Analysis and detection method of 6-methyl nicotine

Abstract

The invention belongs to the technical field of analytical chemistry, and relates to a method for analyzing and detecting 6-methyl nicotine. The method comprises the following steps: (1) obtaining a detection solution containing 6-methyl nicotine; (2) carrying out high performance liquid chromatography analysis on the detection liquid; wherein the conditions of the high performance liquid chromatography are as follows: in a mobile phase, a triethylamine-containing disodium hydrogen phosphate aqueous solution is taken as a mobile phase A, and methanol is taken as a mobile phase B; a chromatographic column takes octadecyl as a filling agent; the elution mode is isocratic elution; the detection mode is ultraviolet detection; and (3) calculating the content of the 6-methyl nicotine in the detection solution. According to the method, the content of 6-methyl nicotine in the detection liquid is accurately and quantitatively analyzed through high performance liquid chromatography, and the method is good in specificity, high in sensitivity and accurate and reliable in result.

Description

Technical Field

[0001] The present application relates to the technical field of analytical chemistry, and more specifically, to an analytical detection method for 6-methylnicotine. Background Art

[0002] In recent years, new products such as electronic atomizers have developed rapidly in the European and American markets. According to IMARC Group, the global electronic atomizer market will reach US$23.3 billion in 2023. With the continuous emergence of new technologies and new products, WHO has warned that manufacturers may use non-nicotine tobacco alkaloids or other synthetic nicotine analogs to bypass the regulatory plans of various countries that only focus on nicotine. At the end of 2023, Charlie's Holdings Inc., an American e-cigarette company, launched the disposable atomizer product Spree Bar, whose active ingredient is a substance with the trademark name "Metatine" instead of nicotine. The active substance of this product is actually 6-methylnicotine, a nicotine analog.

[0003] Among them, the molecular formula of 6-methylnicotinamide is C 11 H 16 N2, molecular weight is 176.26, melting point is 20.9℃, boiling point is 259℃, density is 1.10g / cm3, solubility in water is 0.849mol / L, and physical and chemical properties are close to nicotine. Studies have found that compared with nicotine, 6-methylnicotine has higher cytotoxicity in the human bronchial epithelial cell line (BEAS-2B), but the effect of increasing lung cancer-related proteins is relatively mild. In the sensory evaluation, 1mg / mL of 6-methylnicotine in the atomized liquid has a similar "throat hit" compared to 3mg / mL nicotine, which indicates that 6-methylnicotine has the potential to replace nicotine in electronic atomization. At present, there are few reports on related research on testing 6-methylnicotine. Accurate detection of the content of 6-methylnicotine in the atomized liquid plays a key role in product production and research and development. Summary of the invention

[0004] The embodiment of the present application provides a method for analyzing and detecting 6-methylnicotine, which is used to solve the problem in the prior art of how to quantitatively detect and analyze the content of 6-methylnicotine in the atomizer of an electronic cigarette.

[0005] In order to solve the above technical problems, the present application provides a method for analyzing and detecting 6-methylnicotine, which adopts the following technical solution:

[0006] A method for analyzing and detecting 6-methylnicotine, the method comprising:

[0007] (1) obtaining a test solution containing 6-methylnicotine;

[0008] (2) The test solution is analyzed by high performance liquid chromatography; wherein the conditions of the high performance liquid chromatography analysis are as follows: mobile phase: sodium hydrogen phosphate aqueous solution containing triethylamine as mobile phase A, methanol as mobile phase B; chromatographic column: octadecyl as filler; elution method: isocratic elution; detection method: ultraviolet detection;

[0009] (3) Calculating the content of 6-methylnicotine in the test solution.

[0010] Furthermore, in step (2), the pH value of the mobile phase A is 4 to 5.5, and the concentration of the sodium hydrogen phosphate aqueous solution in the mobile phase A is 0.01 mol / L;

[0011] The mobile phase A is configured as follows: 1.42 g of disodium hydrogen phosphate is added with 1000 ml of water, and the pH value is adjusted to 4-5.5 with triethylamine.

[0012] Furthermore, in step (2), the detection wavelength of the ultraviolet detection is 258nm to 268nm.

