High performance liquid chromatography for detecting 6-methylnicotine and impurities thereof

Abstract

The invention discloses a high performance liquid chromatography method for detecting 6-methylnicotine and impurities thereof, the technical scheme is as follows: the determination target of the high performance liquid chromatography is 6-methylnicotine and impurities thereof, and the method comprises the following steps: S1, preparing a test solution, taking a 6-methylnicotine raw material as a test, adding ultrapure water, mixing, and standing; preparing a solution with the concentration of 0.1 to 2mg / mL to obtain a test solution; s2, preparing a reference substance solution, namely mixing a 6-methylnicotine raw material with ultrapure water to prepare a solution with the concentration of 0.1-2mg / mL, so as to obtain the reference substance solution; and S3, detecting the test solution and the reference solution by adopting high performance liquid chromatography. According to the method, the method capable of effectively separating the 6-methylnicotine and the impurities thereof is determined by exploring college liquid chromatography conditions, the impurities such as the 6-methylnicotine and degradation products thereof are detected at the same time, the operation is simple and convenient, and the reproducibility is good.

Description

Technical Field

[0001] The invention relates to the field of detection technology, and in particular to a method for simultaneous determination using a high performance liquid chromatography gradient method. Background Art

[0002] 6-Methylnicotine is a basic compound closely related to nicotine, with a molecular formula of C 11 H 16 N2, having a structure shown in formula (I), has the effect of stimulating the central nervous system. Because 6-methyl nicotine contains a 6-methyl lipophilic group, it is considered to be more easily absorbed by the human body than nicotine, thereby obtaining more satisfaction. However, products such as 6-methyl nicotine salts, electronic cigarettes, and nicotine bags prepared using 6-methyl nicotine have problems such as poor taste and quality stability, which are closely related to their quality, especially the type and content of impurities. From the perspective of the prior art, there is currently no relevant detection method to control the quality of 6-methyl nicotine. Therefore, we propose a high-performance liquid chromatography method for detecting 6-methyl nicotine and its impurities.

[0003] Summary of the invention

[0004] The purpose of the present invention is to solve the shortcomings of the prior art that the existing detection technology has no definite analysis method for 6-methyl nicotine and its impurities, and cannot control the quality of 6-methyl nicotine through detection, and proposes a high performance liquid chromatography method for detecting 6-methyl nicotine and its impurities.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A high performance liquid chromatography method for detecting 6-methyl nicotine and its impurities, wherein the determination targets of the high performance liquid chromatography method are 6-methyl nicotine and its impurities.

[0007] The following steps are involved:

[0008] S1: Prepare a test solution, take 6-methylnicotine raw material as a test sample, add ultrapure water and mix to prepare a solution with a concentration of 0.1-2 mg / mL to obtain a test solution;

[0009] S2: preparing a reference solution, taking 6-methylnicotine raw material and mixing it with ultrapure water to prepare a solution with a concentration of 0.1 to 2 mg / mL, thereby obtaining a reference solution;

[0010] S3: Detect the test solution and reference solution using high performance liquid chromatography

[0011] The test conditions are:

[0012] Chromatographic column: ZORBAX Extend C18 column;

[0013] Column temperature: 25-40°C;

[0014] Detection wavelength: 266±20nm;

[0015] Flow rate: 0.8~1.3ml / min;

[0016] Mobile phase: acetic acid-ammonia buffer as mobile phase A, acetonitrile as mobile phase B, gradient elution.

[0017] Further, the gradient elution is:

[0018] From 0 to 6 minutes, linear gradient elution was performed, with mobile phase A maintaining a constant ratio of 95% to 100% and mobile phase B maintaining a constant ratio of 0% to 5%;

[0019] From 6 to 41 minutes, linear gradient elution was used, with the proportion of mobile phase A changing from 95-100% to 60%, and the proportion of mobile phase B changing from 0-5% to 40%;

[0020] From 41 to 45 minutes, linear gradient elution was performed, with mobile phase A maintaining a constant proportion of 60% and mobile phase B maintaining a constant proportion of 40%;

[0021] From 45 to 45.1 min, linear gradient elution was performed, the proportion of mobile phase A changed from 60% to 95-100%, and the proportion of mobile phase B changed from 40% to 0-5%;

[0022] From 45.1 to 49 minutes, linear gradient elution was performed, with the mobile phase A maintaining a constant proportion of 95 to 100%, and the mobile phase B maintaining a constant proportion of 0 to 5%.

