Analysis method for simultaneously determining contents of nicotine and three cooling agents in buccal tobacco

Abstract

The invention relates to the field of analytical chemistry, in particular to an analysis method for simultaneously determining the content of nicotine and three cooling agents in buccal tobacco, which comprises the following steps: (1) sample pretreatment: extracting nicotine and cooling agents in a buccal tobacco sample by adopting a Y-type gas-liquid-liquid extraction technology; (2) gas chromatography-mass spectrometry analysis: injecting the pretreated sample solution into a gas chromatography-mass spectrometer for analysis; (3) determining the nature according to the retention time of the standard substance and the characteristic fragment ions, quantifying through an external standard method, and calculating the content of nicotine and the cooling agent in the sample; wherein the cooling agents comprise menthol, WS-23 and WS-3. According to the method provided by the invention, the content of nicotine and the content of the three cooling agents in the buccal cigarette can be measured at the same time, the analysis frequency is reduced, the analysis efficiency is improved, and the analysis cost is reduced; a Y-shaped gas-liquid-liquid extraction technology is adopted for sample pretreatment, so that the operation process is simplified, and the extraction efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the field of analytical chemistry, and particularly to an analytical method for simultaneously determining the contents of nicotine and three coolants in oral tobacco products. Background Art

[0002] With the development of e-cigarettes and new tobacco products, oral tobacco products have become the new favorites in the market. Oral tobacco products often contain nicotine and various coolants. To ensure the quality and safety of products, it is necessary to accurately determine these active ingredients.

[0003] Traditional analytical methods often require separate determination of nicotine and coolants, with cumbersome operations, long time consumption, and high analysis costs. Among them, nicotine is usually determined by gas chromatography, while coolants are mostly determined by liquid chromatography, which makes the entire analysis process require different instruments and methods, greatly reducing the analysis efficiency.

[0004] The existing patent CN201610067341 discloses a method for determining coolants in tobacco products, but it can only be used for the determination of coolants; the patent CN202111005032 discloses a method for determining the nicotine content in gasoline, but it is only applicable to nicotine. At present, there is no simple and efficient method that can simultaneously determine the contents of nicotine and coolants in oral tobacco products.

[0005] Therefore, there is an urgent need to develop a simple, rapid, and efficient analytical method that can simultaneously determine the contents of nicotine and multiple coolants in oral tobacco products. Summary of the Invention

[0006] The purpose of the present invention is to provide an analytical method for simultaneously determining the contents of nicotine and three coolants in oral tobacco products, and to solve the deficiencies of traditional law enforcement shields in terms of concealment, function integration, and environmental adaptability through the deep integration of materials science, optical engineering, and equipment intelligence.

[0007] To achieve the above purpose, the present invention provides an analytical method for simultaneously determining the contents of nicotine and three coolants in oral tobacco products, including the following steps: (1) Sample pretreatment: Extract nicotine and coolants in the oral tobacco product sample by using the Y-type gas-liquid-liquid extraction technique; (2) Gas chromatography-mass spectrometry analysis: Inject the pretreated sample solution into a gas chromatography-mass spectrometry instrument for analysis; (3) Qualify according to the retention time and characteristic fragment ions of the standard product, and quantify by the external standard method to calculate the contents of nicotine and coolants in the sample; wherein, the coolants include menthol, WS-23, and WS-3.

[0008] Preferably, the Y-type gas-liquid-liquid extraction in step (1) specifically includes the following steps: (a) Take an appropriate amount of oral tobacco sample and weigh it precisely; (b) Add an appropriate amount of extraction solvent and perform ultrasonic extraction; (c) Centrifuge and separate, and take the supernatant; (d) Filter the supernatant and collect the filtrate for standby.

[0009] Preferably, the gas chromatography conditions in step (2) are as follows: The chromatographic column is

[0010] HP-5MS (60m × 0.25mm × 0.25μm); The carrier gas is helium, and the flow rate is 1 mL / min; The column temperature program is: The initial temperature is 90°C, hold for 5 min, increase to 200°C at a rate of 40°C / min, hold for 5 min, then increase to 280°C at a rate of 40°C / min, and hold for 5 min; The total analysis time is 45 min.

[0011] Preferably, the mass spectrometry conditions in step (2) are as follows: The ion source is an electron impact ion source (EI); The ionization voltage is 70 eV; The transfer line temperature is 280°C; The ion source temperature is 230°C; The quadrupole temperature is 150°C; Detection is carried out in the selected ion monitoring (SIM) mode.