[0013] Furthermore, in step (2), the volume ratio of the mobile phase B in the mobile phase is 10% to 30%; optionally, the volume ratio of the mobile phase B in the mobile phase is 20%.

[0014] Furthermore, in step (2), the column temperature of the chromatographic column is 30-40° C.; and the flow rate of the mobile phase is 0.9-1.1 mL / min.

[0015] Furthermore, the step (1) specifically comprises:

[0016] Weigh the electronic cigarette atomized liquid, add the extractant, and perform extraction by ultrasonic oscillation to obtain an extract;

[0017] The extract is filtered to obtain the test solution.

[0018] Furthermore, the extract is a 50% methanol aqueous solution; the step of "weighing the electronic cigarette atomizer liquid, adding the extractant, and extracting by ultrasonic oscillation to obtain the extract" specifically includes: weighing 0.1g of the electronic cigarette atomizer liquid sample into a 10mL volumetric flask, adding 50% methanol aqueous solution to the mark, and then ultrasonically oscillating for 10 minutes to obtain the extract;

[0019] The step of "filtering the extract" specifically includes: filtering the extract through a 0.22 μm filter membrane.

[0020] Furthermore, the concentration range of the 6-methylnicotine in the detection solution is 0.05 to 0.2 mg / mL.

[0021] Furthermore, the step (3) specifically includes:

[0022] Weigh 10 mg of 6-methylnicotine into a 10 mL volumetric flask, add 50% methanol aqueous solution and dilute to the mark to obtain a standard stock solution with a concentration of 1 mg / mL, and store it in a sealed container at 4°C away from light;

[0023] 50, 100, 200, 500, 1000, and 2000 μL of the standard stock solution were respectively transferred into 6 10 mL volumetric flasks, and diluted with 50% methanol aqueous solution and fixed to the mark to prepare a series of standard working solutions, wherein the concentration of 6-methylnicotine in each of the standard working solutions was 5, 10, 20, 50, 100, and 200 μg / mL, respectively;

[0024] Analyzing each of the standard working solutions by high performance liquid chromatography to obtain the peak area of ​​6-methylnicotine in each of the standard working solutions, and establishing a regression curve equation;

[0025] Substituting the analysis result of the step (2) into the regression curve equation to obtain the concentration of the 6-methylnicotine in the test solution; calculating the dilution factor according to the concentration of the standard stock solution; and then calculating the content of the 6-methylnicotine in the test solution according to the dilution factor.

[0026] Furthermore, the chromatographic column is a C18 column, the injection volume of the chromatographic column is 10-50 μL, the length of the chromatographic column is 250 mm, the diameter is 4.6 mm, and the filler particle size is 5 μm.

[0027] Compared with the prior art, the embodiments of the present application have the following beneficial effects: the content of 6-methylnicotine in the test solution is accurately and quantitatively analyzed by high performance liquid chromatography, with good specificity, high sensitivity, and accurate and reliable results. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the scheme of the present application, a brief introduction is given below to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 It is a flow chart of an embodiment of a method for analyzing and detecting 6-methylnicotine in the present application;

[0030] Figure 2 is the ultraviolet absorption spectrum of 6-methylnicotine;

[0031] Figure 3 The chromatograms of 6-methylnicotine under mobile phase A with different pH values;

[0032] Figure 4 The chromatograms are compared when the pH value of mobile phase A is 5 and the volume proportion of mobile phase B in the mobile phase is different;

[0033] Figure 5 The chromatograms are compared when the pH value of mobile phase A is 4 and the volume proportion of mobile phase B in the mobile phase is different;

[0034] Figure 6 It is a comparative chromatogram of different test solutions tested under the same HPLC conditions;

[0035] Figure 7 This is the chromatogram of 6-methylnicotine when mobile phase B is acetonitrile.

