Method for determining content of key impurities in tobacco extract
Through the pretreatment method of non-polar solvent and acid extraction, combined with HPLC technology, the problem of key impurities detection in tobacco extract is solved, and the accurate detection of impurities A, D, and F in nicotine is achieved, ensuring the quality control of nicotine raw materials.
Patent Information
- Application Number
- CN202510741534.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-05
- Publication Date
- 2025-08-12
AI Technical Summary
There is a lack of effective methods in the prior art to detect the content of key impurities A (008-Z1), D (008-Z4) and F (008-Z6) in tobacco extracts, making it difficult to achieve quality control of nicotine raw materials.
The pretreatment method of extracting tobacco extract with non-polar solvent and acid was used, and then the determination was performed using HPLC. The specific steps include adding non-polar solvent A, acid-dividing water layer, adding non-polar solvent B and then diluting the test sample solution.
Through this method, plant tissue debris and alkane solvents in the tobacco extract were removed and converted into a uniform aqueous system, achieving accurate quantitative analysis of key impurities.
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Figure CN120468341A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of impurity detection, in particular to a method for determining the content of key impurities in tobacco extract. Background Art
[0002] Nicotine, also known as nicotine, is an alkaloid found in plants of the Solanaceae family (Solanum genus) and is also a key component of tobacco. Nicotine is often made into chewable tablets, patches, etc. as an alternative therapy to help patients quit smoking, and is also a key raw material for the preparation of e-cigarettes. On April 8, 2022, the National Standardization Administration of China issued the national standard for e-cigarettes (standard number: GB41700-2022). The standard clearly stipulates that "nicotine extracted from tobacco must be used, and the purity should not be less than 99% (mass fraction)", so extracting nicotine from tobacco has become the only officially permitted preparation method.
[0003] Nicotine content in tobacco is as low as 0.5% and contains hundreds of impurities. In the API process for extracting nicotine, tobacco plants are extracted with non-polar solvents such as alkanes to produce a tobacco extract. This not only extracts nicotine but also removes most polar impurities. This simultaneously transforms the raw material from its plant form into a processable and detectable extract. This extract is a key intermediate in the API route and is crucial for subsequent impurity research and control.
[0004] Tobacco extract is a dark brown to black viscous paste extracted with petroleum ether. Nicotine and various weakly polar impurities present in the plant are extracted, and a small amount of tobacco debris may remain. Impurities A (008-Z1), D (008-Z4), and F (008-Z6) are known impurities listed in the European Pharmacopoeia (EP11) and the United States Pharmacopoeia (USP44) for nicotine and are also key known impurities in tobacco extract. To effectively control the quality of nicotine APIs, it is necessary to develop analytical methods for the determination of these three key impurities in tobacco extract.
[0005]
[0006] Depending on the production process, tobacco extracts have a nicotine content of approximately 5% to 15%. They are also highly viscous and contain some plant tissue fragments. Furthermore, the extracts contain a large amount of residual alkane solvents, making them unsuitable for direct analysis using high-performance liquid chromatography (HPLC). Currently, no analytical methods for specific impurities in nicotine extracts have been documented. Summary of the Invention
[0007] Problems to be solved by the invention
[0008] In view of the above problems existing in the prior art, the purpose of the present invention is to provide a method for detecting three key impurities in tobacco extract.
[0009] Solutions for solving problems
[0010] The present invention provides a method for determining the content of key impurities in tobacco extract, the method comprising: pre-treating the tobacco extract and then determining the content of key impurities using HPLC;
[0011] Wherein, the pretreatment includes: extracting tobacco extract using a non-polar solvent and acid.
[0012] Preferably, the pretreatment comprises the following steps:
[0013] (1) Weigh tobacco extract and add non-polar solvent A;
[0014] (2) adding acid and separating the aqueous layer;
[0015] (3) Add non-polar solvent B and separate the aqueous layer;
[0016] (4) After dilution, the tobacco extract test solution is obtained.
[0017] Preferably, the ratio of the tobacco extract to the non-polar solvent A in step (1) is 1 g of tobacco extract: 3 to 10 ml of non-polar solvent A. More preferably, the ratio of the tobacco extract to the non-polar solvent A in step (1) is 1 g of tobacco extract: 5 ml of non-polar solvent A.
