A method for identifying 6-methyl nicotine in an electronic cigarette liquid

Abstract

The present application belongs to the technical field of electronic cigarette product safety detection, and particularly relates to a method for identifying 6-methyl nicotine in electronic cigarette liquid. The method comprises: taking standard samples of 6-methyl nicotine, nicotine and anatabine, and respectively performing liquid chromatography-high resolution mass spectrometry analysis under the same conditions to obtain retention time on an Amide chromatographic column, a first accurate mass number and a second fragment spectrum; under the same liquid chromatography-high resolution mass spectrometry conditions, analyzing a to-be-tested solution, preliminarily screening according to the accurate mass number of 6-methyl nicotine, and then verifying by comparing the retention time on the Amide chromatographic column and the second fragment spectrum. The method can effectively eliminate the interference of nicotine, anatabine and other compounds of the same type, and especially under the premise that a large nicotine concentration generally exists in electronic cigarette liquid, the specific identification of 6-methyl nicotine can be realized.

Description

Technical Field

[0001] This invention belongs to the field of electronic cigarette product safety testing technology, specifically relating to a method for identifying 6-methylnicotine in electronic cigarette liquid. Background Technology

[0002] 6-Methylnicotinic acid is a methylated analogue of nicotine, which may have unknown pharmacological and toxicological effects and is not listed in the national standard for additives in e-cigarettes. Accurate identification of 6-methylnicotinic acid in e-cigarette liquids is of great significance for the quality supervision of e-cigarette products.

[0003] Chinese invention patent application CN 118671047 A, published on September 20, 2024, discloses a method for rapid detection of 6-methylnicotine in e-cigarette liquid. The method involves preparing reducing dithiothreitol-carbon dots (DTT-CDs) and further using these carbon dots to reduce chloroauric acid and silver nitrate, thus preparing a carbon dot-based / silver nanoparticle (Au@Ag / DTT-CDs) enhancer with excellent surface-enhanced Raman spectroscopy (SERS) signal enhancement. Au@Ag / DTT-CDs can interact with 6-methylnicotine, significantly enhancing its SERS signal. Based on the 6-methylnicotine concentration and 820 cm⁻¹... -1 The linear relationship between the SERS signal intensity at different locations enabled the SERS detection of 6-methylnicotine in e-liquid.

[0004] Chinese invention patent application CN 119936260 A, published on May 6, 2025, discloses an analytical method for 6-methylnicotinic acid. The method employs high performance liquid chromatography (HPLC) with a mobile phase A of disodium hydrogen phosphate containing triethylamine and a mobile phase B of methanol. The quantitative analysis and detection of 6-methylnicotinic acid are achieved using a C18 column and ultraviolet detection mode.

[0005] Neither the surface-enhanced Raman spectroscopy nor the high-performance liquid chromatography-ultraviolet method can match the detection sensitivity and analytical reliability of high-resolution mass spectrometry. However, because the structure of 6-methylnicotinic acid is similar to that of nicotine and neonicotinoids, it is very easy to miss or misjudge when using high-resolution mass spectrometry for the specific identification of 6-methylnicotinic acid. Summary of the Invention

[0006] The purpose of this invention is to provide a method for identifying 6-methylnicotine in e-cigarette liquids, in order to solve the problem that when using high-resolution mass spectrometry to identify 6-methylnicotine in e-cigarette liquids, it may cause missed detection or misjudgment due to interference from similar compounds such as nicotine and neonicotinoids.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for identifying 6-methylnicotine in e-cigarette liquid, comprising the following steps: (1) Standards of 6-methylnicotine, nicotine and neonicotine were taken and analyzed by liquid chromatography-high resolution mass spectrometry under the same conditions. The retention time of 6-methylnicotine on the Amide column was 5.89±0.2 min, the first-order exact mass number was 177.1386±0.0009, and the characteristic fragment ions m / z of the second-order fragment spectrum were 146.0964, 144.0799 and 120.0803. (2) Pretreatment of the e-cigarette liquid sample to be tested to obtain the test solution; (3) The test solution was analyzed under the same liquid chromatography-high resolution mass spectrometry conditions. The initial screening was performed based on the precise mass number of 6-methylnicotinic acid, and then the Amide column retention time and secondary fragment spectrum were compared for confirmation.

