A method for chromatographic separation of 5-methylnicotine and 6-methylnicotine and applications thereof

Abstract

The application discloses a method for chromatographic separation and application of 5-methylnicotine and 6-methylnicotine. The method comprises the following steps: injecting a to-be-detected liquid into a gas chromatograph-mass spectrometer for detection; at a chromatographic separation end, a polar capillary column is adopted, a temperature rising program is set, and the temperature is raised at a temperature rising rate of not higher than 5 DEG C / min in a temperature interval in which target isomers are eluted, so that a chromatographic retention time difference of 5-methylnicotine and 6-methylnicotine is greater than 1 min; at a mass spectrometric detection end, an electron impact ion source is adopted to detect the separated effluent, and 5-methylnicotine and 6-methylnicotine are distinguished and qualitatively determined according to the retention time difference generated by the two. The technical scheme of the application utilizes the synergistic effect of a polar stationary phase and slow temperature rising, greatly amplifies the weak polarity difference of 5-methylnicotine and 6-methylnicotine, realizes baseline separation, and thus accurately determines the quality according to the retention time difference, and realizes the simultaneous distinguishing detection of the two.

Description

Technical Field

[0001] This application relates to the field of novel tobacco product analysis and detection technology, and in particular to a chromatographic method for distinguishing between 5-methylnicotine and 6-methylnicotine and its application. Background Technology

[0002] With the rapid development of new tobacco products (such as e-liquids), the accurate detection of nicotine and its derivatives is crucial for product quality control and safety assessment. 5-Methylnicotine and 6-methylnicotine, as methylated isomers of nicotine, are increasingly used in new tobacco formulations due to their differences in sensory experience and physiological effects. Therefore, establishing a method that can accurately distinguish and determine these two isomers is of significant practical importance.

[0003] Currently, gas chromatography-mass spectrometry (GC-MS) is the mainstream method for detecting nicotine-related substances in the industry. However, 5-methylnicotine and 6-methylnicotine are positional isomers with identical molecular formulas. Under the commonly used electron impact ionization (EI) source in GC-MS, their mass spectrometric fragmentation behavior is identical, producing the same quantitative and qualitative ions, such as... Figure 1 and Figure 2 As shown. This means that it is absolutely impossible to distinguish between the two using characteristic fragment ions at the mass spectrometry detection end. Since they co-elute on the column, the mass spectrometer will become ineffective, inevitably leading to false positives in qualitative analysis or severely distorted quantitative results. Conventional gas chromatography-mass spectrometry (GC-MS) cannot achieve the distinguishing detection of these two components.

[0004] Therefore, there is an urgent need for a detection method that can accurately distinguish between the two. Summary of the Invention

[0005] This application provides a chromatographic method and application for distinguishing 5-methylnicotine and 6-methylnicotine, aiming to solve the problem in the prior art that 5-methylnicotine and 6-methylnicotine cannot be simultaneously distinguished and detected due to the consistency of mass spectrometry fragments.

[0006] To achieve the above objectives, this application proposes a chromatographic method for distinguishing between 5-methylnicotine and 6-methylnicotine, comprising: The test solution was injected into a gas chromatograph-mass spectrometer for detection. At the chromatographic separation end, a polar capillary column was used, and a temperature program was set. The temperature was increased at a rate not exceeding 5℃ / min within the temperature range for eluting the target isomer, so that the difference in chromatographic retention time between 5-methylnicotine and 6-methylnicotine was greater than 1 min. At the mass spectrometry detection end, an electron impact ion source is used to detect the separated effluent, and the two are distinguished and identified based on the difference in retention time between 5-methylnicotine and 6-methylnicotine.

[0007] In some embodiments, the temperature range for eluting the target isomer at a heating rate not exceeding 5°C / min is 180°C to 200°C.

[0008] In some embodiments, the temperature is increased at a rate of 1.5 to 3.5 °C / min within the temperature range for eluting the target isomer.

[0009] In some embodiments, the heating process is a multi-stage heating process: before reaching the temperature range for eluting the target isomer, the temperature is increased at a first heating rate of not less than 10 °C / min; within the temperature range for eluting the target isomer, the temperature is increased at a second heating rate of not more than 5 °C / min; and after the temperature range for eluting the target isomer, the temperature is increased at a third heating rate of not less than 10 °C / min.

