An analysis method of non-volatile organic acids in electronic cigarette liquid

Abstract

The application discloses an analysis method of non-volatile organic acids in electronic cigarette liquid, comprising the following steps: adding a to-be-tested sample of electronic cigarette liquid into an internal standard stock solution and a formic acid-methanol aqueous solution, sealing and oscillating, diluting with an internal standard solution to obtain a to-be-tested sample solution; mixing single standard stock solutions prepared by different target standard samples, diluting into a mixed standard intermediate solution, taking different volumes of the mixed standard intermediate solution, adding an internal standard stock solution and a formic acid-methanol aqueous solution, and configuring into standard working solutions with different concentrations; respectively performing ultra-high performance liquid chromatography-tandem mass spectrometry analysis on the to-be-tested sample solution and the standard working solution; and according to the analysis result, the content of the target in the to-be-tested sample is calculated by using an internal standard standard curve method. The application can realize simultaneous analysis and chromatographic separation of six non-volatile organic acids in electronic cigarette liquid, has the advantages of accurate quantification, high flux, good separation degree, simple operation, high sensitivity, good recovery rate and good repeatability, etc.

Description

Technical Field

[0001] This invention belongs to the field of tobacco chemical analysis technology, and more specifically, relates to an analytical method for non-volatile organic acids in e-cigarette liquid. Background Technology

[0002] Electronic cigarettes are electronic delivery systems used to generate aerosols for inhalation. Benzoic acid, lactic acid, levulinic acid, and other non-volatile organic acids are commonly added ingredients in e-cigarette liquids. They can combine with nicotine to form nicotine salt structures, altering the form of nicotine in the aerosol, reducing nicotine irritation, and improving the vaping experience. In recent years, the development of e-cigarettes has been rapid, and regulations vary across countries and regions. my country issued and implemented the mandatory national standard GB 41700-2022 "E-cigarettes" in 2022, which specifies the permitted additives in e-cigarette liquids and their maximum usage amounts. The non-volatile organic acids include D,L-tartaric acid, malic acid, lactic acid, citric acid, levulinic acid, and benzoic acid, with maximum usage amounts of 10, 24, 20, 50, 28, and 26 mg / g, respectively, and cover the types of organic acids commonly used in e-cigarette liquids for nicotine salts. Therefore, rapid and accurate analysis of non-volatile organic acids in e-cigarette liquids is of great significance.

[0003] Currently, the main methods for determining non-volatile organic acids in food and tobacco include ion chromatography, high-performance liquid chromatography, gas chromatography, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS). Ion chromatography and GC-MS are easily affected by impurities under complex matrix conditions, which can affect the quantification of target substances. Gas chromatography and GC-MS require long-term high-temperature derivatization, and methyl esterification is a commonly used derivatization method, but the pretreatment process is relatively cumbersome. In addition, methyl benzoate, a benzoic acid derivative, is also on the list of e-cigarette additives, which affects the quantification of benzoic acid. Summary of the Invention

[0004] The purpose of this invention is to address the above-mentioned shortcomings by providing an analytical method for non-volatile organic acids in e-cigarette liquids. Based on UPLC-MS / MS technology, using a 0.1% formic acid-10% methanol aqueous solution, and through optimization of instrument liquid phase conditions and sample pretreatment experimental conditions, chromatographic separation of tartaric acid, malic acid, lactic acid, citric acid, levulinic acid, and benzoic acid is achieved. This method has advantages such as accurate quantification, high throughput, good separation, simple operation, high sensitivity, good recovery rate, and good repeatability, making it suitable for the analysis of non-volatile organic acids in e-cigarette liquids.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0006] A method for analyzing non-volatile organic acids in e-cigarette liquids, comprising the following steps:

[0007] Add the test sample of e-cigarette liquid to internal standard stock solution and formic acid-methanol aqueous solution, seal and shake, then dilute with internal standard solution to obtain the test sample solution;

[0008] Single standard stock solutions prepared from different target analyte standards were mixed and diluted to form mixed standard intermediate solutions. Different volumes of mixed standard intermediate solutions were taken and added to internal standard stock solutions and formic acid-methanol aqueous solutions to prepare standard working solutions of different concentrations.

