CN114593969A - Pretreatment method for nicotine detection in mainstream smoke and application thereof - Google Patents

Pretreatment method for nicotine detection in mainstream smoke and application thereof Download PDF

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CN114593969A
CN114593969A CN202210250073.0A CN202210250073A CN114593969A CN 114593969 A CN114593969 A CN 114593969A CN 202210250073 A CN202210250073 A CN 202210250073A CN 114593969 A CN114593969 A CN 114593969A
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nicotine
extraction
pretreatment method
detection
mainstream smoke
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陈满堂
万丽斌
谢复炜
秦亚琼
刘绍锋
王昇
赵阁
郭军伟
华辰凤
蔡君兰
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Henan Business Research Institute Co ltd
Zhengzhou Tobacco Research Institute of CNTC
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Zhengzhou Tobacco Research Institute of CNTC
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    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • 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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    • G01N30/02—Column chromatography
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Abstract

The invention relates to the technical field of chemical substance analysis and detection, and particularly discloses a pretreatment method for nicotine detection in mainstream smoke and application thereof. The method comprises the following steps: (1) smoking tobacco products on a smoking machine, and trapping total particulate matters of the mainstream smoke by using a Cambridge filter disc; (2) mixing the Cambridge filter with a solvent, extracting nicotine in the Cambridge filter, filtering after extraction, and collecting filtrate; (3) and (3) taking the filtrate obtained in the step (2) as a donor phase, and extracting nicotine in the filtrate by adopting an electrified auxiliary liquid membrane extraction technology to obtain a purified nicotine extract. The sample pretreatment method integrates the separation and purification steps, is simple to operate, has small organic solvent consumption and outstanding sample purification capacity, can reduce the interference of other impurities in the mainstream smoke on nicotine detection, greatly improves the detection accuracy and precision of nicotine, and can realize high-throughput detection.

Description

Pretreatment method for nicotine detection in mainstream smoke and application thereof
Technical Field
The invention belongs to the technical field of chemical substance analysis and detection, and particularly relates to a pretreatment method for nicotine detection in mainstream smoke and application thereof.
Background
Nicotine is one of the unique substances in tobacco, and accounts for more than 90% of the total alkaloid content in tobacco. The nicotine has special tobacco flavor, and can excite sympathetic nerve while increasing the tobacco flavor, so that people feel excitation and stimulation. Studies have shown that the presence of nicotine is a major cause of addiction in tobacco products. The nicotine content in the mainstream smoke is an important index of the tobacco products, which not only influences the quality of the tobacco products, but also is related to the human health, so that the rapid and accurate determination of the nicotine content has important significance.
The detection method of nicotine mainly comprises ultraviolet spectrophotometry, gas chromatography, high performance liquid chromatography, gas chromatography-mass spectrometry combined method, liquid chromatography-mass spectrometry combined method and the like. The ultraviolet spectrophotometry method is simple to operate, but is easily interfered by coexisting substances, and has low accuracy. GB/T23355-2009 adopts gas chromatography to measure the content of nicotine in the total particulate matter of cigarette smoke. When the gas chromatography and the gas chromatography-mass spectrometry are combined, a large amount of organic solvents such as ethyl acetate, dichloromethane and the like are required for extracting nicotine, and a complicated sample pretreatment process is required. The high performance liquid chromatography has simple operation and good reproducibility, is suitable for analyzing a large number of conventional samples, but has high requirements on the samples and long time consumption for sample pretreatment. The liquid chromatography-mass spectrometry combined method can reduce the requirements on sample pretreatment, but the operation is complex, the cost is high, and the application is limited to a certain extent.
The components in the cigarette smoke are very complex, so that serious matrix interference is caused to the detection of a follow-up instrument, the accuracy of a detection result is low, the pretreatment steps of nicotine detection in the existing mainstream smoke are complex, the consumed time is long, the working efficiency of detection is influenced, and a large amount of time and economic cost are consumed.
Disclosure of Invention
The invention aims to provide a pretreatment method for detecting nicotine in mainstream smoke and application thereof, aiming at the existing problems.
In order to achieve the purpose, the invention provides the following technical scheme:
a pretreatment method for nicotine detection in mainstream smoke comprises the following steps:
1) smoking tobacco products on a smoking machine, and trapping total particulate matters of the mainstream smoke by using a Cambridge filter disc;
2) mixing the Cambridge filter with a solvent, extracting nicotine in the Cambridge filter, filtering after extraction, and collecting filtrate;
3) and (2) taking the obtained filtrate as a donor phase, extracting nicotine in the filtrate by adopting an electric auxiliary liquid film extraction technology to obtain a purified nicotine extract, wherein the adopted membrane is a porous membrane, an extraction solvent is loaded on the porous membrane, the extraction solvent is a mixed solution of di (2-ethylhexyl) phosphate and 2-nitrophenyloctyl ether, and a receiving phase adopted in the electric auxiliary liquid film extraction technology is a hydrochloric acid solution with the pH value of 1-3.