[0012] Preferably, the retention times and characteristic fragment ions of the target compounds in step (3) are: (i) Menthol: The retention time is 7.613 min, and the characteristic fragment ion is m / z71; (ii) WS-23: The retention time is 9.427 min, and the characteristic fragment ion is m / z114; (iii) WS-3: The retention time is 10.716 min, and the characteristic fragment ion is m / z100; (iv) Nicotine: The retention time is 15.739 min, and the characteristic fragment ion is m / z84.

[0013] Preferably, the extraction solvent in step (b) is acetonitrile.

[0014] Preferably, the ultrasonic extraction time in step (b) is 10 min, and the ultrasonic temperature is room temperature.

[0015] Preferably, the centrifugation conditions in step (c) are: The centrifugation speed is 3500 r / min, and the centrifugation time is 10 min.

[0016] Preferably, in step (d), a 0.22μm organic phase filter membrane is used for filtration.

[0017] Preferably, the calibration curve concentration range of the external standard method in step (3) is: Nicotine: 0.5 - 100 μg / mL; Menthol: 0.5 - 100 μg / mL; WS-23: 0.1 - 20 μg / mL; WS-3: 0.1 - 20 μg / mL.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The method provided by the present invention can simultaneously determine the contents of nicotine and three coolants in the oral tobacco, reducing the number of analyses, improving the analysis efficiency, and reducing the analysis cost;

[0020] 2. The Y-type gas-liquid - liquid extraction technology is used for sample pretreatment, simplifying the operation process, improving the extraction efficiency, and reducing matrix interference;

[0021] 3. The optimized gas chromatography - mass spectrometry conditions enable good separation and detection of target compounds, improving the accuracy and reliability of the analysis;

[0022] 4. This method is simple, rapid, and efficient, and is applicable to the quality control and R & D process of oral tobacco products. Description of the Drawings

[0023] Figure 1 It is the spectrum diagram of the present invention. Detailed Embodiments

[0024] The following further elaborates the present invention in detail with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0025] Example 1:

[0026] An analytical method for simultaneously determining the contents of nicotine and three coolants in oral tobacco, comprising the following steps:

[0027] (1) Sample pretreatment: (a) Take about 1 g of the oral tobacco sample and weigh it accurately; (b) Add 10 mL of acetonitrile and ultrasonically extract for 10 min at room temperature; (c) Centrifuge at 3500 r / min for 10 min and take the supernatant; (d) Filter the supernatant with a 0.22 μm organic phase filter membrane and collect the filtrate for standby.

[0028] (2) Gas chromatography - mass spectrometry analysis: Analyze using a gas chromatography - mass spectrometer. The chromatographic conditions are as follows: Chromatographic column: HP-5MS (60 m × 0.25 mm × 0.25 μm); Carrier gas: helium, flow rate 1 mL / min; Column temperature program: Initial temperature 90 °C, hold for 5 min, increase to 200 °C at 40 °C / min, hold for 5 min, then increase to 280 °C at 40 °C / min, hold for 5 min; Total analysis time: 45 min.

[0029] The mass spectrometry conditions are as follows: Ion source: Electron impact ionization source (EI); Ionization voltage: 70 eV; Transfer line temperature: 280 °C; Ion source temperature: 230 °C; Quadrupole temperature: 150 °C; Detection mode: SIM mode.

[0030] (3) Qualitative and quantitative analysis: Qualitative analysis is performed based on the retention times and characteristic fragment ions of the reference standards. The retention times and characteristic fragment ions of each target compound are as follows: (i) Menthol: retention time is 7.613 min, characteristic fragment ion is m / z 71; (ii) WS-23: retention time is 9.427 min, characteristic fragment ion is m / z 114; (iii) WS-3: retention time is 10.716 min, characteristic fragment ion is m / z 100; (iv) Nicotine: retention time is 15.739 min, characteristic fragment ion is m / z 84.

[0031] Quantitative analysis is carried out by the external standard method. The calibration curve concentration ranges for the external standard method are as follows: Nicotine: 0.5 - 100 μg / mL; Menthol: 0.5 - 100 μg / mL; WS-23: 0.1 - 20 μg / mL; WS-3: 0.1 - 20 μg / mL.

[0032] Example 2:

[0033] An analytical method for simultaneously determining the contents of nicotine and three coolants in a nicotine pouch, comprising the following steps:

[0034] (1) Sample pretreatment: (a) Take about 0.5 g of the nicotine pouch sample and weigh it accurately; (b) Add 5 mL of acetonitrile and ultrasonically extract for 8 min at room temperature; (c) Centrifuge at 3000 r / min for 8 min and take the supernatant; (d) Filter the supernatant using a 0.22 μm organic phase filter membrane and collect the filtrate for standby.