[0009] The above-mentioned sample solutions and standard working solutions were analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS).

[0010] Based on the analytical results of the above ultra-high performance liquid chromatography-tandem mass spectrometry analysis, the content of the target analyte in the sample was calculated using the internal standard curve method.

[0011] Preferably, the internal standard used in this invention is benzoic acid-d5 and succinic acid-d6; the internal standard stock solution is a methanol mixture of benzoic acid-d5 and succinic acid-d6 with a concentration of 1 mg / mL.

[0012] Preferably, the formic acid-methanol aqueous solution is a 0.1% formic acid-10% methanol aqueous solution, wherein the percentage content is a volume fraction. The volume of the formic acid-methanol aqueous solution used in the preparation of the test sample solution is 10 mL, the shaking time is 20 min, and after shaking, it is diluted 20 times with an internal standard solution.

[0013] Preferably, the internal standard solution is a 0.1% formic acid-10% methanol-water mixture of benzoic acid-d5 and succinic acid-d6, with a concentration of 0.005 mg / mL.

[0014] Preferably, the non-volatile organic acids in the e-cigarette liquid include tartaric acid, malic acid, lactic acid, citric acid, acetylpropionic acid, and benzoic acid.

[0015] Preferably, the concentration range of each target analyte in the standard working solution is as follows: tartaric acid, malic acid, citric acid, levulinic acid and benzoic acid are 0.05-15 μg / mL, and lactic acid is 0.4-15 μg / mL.

[0016] Preferably, in the ultra-high performance liquid chromatography-tandem mass spectrometry analysis, the ultra-high performance liquid chromatography uses a Waters HSS T3 column, 150 mm × 2.1 mm, 1.8 μm; mobile phase A is 5 mmol / L ammonium formate aqueous solution (pH adjusted to 2.5 with formic acid), and mobile phase B is 0.1% (v / v) formic acid methanol solution. Column temperature: 30℃; flow rate: 0.3 mL / min.

[0017] Preferably, in the ultra-high performance liquid chromatography-tandem mass spectrometry analysis, the ultra-high performance liquid chromatography uses gradient elution, with the elution program as follows: 0–3.5 min, phase A remains at 98%; 3.5–11 min, phase A decreases from 98% to 2%; 11–14 min, phase A remains at 2%; 14–15 min, phase A remains unchanged at 98% (at 14 min, it directly increases from 2% to 98% and remains there for 1 min); the injection volume is 1 μL.

[0018] Preferably, in the ultra-high performance liquid chromatography-tandem mass spectrometry analysis, the mass spectrometry conditions are as follows:

[0019] The ion source was an electrospray ionization (ESI) source; the ionization mode was negative ion mode; the electrospray voltage was -4500V; the ion source temperature was 500℃; and the scanning method was multiple reaction monitoring (MRM).

[0020] Preferably, the MRM parameters of the above 6 non-volatile organic acids and 2 internal standards are shown in Table 1:

[0021] Table 1. MRM parameters of 6 non-volatile organic acids and 2 internal standards

[0022]

[0023]

[0024] Note: "*" indicates quantitative ions.

[0025] Preferably, the step of calculating the content of the target analyte in the sample using the internal standard curve method includes:

[0026] After the standard working solutions of different concentrations were detected and analyzed by the ultra-high performance liquid chromatography-tandem mass spectrometry, a standard working curve was prepared with the ratio of the quantitative ion peak area of ​​the target analyte to that of the internal standard as the ordinate and the content of the target analyte as the abscissa.

[0027] After the sample solution is analyzed by the ultra-high performance liquid chromatography-tandem mass spectrometry instrument, the content of the target substances in the sample solution is obtained, and then the content of each target substance in the sample is calculated.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] This invention, based on UPLC-MS / MS technology, establishes an analytical method for six non-volatile organic acids in e-cigarette liquids by optimizing instrument analysis conditions and sample pretreatment conditions. It achieves simultaneous analysis and chromatographic separation of tartaric acid, malic acid, lactic acid, citric acid, levulinic acid, and benzoic acid. This invention offers advantages such as accurate quantification, high throughput, good separation, simple operation, high sensitivity, good recovery rate, and good repeatability, making it suitable for the analysis of non-volatile organic acids in e-cigarette liquids. Attached Figure Description

[0030] Picture 1 This is a multiple reaction monitoring (MRM) chromatogram of the target analyte and internal standard in the standard working solution described in this embodiment of the invention. Detailed Implementation

[0031] Preferred embodiments of the present invention will now be described in more detail with reference to the accompanying drawings and specific examples.