[0023] Furthermore, the test sample is 6-methylnicotine raw material, including but not limited to one or more of the following impurities: oxidation products of 6-methylnicotine, 6-methylcotinine, 6-methylmysamine and impurities 1-8.

[0024] Furthermore, in step S3, the acetic acid-ammonia buffer solution is prepared by adding 25 ml of 1 mol / L acetic acid to 900 ml of ultrapure water, adjusting the pH to 9.0-11.0 with ammonia water, and then making up the volume to 1000 mL with ultrapure water.

[0025] Furthermore, in step S3, acetic acid and ammonia water are both of HPLC grade.

[0026] Furthermore, in step S3, the column temperature used in the method is 30°C, the flow rate is 1.0 mL / min, and the detection wavelength is 266 nm.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] In the present invention, a method for effectively separating 6-methyl nicotine and its impurities is determined by exploring chromatographic conditions, and the simultaneous detection of 6-methyl nicotine and its degradation products and other impurities is achieved. The operation is simple and the reproducibility is good, and the method can be used to control the quality of 6-methyl nicotine and related products, which is of great significance for evaluating the quality of 6-methyl nicotine and related product processes, product quality control, and storage stability detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A chromatogram of a blank solvent in a high performance liquid chromatography method for detecting 6-methylnicotine and its impurities proposed by the present invention;

[0030] Figure 2 A chromatogram of an undestroyed control sample in a high performance liquid chromatography method for detecting 6-methylnicotine and its impurities proposed by the present invention;

[0031] Figure 3 A chromatogram of an acid-degraded sample in a high performance liquid chromatography method for detecting 6-methylnicotine and its impurities proposed by the present invention;

[0032] Figure 4 A chromatogram of an alkaline-degraded sample in a high performance liquid chromatography method for detecting 6-methylnicotine and its impurities proposed by the present invention;

[0033] Figure 5 A chromatogram of an oxidative degradation sample in a high performance liquid chromatography method for detecting 6-methylnicotine and its impurities proposed by the present invention;

[0034] Figure 6 A chromatogram of a photodegraded sample in a high performance liquid chromatography method for detecting 6-methylnicotine and its impurities proposed by the present invention;

[0035] Figure 7 A chromatogram of a high temperature degradation sample in a high performance liquid chromatography method for detecting 6-methyl nicotine and its impurities proposed by the present invention;

[0036] Figure 8 The present invention provides a chromatogram of a sample blank solution in a high performance liquid chromatography method for detecting 6-methylnicotine and its impurities. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical embodiments and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0038] The application principle of the present invention is described in detail below in conjunction with the accompanying drawings.

[0039] Embodiment 1

[0040] like Figure 1-8 , a high performance liquid chromatography method for detecting 6-methyl nicotine and its impurities, wherein the determination target of the high performance liquid chromatography method is 6-methyl nicotine and its impurities, comprising the following steps:

[0041] S1: Degradation test sample preparation method

[0042] Photodegradation sample: Take about 1g (accurate to 0.0001g) of 6-methylnicotine raw material, place it in a 10ml volumetric flask, place it under normal light conditions for 6 days, then take it out, add ultrapure water to make up to 10mL, and shake to mix;

[0043] For the oxidative degradation sample, take about 1g (accurate to 0.0001g) of 6-methylnicotine raw material, place it in a 10ml volumetric flask, add 30uL of hydrogen peroxide, shake and mix, place it in a sealed, room temperature, light-proof condition for 1 hour, take it out, add ultrapure water to make up to 10mL, shake and mix to obtain the sample;

[0044] For acid degradation samples, take about 1g (accurate to 0.0001g) of 6-methylnicotine raw material, place it in a 10ml volumetric flask, add 1mL of 1mol / L hydrochloric acid solution, shake and mix, place in a sealed, 60℃ water bath for 2 hours, take it out, cool to room temperature, add ultrapure water to 10mL, shake and mix to obtain the sample;