[0010] In some embodiments, the polar capillary column is a polyethylene glycol capillary column.

[0011] In some embodiments, when injecting the test solution into the gas chromatograph-mass spectrometer, the injection mode is split injection with a split ratio of (10-50):1; and / or, the solvent delay time for mass spectrometry detection is 5-10 min.

[0012] In some embodiments, a liquid sample pretreatment step is included before injecting the test liquid into the gas chromatograph-mass spectrometer: weighing the electronic vaporization liquid sample, diluting and making up to volume with a methanol solution containing an internal standard, mixing and filtering to obtain the test liquid.

[0013] In some embodiments, a gaseous sample pretreatment step is included before injecting the test solution into the gas chromatograph-mass spectrometer: under conditions simulating manual aspiration, an electronic atomizer is aspirated using a smoke extractor, and aerosols are captured through a Cambridge filter; the captured Cambridge filter is placed in a methanol solution containing an internal standard for vortex extraction, and the test solution is obtained after filtration.

[0014] In some embodiments, the internal standard is n-heptadecane, and the concentration of n-heptadecane in the methanol solution containing the internal standard is 15~25 mg / L.

[0015] This application also provides an application of a chromatographic method for distinguishing 5-methylnicotine from 6-methylnicotine in quality control.

[0016] This application proposes a chromatographic method and its application for distinguishing 5-methylnicotine from 6-methylnicotine. The method includes the following steps: injecting the test solution into a gas chromatography-mass spectrometry (GC-MS) instrument for detection; at the chromatographic separation end, using a polar capillary column, setting a temperature program, and heating at a rate not exceeding 5℃ / min within the elution temperature range of the target isomer, so that the chromatographic retention time difference between 5-methylnicotine and 6-methylnicotine is greater than 1 min; at the mass spectrometry detection end, using an electron impact ion source to detect the separated effluent, and distinguishing and qualitatively identifying the two based on the retention time difference. This application's technical solution has the following beneficial effects: A polar capillary column was used with a slow heating program of no more than 5°C / min within the elution temperature range of the target isomer, which increased the chromatographic retention time difference between the two isomers to more than 1 min. Based on the synergistic effect of the polar stationary phase and the slow heating, the slight polarity difference between the isomers was greatly amplified, and baseline separation between the two was achieved in physical space. Thus, accurate identification was performed based on the retention time difference, avoiding false positives and abnormally high quantitative results caused by co-elution, and achieving simultaneous differentiation and detection of this nicotine isomer in the industry. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort, wherein: Figure 1 This is a schematic diagram of the mass spectrometry information of 5-methylnicotine in this application; Figure 2 This is a schematic diagram of the mass spectrometry information of 6-methylnicotine in this application; Figure 3 This is a schematic diagram showing the qualitative results of 5-methylnicotine and 6-methylnicotine in this application; Figure 4 A schematic diagram of the linear regression standard curve for 5-methylnicotine using the chromatographic differentiation method of this application; Figure 5 A schematic diagram of the linear regression standard curve for 6-methylnicotine using the chromatographic differentiation method of this application; Figure 6 This is a schematic diagram of the TIC of 5-methylnicotine and 6-methylnicotine in this application. Detailed Implementation

[0018] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0019] It should be noted that, unless otherwise stated or limited, all directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of this application are only used to explain the relative positional relationship and movement of each component in a certain specific posture (as shown in the figure). If the specific posture changes, the directional indication will also change accordingly.

[0020] It should also be noted that, unless otherwise stated or limited, when an element is referred to as "fixed to" or "set on" another element, it may be directly on the other element or there may be an intervening element present. When an element is referred to as "connected to" another element, it may be directly connected to the other element or there may be an intervening element present.

[0021] Furthermore, unless otherwise stated or limited, the descriptions involving "first," "second," etc., in this application are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. Additionally, the technical solutions of the various embodiments can be combined with each other, but only on the basis of being achievable by those skilled in the art. When the combination of technical solutions is contradictory or impossible to implement, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed in this application.