[0036] Figure 8 This is the chromatogram of 6-methylnicotine when mobile phase B is methanol. DETAILED DESCRIPTION

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of this application; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of this application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0038] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0039] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0040] The present application embodiment provides a method for analyzing and detecting 6-methylnicotine, such as Figure 1 As shown, the method includes:

[0041] (1) obtaining a test solution containing 6-methylnicotine;

[0042] (2) The test solution is analyzed by high performance liquid chromatography; wherein the conditions of the high performance liquid chromatography analysis are as follows: mobile phase: sodium hydrogen phosphate aqueous solution containing triethylamine as mobile phase A, methanol as mobile phase B; chromatographic column: octadecyl as filler; elution method: isocratic elution; detection method: ultraviolet detection;

[0043] (3) Calculating the content of 6-methylnicotine in the test solution.

[0044] In this embodiment, the mobile phase A contains triethylamine, which is weakly alkaline and can adjust the pH value of the mobile phase A, thereby optimizing the chromatographic separation conditions, helping to reduce peak tailing, and improving the symmetry of the peak shape; moreover, triethylamine can improve the adsorption behavior of 6-methylnicotine on the chromatographic column, making the peak shape sharper and more symmetrical, thereby improving the resolution.

[0045] In this embodiment, the content of 6-methylnicotine in the test solution is accurately and quantitatively analyzed by high performance liquid chromatography, which has good specificity, high sensitivity, and accurate and reliable results.

[0046] Furthermore, in step (2), the pH value of the mobile phase A is 4 to 5.5, and the concentration of the sodium hydrogen phosphate aqueous solution in the mobile phase A is 0.01 mol / L; the mobile phase A is configured as follows: take 1.42 g of sodium hydrogen phosphate, add 1000 ml of water, and adjust the pH value to 4 to 5.5 with triethylamine.

[0047] In this embodiment, in the high performance liquid chromatography analysis, the tailing factor T should be between 0.95 and 1.05, and when the pH value of the mobile phase A is between 4 and 5.5, the quantitative separation requirements of the target peak are met.

[0048] In this embodiment, since triethylamine is weakly alkaline, after preparing the sodium hydrogen phosphate aqueous solution, triethylamine is used to adjust the pH value, so that the pH value can be adjusted to an appropriate range.

[0049] In some optional implementations of this embodiment, the pH value of the mobile phase A is any one of 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.5, etc., or a range between any two of them.

[0050] Preferably, the pH value of the mobile phase A is 5.

[0051] Furthermore, in step (2), the detection wavelength of the ultraviolet detection is 258nm to 268nm.

[0052] In this embodiment, the peak area of ​​6-methylnicotine is relatively large under the detection wavelength of 258nm to 268nm under ultraviolet detection, and it is not easily interfered by impurity peaks.

[0053] Preferably, the detection wavelength of the ultraviolet detection is 263 nm.

[0054] In some optional implementations of this embodiment, the detection wavelength of the ultraviolet detection is any one of 258nm, 259nm, 260nm, 261nm, 262nm, 263nm, 264nm, 265nm, 266nm, 267nm, 268nm, etc., or a range between any two of them.

[0055] Furthermore, in step (2), the volume ratio of the mobile phase B in the mobile phase is 10% to 30%; optionally, the volume ratio of the mobile phase B in the mobile phase is 20%.

[0056] In this embodiment, when the volume ratio of mobile phase B in the mobile phase is 10% to 30%, it can avoid that the chromatographic peak may appear too quickly, thereby avoiding overlap with the impurity peak; it can also further expand the ability to simultaneously detect nicotine and 6-methylnicotine in the test solution; when the volume ratio of mobile phase B in the mobile phase is 20%, the peak shape and separation of 6-methylnicotine and nicotine are better, and the analysis time is faster.

[0057] Furthermore, in step (2), the column temperature of the chromatographic column is 30-40° C.; and the flow rate of the mobile phase is 0.9-1.1 mL / min.

[0058] In this embodiment, the column temperature of the chromatographic column is 30-40° C., which can avoid damage to the chromatographic column.

[0059] In this embodiment, the flow rate of the mobile phase is 0.9-1.1 mL / min, which can avoid excessively long analysis time.

[0060] Furthermore, the step (1) specifically comprises:

[0061] Weigh the electronic cigarette atomized liquid, add the extractant, and perform extraction by ultrasonic oscillation to obtain an extract;

[0062] The extract is filtered to obtain the test solution.