The tobacco products are traditional cigarettes, heating non-combustible cigarettes and electronic cigarettes.
The solvent in the step 2) is phosphate buffer solution with pH value of 5-6.
The extraction mode in the step 2) is dipping, ultrasonic, vortex oscillation and the like, and ultrasonic extraction is preferred.
The porous membrane is a porous polypropylene membrane, a porous polyvinylidene fluoride fiber membrane and the like, preferably a porous polypropylene membrane, and the porous membrane has a pore diameter of 0.2 μm and a thickness of 100 μm to 200 μm.
The volume percentage of the di (2-ethylhexyl) phosphate in the mixed solution is 1% to 10%, more preferably 3% to 10%, and particularly preferably 5%.
In the electrically assisted liquid membrane extraction technique, the voltage at which the electric field is applied between the donor phase and the acceptor phase is 5V to 80V, more preferably 40V to 80V, and particularly preferably 60V.
The extraction time in step 3) is 5 min to 25 min, preferably 15 min to 25 min, and more preferably 20 min.
The invention also provides application of the pretreatment method in nicotine detection in mainstream smoke of tobacco products, and the nicotine detection technology is ultraviolet spectrophotometry, gas chromatography, high performance liquid chromatography, gas chromatography-mass spectrometry combined method and liquid chromatography-mass spectrometry combined method, preferably high performance liquid chromatography.
The liquid chromatogram conditions during detection are as follows: a chromatographic column: xtimate C18 (4.6 × 150 mm, 3 μm); mobile phase A is 10 mmol/L phosphate buffer solution (containing 0.1% triethylamine) with pH 6.5, mobile phase B is methanol, and the ratio of the two is 40/60; flow rate: 1.0 mL/min; sample introduction amount: 5 mu L of the solution; column temperature: 35 ℃; detection wavelength: 259 nm.
The operation method for extracting nicotine in filtrate by adopting the electrification-assisted liquid film extraction technology comprises the following steps: a) selecting a tubular receiving phase container with two open ends, sealing and fixing the porous polypropylene film at one end of the container at high temperature of an electric iron, and loading an extraction solvent on the porous polypropylene film; b) injecting a receiving phase into the inner cavity of the receiving compatilizer; c) immersing the lower end of the receiving phase container processed in the step b) in a donor phase (the filtrate obtained in the step S002), inserting a working electrode into the donor phase, inserting a counter electrode into the receiving phase, connecting the working electrode with a positive electrode of a power supply, connecting the counter electrode with a negative electrode of the power supply, applying voltage to the working electrode and the counter electrode to form an electric field, and extracting nicotine in the donor phase under the action of the electric field.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the invention provides a pretreatment method for nicotine detection in mainstream smoke, which adopts an electrified auxiliary liquid film extraction technology to extract nicotine in mainstream smoke, wherein the mass transfer process of nicotine mainly takes electromigration as a main part in the extraction process, thereby greatly shortening the extraction time; in the extraction process, the interference of other impurities in the main stream smoke on nicotine detection is greatly reduced through the selectivity of the membrane and the extraction solvent loaded on the membrane; the separation and purification steps are integrated in the pretreatment method process, intermediate transfer and blow-drying redissolution are not needed, the pretreatment time is shortened, the sample loss caused in the phase transfer process is effectively prevented, and the detection accuracy and precision of nicotine are improved.
(2) According to the invention, a phosphate buffer solution with pH of 5-6 is used as a solvent to prepare a donor phase solution, so that nicotine exists in a form of single charged ions, and extraction and separation of nicotine are facilitated under the action of electric field migration.
(3) When the nicotine is extracted, the hydrochloric acid solution with the pH value of 1-3 is used as a receiving phase, so that the electric field migration effect is a main mass transfer driving force in the process of the power-on assisted liquid film extraction technology, and the acidic solvent can enable the nicotine to exist in the form of positive charge ions, so that the nicotine is favorably extracted into the receiving phase under the electric field migration effect.
(4) The invention loads the extraction solvent on the porous polypropylene film, the dosage of the extraction solvent is less and is only 10 mu L, and the problems of large organic solvent consumption and harm to the environment in the traditional extraction technology are avoided.
(5) The detection method is suitable for detecting nicotine in main stream smoke of different tobacco products, and has wide application prospect.