[0008] This invention is an improved invention. It utilizes the Amide chromatographic column to achieve baseline separation of 6-methylnicotinic acid, nicotine, and neonicotinoids, solving the problem of unsatisfactory separation effect of C18 column and providing a reliable basis for subsequent retention time comparison. For specific identification, the candidate peaks of 6-methylnicotinic acid corresponding to the mass number are first screened out, and then the retention time of Amide column is compared and secondary fragments are confirmed, which can accurately identify 6-methylnicotinic acid.

[0009] This method effectively eliminates interference from similar compounds such as nicotine and neonicotinoids. Particularly noteworthy is its ability to specifically identify 6-methylnicotinoids, a feat difficult to achieve with currently reported high-performance liquid chromatography-ultraviolet (HPLC-UV) and surface-enhanced Raman spectroscopy (SMR). Furthermore, this method features simple pretreatment and short analysis time, making it suitable for routine screening of large batches of samples.

[0010] Preferably, during the initial screening, chromatographic peaks with a deviation of less than 5 ppm from the exact mass number of 6-methylnicotine are considered candidate peaks for 6-methylnicotine. High-resolution mass spectrometry is used to determine the first-order exact mass number, and a deviation of ≤5 ppm is set for preliminary screening to initially eliminate interference from nicotine and neonicotinoids.

[0011] More preferably, when comparing the retention times of the Amide column, the retention time of the candidate peak on the Amide column is compared with the retention time of the 6-methylnicotinic acid standard. If the deviation is ≤0.2 min, further confirmation is provided. The hydrophilic Amide column utilizes the difference in hydrophilicity between 6-methylnicotinic acid, nicotine, and neonicotinoids to achieve baseline separation of the three, providing a reliable basis for retention time comparison.

[0012] More preferably, when comparing secondary fragment spectra, the secondary fragment spectra of candidate peaks are extracted and compared with the secondary fragment spectra of 6-methylnicotinic acid standards. If the characteristic fragment ions and their abundances match, it is identified as 6-methylnicotinic acid. By identifying the characteristic fragment ions of 6-methylnicotinic acid using standards, the secondary fragments of 6-methylnicotinic acid can be further distinguished from those of nicotine and neonicotinoids, thus improving the accuracy of identification.

[0013] Most preferably, the characteristic fragment ions of nicotine have m / z values ​​of 132.0813, 130.0652, and 106.0654; the characteristic fragment ions of neonicotine have m / z values ​​of 144.0800 and 107.0600. These characteristic fragment ions are obtained by effectively separating 6-methylnicotine, nicotine, and neonicotine using standards, and can be used for the specific identification of 6-methylnicotine.

[0014] If necessary, 6-methylnicotinic acid standard can be added for confirmation to ensure the reliability of the identification results. At this time, after comparing the retention time and secondary fragment spectrum of the Amide column in step (3), 6-methylnicotinic acid standard is added to the sample. If the target peak intensity increases and the retention time remains unchanged, the final confirmation is achieved.

[0015] To further strengthen the confirmatory power of 6-methylnicotine, preferably, in step (3), the retention time of the 6-methylnicotine standard solution is tested simultaneously. Confirmation of 6-methylnicotine is achieved by comparing the retention times of the test solution and the 6-methylnicotine standard solution. For example, if the retention time deviation between the test solution and the simultaneously tested 6-methylnicotine standard solution is ≤0.2 min, it can be further confirmed as 6-methylnicotine. The concentration of the simultaneously tested 6-methylnicotine standard solution can be 1~2 mg / g; it is prepared using 6-methylnicotine standard and methanol.