[0063] In this embodiment, the electronic cigarette atomizer liquid is weighed, and after adding the extractant, ultrasonic oscillation is used for extraction to obtain an extract liquid, which can improve the separation degree of 6-methylnicotine in the electronic cigarette atomizer liquid; the extract liquid is filtered to obtain a detection liquid, which reduces the particulate impurities in the detection liquid and improves the detection accuracy.

[0064] Specifically, the extract is a 50% methanol aqueous solution; the step of "weighing the electronic cigarette atomizer liquid, adding the extractant, and extracting by ultrasonic oscillation to obtain the extract" specifically includes: weighing 0.1g of the electronic cigarette atomizer liquid sample into a 10mL volumetric flask, adding 50% methanol aqueous solution to the mark, and then ultrasonically oscillating for 10 minutes to obtain the extract.

[0065] Specifically, the step of "filtering the extract" comprises: filtering the extract through a 0.22 μm filter membrane.

[0066] Furthermore, the concentration range of the 6-methylnicotine in the detection solution is 0.05 to 0.2 mg / mL.

[0067] In this embodiment, the concentration range of 6-methylnicotine in the detection solution is 0.05-0.2 mg / mL, so that the instrument can detect 6-methylnicotine, and can avoid the situation where the instrument response is too large due to the high concentration of 6-methylnicotine, thereby causing asymmetric chromatographic peaks, and can also avoid residual contamination of the detection instrument.

[0068] Specifically, the step (3) comprises:

[0069] Weigh 10 mg of 6-methylnicotine into a 10 mL volumetric flask, add 50% methanol aqueous solution and dilute to the mark to obtain a standard stock solution with a concentration of 1 mg / mL, and store it in a sealed container at 4°C away from light;

[0070] 50, 100, 200, 500, 1000, and 2000 μL of the standard stock solution were respectively transferred into 6 10 mL volumetric flasks, and diluted with 50% methanol aqueous solution and fixed to the mark to prepare a series of standard working solutions, wherein the concentration of 6-methylnicotine in each of the standard working solutions was 5, 10, 20, 50, 100, and 200 μg / mL, respectively;

[0071] Analyzing each of the standard working solutions by high performance liquid chromatography to obtain the peak area of ​​6-methylnicotine in each of the standard working solutions, and establishing a regression curve equation;

[0072] Substituting the analysis result of the step (2) into the regression curve equation to obtain the concentration of the 6-methylnicotine in the test solution; calculating the dilution factor according to the concentration of the standard stock solution; and then calculating the content of the 6-methylnicotine in the test solution according to the dilution factor.

[0073] For example, if the analysis result of step (2) is substituted into the regression curve equation, the concentration of 6-methylnicotine in the test solution is 10 μg / mL, and the concentration of the standard stock solution is 1 mg / mL, then the dilution factor is 100, that is, the concentration of 6-methylnicotine in the test solution is 0.01 mg / mL. Finally, 0.01 mg / mL is multiplied by the volume of the test solution: 10 mL, and the content of 6-methylnicotine in 0.1 g of the electronic cigarette atomizer liquid sample is calculated to be 0.1 mg.

[0074] For example, if the analysis result of step (2) is substituted into the regression curve equation, the concentration of 6-methylnicotine in the test solution is 200 μg / mL, and the concentration of the standard stock solution is 1 mg / mL, then the dilution factor is 5, that is, the concentration of 6-methylnicotine in the test solution is 0.2 mg / mL. Finally, 0.2 mg / mL is multiplied by the volume of the test solution: 10 mL, and the content of 6-methylnicotine in 0.1 g of the electronic cigarette atomizer liquid sample is calculated to be 2 mg.

[0075] Furthermore, the chromatographic column is a C18 column, the injection volume of the chromatographic column is 10-50 μL, the length of the chromatographic column is 250 mm, the diameter is 4.6 mm, and the filler particle size is 5 μm.