Drawings
FIG. 1 is a schematic view of an electrically assisted liquid membrane extraction apparatus in example 1 of the present invention;
FIG. 2 is a chromatogram before and after purification in example 5-1 of the present invention;
FIG. 3 is a chromatogram before and after purification in example 5-2 of the present invention;
FIG. 4 is a chromatogram before and after purification in example 5-3 of the present invention;
figure 5 is a standard operating curve for nicotine from example 6 of the present invention.
Detailed Description
The following description of the embodiments of the present invention is provided with reference to the accompanying drawings, but the invention is not limited thereto.
(I) optimization of extraction conditions for nicotine extraction by electrically-assisted liquid membrane
Example 1: screening experiment of extraction Voltage
In order to discuss the influence of different extraction voltages on the nicotine separation effect, the method carries out power-on auxiliary liquid membrane extraction at different voltages, calculates the recovery rate of nicotine by detecting the concentration of nicotine in a receiving phase after extraction is finished, and screens the extraction voltages according to the recovery rate. For details of the experiment, see the experiments of examples 1-1 to 1-6 below.
Example 1-1:
(1) preparing a 100 mg/L nicotine standard solution: taking a nicotine standard substance, taking 10 mmol/L phosphate buffer solution with pH of 5.6 as a solvent to prepare a standard solution containing 100 mg/L nicotine, taking the standard solution as a donor phase, and carrying out electro-assisted liquid membrane extraction.
(2) And (3) construction and extraction of an electric auxiliary liquid film extraction device:
a) selecting a 1 mL pipette suction head, fixing a porous polypropylene film with the aperture of 0.2 mu m and the thickness of 200 mu m at the wide-mouth end of the pipette suction head at the high temperature of an electric iron, and cutting off part of the length of the other end of the suction head to obtain a receiving phase container.
b) And coating 10 mu L of extraction solvent on the surface of the porous polypropylene film, wherein the extraction solvent is a mixed solution of bis (2-ethylhexyl) phosphate and 2-nitrophenyloctyl ether, and the volume percentage of the bis (2-ethylhexyl) phosphate in the mixed solution is 5%.
c) Injecting 200 mu L of receiving phase into the receiving compatilizer; the receiving phase is a hydrochloric acid solution with pH 2; taking a sample bottle, and adding 1000 mu L of a donor phase, wherein the donor phase is the standard solution containing 100 mg/L nicotine prepared in the step (1); inserting a working electrode into the donor phase, inserting a counter electrode into the receiving phase, then placing the receiving phase compatilizer into the sample bottle, enabling the porous polypropylene film to be just contacted with the liquid level of the donor phase solution of the sample bottle, connecting the working electrode with the positive electrode of the electrophoresis apparatus power supply, and connecting the counter electrode with the negative electrode of the electrophoresis apparatus power supply. The structure of the electric auxiliary liquid membrane extraction device is schematically shown in figure 1.
(3) Separation of nicotine:
the sample bottle is placed on a constant-temperature blending instrument, the electrophoresis instrument and the blending instrument power supply are started, and under the driving of external voltage and the auxiliary action of oscillation, extraction of nicotine is carried out. The voltage of the power supply of the electrophoresis apparatus is 5V, the rotating speed of the constant-temperature blending apparatus is 1000 rpm, and the extraction time is 20 min.
Examples 1 to 2:
the contents of example 1-2 are substantially the same as those of example 1-1, except that:
and (4) the voltage of the power supply of the electrophoresis apparatus in the step (3) is 10V.
Examples 1 to 3:
examples 1 to 3 are substantially the same as examples 1 to 1 except that:
and (4) the voltage of the power supply of the electrophoresis apparatus in the step (3) is 20V.
Examples 1 to 4:
examples 1 to 4 are substantially the same as examples 1 to 1 except that:
and (4) the voltage of the power supply of the electrophoresis apparatus in the step (3) is 40V.
Examples 1 to 5:
examples 1 to 5 are substantially the same as examples 1 to 1 except that:
and (4) the voltage of the power supply of the electrophoresis apparatus in the step (3) is 60V.
Examples 1 to 6:
examples 1 to 6 are substantially the same as examples 1 to 1 except that:
and (4) the voltage of the power supply of the electrophoresis apparatus in the step (3) is 80V.
The concentration of nicotine in the receiving phase after the extraction of examples 1-1 to 1-6 was completed was measured by a high performance liquid chromatography mass spectrometer, and the recovery rate of nicotine was calculated. The detection conditions of the high performance liquid chromatography mass spectrometer are as follows: the column was an Xtimate C18 column, having a size of 4.6X 150 mm and a particle size of 3.0. mu.m. The mobile phase was 40% 10 mmol/L phosphate buffer pH =6.5 (containing 0.1% triethylamine) -60% methanol, flow rate 1.0 mL/min, sample size 5.0 μ L, detection wavelength 259 nm. Specific results are shown in table 1.