[0022] This application proposes a chromatographic method for distinguishing between 5-methylnicotine and 6-methylnicotine, which mainly includes the following steps: S1. Sample pretreatment to obtain the test solution; For liquid samples (e-cigarette vaporizer): Weigh the e-cigarette vaporizer sample, dilute it directly to volume with a methanol solution containing an internal standard, mix well, filter, and obtain the test solution. This eliminates the need for cumbersome purification steps such as solid-phase extraction, and significantly improves the detection efficiency of liquid samples by relying on the excellent separation and anti-interference capabilities of the subsequent instrument.

[0023] For gaseous samples (e-cigarette aerosol): Under simulated manual inhalation conditions, the e-cigarette was inhaled using a vaping device, and the aerosol was captured through a Cambridge filter. The captured Cambridge filter was then placed in a methanol solution containing an internal standard for shaking extraction, followed by filtration to obtain the test solution. The inhalation test parameters for this e-cigarette are shown in Table 1. Table 1. Electronic atomizer inhalation test parameters

[0024] Meanwhile, during the vaping test, the tilt angle α of the e-cigarette tank is set to -30° to -60°, preferably -45°, which ensures that the e-liquid is always fully wetted by the wicking cotton during the vaping process, avoiding isomer degradation or release distortion caused by dry burning, thereby accurately obtaining the true release amount of the target substance in the aerosol.

[0025] For both liquid and gaseous samples, n-heptadecane is the preferred internal standard, with a concentration of 15–25 mg / L, preferably 20 mg / L, in a methanol solution containing the internal standard. Heptadecane has a later chromatographic retention time than the target analyte, avoiding cross-interference and offering low cost. Setting a concentration range that matches the actual target analyte concentration in the sample prevents calibration deviations due to excessively low internal standard concentrations or mass spectrometry signal saturation due to excessively high concentrations, ensuring the quantitative accuracy of internal standard calibration.

[0026] S2. Inject the test solution into the gas chromatograph-mass spectrometer for detection.

[0027] At the chromatographic separation end, a polar capillary column was used, and a temperature program was set. The temperature was increased at a rate not exceeding 5℃ / min within the temperature range for eluting the target isomer, so that the difference in chromatographic retention time between 5-methylnicotine and 6-methylnicotine was greater than 1 min.

[0028] For column selection, polyethylene glycol (PEG) capillary columns (such as DB-WAX) are preferred polar capillary columns with dimensions of 60m × 0.25mm × 0.25μm or equivalent. 5-Methylnicotine and 6-methylnicotine exhibit a slight polarity difference; using a long-column (60m) PEG polar stationary phase provides a higher theoretical plate number, enabling differentiated hydrogen bonding and dipole interactions with both, thus achieving sufficient retention and separation based on their polarity differences.

[0029] For carrier gas flow rate control, helium was used, and the column flow rate was set to 1.0~2.0 mL / min, preferably 1.5 mL / min, at a constant flow rate. This moderate, constant flow rate ensures analytical speed while providing sufficient retention time for isomers to undergo multiple partitions between the stationary and mobile phases, balancing resolution and analytical efficiency.

[0030] Regarding the injection conditions, the injection port temperature was set to 220–280°C, preferably 250°C, and the injection volume was 1 µL. Split injection was used, with a split ratio set to (10–50):1, preferably 20:1. An injection port temperature of 250°C ensures that high-boiling-point isomers are instantly and completely vaporized, avoiding thermal degradation or discrimination effects. A suitable split ratio prevents a large influx of solvent and matrix into the column, thus avoiding column overload, effectively protecting column efficiency and reducing contamination at the mass spectrometer end.

[0031] In terms of temperature program settings, the elution temperature range for the target isomer is 180℃~200℃. Within this temperature range, the temperature is increased at a rate not exceeding 5℃ / min, more preferably 1.5~3.5℃ / min, and most preferably 2℃ / min. Implementing slow heating within a very narrow temperature range can prolong the residence time of the isomer in the chromatographic column, allowing the difference in polarity between the two isomers and the stationary phase to be fully amplified and accumulated, significantly increasing the chromatographic retention time between the two isomers, making the retention time difference greater than 1 min, thereby achieving baseline separation.