[0076] The above scheme is further described below in conjunction with specific implementation examples. The preferred embodiments of the present invention are described in detail as follows:

[0077] Example 1: Selection of detection wavelength for ultraviolet detection

[0078] The United States Pharmacopoeia USP40 stipulates that the detection wavelength of nicotine organic impurities is 254nm. 6-methylnicotine has a similar structure to nicotine. A test solution containing 6-methylnicotine is prepared. The concentration of 6-methylnicotine in the test solution is 100μg / mL. The ultraviolet absorption wavelength is detected at 190nm~400nm. The test results are shown in Figure 2 There are large absorption peaks at 209nm and 263nm, but there is easy interference from solvent peaks at low bands. The electronic cigarette atomizer may contain a variety of solvents, resulting in low-band absorption interference. Therefore, 263nm is preferred for further testing. The peak areas of multiple different absorption wavelengths are detected at 250nm~275nm. The test results are shown in Table 1. It can be seen that there is a better absorption wavelength at 258~268nm, and the maximum absorption wavelength in this band is 263nm.

[0079] Table 1

[0080] Absorption wavelength Peak area 250 717394 254 893795 258 1081651 259 1123091 260 1158852 261 1187302 262 1207373 263 1222911 264 1219492 265 1209794 266 1188488 267 1155266 268 1110615 270 991403 272 838043 275 568954

[0081] Example 2: Selection of pH value of mobile phase A

[0082] Prepare a test solution containing 6-methylnicotine, the concentration of 6-methylnicotine in the test solution is 100μg / mL; use a high performance liquid chromatograph UV-visible detector, the wavelength of the UV detector is 263nm, and select a chromatographic column with octadecyl bonded silica particles with a particle size of 5μm as a filler. Mobile phase A is a sodium dihydrogen phosphate aqueous solution containing triethylamine, and mobile phase B is methanol. The pH value of mobile phase A is adjusted by triethylamine, and the pH values ​​are 3, 4, 4.5, 5, 5.5, 5.8, 6, and 8, respectively. The chromatograms of 6-methylnicotine under mobile phase A with different pH values ​​are shown in Figure 2. Figure 3 ; The black line represents the chromatographic curve of 6-methylnicotine when the pH value of mobile phase A is 3; the purple line represents the chromatographic curve of 6-methylnicotine when the pH value of mobile phase A is 4; the blue line represents the chromatographic curve of 6-methylnicotine when the pH value of mobile phase A is 4.5; the brown line represents the chromatographic curve of 6-methylnicotine when the pH value of mobile phase A is 5; the green line represents the chromatographic curve of 6-methylnicotine when the pH value of mobile phase A is 5.5; the dark blue line represents the chromatographic curve of 6-methylnicotine when the pH value of mobile phase A is 5.8; the yellow line represents the chromatographic curve of 6-methylnicotine when the pH value of mobile phase A is 6.

[0083] The peak time and tailing factor detection results of 6-methylnicotine under mobile phase A with different pH values ​​are shown in Table 2. The results show that the larger the pH value, the later the peak time of 6-methylnicotine. When the pH value is 8, the peak time is 49.56min, and the analysis time is too long; when pH ≤ 5.5, the peak time is less than 5min; when the pH is 3, the solvent peak is easily covered.

[0084] In addition, in HPLC analysis, the tailing factor T should be between 0.95 and 1.05. When the pH value is greater than 5, the tailing factor is greater than 1.05. The tailing factor increases with the increase of pH value. When the pH value is ≤3, the tailing factor is greater than 1.05. Therefore, the pH range of 4.0 to 5.5 meets the quantitative requirements for separation of the target peak.

[0085] Table 2

[0086]

[0087]

[0088] Example 3: Selection of the volume ratio of mobile phase B in the mobile phase

[0089] A test solution containing nicotine and 6-methylnicotine was prepared, the concentrations of nicotine and 6-methylnicotine in the test solution were 100 μg / mL, respectively. A high performance liquid chromatograph UV-visible detector was used, the wavelength of the UV detector was 263 nm, and a chromatographic column with octadecyl bonded silica particles with a particle size of 5 μm was selected as a filler. Mobile phase A was a sodium dihydrogen phosphate aqueous solution containing triethylamine, and mobile phase B was methanol. The proportion of mobile phase B was adjusted to 10%, 20%, 25%, and 30%, and the differences in mobile phase A under different pH conditions were compared. The test results are shown in FIG. Figure 4 and Figure 5 .