TABLE 1 Effect of Voltage on Nicotine separation Effect
Examples Voltage (V) Nicotine recovery (%)
Examples 1 to 1 5 22
Examples 1 to 2 10 52
Examples 1 to 3 20 85
Examples 1 to 4 40 96
Examples 1 to 5 60 97
Examples 1 to 6 80 96
As can be seen from table 1, as the extraction voltage is increased from 5V to 40V, the recovery rate of nicotine is increased from 22% to 96%, and the recovery rate of nicotine is substantially maintained by continuously increasing the extraction voltage. For robustness of the extraction process, the extraction voltage is preferably 60V.
Example 2: screening experiment for extraction time
In order to discuss the influence of different extraction times on the nicotine separation effect, the method carries out power-on auxiliary liquid membrane extraction at different times, calculates the recovery rate of nicotine by detecting the concentration of the nicotine in a receiving phase after extraction is finished, and screens the extraction time according to the recovery rate. For details of the experiment, see the experiment of example 2-1 to example 2-5 below.
Example 2-1:
(1) preparing a 100 mg/L nicotine standard solution: taking a nicotine standard substance, taking 10 mmol/L phosphate buffer solution with pH of 5.6 as a solvent to prepare a standard solution containing 100 mg/L nicotine, taking the standard solution as a donor phase, and carrying out electro-assisted liquid membrane extraction.
(2) And (3) construction and extraction of an electric auxiliary liquid film extraction device:
a) selecting a 1 mL pipette suction head, fixing a porous polypropylene film with the aperture of 0.2 mu m and the thickness of 200 mu m at the wide-mouth end of the pipette suction head at the high temperature of an electric iron, and cutting off part of the length of the other end of the suction head to obtain a receiving phase container.
b) And coating 10 mu L of extraction solvent on the surface of the porous polypropylene film, wherein the extraction solvent is a mixed solution of bis (2-ethylhexyl) phosphate and 2-nitrophenyloctyl ether, and the volume percentage of the bis (2-ethylhexyl) phosphate in the mixed solution is 5%.
c) Injecting 200 mu L of receiving phase into the receiving compatilizer; the receiving phase is a hydrochloric acid solution with pH 2; taking a sample bottle, and adding 1000 mu L of a donor phase, wherein the donor phase is the standard solution containing 100 mg/L nicotine prepared in the step (1); inserting a working electrode into the donor phase, inserting a counter electrode into the receiving phase, then placing the receiving phase compatilizer into the sample bottle, enabling the porous polypropylene film to be just contacted with the liquid level of the donor phase solution of the sample bottle, connecting the working electrode with the positive electrode of the electrophoresis apparatus power supply, and connecting the counter electrode with the negative electrode of the electrophoresis apparatus power supply. The structure of the electric auxiliary liquid membrane extraction device is schematically shown in figure 1.
(3) Separation of nicotine:
the sample bottle is placed on a constant-temperature blending instrument, the electrophoresis instrument and the blending instrument power supply are started, and under the driving of external voltage and the auxiliary action of oscillation, extraction of nicotine is carried out. The voltage of the power supply of the electrophoresis apparatus is 60V, the rotating speed of the constant-temperature blending apparatus is 1000 rpm, and the extraction time is 5 min.
Example 2-2:
the contents of example 2-2 are substantially the same as those of example 2-1 except that:
the extraction time in the step (3) is 10 min.
Examples 2 to 3:
the contents of example 2-3 are substantially the same as example 2-1 except that:
the extraction time in the step (3) is 15 min.
Examples 2 to 4:
examples 2 to 4 are substantially the same as example 2 to 1 except that:
the extraction time in the step (3) is 20 min.
Examples 2 to 5:
examples 2 to 5 are substantially the same as example 2 to 1 except that:
the extraction time in the step (3) is 25 min.
The concentration of nicotine in the receiving phase after the extraction of examples 2-1 to 2-5 was completed was measured by a high performance liquid chromatography mass spectrometer, and the recovery rate of nicotine was calculated. The detection conditions of the high performance liquid chromatography mass spectrometer are as follows: the column was an Xtimate C18 column, having a size of 4.6X 150 mm and a particle size of 3.0. mu.m. The mobile phase was 40% 10 mmol/L phosphate buffer pH =6.5 (containing 0.1% triethylamine) -60% methanol, flow rate 1.0 mL/min, sample size 5.0 μ L, detection wavelength 259 nm. Specific results are shown in table 2.