[0035] (2) Gas chromatography - mass spectrometry (GC - MS) analysis: Perform analysis using a gas chromatography - mass spectrometry instrument. The chromatographic conditions are as follows: Chromatographic column: HP-5MS (60 m × 0.25 mm × 0.25 μm); Carrier gas: helium, flow rate 1 mL / min; Column temperature program: Initial temperature 90°C, hold for 5 min, increase to 200°C at 40°C / min, hold for 5 min, then increase to 280°C at 40°C / min, hold for 5 min; Total analysis time: 45 min.

[0036] The mass spectrometry conditions are the same as those in Example 1.

[0037] (3) Qualitative and quantitative analysis: The qualitative and quantitative methods are the same as those in Example 1.

[0038] Example 3:

[0039] An analytical method for simultaneously determining the contents of nicotine and three coolants in a nicotine pouch, comprising the following steps:

[0040] (1) Sample pretreatment: (a) Take about 2 g of the oral tobacco sample and weigh it precisely. (b) Add 15 mL of acetonitrile and ultrasonically extract for 12 min at room temperature. (c) Centrifuge at 4000 r / min for 12 min and take the supernatant. (d) Filter the supernatant using a 0.45 μm organic phase filter membrane and collect the filtrate for standby.

[0041] (2) Gas chromatography - mass spectrometry analysis: The chromatographic conditions are the same as those in Example 1, and the mass spectrometry conditions are also the same as those in Example 1.

[0042] (3) Qualitative and quantitative analysis: The qualitative and quantitative methods are the same as those in Example 1.

[0043] Example 4:

[0044] This example investigated the influence of different extraction solvents on the extraction efficiency. Acetonitrile, methanol, and dichloromethane were used as extraction solvents respectively, and other conditions were the same as those in Example 1. The results showed that when acetonitrile was used as the extraction solvent, the extraction efficiency for nicotine and three coolants was the highest, and the matrix interference was the smallest. This might be because acetonitrile has a moderate polarity, which can not only effectively extract the target compounds but also reduce the matrix interference.

[0045] Example 5:

[0046] This example investigated the influence of the ultrasonic extraction time on the extraction efficiency. The ultrasonic extraction times were set to 5 min, 10 min, and 15 min respectively, and other conditions were the same as those in Example 1. The results showed that when the ultrasonic extraction time was 10 min, the extraction efficiency for nicotine and three coolants reached the best balance, and further extending the extraction time did not significantly improve the extraction efficiency. This indicates that a 10 - minute ultrasonic extraction time is sufficient to effectively extract the target compounds and also ensure the analysis efficiency.

[0047] Example 6:

[0048] This example investigated the influence of the chromatographic column length on the separation effect. HP - 5MS chromatographic columns with lengths of 30 m, 60 m, and 80 m were used respectively, and other conditions were the same as those in Example 1. The results showed that the 60 - m chromatographic column could provide sufficient separation efficiency to achieve good separation of the target compounds. The 30 - m chromatographic column had an unsatisfactory separation for some compounds, and although the 80 - m chromatographic column had a better separation effect, the analysis time was significantly prolonged. Considering the separation effect and analysis efficiency comprehensively, the 60 - m chromatographic column is the best choice.

[0049] Example 7:

[0050] This example investigated the influence of the column temperature program on the separation effect. Different initial temperatures and heating rates were set, and other conditions were the same as in Example 1. The results showed that an initial temperature of 90 °C and a heating rate of 40 °C / min could achieve good separation of the target compounds in a relatively short time, ensuring a balance between analysis efficiency and separation effect. This column temperature program design considered the boiling point and polarity differences of the target compounds, ensuring the effective separation and detection of all target compounds in a single analysis.

[0051] Example 8:

[0052] This example investigated the linear range, precision, and accuracy of the method. By preparing standard solutions with different concentrations, a calibration curve was established to evaluate the linear range of the method; by performing multiple repeated determinations on the same sample, the precision of the method was evaluated; and by the standard addition recovery experiment, the accuracy of the method was evaluated. The results showed that the method had a good linear relationship (correlation coefficient r > 0.999) within the concentration range of the above calibration curve, and the precision (RSD < 3%) and accuracy (recovery rate of 95% - 105%) of the method both met the requirements of the analytical method validation. These results demonstrated the reliability and accuracy of the method and were suitable for the determination of nicotine and cooling agent contents in oral tobacco products.