[0045] For alkaline degradation samples, take about 1g (accurate to 0.0001g) of 6-methylnicotine raw material, place it in a 10ml volumetric flask, add 1mL of 1mol / L sodium hydroxide solution, shake and mix, place in a sealed, 60℃ water bath for 2 hours, take it out, cool to room temperature, add ultrapure water to 10mL, shake and mix to obtain the product;

[0046] For high temperature degradation samples, take about 1g (accurate to 0.0001g) of 6-methylnicotine raw material, put it in a 10ml volumetric flask, seal it and place it in a 90℃ water bath for 12 hours, then take it out, cool it to room temperature, add ultrapure water to make up to 10mL, and shake to mix.

[0047] S2: Use high performance liquid chromatography to detect the test solution and the reference solution

[0048] For the control sample, take about 1 g (accurate to 0.0001 g) of 6-methyl nicotine raw material, place it in a 10 mL volumetric flask, add ultrapure water to make up to 10 mL, shake and mix well, and use it as the control sample solution;

[0049] S3: Use high performance liquid chromatography to detect the test solution and the reference solution. The detection conditions are:

[0050] Chromatographic column: ZORBAX Extend C18 column;

[0051] Column temperature: 30°C;

[0052] Detection wavelength: 266nm;

[0053] Flow rate: 1ml / min;

[0054] Injection volume: 20uL;

[0055] Mobile phase: acetic acid-ammonia buffer as mobile phase A, acetonitrile as mobile phase B, gradient elution, pH of acetic acid-ammonia buffer was 9.0-11.0.

[0056] The experimental results show that:

[0057] 1. For the control sample solution, take about 1 mg of 6-methylnicotine raw material, place it in a 10 ml volumetric flask, add ultrapure water to dissolve and dilute to the mark, shake well and dilute 100 times, filter and obtain. Record the chromatogram as follows: Figure 2 shown.

[0058] Results The main impurities were: impurity 4 (0.0332%), 6-methylnicotine nitrogen oxide 2 (0.1125%), impurity 8 (0.0487%), 6-methylcotinine (0.0422%), 6-methylmysamine (0.1029%);

[0059] The main peak is 6-methylnicotine (99.6550%).

[0060] 2. Acid destruction of the test solution:

[0061] Take about 1 mg of 6-methyl nicotine raw material, place it in a 10 ml volumetric flask, add 1 mL of 1 mol / L hydrochloric acid solution, add 1 mL of 1 mol / L sodium hydroxide solution after 2 hours, dissolve it in ultrapure water and make up to the scale, shake well and dilute it 100 times, filter it and you are done. Record the chromatogram as follows Figure 3 shown.

[0062] Results The main impurities were: impurity 1 (0.0084%), impurity 4 (0.0148%), 6-methylnicotine nitrogen oxide 2 (0.0126%), impurity 8 (0.0097%), 6-methylcotinine (0.0346%), 6-methylmysamine (0.0720%);

[0063] The main peak is 6-methylnicotine (99.8479%).

[0064] 3. Alkali destruction of test solution:

[0065] Take about 1 mg of 6-methyl nicotine raw material, place it in a 10 ml volumetric flask, add 1 mL of 1 mol / L hydrochloric acid solution, shake to mix, and let stand for 2 hours, then add 1 mL of 1 mol / L sodium hydroxide solution, dissolve it in ultrapure water and make up to the scale, shake well, dilute it 100 times, and filter it. Record the chromatogram as follows: Figure 4 shown.

[0066] Results The main impurities were: impurity 1 (0.0056%), impurity 4 (0.0171%), 6-methylnicotine nitrogen oxide 2 (0.0182%), impurity 8 (0.0118%), 6-methylcotinine (0.0344%), 6-methylmysamine (0.0736%);

[0067] The main peak is 6-methylnicotine (99.8392%).