TABLE 2 Effect of extraction time on nicotine separation Effect
Examples Extraction time (min) Nicotine recovery (%)
Example 2-1 5 62
Examples 2 to 2 10 83
Examples 2 to 3 15 93
Examples 2 to 4 20 97
Examples 2 to 5 25 97
As can be seen from Table 2, the recovery rate of nicotine increased from 62% to 97% as the extraction time increased from 5 min to 20min, and the recovery rate of nicotine remained unchanged as the extraction time continued to increase. Therefore, the extraction time is preferably 20 min.
Example 3: screening experiment of extraction solvent
In order to discuss the influence of different extraction solvents on the nicotine separation effect, the method carries out power-on auxiliary liquid membrane extraction under different extraction solvents, calculates the recovery rate of nicotine by detecting the concentration of nicotine in a receiving phase after extraction is finished, and screens the extraction solvents according to the recovery rate. For details of the experiment, see the experiment of example 3-1 to example 3-5 below.
Example 3-1:
(1) preparing a 100 mg/L nicotine standard solution: taking a nicotine standard substance, taking 10 mmol/L phosphate buffer solution with pH of 5.6 as a solvent to prepare a standard solution containing 100 mg/L nicotine, taking the standard solution as a donor phase, and carrying out electro-assisted liquid membrane extraction.
(2) And (3) construction and extraction of an electric auxiliary liquid film extraction device:
a) selecting a 1 mL pipette suction head, fixing a porous polypropylene film with the aperture of 0.2 mu m and the thickness of 200 mu m at the wide-mouth end of the pipette suction head at the high temperature of an electric iron, and cutting off part of the length of the other end of the suction head to obtain a receiving phase container.
b) And coating 10 mu L of extraction solvent on the surface of the porous polypropylene membrane, wherein the extraction solvent is a mixed solution of bis (2-ethylhexyl) phosphate and 2-nitrophenyloctyl ether, and the volume percentage of the bis (2-ethylhexyl) phosphate in the mixed solution is 1%.
c) Injecting 200 mu L of receiving phase into the receiving compatilizer; the receiving phase is a hydrochloric acid solution with pH 2; taking a sample bottle, and adding 1000 mu L of a donor phase, wherein the donor phase is the standard solution containing 100 mg/L nicotine prepared in the step (1); inserting a working electrode into the donor phase, inserting a counter electrode into the receiving phase, then placing the receiving phase compatilizer into the sample bottle, enabling the porous polypropylene film to be just contacted with the liquid level of the donor phase solution of the sample bottle, connecting the working electrode with the positive electrode of the electrophoresis apparatus power supply, and connecting the counter electrode with the negative electrode of the electrophoresis apparatus power supply. The structure of the electric auxiliary liquid membrane extraction device is schematically shown in figure 1.
(3) Separation of nicotine:
the sample bottle is placed on a constant-temperature blending instrument, the electrophoresis instrument and the blending instrument power supply are started, and under the driving of external voltage and the auxiliary action of oscillation, extraction of nicotine is carried out. The voltage of the power supply of the electrophoresis apparatus is 60V, the rotating speed of the constant-temperature blending apparatus is 1000 rpm, and the extraction time is 20 min.
Example 3-2:
the contents of example 3-2 are substantially the same as those of example 3-1 except that:
the extraction solvent in the step (3) is a mixed solution of di (2-ethylhexyl) phosphate and 2-nitrophenyloctyl ether, and the volume percentage of the di (2-ethylhexyl) phosphate in the mixed solution is 2%.
Examples 3 to 3:
the contents of example 3-3 are substantially the same as those of example 3-1 except that:
in the step (3), the extraction solvent is a mixed solution of bis (2-ethylhexyl) phosphate and 2-nitrophenyloctyl ether, and the volume percentage of the bis (2-ethylhexyl) phosphate in the mixed solution is 3%.
Examples 3 to 4:
the contents of example 3-4 are substantially the same as example 3-1 except that:
the extraction solvent in the step (3) is a mixed solution of bis (2-ethylhexyl) phosphate and 2-nitrophenyloctyl ether, and the volume percentage of the bis (2-ethylhexyl) phosphate in the mixed solution is 5%.
Examples 3 to 5:
the contents of examples 3 to 5 are substantially the same as those of example 3 to 1 except that:
the extraction solvent in the step (3) is a mixed solution of bis (2-ethylhexyl) phosphate and 2-nitrophenyloctyl ether, and the volume percentage of the bis (2-ethylhexyl) phosphate in the mixed solution is 10%.