[0018] Preferably, the non-polar solvent A in step (1) is selected from n-hexane and / or n-heptane, and more preferably, the non-polar solvent A in step (1) is n-hexane.
[0019] Preferably, the ratio of the tobacco extract in step (1) to the acid in step (2) is 1 g tobacco extract: 15-35 ml acid. Preferably, the ratio of the tobacco extract in step (1) to the acid in step (2) is 1 g tobacco extract: 25 ml acid.
[0020] Preferably, the acid in step (2) is sulfuric acid and / or hydrochloric acid;
[0021] Preferably, the concentration of the sulfuric acid is 0.2 to 1.2 mol / L, more preferably, the concentration of the sulfuric acid is 0.4 to 0.8 mol / L.
[0022] Preferably, the ratio of the tobacco extract in step (1) to the non-polar solvent B in step (3) is 1 g of tobacco extract: 3 to 10 ml of non-polar solvent B. More preferably, the ratio of the tobacco extract in step (1) to the non-polar solvent B in step (3) is 1 g of tobacco extract: 5 ml of non-polar solvent B.
[0023] Preferably, the non-polar solvent B in step (3) is selected from one or more of n-hexane, n-heptane, and ethyl acetate. More preferably, the non-polar solvent B in step (3) is ethyl acetate.
[0024] Preferably, the pH of the aqueous layer in step (3) is 1 to 4;
[0025] More preferably, the pH of the aqueous layer in step (3) is 2-3.
[0026] Preferably, the HPLC comprises the steps of:
[0027] (1) preparing a solution containing the key impurity as an impurity reference substance mixed solution;
[0028] (II) preparing a solution containing nicotine and the key impurities as a system suitability solution;
[0029] (III) preparing a tobacco extract test solution according to the method of any one of claims 1 to 6;
[0030] (IV) Using HPLC, the impurity reference substance mixed solution, the system suitability solution, and the tobacco extract test solution were respectively measured.
[0031] Preferably, the HPLC column in step (IV) is Waters XTERRA RP18, 4.6 mm × 150 mm, 5 μm;
[0032] Preferably, the mobile phase A of the HPLC in step (IV) is an acetic acid-ammonia buffer;
[0033] Preferably, the mobile phase B of the HPLC in step (IV) is acetonitrile;
[0034] Preferably, the elution gradient of the HPLC in step (IV) is:
[0035]
[0036] Preferably, the preparation method of the mobile phase A comprises: measuring 1.5 ml of acetic acid and 6 ml of concentrated ammonia water to 1000 ml of water, and adjusting the pH value to 9.5-10.5 with concentrated ammonia water;
[0037] More preferably, the pH value is 10.0.
[0038] Preferably, the key impurities include:
[0039]
[0040] Effects of the Invention
[0041] The method for detecting key impurities in tobacco extract of the present invention performs a certain number of pretreatment steps on the sample. After the treatment, the sample solution is substantially free of plant tissue debris, and the main alkane solvent is removed. The solution is converted into a uniform aqueous phase system. The impurities can be quantitatively analyzed using a high performance liquid chromatograph using a reference substance external standard method, and the contents of impurities A (008-Z1), impurity D (008-Z4), and impurity F (008-Z6) in nicotine in the tobacco extract can be accurately detected. BRIEF DESCRIPTION OF THE DRAWINGS
[0042] Figure 1 This is a typical spectrum of blank solution.
[0043] Figure 2 This is a typical spectrum of the test solution.
[0044] Figure 3 This is a typical spectrum of system suitability solution.
[0045] Figure 4 This is a typical spectrum of the spiked test solution. DETAILED DESCRIPTION
[0046] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate the details of the local features. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.
[0047] The present invention provides a method for determining the content of key impurities in tobacco extract, the method comprising: pre-treating the tobacco extract and then determining the content of key impurities using HPLC;
[0048] Wherein, the pretreatment includes: extracting tobacco extract using a non-polar solvent and acid.