[0090] Figure 4 The black line in the middle is the chromatographic curve when the pH value of mobile phase A is 5 and the volume proportion of mobile phase B in the mobile phase is 10%; Figure 4 The purple line in the middle is the chromatographic curve when the pH value of mobile phase A is 5 and the volume proportion of mobile phase B in the mobile phase is 20%; Figure 4 The blue line in the middle is the chromatographic curve when the pH value of mobile phase A is 5 and the volume proportion of mobile phase B in the mobile phase is 25%; Figure 4 The brown line in the middle is the chromatographic curve when the pH value of mobile phase A is 5 and the volume proportion of mobile phase B in the mobile phase is 30%.

[0091] Figure 5 The black line in the middle is the chromatographic curve when the pH value of mobile phase A is 4 and the volume proportion of mobile phase B in the mobile phase is 10%; Figure 4 The purple line in the middle is the chromatographic curve when the pH value of mobile phase A is 4 and the volume proportion of mobile phase B in the mobile phase is 20%; Figure 4 The blue line in the middle is the chromatographic curve when the pH value of mobile phase A is 4 and the volume proportion of mobile phase B in the mobile phase is 25%; Figure 4 The brown line in the middle is the chromatographic curve when the pH value of mobile phase A is 4 and the volume proportion of mobile phase B in the mobile phase is 30%.

[0092] It can be seen that 6-methylnicotine can be effectively separated when the pH value of mobile phase A is 4 or 5; and the higher the proportion of mobile phase B, the faster the peak elution, but nicotine has a front peak and a shoulder peak when the pH value of mobile phase A is 4, which is not suitable for the simultaneous detection of nicotine and 6-methylnicotine; when the pH value of mobile phase A is 5, and when the ratio of mobile phase B: methanol is 10% to 30%, the ability to simultaneously detect nicotine and 6-methylnicotine in the liquid can be further expanded. When the proportion of mobile phase B is 20%, the peak shape and separation of 6-methylnicotine and nicotine are better, and the analysis time is faster.

[0093] Example 4: Specificity Experiment

[0094] Electronic cigarette atomizers generally contain propylene glycol, glycerol, nicotine and flavors. Nicotine and other flavor ingredients may interfere with the detection of 6-methylnicotine. Select electronic cigarette atomizers with flavors of grape apple (nicotine concentration is 30mg / mL), frozen mint (nicotine concentration is 30mg / mL), strawberry milkshake (nicotine concentration is 30mg / mL), Slurpee cola (nicotine concentration is 30mg / mL), and tobacco (nicotine concentration is 18mg / mL) containing nicotine, and prepare the detection liquid respectively. The concentration of nicotine in the detection liquid is 0.3mg / mL. And prepare the detection liquid containing 6-methylnicotine, and the concentration of 6-methylnicotine in the detection liquid is 0.3mg / mL.

[0095] Tested under the same HPLC conditions, the test results are shown in Figure 6 ; Specifically, the HPLC conditions are: mobile phase A: 0.01 mol / L sodium dihydrogen phosphate aqueous solution containing triethylamine (pH = 5), mobile phase B: methanol, the volume ratio of mobile phase B in the mobile phase is 20%; elution mode: isocratic elution; detection wavelength of UV detection: 263 nm; column temperature of the chromatographic column: 35°C; injection volume of the chromatographic column: 10 μL; mobile phase flow rate is flow rate: 1 mL / min.

[0096] Figure 6 In the figure, the black line is the chromatographic curve of the grape apple flavored electronic cigarette aerosol liquid, the purple line is the chromatographic curve of the frozen mint flavored detection liquid, the blue line is the chromatographic curve of the strawberry milkshake flavored detection liquid, the brown line is the chromatographic curve of the Slurpee Cola flavored detection liquid, the green line is the chromatographic curve of the tobacco flavored detection liquid, and the dark blue line is the chromatographic curve of the detection liquid containing 6-methylnicotine; the results show that the nicotine and 6-methylnicotine in all the test samples are well separated, and other compounds with ultraviolet absorption do not interfere with the detection of 6-methylnicotine, indicating good specificity.