The concentration of nicotine in the receiving phase after the extraction of examples 3-1 to 3-5 was completed was measured by a high performance liquid chromatography mass spectrometer, and the recovery rate of nicotine was calculated. The detection conditions of the high performance liquid chromatography mass spectrometer are as follows: the column was an Xtimate C18 column, having a size of 4.6X 150 mm and a particle size of 3.0. mu.m. The mobile phase was 40% 10 mmol/L phosphate buffer pH =6.5 (containing 0.1% triethylamine) -60% methanol, flow rate 1.0 mL/min, sample size 5.0 μ L, detection wavelength 259 nm. Specific results are shown in table 3.
TABLE 3 Effect of extraction solvent on nicotine separation Effect
Examples Volume percent of bis (2-ethylhexyl) phosphate (%) Nicotine recovery (%)
Example 2-1 1 38
Examples 2 to 2 2 79
Examples 2 to 3 3 97
Examples 2 to 4 5 97
Examples 2 to 5 10 97
As can be seen from Table 1, as the volume percentage of di (2-ethylhexyl) phosphate in the extraction solvent is increased from 1% to 3%, the recovery rate of nicotine is increased from 38% to 97%, and the recovery rate of nicotine is substantially maintained by continuously increasing the extraction voltage. For robustness of the extraction process, the volume percentage of di (2-ethylhexyl) phosphate in the extraction solvent is preferably 5%.
(II) separation and purification of nicotine from mainstream smoke
Example 4: pretreatment method for nicotine detection in mainstream smoke
Under the optimal extraction conditions obtained by optimization in the embodiment (I), nicotine in the main stream smoke is separated and purified, and a pretreatment method for detecting nicotine in the main stream smoke is further provided. For details of the experiment, see the experiment of example 4-1 to example 4-3 below.
Example 4-1:
a pretreatment method for nicotine detection in mainstream smoke comprises the following steps:
(1) trapping of mainstream smoke of traditional cigarettes:
according to the requirements of GB/T16447-2004, balancing the traditional cigarette for 48 hours in the environment of (22 +/-1) DEG C and relative humidity (60 +/-3)% to select the mass (average mass +/-0.019) and the suction resistance (average suction resistance +/-49 Pa); 4 cigarettes were smoked under the ISO smoking mode, i.e. 2 s smoking time, 58 s smoking interval and 35 mL smoking capacity, and Cambridge filter discs were used to capture the total particulate matter in the mainstream smoke of the cigarettes.
(2) Extraction of crude nicotine extract in mainstream smoke:
and (2) putting the Cambridge filter disc trapping the total particulate matters of the mainstream smoke in the step (1) into a 250 mL conical flask, adding 100 mL 10 mmol/L phosphate buffer solution with pH of 5.6, carrying out ultrasonic extraction for 20min, and filtering the extracting solution by using a 0.45-micrometer microporous filter membrane to obtain a nicotine crude extract solution in the mainstream smoke.
(3) And (3) adding electric auxiliary liquid membrane extraction:
and (3) taking the nicotine crude extract solution obtained in the step (2) as a donor phase, and extracting nicotine in the nicotine crude extract by adopting an electrified auxiliary liquid membrane extraction technology. The specific operation of the electric assisted liquid membrane extraction is as follows:
a) selecting a 1 mL pipette suction head, fixing a porous polypropylene film with the aperture of 0.2 mu m and the thickness of 200 mu m at the wide-mouth end of the pipette suction head at the high temperature of an electric iron, and cutting off part of the length of the other end of the suction head to obtain a receiving phase container.
b) And coating 10 mu L of extraction solvent on the surface of the porous polypropylene film, wherein the extraction solvent is a mixed solution of bis (2-ethylhexyl) phosphate and 2-nitrophenyloctyl ether, and the volume percentage of the bis (2-ethylhexyl) phosphate in the mixed solution is 5%.
c) Injecting 200 mu L of receiving phase into a receiving compatilizer, adding 1000 mu L of donor phase solution into a sample bottle, inserting a working electrode into the donor phase, inserting a counter electrode into the receiving phase, then placing the receiving compatilizer into the sample bottle, enabling the porous polypropylene film to be just contacted with the liquid level of the donor phase solution of the sample bottle, connecting the working electrode with the positive electrode of an electrophoresis apparatus power supply, and connecting the counter electrode with the negative electrode of the electrophoresis apparatus power supply.
d) The sample bottle is placed on a constant-temperature blending instrument, the electrophoresis apparatus and the blending instrument power supply are started, and under the driving of external voltage and the auxiliary action of oscillation, extraction of nicotine is carried out. The voltage of the power supply of the electrophoresis apparatus is 60V, the rotating speed of the constant-temperature blending apparatus is 1000 rpm, and the extraction time is 20 min.
e) And after extraction is finished, taking out the receiving phase solution in the receiving phase container, namely the purified nicotine extract.