[0032] The following are the instruments and reagents used in the examples:

[0033] 1290 ultra-high performance liquid chromatograph (Agilent Technologies, USA), AB5500 triple quadrupole mass spectrometer (SCIEX, USA); vortex mixer (DATI Instrument Technology Co., Ltd.); electronic balance (Sartorius, Germany); pH meter (Mettler, Switzerland).

[0034] DL-tartaric acid (99.7% purity), lactic acid (90.2% purity) standards (Beijing Manhag Biotechnology Co., Ltd.), citric acid (99.7% purity) standards (Beijing Tanmo Quality Inspection and Standard Material Center), levulinic acid (99% purity) standards (Shanghai Anpu Cuishi Standard Technology Service Co., Ltd.), malic acid (99.8% purity), benzoic acid (99.5% purity) standards (Dr. Ehrenstorfer, Germany), benzoic acid-d5 (99% purity) and succinic acid-d6 (99% purity) (Sigma-Aldrich, USA). Methanol (chromatographic grade, Shanghai Keyi Chemical Technology Co., Ltd.); formic acid and ammonium formate (chromatographic grade, Beijing Inokai Technology Co., Ltd.). Ultrapure water was used in the experiments.

[0035] This invention provides a method for analyzing non-volatile organic acids in e-cigarette liquids, comprising the following steps:

[0036] Add the test sample of e-cigarette liquid to internal standard stock solution and formic acid-methanol aqueous solution, seal and shake, then dilute with internal standard solution to obtain the test sample solution;

[0037] Single standard stock solutions prepared from different target analyte standards were mixed and diluted to form mixed standard intermediate solutions. Different volumes of mixed standard intermediate solutions were taken and added to internal standard stock solutions and formic acid-methanol aqueous solutions to prepare standard working solutions of different concentrations.

[0038] The above-mentioned sample solutions and standard working solutions were analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry, respectively.

[0039] Based on the analytical results of the above ultra-high performance liquid chromatography-tandem mass spectrometry analysis, the content of the target analyte in the sample was calculated using the internal standard curve method.

[0040] The specific experimental steps are described in the following examples.

[0041] Example 1

[0042] A method for analyzing non-volatile organic acids in e-cigarette liquids, comprising the following steps:

[0043] 1. Sample pretreatment

[0044] Weigh 0.1 g of the sample to be tested (e-cigarette liquid) into a 50 mL stoppered plastic centrifuge tube, add 0.05 mL of internal standard stock solution and 10 mL of 0.1% formic acid-10% methanol aqueous solution, seal the tube, and vortex for 20 min to obtain the extraction solution. Dilute the extraction solution 20 times with the internal standard solution, filter it through an organic phase filter membrane, and obtain the sample solution to be tested for liquid chromatography-tandem mass spectrometry analysis.

[0045] The preparation method of the internal standard stock solution is as follows:

[0046] Accurately weigh 0.1 g of internal standards (benzoic acid-d5 and succinic acid-d6), dissolve in methanol, and dilute to a 100 mL volumetric flask to prepare an internal standard stock solution with a concentration of 1 mg / mL. Store at 0℃~4℃ protected from light; shelf life is 3 months.

[0047] The preparation method of 0.1% formic acid-10% methanol aqueous solution is as follows:

[0048] Transfer 100 mL of methanol and 1 mL of formic acid into a 1 L volumetric flask, and dilute to the mark with ultrapure water.

[0049] The method for preparing the internal standard solution is as follows:

[0050] Accurately transfer 5 mL of the internal standard stock solution and dilute it to a 1000 mL volumetric flask with 0.1% formic acid-10% methanol aqueous solution to prepare an internal standard solution with a concentration of 0.005 mg / mL. Store at 0℃~4℃ protected from light. Shelf life is 3 months.