[0032] Understandably, the temperature program employs a multi-stage setting: before reaching the elution temperature range of the target isomer, the temperature is increased at a first rate of not less than 10°C / min; within the elution temperature range, the temperature is increased at a second rate of not more than 5°C / min; and after the elution temperature range, the temperature is increased at a third rate of not less than 10°C / min. For example, the initial temperature is 80°C, held for 1 min; then increased to 180°C at 15°C / min. At this point, the target analyte has not yet eluted; rapid heating allows the column temperature to quickly reach the critical elution temperature, effectively shortening the analysis waiting time; then increased to 200°C at 2°C / min. At this point, the target analyte is eluting; slow heating ensures complete separation of the isomers; finally, increased to 245°C at 30°C / min, held for 5 min. At this point, the target analyte has been detected; rapid heating quickly purges high-boiling-point impurities remaining in the column, avoiding column efficiency degradation due to impurity residue and ensuring reproducibility of continuous injections.

[0033] At the mass spectrometry detection end, an electron impact ion source is used to detect the separated effluent, and the two are distinguished and identified based on the difference in retention time between 5-methylnicotine and 6-methylnicotine.

[0034] Regarding the mass spectrometry parameter settings, the transfer line temperature is set to 220–280℃, preferably 250℃; the ion source temperature is set to 200–250℃, preferably 230℃; and the quadrupole temperature is set to 100–200℃, preferably 150℃. The coordinated setting of the transfer line and ion source temperatures ensures that the chromatographic effluent does not condense after entering the mass spectrometer, guaranteeing ionization efficiency; the quadrupole temperature setting ensures stable operation of the mass analyzer. Simultaneously, the solvent delay time for mass spectrometry detection is set to 5–10 min, preferably 8 min. The e-cigarette vaporizing liquid contains a large amount of propylene glycol and glycerol; the solvent delay allows the filament to shut off when the solvent peak passes, effectively protecting the mass spectrometer filament from damage.

[0035] In the qualitative analysis, selected ion monitoring (SIM) was used for the acquisition. The specific mass spectrometry parameters monitored are shown in Table 2.

[0036] Table 2 Mass Spectrometry Parameters

[0037] By setting these characteristic ions for monitoring, background interference from complex matrices can be eliminated to the greatest extent, thereby improving detection sensitivity.

[0038] Because the quantitative and qualitative ions of 5-methylnicotine and 6-methylnicotine completely overlap, mass spectrometry cannot distinguish them. However, under the specific chromatographic conditions established in this application, the retention time of 6-methylnicotine is approximately 15.4 min, and the retention time of 5-methylnicotine is approximately 17.1 min, with a retention time difference of approximately 1.7 min, achieving good baseline separation. Figure 3 As shown. Therefore, accurate qualitative analysis based on individual chromatographic retention times can avoid false positives caused by co-elution.

[0039] In the quantitative step, the content X of 5-methylnicotine or 6-methylnicotine in the sample, expressed in mg / g, is calculated using the following formula:

[0040] Where: Ci - concentration of 5-methylnicotine or 6-methylnicotine in the test solution (unit: mg / L); Co - concentration of 5-methylnicotine or 6-methylnicotine in the blank solution (unit: mg / L); V - sample volume (unit: mL); f - dilution factor; m - sample mass (unit: g); 1000 - conversion factor, dimensionless.

[0041] Using the internal standard method in conjunction with the above formula for quantitative calculation can effectively eliminate errors caused by small fluctuations in injection volume and instrument signal drift, ensuring the accuracy and reproducibility of quantitative results for trace isomers in complex matrices.

[0042] To verify the effectiveness of the technical solution of this application, the following specific embodiments are provided: Example 1 (Linearity Verification) 1. Preparation of standard solutions: Weigh out 5-methylnicotine and 6-methylnicotine standards, dissolve and dilute them in methanol solution containing 20 mg / L n-heptadecane to prepare primary standard stock solutions. Prepare standard working solutions with concentrations of 80 mg / L, 500 mg / L, 1000 mg / L, 2000 mg / L, and 3000 mg / L through serial dilution.