[0016] Preferably, in step (1), 6-methylnicotine standard, nicotine standard, neonicotine standard, and methanol are mixed to prepare a working solution with a concentration of 1~3 μg / mL for the liquid chromatography-high resolution mass spectrometry analysis. The preparation of the working solution using the above method is simple and creates favorable conditions for subsequent liquid chromatography-high resolution mass spectrometry analysis.

[0017] Preferably, the chromatographic conditions for liquid chromatography-high resolution mass spectrometry analysis in steps (1) and (3) are as follows: WatersAcquity UPLC BEH Amide, mobile phase A is a 50% acetonitrile aqueous solution containing 5 mM ammonium acetate and 0.1% formic acid, mobile phase B is a 95% acetonitrile aqueous solution containing 5 mM ammonium acetate and 0.1% formic acid, and the gradient elution program is: 0 min, 95% B; 0.5 min, 95% B; 10 min, 55% B; 12 min, 30% B; 13 min, 30% B; 13.1 min, 95% B; 20 min, 95% B. Under the above chromatographic conditions, effective separation of 6-methylnicotine, nicotine, and neonicotinoids is achieved, avoiding qualitative interference from the C18 column.

[0018] More preferably, the mass spectrometry conditions are as follows: heated electrospray ion source, positive ion mode, Full MS / dd-MS2 scan, Full MS resolution 60000, dd-MS2 resolution 15000, scan range m / z 70-1050, spray voltage 3000 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 350℃, auxiliary gas temperature 320℃, and collision energy gradients of 20, 40, and 60 eV. Attached Figure Description

[0019] Figure 1 This is the extraction ion chromatogram (C18 column) of 6-methylnicotinic acid, nicotine and neonicotinoids in Example 1 of the present invention. Figure 2 This is the extraction ion chromatogram (Amide column) of 6-methylnicotinic acid, nicotine and neonicotinoids in Example 1 of the present invention. Figure 3 The mass spectrum (C18 column) of the parent ions of 6-methylnicotinic acid, nicotine and neonicotinoid in Example 1 of the present invention. Figure 4 The mass spectrum of the parent ions of 6-methylnicotinic acid, nicotine and neonicotinoid in Example 1 of this invention (Amide column). Figure 5 The mass spectra of the daughter ions of 6-methylnicotinic acid, nicotine and neonicotinoid in Example 1 of this invention are shown. Detailed Implementation

[0020] (a) Preferred embodiment of the method for identifying 6-methylnicotine in electronic cigarette liquid of the present invention This invention investigated the chromatographic behavior of 6-methylnicotine, nicotine, and neonicotinoids on C18 and Amide columns, clarifying the key characteristic that C18 columns provide unsatisfactory separation while Amide columns achieve effective separation, providing a scientific basis for method selection. Based on this, the precise mass number of 6-methylnicotine (177.1386) and its difference from nicotine (163.1230) and neonicotinoids (161.1073) were used for initial screening based on precise mass number. Then, by comparing retention times on Amide columns and distinguishing characteristic secondary fragment ions (m / z 146.0964, 144.0799, 120.0803, etc.), specific identification of 6-methylnicotine was achieved.

[0021] The working principle of the above-mentioned triple identification method—precise mass number initial screening + Amide column retention time comparison + feature fragment confirmation—is as follows: Preliminary screening using precise mass numbers: 6-methylnicotine has the molecular formula C11H16N2, with a precise mass number ([M+H]+) of 177.1386; nicotine has the molecular formula C10H14N2, with a precise mass number ([M+H]+) of 163.1230; and neonicotine has the molecular formula C10H12N2, with a precise mass number ([M+H]+) of 161.1073. The precise mass numbers of these three substances differ significantly (mass differences of 14.0156 and 16.0313, respectively). Therefore, by determining the first-order precise mass number using high-resolution mass spectrometry and setting a strict mass deviation (≤5 ppm), candidate peaks potentially belonging to 6-methylnicotine can be preliminarily screened, eliminating interference from nicotine and neonicotine.