[0049] In certain embodiments, the pretreatment comprises the steps of:
[0050] (1) Weigh tobacco extract and add non-polar solvent A;
[0051] (2) adding acid and separating the aqueous layer;
[0052] (3) Add non-polar solvent B and separate the aqueous layer;
[0053] (4) After dilution, the tobacco extract test solution is obtained.
[0054] In certain embodiments, the ratio of the tobacco extract to the non-polar solvent A in step (1) is 1 g tobacco extract: 3 to 10 ml non-polar solvent A.
[0055] In certain embodiments, the ratio of the tobacco extract to the non-polar solvent A in step (1) is 1g tobacco extract: 3ml non-polar solvent A, or 1g tobacco extract: 4ml non-polar solvent A, or 1g tobacco extract: 5ml non-polar solvent A, or 1g tobacco extract: 6ml non-polar solvent A, or 1g tobacco extract: 7ml non-polar solvent A, or 1g tobacco extract: 8ml non-polar solvent A, or 1g tobacco extract: 9ml non-polar solvent A, or 1g tobacco extract: 10ml non-polar solvent A.
[0056] In certain embodiments, the ratio of the tobacco extract to the non-polar solvent A in step (1) is 1 g tobacco extract: 5 ml non-polar solvent A.
[0057] In certain embodiments, the non-polar solvent A in step (1) is selected from n-hexane and / or n-heptane.
[0058] In certain embodiments, the non-polar solvent A in step (1) is n-hexane or n-heptane.
[0059] In certain embodiments, the non-polar solvent A in step (1) is n-hexane.
[0060] In certain embodiments, the non-polar solvent A in step (1) is n-heptane.
[0061] In certain embodiments, the ratio of the tobacco extract in step (1) to the acid in step (2) is 1 g tobacco extract: 15-35 ml acid.
[0062] In some embodiments, the ratio of the tobacco extract of step (1) to the acid of step (2) is 1g tobacco extract: 15ml acid, or 1g tobacco extract: 16ml acid, or 1g tobacco extract: 17ml acid, or 1g tobacco extract: 18ml acid, or 1g tobacco extract: 19ml acid, or 1g tobacco extract: 20ml acid, or 1g tobacco extract: 21ml acid, or 1g tobacco extract: 22ml acid, or 1g tobacco extract: 23ml acid, or 1g tobacco extract: 24ml acid, or 1g tobacco extract: 25ml acid, or 1g tobacco extract: 26ml acid, or 1g tobacco extract: 27ml acid, or 1g tobacco extract: 28ml acid, or 1g tobacco extract: 29ml acid, or 1g tobacco extract: 30ml acid.
[0063] In certain embodiments, the ratio of the tobacco extract in step (1) to the acid in step (2) is 1 g tobacco extract: 25 ml acid.
[0064] In certain embodiments, the acid in step (2) is sulfuric acid and / or hydrochloric acid.
[0065] In certain embodiments, the acid in step (2) is sulfuric acid or hydrochloric acid.
[0066] In certain embodiments, the acid in step (2) is sulfuric acid.
[0067] In certain embodiments, the acid in step (2) is hydrochloric acid.
[0068] In certain embodiments, the concentration of sulfuric acid is 0.2 to 1.2 mol / L.
[0069] In certain embodiments, the concentration of sulfuric acid is 0.2 mol / L, or 0.25 mol / L, or 0.3 mol / L, or 0.35 mol / L, or 0.4 mol / L, or 0.45 mol / L, or 0.5 mol / L, or 0.55 mol / L, or 0.6 mol / L, or 0.65 mol / L, or 0.7 mol / L, or 0.75 mol / L, or 0.8 mol / L, or 0.85 mol / L, or 0.9 mol / L, or 0.95 mol / L, or 1 mol / L, or 1.05 mol / L, or 1.1 mol / L, or 1.15 mol / L, or 1.2 mol / L.
[0070] In certain embodiments, the concentration of sulfuric acid is 0.2 to 0.9 mol / L.
[0071] In certain embodiments, the concentration of sulfuric acid is 0.4-0.8 mol / L.
[0072] In some embodiments, the concentration of sulfuric acid is 0.5 mol / L
[0073] In certain embodiments, the ratio of the tobacco extract in step (1) to the non-polar solvent B in step (3) is 1 g tobacco extract: 3 to 10 ml non-polar solvent B.