[0043] 2. Instrument conditions: Gas chromatography-mass spectrometry (GC-MS): Agilent 8860; Ion source: EI; Column: DB-WAX, 60m × 0.25mm × 0.25μm; Carrier gas: Helium; Injector temperature: 250℃; Injection mode: split injection, split ratio 20:1; Column flow rate: 1.5mL / min, constant flow rate; Injection volume: 1µL; Solvent delay: 8min; Transfer line temperature: 250℃; Ion source temperature: 230℃; Quadrupole temperature: 150℃; Acquisition mode: SIM.

[0044] Qualitative and quantitative ions of the target analytes: heptadecane quantitative ion m / z 57, qualitative ions m / z 71 and m / z 85; 5-methylnicotine and 6-methylnicotine quantitative ion m / z 84, qualitative ions m / z 147 and m / z 176.

[0045] Temperature rise program: Initial temperature 80℃, hold for 1 min; rise at 15℃ / min to 180℃, hold for 0 min; rise at 2℃ / min to 200℃, hold for 0 min; rise at 30℃ / min to 245℃, hold for 5 min.

[0046] Five standard working solutions at different concentration levels were injected and tested, with each concentration level tested in triplicate. A linear regression analysis was performed with the target analyte concentration on the x-axis and the peak area response value on the y-axis to plot a standard curve, as shown below. Figure 4 and Figure 5 As shown; the linear test results are shown in Table 3: Table 3 Linearity Test Results

[0047] Under these conditions, the retention time of 6-methylnicotine was approximately 15.4 min, and that of 5-methylnicotine was approximately 17.1 min, with a retention time difference of 1.7 min, achieving baseline separation. The linear correlation coefficients (R²) for both 5-methylnicotine and 6-methylnicotine were greater than 0.999 within the concentration range of 80–3000 mg / L, meeting the requirements for accurate quantification over a wide concentration range.

[0048] Furthermore, according to the chromatography workstation, the chromatographic separation Rs=11 is much greater than the quantitative requirement of 1.5, achieving complete baseline separation and eliminating the mutual interference of isomers at the mass spectrometry detection end in physical space.

[0049] Example 2 (Precision verification, liquid atomized matrix) Under specified testing conditions, the degree of similarity between the results obtained from multiple sampling and testing of the same homogeneous sample is used to estimate the impact of random errors in the detection method on the experimental results, that is, to assess the dispersion of the experimental data.

[0050] Specifically, mint-flavored e-liquid samples were selected, and seven parallel measurements were taken, each containing 0.25 g of 5-methylnicotine and 0.25 g of 6-methylnicotine, into 5 mL volumetric flasks. The flasks were then diluted to the mark with a methanol solution containing 20 mg / L n-heptadecane (V = 5 mL), vortexed for 2 min to mix, and filtered through a 0.22 μm filter. The mixture was then measured seven times in parallel under the instrument conditions described in Example 1. The content was calculated using the quantitative formula X = (Ci - Co) × V × f / (m × 1000) (where Co is 0 and f is 1). The results are shown in Table 4.

[0051] Table 4 Precision Test Results of Mint-Flavored E-Liquid

[0052] Precision (relative standard deviation) = standard deviation / arithmetic mean of the calculated results * 100%.

[0053] When tested according to the procedure of this method, the precision of 5-methylnicotine is 0.32% (n=7), and the precision of 6-methylnicotine is 0.34% (n=7); precision refers to the degree of similarity of the results obtained from multiple measurements of the same homogeneous sample under specified test conditions.

[0054] As can be seen from the above, this test method exhibits good precision when testing for 5-methylnicotine and 6-methylnicotine. Furthermore, through… Figure 6 The TIC plot shows that the peak of 5-methylnicotine is not affected by 6-methylnicotine, indicating that there are no extraneous peaks near the target peak. This proves that the direct dilution method combined with the polar column successfully resists the interference of complex fragrance matrix and has extremely high specificity.