[0022] Amide column retention time comparison: Although the C18 reversed-phase column is not ideal for separating 6-methylnicotinic acid, nicotine, and neonicotinoids, as their retention times are extremely similar, making baseline separation difficult, the hydrophilic interaction Amide column can achieve effective separation by utilizing the differences in their hydrophilicity. Further confirmation can be obtained by measuring the retention time of 6-methylnicotinic acid on the Amide column using standards and comparing it with the retention times of candidate peaks in the sample (deviation ≤ 0.2 min).

[0023] Characteristic secondary fragment ion identification: Through standard studies, characteristic fragment ions of 6-methylnicotine (e.g., m / z 146.0964, 144.0799, 120.0803, etc.) are identified and distinguished from characteristic fragments of nicotine (m / z 132.0813, 130.0652, 106.0654, etc.) and neonicotine (m / z 144.0800, 107.0600, etc.). Final confirmation is achieved when the secondary fragment ion of a candidate peak in the sample matches the spectral density of the 6-methylnicotine standard with ≥80%.

[0024] Comprehensive judgment: When a chromatographic peak in a sample simultaneously meets the three conditions of accurate mass number matching (m / z 177.1386, deviation ≤ 5 ppm), Amide column retention time matching (deviation ≤ 0.2 min), and secondary fragment matching (matching degree ≥ 80%), it can be accurately identified as 6-methylnicotinic acid.

[0025] In addition, although the separation effect of C18 column is not ideal, it can be used as a dual-column complementary verification, which can help provide chromatographic behavior information and enhance the reliability of identification.

[0026] The specific implementation steps of the above-mentioned method for identifying 6-methylnicotine in e-cigarette liquid are as follows: (1) Obtain 6-methylnicotine standard, and determine its retention time, primary exact mass number and secondary fragment chromatogram on C18 and Amide columns under the set liquid chromatography-high resolution mass spectrometry conditions as reference data. At the same time, obtain data of nicotine and neonicotinoid standards under the same conditions to help distinguish them.

[0027] (2) The e-cigarette liquid sample to be tested is pretreated to obtain the solution to be tested.

[0028] During pretreatment, the e-cigarette liquid and a 50-70% (v / v) methanol aqueous solution are shaken and mixed, centrifuged, and the supernatant is collected and filtered to obtain the solution to be tested. The preferred volume ratio of e-cigarette liquid to the 50-70% (v / v) methanol aqueous solution is 1:(45-55) to better balance matrix interference elimination and quantitative accuracy.

[0029] (3) The test solution was analyzed under the same liquid chromatography-high resolution mass spectrometry conditions to obtain the retention time, first-order exact mass number and second-order fragment spectrum of each component.

[0030] (4) Preliminary screening of precise mass number: Extraction ion chromatogram with precise mass number of 177.1386±0.0009 (corresponding to 5 ppm deviation) is extracted, and the chromatographic peak corresponding to this mass number is screened as 6-methylnicotinic acid candidate peak.

[0031] (5) Amide column retention time comparison: Compare the retention time of the candidate peak on the Amide column with the retention time of the 6-methylnicotinic acid standard. If the deviation is ≤0.2 min, proceed to the next step.

[0032] (6) Secondary fragment confirmation: The secondary mass spectrum of the candidate peak is extracted and compared with the secondary fragment spectrum of the 6-methylnicotinic acid standard. If the characteristic fragment ions (m / z 146.0964, 144.0799, 120.0803, etc.) and their abundance match (matching degree ≥80%), then it is identified as 6-methylnicotinic acid. Here, the matching degree refers to the comprehensive matching degree of secondary fragments and abundance. A comprehensive matching degree ≥80% is considered a match between the secondary fragment spectra.

[0033] (7) If necessary, the standard addition method is used for verification: 6-methylnicotinic acid standard is added to the sample. If the target peak intensity increases and the retention time remains unchanged, the verification is finally confirmed.

[0034] The preferred embodiments described above will be illustrated below with reference to examples.