[0051] 2. Preparation of standard working solutions

[0052] 2.1 Preparation of Single Standard Stock Solution: Accurately weigh 0.1 g of the target standard, dissolve it in methanol, and dilute to 10 mL in volumetric flasks to prepare a single standard stock solution with a concentration of 10 mg / mL. Store at 0℃~4℃ protected from light. Shelf life is 3 months.

[0053] 2.2 Preparation of mixed standard intermediate solution: Accurately transfer 0.1 mL of the single standard stock solution of each target analyte into a 10 mL volumetric flask, dilute with 0.1% formic acid-10% methanol aqueous solution, and bring the volume to 10 mL to prepare a mixed standard intermediate solution with a concentration of 0.1 mg / mL. Store at 0℃~4℃ protected from light; shelf life is 3 months.

[0054] 2.3 Preparation of Standard Working Solutions

[0055] Transfer 0.005 mL, 0.04 mL, 0.1 mL, 0.2 mL, 0.5 mL, 1.0 mL, 2.0 mL, and 3.0 mL of the mixed standard intermediate solution into 10 mL volumetric flasks, respectively. Add 0.05 mL of internal standard stock solution to each flask and dilute to the mark with 0.1% formic acid-10% methanol aqueous solution to obtain standard working solutions of different concentrations. Prepare and use immediately.

[0056] 3. Analysis and determination

[0057] The sample solution obtained in step 1 and the standard working solutions of different concentrations obtained in step 2 were analyzed by UPLC-MS / MS.

[0058] In the UPLC-MS / MS analysis of this embodiment, the instrument operating conditions used are as follows:

[0059] Chromatographic conditions: Column: Waters HSS T3 column, 150 mm × 2.1 mm, 1.8 μm; Mobile phase A: 5 mmol / L ammonium formate aqueous solution (pH adjusted to 2.5 with formic acid); Mobile phase B: 0.1% formic acid methanol solution; Gradient elution program: 0–3.5 min, phase A maintained at 98%; 3.5–11 min, phase A decreased from 98% to 2%; 11–14 min, phase A maintained at 2%; 14–15 min, phase A maintained at 98%; Column temperature: 30 °C; Flow rate: 0.3 mL / min; Injection volume: 1 μL.

[0060] Mass spectrometry conditions: Electrospray ionization (ESI); Ionization mode: negative ion mode; Electrospray voltage: -4500V; Ion source temperature: 500℃; Scan mode: Multiple reaction monitoring (MRM).

[0061] The non-volatile organic acids to be detected in e-cigarette liquids in this invention include tartaric acid, malic acid, lactic acid, citric acid, levulinic acid, and benzoic acid. The MRM parameters of the six non-volatile organic acids and two internal standards are shown in Table 1.

[0062] Table 1. MRM parameters of 6 non-volatile organic acids and 2 internal standards

[0063]

[0064]

[0065] Note: "*" indicates quantitative ions.

[0066] After analysis, the multiple reaction monitoring (MRM) chromatograms of the target analyte and its internal standard in the standard working solution are as follows: Picture 1 As shown in the figure. 1. Tartaric acid; 2. Malic acid; 3. Lactic acid; 4. Citric acid; 5. Succinic acid-d6; 6. Acetylacetic acid; 7. Benzoic acid-d5; 8. Benzoic acid.

[0067] 4. Calculation of analyte content

[0068] Based on the analysis results of step 3, a standard working curve is constructed with the ratio of the quantitative ion peak area of ​​the target analyte to the internal standard in each standard working solution as the ordinate and the content of the target analyte in each standard working solution as the abscissa. After the sample solution to be tested is detected and analyzed by the ultra-high performance liquid chromatography-tandem mass spectrometry instrument, the content of the target analyte in the sample solution to be tested is obtained, and then the content of each target analyte in the sample to be tested is further calculated.