[0055] The precision of 5-methylnicotine was 0.32%, and that of 6-methylnicotine was 0.34%. These relative standard deviations are well below the 2% acceptance limit in conventional gas chromatography-mass spectrometry analysis, demonstrating that this method still possesses extremely high reproducibility and anti-interference capability for complex liquid e-liquid matrices containing significant flavor interference.

[0056] Example 3 (Precision verification, gaseous-electronic cigarette) Seven blueberry and raspberry flavored e-cigarette samples were selected, and Cambridge filters and traps were installed on each. The target samples were generated by taking 20 puffs using a cigarette applicator. The filter with the target sample was removed, and the inner wall of the front cover of the trap was wiped with another filter. All the filters were placed into a 50mL stoppered conical flask, and 10mL of a heptadecanol solution containing 20mg / L was added. The stoppered conical flask was placed in a vortex mixer and shaken for 30min for extraction. After filtration through an organic filter membrane, the samples were tested using the same instrument conditions as in Example 1.

[0057] The e-cigarette had 20 puffs (range 20-50 puffs) and the e-cigarette tank tilt angle α = -45° was used during the puff test, with the top mouthpiece aligned with the device's axis. The relevant test results are shown in Table 5 below. Table 5. Test results of nicotine content in blueberry and raspberry flavored e-cigarettes

[0058] As demonstrated by the above embodiments, when tested according to the proposed method, the precision of 5-methylnicotine is 0.25% (n=7), and the precision of 6-methylnicotine is 0.22% (n=7), both less than 0.25%. This proves that the chromatographic system and pretreatment process of this application can offset the fluctuations caused by the physical suction process, and exhibits good precision when testing for 5-methylnicotine and 6-methylnicotine. It also demonstrates that the pretreatment and chromatographic system are highly adaptable to changes in physical state.

[0059] Example 4: Accuracy Verification (Liquid-Atomized Matrix) Given that there are currently no commercially available liquid quality control samples containing both 5-methyl and 6-methylnicotine with accurate values, negative matrix spiked recovery tests are typically used in the art to verify the accuracy of the method. Therefore, using the standard curve established in Example 1, negative e-liquid samples were spiked at low, medium, and high concentrations, and the results were examined separately according to the following method.

[0060] Specifically, in the embodiments of this application, an e-cigarette vaporizer (flavor: tropical fruit) that does not contain 6-methylnicotine and 5-methylnicotine was selected. 0.25g of the sample was weighed into a 5mL volumetric flask, and the volume was adjusted to the mark with a heptadecanoethanol solution containing 20mg / L. The mixture was vortexed on a vortex mixer for 2min until well mixed, filtered through a 0.22μm filter membrane, and then placed in a chromatographic vial for instrumental testing.

[0061] The processed samples were tested according to the instrumental method of Example 1. The three spiked recovery results are shown in Tables 6, 7 and 8 below. Table 6. Recovery rates of low-concentration spiked tropical fruits

[0062] Table 7. Results of Spike Recovery in Tropical Fruits

[0063] Table 8. Results of high-concentration spiked recovery rates of tropical fruits

[0064] As can be seen from the above examples, when tested according to the method, the recovery rate of 5-methylnicotine ranges from 99.2% to 101.6%, and the recovery rate of 6-methylnicotine ranges from 96.6% to 105.3%. The recovery rates are concentrated between 96.6% and 105.3%, with no obvious systematic error (too high or too low). The data are highly concentrated around 100%, proving that direct volume adjustment with methanol did not cause adsorption loss of the target substance, and the quantitative results are accurate and reliable.

[0065] Accuracy refers to the degree of agreement between the test results and the received reference value. This experiment uses the spiked recovery rate to verify the accuracy of the method. The results above show that this test method has good accuracy.

[0066] Example 5 (Blind Test Verification) Multiple flavors of e-cigarette products were selected, and the e-cigarette atomizing liquid samples were tested and analyzed using the detection method described in Example 2 of this application, while the e-cigarette samples were tested and analyzed using the detection method described in Example 3 of this application.