[0035] Example 1 The method for identifying 6-methylnicotine in e-cigarette liquid in this embodiment details the chromatographic behavior and mass spectrometric characteristics of 6-methylnicotine, nicotine, and neonicotinoid standards: 1. Preparation of standard solutions: Weigh 10 mg each of 6-methylnicotine, nicotine and neonicotine standards, dissolve and dilute with methanol to 10 mL to obtain a 1000 μg / mL stock solution; then dilute with methanol to 1 μg / mL working solution.

[0036] 2. Take the working solution for UHPLC-Orbitrap-MS analysis.

[0037] Column 1 (C18 column): Thermo scientific Hypersil GOLD VANQUISH (100 mm × 2.1 mm, 1.9 μm), mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile, gradient elution program: 0 min, 5% B; 0.5 min, 5% B; 10 min, 95% B; 12 min, 95% B; 12.1 min, 5% B; 15 min, 5% B; flow rate: 0.3 mL / min, column temperature: 40℃, injection volume: 2 μL. All percentages (%) in the chromatographic conditions are volume percentages.

[0038] Column 2 (Amide column): Waters Acquity UPLC BEH Amide (150 mm × 2.1 mm, 1.7 μm). Mobile phase A was a 50% aqueous solution of acetonitrile containing 5 mM ammonium acetate and 0.1% formic acid; mobile phase B was a 95% aqueous solution of acetonitrile containing 5 mM ammonium acetate and 0.1% formic acid. Gradient elution program: 0 min, 95% B; 0.5 min, 95% B; 10 min, 55% B; 12 min, 30% B; 13 min, 30% B; 13.1 min, 95% B; 20 min, 95% B; flow rate 0.3 mL / min; column temperature 40℃; injection volume 2 μL. All percentages (%) in the chromatographic conditions are volume percentages.

[0039] Mass spectrometry conditions: heated electrospray ionization source (H-ESI), positive ion mode, Full MS / dd-MS2 scan, Full MS resolution 60000, dd-MS2 resolution 15000, scan range m / z 70-1050, spray voltage 3000V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 350℃, auxiliary gas temperature 320℃, collision energy gradient 20, 40, 60 eV.

[0040] Results: The retention times and mass spectrometric characteristics of each component are shown in Table 1. Extraction ion chromatograms for the C18 and Amide columns are shown in Table 1. Figure 1 and Figure 2 As shown, the precursor ion mass spectra of the C18 column and the Amide column are as follows: Figure 3 and Figure 4 As shown, the daughter ion mass spectrum of the Amide column is as follows: Figure 5 As shown.

[0041] Table 1. Chromatographic retention times and mass spectrometric characteristics of 6-methylnicotine and its analogues From Table 1 and Figure 1-5 It can be seen that: Significant differences in precise mass numbers: The precise mass number of 6-methylnicotine (177.1386) differs by 14.0158 from that of nicotine (163.1228) and by 16.0313 from that of neonicotine (161.1073). These differences can be completely distinguished by high-resolution mass spectrometry (resolution ≥60000).

[0042] The separation effect of C18 column is not ideal: On C18 column, the retention times of the three are very close, making it difficult to achieve baseline separation, resulting in co-elution and interference in the mother ion mass spectrum.

[0043] Amide columns can achieve effective separation: on Amide columns, the retention time difference between 6-methylnicotinic acid (5.89 min) and nicotine (6.04 min) is 0.15 min, and the retention time difference between 6-methylnicotinic acid and neonicotinic acid (6.46 min) is 0.57 min, achieving baseline separation or good separation.

[0044] The secondary fragment ions showed significant differences: the characteristic fragment ions of 6-methylnicotine were m / z 146.0964, 144.0799, and 120.0803, etc.; the characteristic fragment ions of nicotine were m / z 132.0813, 130.0652, and 106.0654, etc.; and the characteristic fragment ions of neonicotine were m / z 144.0800 and 107.0600, etc. These characteristic fragment ions can be used to distinguish 6-methylnicotine.