[0068] 4. Oxidative destruction of the test solution:

[0069] Take about 1 mg of 6-methyl nicotine raw material, place it in a 10 ml volumetric flask, add 300 uL of hydrogen peroxide, mix well and let stand for 1 hour, then dissolve it with ultrapure water and dilute to the scale, shake well and dilute 100 times, filter and obtain. Record the chromatogram as follows Figure 5 shown.

[0070] Results The main impurities were: 6-methylnicotine nitrogen oxide 1 (0.1346%), impurity 1 (0.0057%), impurity 4 (0.0143%), 6-methylnicotine nitrogen oxide 2 (1.7020%), impurity 6 (0.0081%), impurity 7 (0.0122%), impurity 8 (0.0489%), 6-methyl cotinine (0.0491%), 6-methyl mysamine (0.0812%), impurity 9 (0.0074%);

[0071] The main peak is 6-methylnicotine (97.9360%).

[0072] 5. Light damage to the test solution:

[0073] Take about 1 mg of 6-methylnicotine raw material, place it in a 10 ml volumetric flask, place it under normal light conditions for 6 days, then dissolve it in ultrapure water and dilute it to the scale, shake it well and dilute it 100 times, and filter it. Record the chromatogram as follows Figure 6 shown.

[0074] Results The main impurities were: impurity 1 (0.0320%), impurity 2 (0.0545%), impurity 3 (0.0307%), impurity 4 (0.0926%), 6-methylnicotine nitrogen oxide 2 (0.6323%), impurity 8 (0.3142%), 6-methyl cotinine (0.3243%), 6-methyl mysamine (0.4172%), impurity 9 (0.0216%);

[0075] The main peak is 6-methylnicotine (98.0806%).

[0076] 6. High temperature destroys the test solution:

[0077] Take about 1 mg of 6-methylnicotine raw material, place it in a 10 ml volumetric flask, place it in a 90°C water bath for 12 hours, then dissolve it in ultrapure water and make up to the mark, shake well and dilute it 100 times, and filter it. Record the chromatogram as follows Figure 7 shown.

[0078] Results The main impurities were: impurity 1 (0.0163%), impurity 2 (0.0103%), impurity 3 (0.0311%), impurity 4 (0.0565%), 6-methylnicotine nitrogen oxide 2 (0.0972%), impurity 5 (0.0466%), impurity 8 (0.1092%), 6-methyl cotinine (0.3502%), 6-methyl mysamine (0.4202%), impurity 9 (0.0219%);

[0079] The main peak is 6-methylnicotine (98.8408%).

[0080] The results of the degradation test show that under the degradation conditions of acid, alkali, light, oxidation, high temperature, etc., the separation between the main peak of 6-methyl nicotine and its impurities and between adjacent impurities is greater than 1.5; the peak purity of 6-methyl nicotine is greater than 990; the signal-to-noise ratio of the sensitivity solution is 8.71; the mobile phase and elution procedure are shown in Table 1, the impurities generated after the test samples are degraded and destroyed under the conditions of light, oxidation, high temperature, acid, alkali, etc. are shown in Table 2, and the peak purity of 6-methyl nicotine and the separation between impurities are shown in Table 3.

[0081] Table 1

[0082] Time (min) Mobile phase A (%) Mobile phase B (%) 0～6 100 0 6～41 100→60 0→40 41～45 60 40 45～45.1 60→100 40→0 45～49 100 0

[0083] Table 2

[0084]

[0085]

[0086] Table 3

[0087]

[0088] Embodiment 2

[0089] like Figure 1 , 8 , a high performance liquid chromatography method for detecting 6-methyl nicotine and its impurities, wherein the determination target of the high performance liquid chromatography method is 6-methyl nicotine and its related substances, comprising the following steps:

[0090] S1: Take 20uL of solvent blank and blank solution and inject them into liquid chromatograph;

[0091] S2: Use high performance liquid chromatography to detect the blank solution. The detection conditions are:

[0092] Chromatographic column: ZORBAX Extend C18 column;

[0093] Column temperature: 30°C;

[0094] Detection wavelength: 266nm;

[0095] Flow rate: 1ml / min;

[0096] Injection volume: 20uL;

[0097] Mobile phase: acetic acid-ammonia buffer as mobile phase A, acetonitrile as mobile phase B, gradient elution, pH of acetic acid-ammonia buffer was 9.0-11.0;

[0098] The experimental results show that: the chromatograms were recorded, and the results showed that no chromatographic peaks were detected in the solvent blank and blank solution at the peaks of 6-methyl nicotine and its impurities, which did not interfere with the detection of 6-methyl nicotine and its impurities. Figure 1 , Figure 8 .