[0067] The test results are shown in Tables 9 and 10. Table 9 Test Results of Electronic Cigarette Fluid Samples

[0068] Table 10 Test Results of Electronic Cigarette Samples

[0069] As shown in the table above, when testing for 5-methylnicotine and 6-methylnicotine in different target samples according to the procedure of this method, the method can effectively qualitatively and quantitatively identify the 5-methylnicotine or 6-methylnicotine contained in the sample without any misjudgment, proving that this method is fully capable of serving as a universal testing standard in the industry.

[0070] As shown in Tables 9 and 10, in real commercial samples with various complex matrices, when only 5-methylnicotine is present (e.g., menthol ice), the detection result for 6-methylnicotine by this method strictly shows "not detected"; conversely, the result is also true when only 5-methylnicotine is present (e.g., blueberry bubble gum). This fully demonstrates the extremely excellent selectivity and specificity of the method of this invention. Since the mass spectrometric fragments of 5-methyl and 6-methyl are completely identical, this method, relying on specific polar chromatographic separation conditions, successfully eliminates the mutual interference of false positives between isomers, proving that this method is fully capable of being applied to commercial blind testing of complex real samples.

[0071] This application further provides the application of a chromatographic method for distinguishing 5-methylnicotine from 6-methylnicotine in quality control. Specifically, it includes the following steps: (1) Obtaining samples of novel tobacco products to be evaluated. In accordance with quality sampling inspection standards, samples of novel tobacco products to be shipped are randomly selected from the production line. The sample forms include liquid e-cigarette atomizing liquid and gaseous e-cigarette aerosol. By covering samples of different forms, the quality consistency of the products before shipment can be comprehensively assessed.

[0072] (2) The content of 5-methylnicotine and / or 6-methylnicotine in the sample is determined using the above method. If the obtained sample is a liquid nebulizer, the aforementioned pretreatment and detection methods for liquid samples are used; if the obtained sample is a gaseous aerosol, the aforementioned suction collection, pretreatment, and detection methods for gaseous samples are used. In this step, relying on the synergistic separation of the polar capillary column and the slow heating program, baseline separation of 5-methylnicotine and 6-methylnicotine can be ensured, eliminating mutual interference caused by the consistency of mass spectrometry fragments. Therefore, regardless of whether only one isomer is added to the product formulation or is added in a specific ratio, this method can accurately determine their true content, avoiding the risk of "false compliance" caused by misjudging the signal of 6-methylnicotine as 5-methylnicotine (or vice versa).

[0073] (3) Compare the measured content results with the preset quality standard threshold. The preset quality standard threshold is set according to the product design formula and relevant safety regulations. For example, for products that are only allowed to contain 5-methylnicotine in the formula, the preset threshold for 6-methylnicotine is "not detectable"; for products that require a specific addition ratio, it is set as a specific content range (e.g., 4.75~5.25 mg / g). Compare the accurate content data measured in step (2) with the preset threshold one by one. Since the measurement results exclude the cross-interference between isomers, the comparison results can truly reflect the compliance status of the target substance in the sample and can eliminate the risk of misjudgment caused by insufficient resolution of the detection method.

[0074] (4) Based on the comparison results, perform the qualified release or unqualified rejection operation for the new tobacco products. If the measured contents of 5-methylnicotine and 6-methylnicotine are both within the preset quality standard threshold range, the batch of new tobacco products is deemed qualified and the release operation is performed; if any indicator exceeds the threshold range (such as the detection of an isomer that should not be detected, or the content of the main component deviating from the formula range), the batch of new tobacco products is deemed unqualified, the rejection operation is performed, and the production line abnormality traceability mechanism is triggered.

[0075] Through the above application steps, the chromatographic differentiation method of this application is deeply integrated into the industrial quality control process. Leveraging its superior isomer separation and accurate quantification capabilities, it can quickly and precisely determine product quality, effectively preventing defective products caused by isomer confusion from entering the market, and significantly improving the reliability and rigor of the quality control system for new tobacco products. This application further proposes embodiments for product quality control: Example 6 (Product Quality Control) A random sample of a batch of e-liquid is taken for inspection according to the company's internal quality control requirements. Only those that pass the inspection can be released from the factory.