[0074] In some embodiments, the ratio of the tobacco extract in step (1) to the non-polar solvent B in step (3) is 1g tobacco extract: 3ml non-polar solvent B, or 1g tobacco extract: 4ml non-polar solvent B, or 1g tobacco extract: 5ml non-polar solvent B, or 1g tobacco extract: 6ml non-polar solvent B, or 1g tobacco extract: 7ml non-polar solvent B, or 1g tobacco extract: 8ml non-polar solvent B, or 1g tobacco extract: 9ml non-polar solvent B, or 1g tobacco extract: 10ml non-polar solvent B.
[0075] In certain embodiments, the ratio of the tobacco extract in step (1) to the non-polar solvent B in step (3) is 1 g tobacco extract: 5 ml non-polar solvent B.
[0076] In certain embodiments, the non-polar solvent B in step (3) is selected from one or more of n-hexane, n-heptane, and ethyl acetate.
[0077] In certain embodiments, the non-polar solvent B in step (3) is n-hexane, n-heptane, and ethyl acetate.
[0078] In certain embodiments, the non-polar solvent B in step (3) is n-hexane or n-heptane.
[0079] In certain embodiments, the non-polar solvent B in step (3) is n-hexane and ethyl acetate.
[0080] In certain embodiments, the non-polar solvent B in step (3) is n-heptane and ethyl acetate.
[0081] In certain embodiments, the non-polar solvent B in step (3) is n-hexane.
[0082] In certain embodiments, the non-polar solvent B in step (3) is n-heptane.
[0083] In certain embodiments, the non-polar solvent B in step (3) is ethyl acetate.
[0084] In certain embodiments, the pH of the aqueous layer in step (3) is 1-4.
[0085] In certain embodiments, the pH of the aqueous layer in step (3) is 1, or 1.5, or 2, or 2.5, or 3, or 3.5, or 4.
[0086] In certain embodiments, the pH of the aqueous layer in step (3) is 2-3.
[0087] In certain embodiments, the HPLC comprises the steps of:
[0088] (1) preparing a solution containing the key impurity as an impurity reference substance mixed solution;
[0089] (II) preparing a solution containing nicotine and the key impurities as a system suitability solution;
[0090] (III) preparing a tobacco extract test solution according to the method of any one of claims 1 to 6;
[0091] (IV) Using HPLC, the impurity reference substance mixed solution, the system suitability solution, and the tobacco extract test solution were respectively measured.
[0092] In certain embodiments, the HPLC column in step (IV) is Waters XTERRA RP18, 4.6 mm×150 mm, 5 μm.
[0093] In certain embodiments, the mobile phase A of the HPLC in step (IV) is an acetic acid-ammonia buffer.
[0094] In certain embodiments, the mobile phase B of the HPLC in step (IV) is acetonitrile.
[0095] In certain embodiments, the elution gradient of the HPLC in step (IV) is:
[0096]
[0097] In certain embodiments, the preparation method of the mobile phase A comprises: measuring 1.5 ml of acetic acid and 6 ml of concentrated ammonia water to 1000 ml of water, and adjusting the pH value to 9.5-10.5 with concentrated ammonia water.
[0098] In certain embodiments, the pH is 9.5, or 9.6, or 9.7, or 9.8, or 9.9, or 10, or 10.1, or 10.2, or 10.3, or 10.4, or 10.5.
[0099] In certain embodiments, the pH is 10.0.
[0100] In certain embodiments, the key impurities include:
[0101]
[0102] In certain embodiments, the key impurities are:
[0103]
[0104] Example 1: Tobacco Extract Key Impurity Detection Method 1
[0105] 1. Prepare reference solution
[0106] Impurity 008-Z1 reference substance stock solution (10 mg / ml): Take 50 mg of impurity 008-Z1 reference substance, place it in a 5 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, and shake well.
[0107] Impurity 008-Z4 reference substance stock solution (10 mg / ml): Take 50 mg of impurity 008-Z4 reference substance, place it in a 5 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, and shake well.
[0108] Impurity 008-Z6 reference substance stock solution (10 mg / ml): Take 50 mg of impurity 008-Z6 reference substance, place it in a 5 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, and shake well.