[0053] Example 9:

[0054] This example applied the method to the analysis of commercially available oral tobacco products. Five different brands of oral tobacco products were randomly selected, and the contents of nicotine and three cooling agents were determined using the method of the present invention. The results showed that there were significant differences in the contents of the target compounds in different brands of oral tobacco products. Among them, the nicotine content was in the range of 5 - 15 mg / g, the menthol content was in the range of 0 - 10 mg / g, the WS-23 content was in the range of 0 - 2 mg / g, and the WS-3 content was in the range of 0 - 1 mg / g. This application example demonstrated the feasibility and practicality of the method in the analysis of actual samples and could provide technical support for the quality control and formulation optimization of oral tobacco products.

[0055] Example 10:

[0056] This example investigated the robustness of the method under different laboratory conditions. The same batch of samples was analyzed under different laboratory environments, different operators, and different instrument conditions. The results showed that even under different conditions, the method could still obtain stable and consistent analytical results, and the RSD between methods was less than 5%. This result verified the robustness and reliability of the method, indicating that it had strong adaptability and could be stably applied under different laboratory conditions.

[0057] As can be seen from the above embodiments, the analytical method provided by the present invention for simultaneously determining the contents of nicotine and three coolants in oral tobacco products is characterized by simple operation, fast analysis speed and high accuracy. Through the optimized sample pretreatment technology and gas chromatography-mass spectrometry coupling conditions, this method realizes the simultaneous determination of nicotine and coolants, and solves the problem of low analysis efficiency caused by the need for separate determination in traditional methods. At the same time, this method has a wide linear range, high precision and accuracy, can meet the actual needs of oral tobacco product analysis, and provides effective technical support for the quality control and research and development of oral tobacco products.

[0058] It should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. An analytical method for simultaneously determining the contents of nicotine and three cooling agents in snus, characterized in that: The following steps are involved: (1) Sample pretreatment: extracting nicotine and cooling agents from the oral tobacco sample using a Y-type gas-liquid-liquid extraction technique; (2) Gas chromatography-mass spectrometry analysis: injecting the pretreated sample solution into a gas chromatography-mass spectrometer for analysis; (3) qualitatively determining the content of nicotine and cooling agents in the sample based on the retention time and characteristic fragment ions of the standard and quantitatively determining the content of nicotine and cooling agents in the sample using an external standard method; wherein the cooling agents include menthol, WS-23, and WS-3.

2. The analysis method according to claim 1, characterized in that The Y-type gas-liquid-liquid extraction in step (1) specifically comprises the following steps: (a) taking an appropriate amount of oral smoke sample and weighing it accurately; (b) adding an appropriate amount of extraction solvent and ultrasonic extraction; (c) centrifuging and taking the supernatant; (d) filtering the supernatant and collecting the filtrate for later use.

3. The analysis method according to claim 1, characterized in that The gas chromatography conditions in step (2) are as follows: the chromatographic column is HP-5MS (60m×0.25mm×0.25μm); the carrier gas is helium with a flow rate of 1mL / min; the column temperature program is as follows: initial temperature 90°C, maintained for 5min, increased to 200°C at 40°C / min, maintained for 5min, then increased to 280°C at 40°C / min, maintained for 5min; the total analysis time is 45min.

4. The analysis method according to claim 1, characterized in that The mass spectrometry conditions in step (2) are as follows: the ion source is an electron impact ion source (EI); the ionization voltage is 70 eV; the transmission line temperature is 280°C; the ion source temperature is 230°C; the quadrupole temperature is 150°C; and the detection is performed in selected ion monitoring (SIM) mode.

5. The analysis method according to claim 1, characterized in that The retention time and characteristic fragment ions of each target compound in step (3) are as follows: (i) menthol: retention time 7.613 min, characteristic fragment ion m / z 71; (ii) WS-23: retention time 9.427 min, characteristic fragment ion m / z 114; (iii) WS-3: retention time 10.716 min, characteristic fragment ion m / z 100; (iv) nicotine: retention time 15.739 min, characteristic fragment ion m / z 84.

6. The analysis method according to claim 2, characterized in that The extraction solvent in the step (b) is acetonitrile.

7. The analysis method according to claim 2, characterized in that The ultrasonic extraction time in step (b) is 10 minutes, and the ultrasonic temperature is room temperature.

8. The analysis method according to claim 2, characterized in that The centrifugal conditions in step (c) are: centrifugal speed 3500 r / min, centrifugal time 10 min.

9. The analysis method according to claim 2, characterized in that In the step (d), a 0.22 μm organic phase filter membrane is used for filtration.

10. The analysis method according to claim 1, characterized in that The concentration ranges of the calibration curve of the external standard method in step (3) are: nicotine: 0.5-100 μg / mL; menthol: 0.5-100 μg / mL; WS-23: 0.1-20 μg / mL; WS-3: 0.1-20 μg / mL.

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