[0045] 3. Differentiation from nicotine-positive samples Select an e-cigarette liquid sample containing only nicotine (without 6-methylnicotine).

[0046] Sample pretreatment: Accurately measure 0.2 mL of e-cigarette liquid into a 50 mL capped centrifuge tube using a pipette, add 10 mL of 60% methanol aqueous solution (volume fraction), place on a vortex mixer and shake at 2000 r / min for 10 min, then centrifuge at 9800 r / min for 10 min, take the supernatant and filter it through a 0.22 μm organic phase filter membrane, discard the first few drops of filtrate, and take the subsequent filtrate as the test solution.

[0047] The test solution was analyzed by UHPLC-Orbitrap-MS under the same conditions, and the precise mass number was used for initial screening: no chromatographic peak was detected in the extract ion chromatogram of m / z 177.1386, and it was determined that it did not contain 6-methylnicotinic acid.

[0048] Example 2 The method for identifying 6-methylnicotine in e-cigarette liquid in this embodiment adopts the following steps: 1. Preparation of simulated samples: Different concentrations of 6-methylnicotine were added to actual e-cigarette liquid (excluding 6-methylnicotine) to prepare a series of simulated samples with final concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20 and 50 mg / g.

[0049] 2. Sample pretreatment: Accurately measure 0.2 mL of e-cigarette liquid (for each final concentration sample mentioned above) into a 50 mL capped centrifuge tube using a pipette, add 10 mL of 60% methanol aqueous solution (diluted 50 times), place on a vortex mixer and shake at 2000 r / min for 10 min, then centrifuge at 9800 r / min for 10 min, take the supernatant and filter it through a 0.22 μm organic phase filter membrane, discard the first few drops of filtrate, and take the subsequent filtrate as the test solution.

[0050] 3. Perform UHPLC-Orbitrap-MS analysis according to the conditions in Example 1.

[0051] 4. Preliminary screening using precise mass numbers: The extracted ion chromatogram (with a deviation of 5 ppm) of m / z 177.1386 was examined on both C18 and Amide columns. A peak was observed at 1.12 min on the C18 column and at 5.72 min on the Amide column.

[0052] 5. Amide column retention time comparison: The retention time of this peak on the Amide column was 5.72 min, which is consistent with the retention time of 5.89 min of the 6-methylnicotinic acid standard initially collected and stored in the database (deviation 0.17 min), meeting the requirements. Furthermore, it is even more consistent with the retention time of 5.77 min of the 6-methylnicotinic acid standard solution (concentration of 1 mg / g) tested simultaneously (deviation 0.05 min), confirming that the retention time meets the requirements.

[0053] 6. Confirmation of secondary fragments: The secondary mass spectrum of this peak was extracted, and the main fragments were m / z 146.0963, 144.0797, and 120.0806, which matched the spectral density of 6-methylnicotinic acid standard by 95.5%. Overall judgment: It was identified as positive for 6-methylnicotinic acid.

[0054] 7. Standard addition verification: Analytical tests on a series of simulated samples showed that the target peak intensity increased with increasing 6-methylnicotinic acid concentration, while the retention time remained constant, confirming detection. In other implementation scenarios, setting 2-3 addition concentrations and determining the trend of 6-methylnicotinic acid target peak intensity and retention time is sufficient for confirmatory judgment.

[0055] As can be seen from the above description, this method can effectively eliminate the interference of structurally similar substances such as nicotine and neonicotinoids in e-cigarette liquids, achieve specific identification of 6-methylnicotinoids, and minimize missed detections or misjudgments.