[0097] Example 5: Experiments on the linearity, precision, reproducibility, recovery, detection limit, quantification limit, etc. of the method

[0098] Preparation of standard working solution: Accurately weigh an appropriate amount of 6-methylnicotine to prepare a series of working solutions with concentrations of 5, 10, 20, 50, 100, and 200 μg / mL.

[0099] Preparation of sample test solution: weigh 0.1g of electronic cigarette atomizer sample into a 10mL volumetric flask, add 50% methanol aqueous solution to the mark, and then ultrasonically oscillate for 10 minutes to obtain an extract, and filter the extract through a 0.22μm filter membrane.

[0100] Chromatographic conditions: Use a high performance liquid chromatograph with a UV-visible detector, the wavelength of the UV detector is 263 nm, and use a chromatographic column with octadecyl bonded silica particles with a particle size of 5 μm as a filler. Mobile phase A is a sodium dihydrogen phosphate aqueous solution containing triethylamine (pH = 5), and mobile phase B is methanol. The flow rate is 1.0 mL / min, and the column temperature of the chromatographic column is 35°C.

[0101] The established high performance liquid chromatography analysis method for detecting 6-methylnicotine content was methodologically validated according to the above-mentioned chromatographic parameters. The validation items included linearity, limit of quantification, limit of detection, precision, accuracy, and solution stability.

[0102] Instrument precision test: The standard working solutions with 6-methylnicotine concentrations of 5, 50, and 200 μg / mL were injected six times in succession, and the RSDs were 0.82%, 0.47%, and 0.21%, respectively.

[0103] Linearity: The linear equation of 6-methylnicotine in the concentration range of 5-200 μg / mL is Y=12335.6X+11730.9; correlation coefficient is 0.9998;

[0104] Detection limit and quantification limit: The concentration of 6-methylnicotine in the detection limit solution is 0.2μg / mL, and the S / N is 3.5. According to the dilution multiple, it is equivalent to 20ppm of 6-methylnicotine in the sample. The concentration of 6-methylnicotine in the quantification limit solution is 0.5μg / mL, and the minimum S / N is 10.23, which is equivalent to 50ppm of 6-methylnicotine in the sample. The quantitative limit solution was injected 6 times continuously, and the RSD of the peak area of ​​6-methylnicotine was 1.09%.

[0105] Accuracy: A spike recovery test was performed with three levels of concentration, where the concentrations of 6-methylnicotine were 1 mg / g, 5 mg / g, and 20 mg / g, respectively. The recovery rates were between 96.4% and 99.1%, and the RSD of the recovery rate was 0.77%.

[0106] Solution stability test: A sample containing 6-methylnicotine at a concentration of about 5 mg / g was pre-treated to obtain a sample test solution, which was placed at 15°C and measured at 0, 24 hours, and 48 hours. The RSD of the three contents was 0.98, and it was considered that the sample test solution was stable within 48 hours under this condition.

[0107] Comparative Example 1

[0108] A high performance liquid chromatograph UV-visible detector was used, the wavelength of the UV detector was 263 nm, and a chromatographic column with octadecyl bonded silica particles with a particle size of 5 μm was selected as a filler. Mobile phase A was water, and no buffer salt was added. Mobile phase B was acetonitrile or methanol, and the volume ratio of mobile phase B in the mobile phase was 30%.

[0109] When mobile phase B is acetonitrile, the chromatogram of 6-methylnicotine is shown in Figure 7 ; When mobile phase B is methanol, the chromatogram of 6-methylnicotine is shown in Figure 8 ; The results showed that when mobile phase B was methanol, the 6-methylnicotine peak had severe tailing; when mobile phase B was acetonitrile, 6-methylnicotine eluted too quickly, the peak shape was tailing, and it co-eluted with the solvent peak, indicating that adding disodium hydrogen phosphate aqueous solution can significantly improve the peak shape.