Example 4-2:
the contents of example 4-2 are substantially the same as those of example 4-1 except that:
the method for trapping the mainstream smoke of the non-burning cigarette by heating in the step (1) comprises the following steps:
according to the requirements of GB/T16447-2004, balancing the heated and non-combusted cigarette for 48 hours in the environment of (22 +/-1) DEG C and relative humidity (60 +/-3)%, and selecting mass (average mass +/-0.019) and draw resistance (average draw resistance +/-49 Pa); the HCI smoking mode, namely the smoking time is 2 s, the smoking interval is 28 s, the smoking capacity is 55 mL, 4 cigarettes which are not burnt under heating are smoked under the condition, and the Cambridge filter disc is used for trapping the total particulate matters in the mainstream smoke of the cigarettes.
Examples 4 to 3:
the contents of example 4-3 are substantially the same as those of example 4-1 except that:
the method for trapping the mainstream smoke of the electronic cigarette in the step (1) comprises the following steps:
one of the smoking modes recommended by the collaborative research and research center for tobacco science (CORESTA), namely 3 s of smoking time, 30 s of smoking interval and 55 mL of smoking capacity, is adopted, 50 mouths of smoking are smoked under the condition, and the Cambridge filter disc is used for capturing the total particulate matters in the mainstream smoke.
Example 5: purification effect of electric auxiliary liquid film extraction technology
Based on the pretreatment method for nicotine detection in mainstream smoke provided in example 4, the purification effect of the pretreatment method provided by the present invention is evaluated by measuring chromatograms of a nicotine crude extract solution and a purified nicotine extract solution by using a high performance liquid chromatography mass spectrometer. The detection conditions of the high performance liquid chromatography mass spectrometer are as follows: the column was an Xtimate C18 column, having a size of 4.6X 150 mm and a particle size of 3.0. mu.m. The mobile phase was 40% 10 mmol/L phosphate buffer pH =6.5 (containing 0.1% triethylamine) -60% methanol, flow rate 1.0 mL/min, sample size 5.0 μ L, detection wavelength 259 nm. For details of the experiment, see the experiment of example 5-1 to example 5-3 below.
Example 5-1:
the chromatograms of the purified nicotine extract solution (diluted 5 times) obtained in step e) of example 4-1 and the nicotine crude extract solution obtained in step (2) of example 4-1 were measured by a high performance liquid chromatography mass spectrometer, and the results are shown in fig. 2. As can be seen from FIG. 2, the sample pretreatment method provided by the invention has outstanding purification capacity for the substrate in the mainstream smoke of the traditional cigarette.
Example 5-2:
the chromatograms of the purified nicotine extract solution (diluted 5 times) obtained in step e) of example 4-2 and the nicotine crude extract solution obtained in step (2) of example 4-2 were measured by high performance liquid chromatography mass spectrometer, and the results are shown in fig. 3. As can be seen from FIG. 3, the sample pretreatment method provided by the invention has outstanding purification capability on the substrate in the mainstream smoke of the heated non-combustible cigarette.
Examples 5 to 3:
chromatograms of the purified nicotine extract solution obtained in step e) of examples 4 to 3 (diluted 5 times) and the nicotine crude extract solution obtained in step 2 of examples 4 to 3 were measured by high performance liquid chromatography mass spectrometry, and the results are shown in fig. 4. As can be seen from FIG. 4, the sample pretreatment method provided by the invention has outstanding purification capability on the substrate in the mainstream smoke of the electronic cigarette.
(III) detection of Nicotine in mainstream Smoke
Example 6: detection of nicotine in mainstream smoke
The pretreatment method for detecting nicotine in mainstream smoke described in example (two) is used for detecting nicotine in mainstream smoke. When the high performance liquid chromatography mass spectrometer is used for measuring the concentration of nicotine in a receiving phase, a working curve of nicotine needs to be firstly made.
The working curve of nicotine is prepared as follows:
taking a nicotine standard substance, and taking 10 mmol/L phosphate buffer solution with pH 5.6 as a solvent to prepare a standard solution containing 1000 mg/L nicotine. The nicotine standard solution with the concentration of 1000 mg/L is diluted by stages by 10 mmol/L phosphate buffer solution with pH 5.6. Series of standard solutions were obtained at concentrations of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 150 and 200 mg/L, respectively. The series of standard solutions are extracted according to the power-on assisted liquid film extraction operation described in step (3) in example 4-1, and after extraction, a high performance liquid chromatography mass spectrometer is used to measure the chromatogram peak area of nicotine in the receiving phase, and a working curve of nicotine is drawn (as shown in fig. 5), and relevant performance parameters of the detection method provided by the invention are obtained, including a linear range, a linear correlation coefficient, a detection limit, a quantification limit, an intra-day deviation and a daytime deviation, as shown in table 4.