[0076] As shown in Table 11 below, the quality inspector randomly selected 3 bottles of each flavor of e-liquid from this batch and then tested them using the method described in this patent. The test results are as follows, and it is determined whether the corresponding flavor of e-liquid in this batch is qualified and whether it is allowed to be released from the factory.

[0077] Table 11 Test Results of Electronic Cigarette Atomized Samples

[0078] As shown in Table 11 above, this method is accurate in detecting the content of 5-methylnicotine and 6-methylnicotine in e-liquid, and they do not interfere with each other. It can quickly help quality inspectors determine whether a batch of e-liquid is qualified and whether it can be released.

[0079] The above description is only a part or preferred embodiment of this application. Neither the text nor the drawings should limit the scope of protection of this application. All equivalent structural transformations made using the content of this application's specification and drawings under the overall concept of this application, or direct / indirect applications in other related technical fields, are included within the scope of protection of this application.

Claims

1. A chromatographic method for distinguishing between 5-methylnicotine and 6-methylnicotine, characterized in that, include: The test solution was injected into a gas chromatograph-mass spectrometer for detection. At the chromatographic separation end, a polar capillary column was used, and a temperature program was set. The temperature was increased at a rate not exceeding 5℃ / min within the temperature range for eluting the target isomer, so that the difference in chromatographic retention time between 5-methylnicotine and 6-methylnicotine was greater than 1 min. At the mass spectrometry detection end, an electron impact ion source is used to detect the separated effluent, and the two are distinguished and identified based on the difference in retention time between 5-methylnicotine and 6-methylnicotine.

2. The chromatographic method for distinguishing 5-methylnicotine and 6-methylnicotine according to claim 1, characterized in that, The temperature range for eluting the target isomer at a heating rate not exceeding 5°C / min is 180°C to 200°C.

3. The chromatographic method for distinguishing 5-methylnicotine and 6-methylnicotine according to claim 2, characterized in that, The temperature was increased at a rate of 1.5 to 3.5 °C / min within the temperature range for eluting the target isomer.

4. The chromatographic method for distinguishing 5-methylnicotine and 6-methylnicotine according to claim 3, characterized in that, The heating process is a multi-stage heating process: before reaching the temperature range for eluting the target isomer, the temperature is increased at a first heating rate of not less than 10°C / min; within the temperature range for eluting the target isomer, the temperature is increased at a second heating rate of not more than 5°C / min; and after reaching the temperature range for eluting the target isomer, the temperature is increased at a third heating rate of not less than 10°C / min.

5. The chromatographic method for distinguishing 5-methylnicotine and 6-methylnicotine according to claim 1, characterized in that, The polar capillary column is a polyethylene glycol capillary column.

6. The chromatographic method for distinguishing 5-methylnicotine and 6-methylnicotine according to claim 1, characterized in that, When injecting the test solution into the gas chromatograph-mass spectrometer, the injection mode is split injection with a split ratio of (10-50):1; and / or the solvent delay time for mass spectrometry detection is 5-10 min.

7. The chromatographic method for distinguishing 5-methylnicotine and 6-methylnicotine according to claim 1, characterized in that, Before injecting the test solution into the gas chromatograph-mass spectrometer, a liquid sample pretreatment step is also included: weigh the electronic cigarette vaporization liquid sample, dilute it with methanol solution containing internal standard, mix and filter to obtain the test solution.

8. The chromatographic method for distinguishing 5-methylnicotine and 6-methylnicotine according to claim 1, characterized in that, Before injecting the test solution into the gas chromatograph-mass spectrometer, a gaseous sample pretreatment step is also included: under conditions simulating manual aspiration, the electronic atomizer is aspirated using a smoke machine, and the aerosol is captured through a Cambridge filter; the captured Cambridge filter is placed in a methanol solution containing an internal standard for shaking extraction, and the test solution is obtained after filtration.

9. The chromatographic method for distinguishing 5-methylnicotine from 6-methylnicotine according to claim 7 or 8, characterized in that, The internal standard is n-heptadecane, and the concentration of n-heptadecane in the methanol solution containing the internal standard is 15~25 mg / L.

10. The application of a chromatographic method for distinguishing 5-methylnicotine from 6-methylnicotine in quality control.

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