[0069] 5. Analysis of actual samples

[0070] Based on the above determination method, 10 e-cigarette liquid samples were selected, and the content of the target substance was measured as shown in Table 2 (unit: mg / g):

[0071] Table 2. Target substance content (mg / g) in typical e-cigarette samples.

[0072] Original item tartaric acid apple acid Lactic acid Cypress acid Etchan-hung acid Choric acid S01 N.D. 0.68 1.76 N.D. N.D. 4.92 S02 N.D. N.D. N.D. N.D. N.D. 12.28 S03 N.D. 0.38 1.01 N.D. 6.18 11.02 S04 N.D. 0.61 1.06 N.D. N.D. 7.22 S05 N.D. 0.38 1.25 N.D. 7.11 10.64 S06 N.D. 0.34 3.02 N.D. 1.04 10.58 S07 N.D. 0.17 N.D. N.D. N.D. 17.95 S08 N.D. N.D. N.D. N.D. N.D. 19.12 S09 N.D. 0.37 0.90 N.D. N.D. 4.50 S10 N.D. 0.42 N.D. N.D. N.D. 13.48

[0073] Note: S01-S10 are different models of electronic cigarette products.

[0074] “ND.” indicates that it was not detected.

[0075] The above results indicate that the present invention can simultaneously analyze six non-volatile organic acids in additives permitted for use in e-cigarette liquids according to the national standard for e-cigarettes, and has the advantages of simple operation, high throughput, and accurate quantification.

[0076] Comparative Example 1

[0077] Non-volatile organic acids, as polar compounds, are poorly retained in reversed-phase chromatography columns. To obtain better separation results, this invention conducted a comparative experiment, comparing four chromatographic columns using the method described in Example 1: Phenomenex Luna C18 column (4.6×150mm, 3μm), Phenomenex Kinetex HILIC column (4.6×150mm, 2.6μm), Thermo AcclaimTrinity P1 column (2.1×100mm, 2.5μm), and Waters HSS T3 column (2.1×150mm, 1.8μm).

[0078] The results showed that malic acid and citric acid were not retained in the Kinetex HILIC and Acclaim Trinity P1 columns, while the six non-volatile organic acids were well retained in the Luna C18 and HSS T3 columns. The Luna C18 column retained all organic acids, but levulinic acid and lactic acid had high baseline noise. The retention times of most organic acids were concentrated, resulting in poor separation. The HSS T3 column met the chromatographic separation requirements.

[0079] Comparative Example 2

[0080] This invention also investigated the effect of different mobile phase systems on the detection method through comparative experiments. The mobile phases were: a 0.1% formic acid aqueous solution-methanol system, a 0.1% formic acid aqueous solution-0.1% formic acid methanol system, a 5 mmol / L ammonium formate aqueous solution-0.1% formic acid methanol system, and a 5 mmol / L ammonium formate aqueous solution-0.1% formic acid methanol system with formic acid adjusted to pH 2.5. The remaining methods are as described in Example 1.

[0081] turn out:

[0082] ① When the mobile phase is a 0.1% formic acid aqueous solution-methanol system, the peak of malic acid in the target analyte splits at the top, and the peaks of the other target analytes have poor shapes and tailing.

[0083] ② When the mobile phase is a 0.1% formic acid aqueous solution-0.1% formic acid methanol system, the peak shape of malic acid in the target analyte is improved, and the tailing phenomenon is not significantly changed.

[0084] ③ When the mobile phase is 5 mmol / L ammonium formate aqueous solution - 0.1% formic acid methanol system, the chromatographic peaks of tartaric acid, malic acid and citric acid are poor in shape and the response is reduced.

[0085] ④ When the mobile phase is a 5 mmol / L ammonium formate aqueous solution with pH adjusted to 2.5 by formic acid - a 0.1% formic acid methanol system, the target compound has a good peak shape without tailing.

[0086] Comparative Example 3

[0087] This invention also investigated the extraction efficiency of different extraction solvents for non-volatile organic acids through comparative experiments. The extraction solvents were: 0.1% formic acid solution, 0.1% formic acid-10% methanol solution, 0.1% formic acid-30% methanol solution, 0.1% formic acid-50% methanol solution, and 0.1% formic acid-75% methanol solution. The remaining methods are the same as in Example 1.