[0099] Embodiment 3

[0100] A high performance liquid chromatography method for detecting 6-methyl nicotine and its impurities, wherein the determination target is 6-methyl nicotine and its impurities, comprising the following steps:

[0101] S1: Accurately weigh about 10 mg of 6-methylnicotine, place it in a 10 mL volumetric flask, add appropriate amount of ultrapure water to dilute to the scale and shake well;

[0102] S2: Precisely dilute and inject 20uL into the liquid chromatograph, record the chromatogram, calculate the signal-to-noise ratio, and the detection conditions are:

[0103] Chromatographic column: ZORBAX Extend C18 column;

[0104] Column temperature: 30°C;

[0105] Detection wavelength: 266nm;

[0106] Flow rate: 1ml / min;

[0107] Injection volume: 20uL;

[0108] Mobile phase: acetic acid-ammonia buffer as mobile phase A, acetonitrile as mobile phase B, gradient elution, pH of acetic acid-ammonia buffer was 9.0-11.0.

[0109] The experimental results show that the test concentration when the signal-to-noise ratio is around 3:1 is used as the detection limit; the test concentration when the signal-to-noise ratio is around 10:1 is used as the quantification limit, and 6 injections are injected continuously, and the RSD of the peak area is ≤10%. The results are shown in Table 4 and Table 5. After detection and analysis, the detection limit concentration of 6-methyl nicotine in this method is 0.1ug / mL, the quantification limit concentration is 0.5ug / mL, and the RSD of repeated injections is 1.15%, which meets the requirements of quantitative limit detection.

[0110] Table 4

[0111]

[0112] Table 5

[0113]

[0114] Embodiment 4

[0115] A high performance liquid chromatography method for detecting 6-methyl nicotine and its impurities, wherein the determination target is 6-methyl nicotine and its impurities, comprising the following steps:

[0116] S1: Prepare linear solution

[0117] 1. Reserve solution configuration:

[0118] Take about 10 mg of 6-methylnicotine reference substance, place it in a 10 mL volumetric flask, add appropriate amount of ultrapure water to dilute to the scale and shake well;

[0119] 2. Linear concentration point configuration:

[0120] Precisely extract an appropriate amount of 6-methylnicotine reference stock solution and prepare it into linear concentration points of 10, 20, 50, 100, and 200 ug / mL respectively;

[0121] S2: Accurately extract 20uL of each of the above solutions and inject them into the liquid chromatograph. The detection conditions are as follows:

[0122] Chromatographic column: ZORBAX Extend C18 column;

[0123] Column temperature: 30°C;

[0124] Detection wavelength: 266nm;

[0125] Flow rate: 1ml / min;

[0126] Injection volume: 20uL;

[0127] Mobile phase: acetic acid-ammonia buffer as mobile phase A, acetonitrile as mobile phase B, gradient elution, pH of acetic acid-ammonia buffer was 9.0-11.0.

[0128] The experimental results show that: with the peak area (A) as the ordinate and the 6-methyl nicotine concentration (C) as the abscissa, the regression equation correlation coefficient is calculated. The results are shown in Table 6. The 6-methyl nicotine linear equation correlation coefficient R>0.990, and the intercept is within 2% of the 100% response value, indicating that the linear relationship of 6-methyl nicotine is good in the range of 10 to 200 ug / mL.

[0129] Table 6

[0130]

[0131] Embodiment 5

[0132] A high performance liquid chromatography method for detecting 6-methyl nicotine and its impurities, wherein the determination target is 6-methyl nicotine and its impurities, comprising the following steps:

[0133] S1: Take the samples for light degradation and oxidation tests, accurately extract 20uL of each and inject into the liquid chromatograph;

[0134] S2: Repeat the injection 6 times and calculate the content of 6-methylnicotine, the largest single impurity and the total impurities according to the peak area normalization method.