Table 4 relevant parameters for nicotine detection method.
Figure DEST_PATH_IMAGE002
Example 7: sample labeling recovery experiment
And selecting mainstream smoke samples of the traditional cigarette, the heating non-combustible cigarette and the electronic cigarette, and carrying out recovery rate test. Respectively adding nicotine solutions with different mass concentration levels of low, medium and high into the nicotine crude extract solution of the sample to respectively prepare standard sample solutions of 2 mg/L, 20 mg/L and 100 mg/L. Respectively extracting the spiked sample solution according to the powered-on auxiliary liquid film extraction operation in the step (3) in the example 4-1, measuring the chromatogram peak area of nicotine in a receiving phase by using a high performance liquid chromatography mass spectrometer after extraction, calculating the concentration of nicotine according to the working curve prepared in the example 6, further calculating the spiked recovery rate, and repeating the steps for 3 times at each level. The recovery and Relative Standard Deviation (RSD) are shown in Table 5.
TABLE 5 test results of sample spiking recovery experiments
Figure DEST_PATH_IMAGE004
As can be seen from the data in Table 5, when the pretreatment method provided by the invention is used for detecting the standard-added sample, the average recovery rate is between 81.0% and 112.8%, and the RSD is not more than 7.7%. Therefore, the detection method established by the pretreatment method provided by the invention has better recovery rate and precision.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (11)

1. A pretreatment method for nicotine detection in mainstream smoke is characterized in that: the pretreatment method comprises the following steps:
1) smoking tobacco products on a smoking machine, and collecting total particulate matters of the mainstream smoke by using a Cambridge filter disc;
2) mixing the Cambridge filter with a solvent, extracting nicotine in the Cambridge filter, filtering after extraction, and collecting filtrate;
3) and (2) taking the obtained filtrate as a donor phase, extracting nicotine in the filtrate by adopting an electric auxiliary liquid film extraction technology to obtain a purified nicotine extract, wherein the adopted membrane is a porous membrane, an extraction solvent is loaded on the porous membrane, the extraction solvent is a mixed solution of di (2-ethylhexyl) phosphate and 2-nitrophenyloctyl ether, and a receiving phase adopted in the electric auxiliary liquid film extraction technology is a hydrochloric acid solution with the pH value of 1-3.
2. The pretreatment method according to claim 1, wherein: the tobacco products are traditional cigarettes, heating non-combustible cigarettes and electronic cigarettes.
3. The pretreatment method according to claim 1, wherein: the solvent in the step 2) is a phosphate buffer solution with pH of 5-6.
4. The pretreatment method according to claim 1, wherein: the extraction mode in the step 2) is dipping, ultrasonic, vortex oscillation and the like, and ultrasonic extraction is preferred.
5. The pretreatment method according to claim 1, wherein: the porous membrane is a porous polypropylene membrane, a porous polyvinylidene fluoride fiber membrane and the like, and the porous polypropylene membrane is preferred.
6. The pretreatment method according to claim 1 or 5, wherein: the porous membrane has a pore diameter of 0.2 μm and a thickness of 100 to 200. mu.m.
7. The pretreatment method according to claim 1, wherein: the volume percentage of the di (2-ethylhexyl) phosphate in the mixed solution is 1% to 10%, more preferably 3% to 10%, and particularly preferably 5%.
8. The pretreatment method according to claim 1, wherein: in the electrically assisted liquid membrane extraction technique, the voltage at which the electric field is applied between the donor phase and the acceptor phase is 5V to 80V, more preferably 40V to 80V, and particularly preferably 60V.
9. The pretreatment method according to claim 1, wherein: the extraction time in step 3) is 5 min to 25 min, preferably 15 min to 25 min, and more preferably 20 min.
10. Use of the pretreatment method of any one of claims 1 to 9 for the detection of nicotine in mainstream smoke of a smoking article.
11. Use according to claim 10, characterized in that: the nicotine detection technology is ultraviolet spectrophotometry, gas chromatography, high performance liquid chromatography, gas chromatography-mass spectrometry combined method and liquid chromatography-mass spectrometry combined method, and preferably high performance liquid chromatography.
CN202210250073.0A 2022-03-15 2022-03-15 Pretreatment method for nicotine detection in mainstream smoke and application thereof Pending CN114593969A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117054209A (en) * 2023-08-11 2023-11-14 宁波镇海质谱技术研究院 Dilution of smoke nicotine and extraction and detection method

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117054209A (en) * 2023-08-11 2023-11-14 宁波镇海质谱技术研究院 Dilution of smoke nicotine and extraction and detection method

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