[0088] The results showed that for e-cigarette liquids, the extraction efficiency of the target compound was basically the same for different proportions of methanol aqueous solution, with no significant difference; however, different extraction solvents affected the peak shape of the target compound. When 0.1% formic acid-10% methanol aqueous solution was used as the extraction solvent, the chromatographic peak of the target compound did not have phenomena such as tip bifurcation or tailing, and the peak shape was good.

[0089] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and principles of the described embodiments, and these modifications and variations should also be considered within the scope of protection of the present invention.

Claims

1. A method for analyzing non-volatile organic acids in e-cigarette liquid, characterized in that, Includes the following steps: The test sample of e-cigarette liquid was added to internal standard stock solution and 0.1% formic acid-10% methanol aqueous solution, sealed and shaken, and then diluted with internal standard solution to obtain the test sample solution; the internal standard stock solution was a methanol mixed solution of benzoic acid-d5 and succinic acid-d6. The internal standard solution is a 0.1% formic acid-10% methanol-water mixture of benzoic acid-d5 and succinic acid-d6; Single standard stock solutions prepared from different target analyte standards were mixed and diluted to form mixed standard intermediate solutions. Different volumes of mixed standard intermediate solutions were taken and added to internal standard stock solutions and formic acid-methanol aqueous solutions to prepare standard working solutions of different concentrations. The above-mentioned sample solutions and standard working solutions were analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry, respectively. Based on the analytical results of the above ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), the content of the target analyte in the sample was calculated using the internal standard curve method. In the UHPLC-MS / MS analysis, the chromatographic column used was a Waters HSS T3 column, 150 mm × 2.1 mm, 1.8 μm; mobile phase A was 5 mmol / L ammonium formate aqueous solution, and mobile phase B was 0.1% formic acid methanol solution. The UHPLC-MS / MS analysis employed gradient elution with the following elution program: 0–3.5 min, phase A maintained at 98%; 3.5–11 min, phase A decreased from 98% to 2%; 11–14 min, phase A maintained at 2%; 14–15 min, phase A remained at 98%; the injection volume was 1 μL. The non-volatile organic acids in the e-cigarette liquid include tartaric acid, malic acid, lactic acid, citric acid, acetylpropionic acid, and benzoic acid.

2. The method for analyzing non-volatile organic acids in electronic cigarette liquid according to claim 1, characterized in that, The concentration of the methanol mixture of benzoic acid-d5 and succinic acid-d6 is 1 mg / mL.

3. The method for analyzing non-volatile organic acids in electronic cigarette liquid according to claim 1, characterized in that, The concentration of the 0.1% formic acid-10% methanol-water mixed solution of benzoic acid-d5 and succinic acid-d6 is 0.005 mg / mL.

4. The method for analyzing non-volatile organic acids in electronic cigarette liquid according to claim 1, characterized in that, The concentration ranges of each target analyte in the standard working solution are as follows: tartaric acid, malic acid, citric acid, levulinic acid, and benzoic acid are 0.05~15 μg / mL, and lactic acid is 0.4~15 μg / mL.

5. The method for analyzing non-volatile organic acids in electronic cigarette liquid according to claim 1, characterized in that, In the ultra-high performance liquid chromatography-tandem mass spectrometry analysis, the mass spectrometry conditions are as follows: The ion source is an electrospray ion source; the ionization mode is negative ion mode; the electrospray voltage is -4500 V; the ion source temperature is 500℃; and the scanning method is multiple reaction monitoring.

6. The method for analyzing non-volatile organic acids in electronic cigarette liquid according to claim 1, characterized in that, The method of calculating the content of the target analyte in the sample using the internal standard curve method includes: After the standard working solutions of different concentrations were detected and analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry, a standard working curve was constructed with the ratio of the quantitative ion peak area of ​​the target analyte to that of the internal standard as the ordinate and the content of the target analyte as the abscissa. After the sample solution is analyzed by the ultra-high performance liquid chromatography-tandem mass spectrometry instrument, the content of the target substances in the sample solution is obtained, and then the content of each target substance in the sample is calculated.

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