[0135] The experimental results show that the contents of 6-methyl nicotine, the largest single impurity and total impurities were calculated as the repeatability test results. The results are shown in Table 7. The RSD of 6-methyl nicotine content measured by this method is less than 1.5%, and the RSD of total impurity content is less than 3%, indicating that this method has good repeatability.

[0136] Table 7

[0137]

[0138]

[0139] Embodiment 6

[0140] A high performance liquid chromatography method for detecting 6-methyl nicotine and its impurities, wherein the determination target is 6-methyl nicotine and its impurities, comprising the following steps:

[0141] S1: Take 6-methylnicotine test sample and measure it with different personnel and equipment according to the repeatability method;

[0142] S2: Calculate the content according to the peak area normalization method;

[0143] The experimental results show that the precision of the content of 6-methyl nicotine, the largest single impurity and the total impurities is calculated as the reproducibility test result. The results are shown in Table 8. Two different experimenters, A and B, respectively operated on different equipment. The RSD of the 6-methyl nicotine content measured in 12 test solutions was 0.0196%, less than 3%; the RSD of the total impurity content was 1.01%, less than 6%, indicating that this method has good reproducibility.

[0144] Table 8

[0145]

[0146] Embodiment 7

[0147] A high performance liquid chromatography method for detecting 6-methyl nicotine and its impurities, wherein the determination target is 6-methyl nicotine and its impurities, comprising the following steps:

[0148] S1: Preparation of test solution

[0149] 1. Prepare a 6-methyl nicotine-containing e-liquid (6-methyl nicotine content: 2 mg / g) as the test sample, weigh about 0.1g (accurate to 0.0001g) of the test sample and place it in a 10mL volumetric flask, add different volumes of 6-methyl nicotine stock solution, dilute to the scale with ultrapure water and shake well, and filter through a 0.22um water filter membrane. Prepare three copies in parallel;

[0150] 2. Test sample spike solution 1: Accurately weigh about 0.1g (accurate to 0.0001g) of the test sample and place it in a 10mL volumetric flask. Add 0.2mL of 6-methylnicotine stock solution (concentration 10mg / mL) to the volumetric flask, add appropriate amount of ultrapure water to dilute to the scale and shake well. Pass through a 0.22um water filter membrane to obtain the solution. Prepare three replicates in parallel;

[0151] 3. Test sample spike solution 2: Accurately weigh about 0.1g (accurate to 0.0001g) of the test sample and place it in a 10mL volumetric flask. Add 0.8mL of 6-methylnicotine stock solution (concentration 10mg / mL) to the volumetric flask, add appropriate amount of ultrapure water to dilute to the scale and shake well. Pass through a 0.22um water filter membrane to obtain the solution. Prepare three replicates in parallel;

[0152] 4. Test sample spike solution 3: Accurately weigh about 0.1g (accurate to 0.0001g) of the test sample and place it in a 10mL volumetric flask. Add 1.5mL of 6-methylnicotine stock solution (concentration 10mg / mL) to the volumetric flask, add appropriate amount of ultrapure water to dilute to the scale and shake well. Pass through a 0.22um water filter membrane to obtain the solution. Prepare three copies in parallel;

[0153] S2: Determination of the test solution: 20uL of the test solution and each test solution spiked with each sample were measured and injected into the liquid chromatograph. The detection conditions were:

[0154] Chromatographic column: ZORBAX Extend C18 column;

[0155] Column temperature: 30°C;

[0156] Detection wavelength: 266nm;

[0157] Flow rate: 1ml / min;

[0158] Injection volume: 20uL;

[0159] Mobile phase: acetic acid-ammonia buffer as mobile phase A, acetonitrile as mobile phase B, gradient elution, pH of acetic acid-ammonia buffer was 9.0-11.0.