[0109] Impurity reference substance mixed solution (each impurity is 2 mg / ml): Take 2 ml of the above impurity reference substance stock solution, place it in the same 10 ml volumetric flask, add water to dilute to the scale, and shake well.
[0110] 2. Prepare system suitability solution (nicotine: 1 mg / ml, each impurity: 0.1 mg / ml)
[0111] Take 20 mg of nicotine reference substance and accurately measure 1 ml of the mixed solution of impurity reference substance, place them in the same 20 ml volumetric flask, add water to dilute to the scale, and shake well.
[0112] 3. Prepare the test solution
[0113] Take approximately 1g of the tobacco extract sample (Hubei Henuo Bioengineering Co., Ltd.) and accurately add 5ml of n-hexane. Stir with a glass rod for 10 minutes. Add 25ml of 0.5mol / L sulfuric acid solution and continue stirring for 10 minutes. Separate the aqueous layer and add 5ml of ethyl acetate. Shake thoroughly and let stand for 3 minutes. Separate the aqueous layer. The pH of the aqueous solution should be 2-3. Accurately measure 1ml of the above aqueous solution into a 100ml volumetric flask, dilute to the mark with water, and shake well.
[0114] Testing: Accurately measure 10 μl of each of the system suitability solution and the test solution and analyze them using a high-performance liquid chromatograph. Testing can be performed using the liquid chromatography conditions specified for nicotine APIs in the European Pharmacopoeia and the United States Pharmacopoeia. The chromatographic conditions are shown in Table 1, and the test results are shown in Table 2.
[0115] Table 1: HPLC conditions
[0116]
[0117] Table 2: Tobacco extract impurity test results
[0118]
[0119]
[0120] The results showed that when the aqueous phase extraction solvent was dichloromethane, emulsification occurred and stratification was impossible. Therefore, ethyl acetate was the preferred extraction solvent. Increasing the amount of n-hexane and ethyl acetate added had no significant effect on the extraction effect. The impurity detection amount in Example 1 was larger, and it was preferred as a method for detecting related substances in tobacco extract. The impurity detection amounts in Examples 2, 3, 5, and 6 were slightly smaller, but they were also suitable for detecting related substances in tobacco extract.
[0121] Example 2: Optimization method 2 for detecting key impurities in tobacco extract
[0122] The system suitability solution and reference solution were prepared in the same manner as in Example 1. The test solution was prepared as follows:
[0123] Take approximately 1g of the tobacco extract sample (Hubei Henuo Bioengineering Co., Ltd.) and accurately add 5ml of n-hexane. Stir with a glass rod for 10 minutes. Add 25ml of 0.2mol / L sulfuric acid solution and continue stirring for 10 minutes. Separate the aqueous layer and add 5ml of ethyl acetate. Shake thoroughly and let stand for 3 minutes. Separate the aqueous layer. The pH of the aqueous solution should be 3-4. Accurately measure 1ml of the above aqueous solution into a 100ml volumetric flask, dilute to the mark with water, and shake well.
[0124] The system suitability solution, reference solution, and test solution were measured using a high performance liquid chromatograph. The chromatographic conditions are shown in Table 1, and the test results are shown in Table 2.
[0125] Example 3: Optimization method for detecting key impurities in tobacco extract 3
[0126] The system suitability solution and reference solution were prepared in the same manner as in Example 1. The test solution was prepared as follows:
[0127] Take approximately 1g of the tobacco extract sample to be tested (Hubei Henuo Bioengineering Co., Ltd.), accurately add 5ml of n-hexane, and stir with a glass rod for 10 minutes. Add 25ml of 0.9mol / L sulfuric acid solution and continue stirring for 10 minutes. Separate the aqueous layer, add 5ml of ethyl acetate, shake thoroughly, let stand for 3 minutes, and separate the aqueous layer. The pH of the aqueous solution should be 1-2. Accurately measure 1ml of the above aqueous solution and place it in a 100ml volumetric flask. Dilute to the mark with water and shake well.
[0128] The system suitability solution, reference solution, and test solution were measured using a high performance liquid chromatograph. The chromatographic conditions are shown in Table 1, and the test results are shown in Table 2.