[0110] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A method for analyzing and detecting 6-methylnicotine, characterized in that: The method comprises: (1) obtaining a test solution containing 6-methylnicotine; (2) The test solution is analyzed by high performance liquid chromatography; wherein the conditions of the high performance liquid chromatography analysis are as follows: mobile phase: sodium hydrogen phosphate aqueous solution containing triethylamine as mobile phase A, methanol as mobile phase B; chromatographic column: octadecyl as filler; elution method: isocratic elution; detection method: ultraviolet detection; (3) Calculating the content of 6-methylnicotine in the test solution.

2. The method for analyzing and detecting 6-methylnicotine according to claim 1, characterized in that: In step (2), the pH value of the mobile phase A is 4 to 5.5, and the concentration of the sodium hydrogen phosphate aqueous solution in the mobile phase A is 0.01 mol / L; The mobile phase A is configured as follows: 1.42 g of disodium hydrogen phosphate is added with 1000 ml of water, and the pH value is adjusted to 4-5.5 with triethylamine.

3. The method for analyzing and detecting 6-methylnicotine according to claim 1, characterized in that: In step (2), the detection wavelength of the ultraviolet detection is 258nm to 268nm.

4. The method for analyzing and detecting 6-methylnicotine according to claim 1, characterized in that: In step (2), the volume ratio of the mobile phase B in the mobile phase is 10% to 30%; optionally, the volume ratio of the mobile phase B in the mobile phase is 20%.

5. The method for analyzing and detecting 6-methylnicotine according to claim 1, characterized in that: In step (2), the column temperature of the chromatographic column is 30-40° C.; the flow rate of the mobile phase is 0.9-1.1 mL / min.

6. The method for analyzing and detecting 6-methylnicotine according to claim 1, characterized in that: The step (1) specifically comprises: Weigh the electronic cigarette atomized liquid, add the extractant, and perform extraction by ultrasonic oscillation to obtain an extract; The extract is filtered to obtain the test solution.

7. The method for analyzing and detecting 6-methylnicotine according to claim 6, characterized in that: The extract is a 50% methanol aqueous solution; the step of "weighing the electronic cigarette atomizer liquid, adding the extractant, and extracting by ultrasonic oscillation to obtain the extract" specifically includes: weighing 0.1g of the electronic cigarette atomizer liquid sample into a 10mL volumetric flask, adding 50% methanol aqueous solution to the mark, and then ultrasonically oscillating for 10 minutes to obtain the extract; The step of "filtering the extract" specifically includes: filtering the extract through a 0.22 μm filter membrane.

8. The method for analyzing and detecting 6-methylnicotine according to claim 6 or 7, characterized in that: The concentration of 6-methylnicotine in the detection solution is in the range of 0.05 to 0.2 mg / mL.

9. The method for analyzing and detecting 6-methylnicotine according to claim 7, characterized in that: The step (3) specifically comprises: Weigh 10 mg of 6-methylnicotine into a 10 mL volumetric flask, add 50% methanol aqueous solution and dilute to the mark to obtain a standard stock solution with a concentration of 1 mg / mL, and store it in a sealed container at 4°C away from light; 50, 100, 200, 500, 1000, and 2000 μL of the standard stock solution were respectively transferred into 6 10 mL volumetric flasks, and diluted with 50% methanol aqueous solution and fixed to the mark to prepare a series of standard working solutions, wherein the concentration of 6-methylnicotine in each of the standard working solutions was 5, 10, 20, 50, 100, and 200 μg / mL, respectively; Analyzing each of the standard working solutions by high performance liquid chromatography to obtain the peak area of ​​6-methylnicotine in each of the standard working solutions, and establishing a regression curve equation; Substituting the analysis result of the step (2) into the regression curve equation to obtain the concentration of the 6-methylnicotine in the test solution; calculating the dilution factor according to the concentration of the standard stock solution; and then calculating the content of the 6-methylnicotine in the test solution according to the dilution factor.

10. The method for analyzing and detecting 6-methylnicotine according to claim 1, characterized in that: The chromatographic column adopts a C18 column, the injection volume of the chromatographic column is 10-50 μL, the length of the chromatographic column is 250 mm, the diameter is 4.6 mm, and the filler particle size is 5 μm.

Images