[0160] The experimental results show that: the 6-methyl nicotine content was quantitatively calculated according to the external standard method, and the 6-methyl nicotine recovery rate was analyzed. The results are shown in Table 8. The 6-methyl nicotine recovery rate was 100.38%, within the range of 92-105%, and the RSD of the 6-methyl nicotine recovery rate in each test sample was 1.79%, less than 2%, indicating that the accuracy of this method is good.

[0161] Table 8

[0162]

[0163] Embodiment 8

[0164] A high performance liquid chromatography method for detecting 6-methyl nicotine and its impurities, wherein the determination target is 6-methyl nicotine and its impurities, comprising the following steps:

[0165] The column temperature ±2°C, flow rate ±0.1 ml / min, and pH ±0.2 in the mobile phase were examined for durability experiments. The suitability solution and sensitivity solution were prepared according to Example 1.3, and then analyzed according to the chromatographic conditions in Example 1.2.

[0166] The experimental results show that when all parameters change slightly, the theoretical plate number of 6-methylnicotine, the separation between the main peak and the impurity peaks and between the impurity peaks, and the main peak signal-to-noise ratio all meet the system suitability requirements. The test results are shown in Table 10.

[0167] Table 10

[0168]

[0169] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A high performance liquid chromatography method for detecting 6-methyl nicotine and its impurities, wherein the target of the high performance liquid chromatography method is 6-methyl nicotine and its impurities, characterized in that: The detection method comprises the following steps: S1: Prepare a test solution, take 6-methylnicotine raw material as a test sample, add ultrapure water and mix to prepare a solution with a concentration of 0.1-2 mg / mL to obtain a test solution; S2: preparing a reference solution, taking 6-methylnicotine raw material and mixing it with ultrapure water to prepare a solution with a concentration of 0.1 to 2 mg / mL, thereby obtaining a reference solution; S3: Detect the test solution and reference solution using high performance liquid chromatography The test conditions are: Chromatographic column: ZORBAX Extend C18 column; Column temperature: 25-40°C; Detection wavelength: 266±20nm; Flow rate: 0.8~1.3ml / min; Mobile phase: acetic acid-ammonia buffer as mobile phase A, acetonitrile as mobile phase B, gradient elution.

2. The high performance liquid chromatography simultaneous determination according to claim 1, characterized in that: The gradient elution is: From 0 to 6 minutes, linear gradient elution was performed, with mobile phase A maintaining a constant ratio of 95% to 100% and mobile phase B maintaining a constant ratio of 0% to 5%; From 6 to 41 minutes, linear gradient elution was used, with the proportion of mobile phase A changing from 95-100% to 60%, and the proportion of mobile phase B changing from 0-5% to 40%; From 41 to 45 minutes, linear gradient elution was performed, with mobile phase A maintaining a constant proportion of 60% and mobile phase B maintaining a constant proportion of 40%; From 45 to 45.1 min, linear gradient elution was performed, the proportion of mobile phase A changed from 60% to 95-100%, and the proportion of mobile phase B changed from 40% to 0-5%; From 45.1 to 49 minutes, linear gradient elution was performed, with the mobile phase A maintaining a constant proportion of 95 to 100%, and the mobile phase B maintaining a constant proportion of 0 to 5%.

3. The simultaneous determination by high performance liquid chromatography according to claim 2, characterized in that: The test sample is 6-methylnicotine raw material, including but not limited to one or more of the following impurities: oxidation products of 6-methylnicotine, 6-methylcotinine, 6-methylmecsamine and impurities 1-8.

4. The simultaneous determination by high performance liquid chromatography according to claim 1, characterized in that: In the above step S3, the acetic acid-ammonia buffer solution is prepared by adding 25 ml of 1 mol / L acetic acid to 900 ml of ultrapure water, adjusting the pH to 9.0-11.0 with ammonia water, and then making the volume to 1000 mL with ultrapure water.

5. The simultaneous determination by high performance liquid chromatography according to claim 1, characterized in that: In the above step S3, acetic acid and ammonia water are both of HPLC grade.

6. The simultaneous determination by high performance liquid chromatography according to claim 1, characterized in that: In the above step S3, the column temperature used in the method is 30°C, the flow rate is 1.0 mL / min, and the detection wavelength is 266 nm.

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