[0056] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for identifying 6-methylnicotine in e-cigarette liquid, characterized in that, Includes the following steps: (1) Standards of 6-methylnicotine, nicotine and neonicotine were taken and analyzed by liquid chromatography-high resolution mass spectrometry under the same conditions. The retention time of 6-methylnicotine on the Amide column was 5.89±0.2 min, the first-order exact mass number was 177.1386±0.0009, and the characteristic fragment ions m / z of the second-order fragment spectrum were 146.0964, 144.0799 and 120.0803. (2) Pretreatment of the e-cigarette liquid sample to be tested to obtain the test solution; (3) The test solution was analyzed under the same liquid chromatography-high resolution mass spectrometry conditions. The initial screening was performed based on the precise mass number of 6-methylnicotinic acid, and then the Amide column retention time and secondary fragment spectrum were compared for confirmation.

2. The method for identifying 6-methylnicotine in electronic cigarette liquid as described in claim 1, characterized in that, During the initial screening, chromatographic peaks with a precise mass deviation of less than 5 ppm from the extracted 6-methylnicotinic acid were considered candidate peaks for 6-methylnicotinic acid.

3. The method for identifying 6-methylnicotine in e-cigarette liquid as described in claim 2, characterized in that, When comparing the retention times of the Amide column, the retention times of the candidate peaks on the Amide column are compared with the retention times of the 6-methylnicotinic acid standard. If the deviation is ≤0.2 min, further confirmation is required.

4. The method for identifying 6-methylnicotine in electronic cigarette liquid as described in claim 3, characterized in that, When comparing secondary fragment spectra, the secondary fragment spectra of the candidate peaks are extracted and compared with the secondary fragment spectra of the 6-methylnicotinic acid standard. If the characteristic fragment ions and their abundances match, they are identified as 6-methylnicotinic acid.

5. The method for identifying 6-methylnicotine in electronic cigarette liquid as described in claim 4, characterized in that, The characteristic fragment ions m / z of nicotine are 132.0813, 130.0652 and 106.0654; the characteristic fragment ions m / z of neonicotine are 144.0800 and 107.0600.

6. The method for identifying 6-methylnicotine in electronic cigarette liquid as described in any one of claims 2 to 5, characterized in that, Step (3) After comparing the retention time and secondary fragment spectrum of the Amide column, add 6-methylnicotinic acid standard to the sample. If the target peak intensity increases and the retention time remains unchanged, the final confirmation is achieved.

7. The method for identifying 6-methylnicotine in electronic cigarette liquid as described in claim 6, characterized in that, In step (3), the retention time of the 6-methylnicotinic acid standard solution is tested simultaneously. The 6-methylnicotinic acid is confirmed by comparing the retention time of the test solution with that of the 6-methylnicotinic acid standard solution.

8. The method for identifying 6-methylnicotine in electronic cigarette liquid as described in claim 1, characterized in that, Step (1) Prepare a working solution with a concentration of 1~3 μg / mL using 6-methylnicotinic acid standard, nicotine standard, neonicotinic acid standard and methanol, and perform the liquid chromatography-high resolution mass spectrometry analysis.

9. The method for identifying 6-methylnicotine in electronic cigarette liquid as described in claim 1, characterized in that, The chromatographic conditions for liquid chromatography-high resolution mass spectrometry analysis in steps (1) and (3) were as follows: Waters Acquity UPLC BEH Amide, mobile phase A was a 50% aqueous solution of acetonitrile containing 5 mM ammonium acetate and 0.1% formic acid, mobile phase B was a 95% aqueous solution of acetonitrile containing 5 mM ammonium acetate and 0.1% formic acid, and the gradient elution program was: 0 min, 95% B; 0.5 min, 95% B; 10 min, 55% B; 12 min, 30% B; 13 min, 30% B; 13.1 min, 95% B; 20 min, 95% B.

10. The method for identifying 6-methylnicotine in electronic cigarette liquid as described in claim 9, characterized in that, The mass spectrometry conditions were as follows: heated electrospray ion source, positive ion mode, Full MS / dd-MS2 scan, Full MS resolution 60000, dd-MS2 resolution 15000, scan range m / z 70-1050, spray voltage 3000 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 350℃, auxiliary gas temperature 320℃, and collision energy gradients of 20, 40, and 60 eV.

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