[0129] Example 4: Optimization method 4 for detecting key impurities in tobacco extract
[0130] The system suitability solution and reference solution were prepared in the same manner as in Example 1. The test solution was prepared as follows:
[0131] Take approximately 1g of the tobacco extract sample to be tested (Hubei Henuo Bioengineering Co., Ltd.), accurately add 5ml of n-hexane, and stir with a glass rod for 10 minutes. Add 25ml of 0.5mol / L sulfuric acid solution and continue stirring for 10 minutes. Separate the aqueous layer, add 5ml of dichloromethane, shake thoroughly, let stand for 3 minutes, and separate the aqueous layer. The pH of the aqueous solution should be 2-3. Accurately measure 1ml of the above aqueous solution and place it in a 100ml volumetric flask. Dilute to the mark with water and shake well.
[0132] The system suitability solution, reference solution, and test solution were measured using a high performance liquid chromatograph. The chromatographic conditions are shown in Table 1, and the test results are shown in Table 2.
[0133] Example 5: Optimization method for detecting key impurities in tobacco extract
[0134] The system suitability solution and reference solution were prepared in the same manner as in Example 1. The test solution was prepared as follows:
[0135] Take approximately 1g of the tobacco extract sample (Hubei Henuo Bioengineering Co., Ltd.) and accurately add 10ml of n-hexane. Stir with a glass rod for 10 minutes. Add 25ml of 0.5mol / L sulfuric acid solution and continue stirring for 10 minutes. Separate the aqueous layer and add 5ml of ethyl acetate. Shake thoroughly and let stand for 3 minutes. Separate the aqueous layer. The pH of the aqueous solution should be 2-3. Accurately measure 1ml of the above aqueous solution into a 100ml volumetric flask, dilute to the mark with water, and shake well.
[0136] The system suitability solution, reference solution, and test solution were measured using a high performance liquid chromatograph. The chromatographic conditions are shown in Table 1, and the test results are shown in Table 2.
[0137] Example 6: Optimization method for detecting key impurities in tobacco extract
[0138] The system suitability solution and reference solution were prepared in the same manner as in Example 1. The test solution was prepared as follows:
[0139] Take approximately 1g of the tobacco extract sample (Hubei Henuo Bioengineering Co., Ltd.) and accurately add 5ml of n-hexane. Stir with a glass rod for 10 minutes. Add 25ml of 0.5mol / L sulfuric acid solution and continue stirring for 10 minutes. Separate the aqueous layer and add 10ml of ethyl acetate. Shake thoroughly and let stand for 3 minutes. Separate the aqueous layer. The pH of the aqueous solution should be 2-3. Accurately measure 1ml of the above aqueous solution into a 100ml volumetric flask, dilute to the mark with water, and shake well.
[0140] The system suitability solution, reference solution, and test solution were measured using a high performance liquid chromatograph. The chromatographic conditions are shown in Table 1, and the test results are shown in Table 2.
[0141] Example 7: Specificity test
[0142] Blank solution: water
[0143] The system suitability solution was prepared in the same manner as in Example 1. 10 μl of each of the blank solution and the system suitability solution were accurately measured and assayed using a high performance liquid chromatograph.
[0144] The separation degree between the known impurities and the main component is greater than 1.5, and the separation degree is good.
[0145] Example 8: Accuracy Test
[0146] Prepare the spiked test solution: Take approximately 1g of the tobacco extract sample (Hubei Henuo Bioengineering Co., Ltd.), accurately add 5ml of n-hexane, and stir with a glass rod for 10 minutes. Add 25ml of 0.5mol / L sulfuric acid solution to 1ml of the impurity reference solution and continue stirring for 10 minutes. Separate the aqueous layer, add 5ml of ethyl acetate, shake thoroughly, let stand for 3 minutes, and separate the aqueous layer. The pH of the aqueous solution should be 2-3. Accurately measure 1ml of the above aqueous solution into a 10ml volumetric flask, dilute to the mark with water, and shake well.
[0147] The preparation of the test solution is the same as that in Example 1.
[0148] Reference solution: Accurately measure 1 ml of the impurity reference solution into a 100 ml volumetric flask, dilute to the mark with water, and shake well.
