 50mL volumetric flask, metering the volume with cyclohexane, and shaking up to prepare an internal standard solution with the concentration of 2.00 mg/mL; 30, 60, 90, 120 and 150 mu L of nicotine standard solution with the concentration of 9.6mg/mL are accurately measured, added into a 50mL volumetric flask, and 250 mu L of the internal standard solution is respectively added to prepare the nicotine standard solution with the concentrations of 5.76, 11.52, 17.28, 23.04 and 28.80 mu g/mL.
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