[0149] The spiked test solution, reference solution, and test solution were measured using a high performance liquid chromatograph. The chromatographic conditions are shown in Table 1, and the test results are shown in Table 3.
[0150] Table 3: Accuracy test results
[0151]
[0152] The results showed that after tobacco extract was pretreated by this method, the recoveries of impurities 008-Z6, 008-Z1 and 008-Z4 in 6 recovery solutions were between 80% and 120%, and the RSDs of the 6 solutions were all less than 10.0%, indicating good accuracy.
[0153] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.
Claims
1. A method for determining the content of key impurities in tobacco extract, characterized in that: The method comprises: pre-treating tobacco extract and then performing determination using HPLC; Wherein, the pretreatment includes: extracting tobacco extract using a non-polar solvent and acid.
2. The method according to claim 1, characterized in that The pre-processing comprises the following steps: (1) Weigh tobacco extract and add non-polar solvent A; (2) adding acid and separating the aqueous layer; (3) Add non-polar solvent B and separate the aqueous layer; (4) After dilution, the tobacco extract test solution is obtained.
3. The method according to claim 2, characterized in that The ratio of the tobacco extract to the non-polar solvent A in step (1) is 1 g of tobacco extract: 3 to 10 ml of non-polar solvent A. Preferably, the ratio of the tobacco extract to the non-polar solvent A in step (1) is 1 g of tobacco extract: 5 ml of non-polar solvent A. Preferably, the non-polar solvent A in step (1) is selected from n-hexane and / or n-heptane, and more preferably, the non-polar solvent A in step (1) is n-hexane.
4. The method according to claim 2, characterized in that The ratio of the tobacco extract in step (1) to the acid in step (2) is 1 g tobacco extract: 15-35 ml acid. Preferably, the ratio of the tobacco extract in step (1) to the acid in step (2) is 1 g tobacco extract: 25 ml acid. Preferably, the acid in step (2) is sulfuric acid and / or hydrochloric acid; Preferably, the concentration of the sulfuric acid is 0.2 to 1.2 mol / L, more preferably, the concentration of the sulfuric acid is 0.4 to 0.8 mol / L.
5. The method according to claim 2, characterized in that The ratio of the tobacco extract in step (1) to the non-polar solvent B in step (3) is 1 g of tobacco extract: 3 to 10 ml of non-polar solvent B. Preferably, the ratio of the tobacco extract in step (1) to the non-polar solvent B in step (3) is 1 g of tobacco extract: 5 ml of non-polar solvent B. Preferably, the non-polar solvent B in step (3) is selected from one or more of n-hexane, n-heptane, and ethyl acetate. More preferably, the non-polar solvent B in step (3) is ethyl acetate.
6. The method according to claim 2, characterized in that The pH of the aqueous layer in step (3) is 1 to 4; Preferably, the pH of the aqueous layer in step (3) is 2-3.
7. The method according to any one of claims 1 to 6, characterized in that Described HPLC comprises the steps: (1) preparing a solution containing the key impurity as an impurity reference substance mixed solution; (II) preparing a solution containing nicotine and the key impurities as a system suitability solution; (III) preparing a tobacco extract test solution according to the method of any one of claims 1 to 6; (IV) Using HPLC, the impurity reference substance mixed solution, the system suitability solution, and the tobacco extract test solution were respectively measured.
8. The method according to claim 7, characterized in that The HPLC column in step (IV) is Waters XTERRA RP18, 4.6 mm × 150 mm, 5 μm; Preferably, the mobile phase A of the HPLC in step (IV) is an acetic acid-ammonia buffer; Preferably, the mobile phase B of the HPLC in step (IV) is acetonitrile; Preferably, the elution gradient of the HPLC in step (IV) is:
9. The method according to claim 8, characterized in that The preparation method of the mobile phase A comprises: measuring 1.5 ml of acetic acid and 6 ml of concentrated ammonia water to 1000 ml of water, and adjusting the pH value to 9.5-10.5 with concentrated ammonia water; Preferably, the pH value is 10.
0.
10. The method according to any one of claims 1 to 9, characterized in that The key impurities include: