Convergence chromatography analysis of caffeine in e-cigarette oil
By using ultra-high efficiency combined chromatography analysis method in electronic cigarette oil, using BEH 2-Ethlpyridine chromatography column and methanol/CO2 mobile phase, the rapid and accurate detection of caffeine in electronic cigarette oil is achieved, and the existing method analysis is solved The problems of long time and high solvent consumption provide scientific basis for the safety detection of e-cigarettes.
Patent Information
- Application Number
- CN202010791393.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2020-08-07
- Publication Date
- 2024-12-24
- Estimated Expiration
- 2040-08-07
AI Technical Summary
The existing caffeine analysis methods exist in electronic cigarette liquor for a long time and consume a lot of organic solvents. They cannot quickly and effectively detect caffeine in electronic cigarette liquor, and cannot meet the needs of electronic cigarette safety detection.
UHE chromatography analysis method was adopted, using ACQUITY UPCC BEH 2-Ethlpyridine chromatography column, methanol and supercritical CO2 as mobile phase, flow rate 0.6mL/min, back pressure 2000psi, PDA detector, 273nm wavelength, to achieve rapid separation and detection Caffeine in e-cigarette liquor.
This method significantly shortens the analysis time, improves the analysis speed, reduces organic solvent consumption, realizes rapid and accurate detection of caffeine in electronic cigarette liquor, is suitable for batch sample analysis, and provides reliable for the safety detection of electronic cigarettes. method.
Smart Images
Figure CN114062527B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of analysis of harmful and potentially harmful components (HPHCs) in electronic cigarette oil, and in particular to a convergence chromatography analysis method for caffeine in electronic cigarette oil. Background Art
[0002] Caffeine is a neuroactive alkaloid widely found in tea, beverages and coffee. Recently, the public has paid more and more attention to the safety of e-cigarettes. Many countries or organizations have clear regulations on the amount of caffeine added to e-cigarette oils, such as the EU Tobacco Products Directive TPD, the French Afnor e-cigarette standard, and the American e-cigarette industry association AEMSA, which explicitly prohibits the addition of caffeine to e-cigarette oils. Common caffeine analysis methods include gas chromatography, liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, etc. Gas chromatography analysis methods often take a long time to analyze, while liquid chromatography often consumes a large amount of organic solvents. Therefore, it is urgent to develop an analytical method that can quickly and sensitively analyze caffeine in e-cigarette oils, so as to achieve rapid and sensitive analysis of caffeine in e-cigarette oils, which is of great significance to the quality safety and control of e-cigarette oils. Summary of the invention
[0003] In view of the problems existing in the above technologies, there is still no efficient and rapid analytical method for determining caffeine in electronic cigarette oil.
[0004] Specifically, a convergence chromatography analysis method for caffeine in electronic cigarette oil is provided, which is used to determine the caffeine in the electronic cigarette oil, and comprises the following steps:
[0005] (1) Preparation of e-cigarette oil samples
[0006] Weigh the electronic cigarette oil, dissolve it in a 10 mL volumetric flask using isopropanol as solvent A, and then filter the dissolved solution using a filter membrane to obtain an electronic cigarette oil sample;
[0007] (2) Preparation of standard working solution
[0008] Weigh the caffeine standard and dilute it to a 25 mL volumetric flask using dichloromethane as a solvent to obtain a mother solution; and dilute the mother solution with dichloromethane to obtain a gradient standard working solution of 1.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 100.0 μg / mL, and 500.0 μg / mL;
[0009] (3) Convergence chromatography analysis
[0010] The standard working solution obtained in step (2) and the electronic cigarette oil sample obtained in step (1) are analyzed for concentration using an ultra-high performance convergence chromatograph, and the PDA detector is used for detection;
[0011] Chromatographic conditions: Use any one of the chromatographic columns ACQUITY UPCC BEH (3.0mm×100mm, 1.7μm), ACQUITY UPCC BEH 2-Ethlpyridine (2.1mm×150mm, 1.7μm), and Viridis HSS C18 SB (2.1mm×150mm, 1.8μm); the chromatographic column temperature is 40°C, the injection volume is 1μL, and the system back pressure is 1800-3000psi; the mobile phase is isocratic elution of co-solvent and supercritical CO2 in a certain proportion, and the flow rate range is 0.5-1.5mL / min.
[0012] Preferably, in step (1), 0.2 μm nylon filter membrane is used for filtration.
[0013] Preferably, in step (1) and step (2), isopropanol and dichloromethane are both of HPLC purity.
[0014] Preferably, in step (3), the co-solvent is one of methanol, ethanol and acetonitrile, and its purity is HPLC purity.
[0015] Preferably, in step (3), a PDA detector is used for detection, and the chromatographic peak area is quantified using an external standard method.
[0016] Preferably, the detection wavelength is 273 nm.
[0017] Preferably, in step (3), the mobile phase is isocratic elution of methanol and supercritical CO2 in a mass ratio of 2%:98% to 7%:93%.
[0018] The beneficial effects of the present invention are: the convergence chromatography analysis method of caffeine in the electronic cigarette oil proposed by the present invention; because caffeine is prohibited from being added to the electronic cigarette oil, it is necessary to quickly detect whether there is caffeine in the electronic cigarette oil production and inspection links; therefore, the present invention establishes a convergence chromatography analysis method for determining caffeine in the electronic cigarette oil for the first time; using convergence chromatography analysis, the analysis time is shorter than that of gas chromatography and liquid chromatography; the optimal chromatographic conditions are BEH2-Ethlpyridine column, 5% methanol 95% CO2 as elution solvent, 0.6mL / min flow rate, 2000psi back pressure. The experiment uses a PDA detector, analyzes at a wavelength of 273nm, has a retention time of 1.65 minutes, and a detection time of within 2 minutes, which can realize the rapid determination of batch samples, and the mobile phase is safe and pollution-free to the environment, providing a method basis for the safety detection of electronic cigarettes; at the same time, it also provides technical support for research in pharmacy, smoking and health; a method for rapid determination of caffeine content in electronic cigarette oil is established, and the analysis method is accurate and rapid. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 The chromatograms of caffeine in Example 1 of the present invention on three different convergence chromatography columns;
[0020] Figure 2 chromatograms of caffeine in Example 2 of the present invention under four different elution solvents;
[0021] Figure 3 This is a convergence chromatogram of caffeine at different flow rates in Example 3 of the present invention;
[0022] Figure 4 This is a convergence chromatogram of caffeine in Example 4 of the present invention under different back pressures. DETAILED DESCRIPTION
[0023] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with embodiments.
[0024] First of all, the original intention and idea of the invention is that many countries or organizations have clear regulations on the amount of caffeine added to e-cigarette oil, such as the EU Tobacco Products Directive TPD, the French Afnor e-cigarette standard, and the American Electronic Cigarette Industry Association AEMSA, which clearly prohibit the addition of caffeine to e-cigarette oil; therefore, the rapid and sensitive analysis of caffeine in e-cigarette oil is crucial to the quality control of e-cigarette oil. Then, as the first time to use the convergence chromatography analysis method to establish an analysis for the determination of caffeine in e-cigarette oil; it should be widely promoted for e-cigarette oil quality detection.
[0025] In the following examples, the instrument used was a Waters ACQULITY ultra-high performance convergence chromatograph equipped with an ACQULITY UPCC diode array detector (PDA); the caffeine standard sample was purchased from Chengdu Munster Biotechnology Co., Ltd. with a purity of 98.6%. Methanol, ethanol, isopropanol, and acetonitrile were all purchased from Merck with HPLC purity.
[0026] Example 1
[0027] (1) Preparation of e-cigarette oil samples
[0028] Weigh 0.2 g of electronic cigarette oil, dilute to 10 mL volumetric flask with isopropanol solvent, and then filter with a 0.2 μm filter membrane to obtain an electronic cigarette oil sample;
[0029] (2) Preparation of standard working solution
[0030] Weigh 25.4 mg of caffeine standard, dilute to a 25 mL volumetric flask with dichloromethane solvent to obtain a mother solution; take 10, 100, 200, 1000, and 5000 μL of the mother solution, respectively, and dilute to a 10 mL volumetric flask with dichloromethane; obtain standard working solutions with concentrations of 1.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 100.0 μg / mL, and 500.0 μg / mL;
[0031] (3) Convergence chromatography analysis
[0032] The standard working solution obtained in step (2) and the electronic cigarette oil sample obtained in step (1) are analyzed for concentration using an ultra-high performance convergence chromatograph, and a diode array detector (PDA) is used for detection;
[0033] For the selection of chromatographic columns, three control groups were set up, using ACQUITY UPCC BEH (3.0mm×100mm, 1.7μm), ACQUITY UPCC BEH 2-Ethlpyridine (2.1mm×150mm, 1.7μm), and Viridis HSSC18 SB (2.1mm×150mm, 1.8μm); the other chromatographic conditions were the same, namely: the chromatographic column temperature was 40°C, the injection volume was 1μL, and the system back pressure was 2000psi; the mobile phase was methanol and supercritical CO2 at a 5%:95% isocratic elution, and the flow rate was 0.6mL / min; the chromatograms were compared as shown in the figure below. Figure 1 As shown in the figure; in convergence chromatography analysis, the chromatographic column has a greater impact on the resolution and peak shape in a shorter analysis time; so Figure 1 It can be seen that caffeine has the best separation effect and the fastest separation time on the BEH 2-Ethlpyridine column.
[0034] Example 2
[0035] (1) Preparation of e-cigarette oil samples
[0036] Weigh 0.2 g of electronic cigarette oil, dilute to 10 mL volumetric flask with isopropanol solvent, and then filter with a 0.22 μm filter membrane to obtain an electronic cigarette oil sample;
[0037] (2) Preparation of standard working solution
[0038] Weigh 25.4 mg of caffeine standard, dilute to a 25 mL volumetric flask with dichloromethane solvent to obtain a mother solution; take 10, 100, 200, 1000, and 5000 μL of the mother solution, respectively, and dilute to a 10 mL volumetric flask with dichloromethane; obtain standard working solutions with concentrations of 1.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 100.0 μg / mL, and 500.0 μg / mL;
[0039] (3) Convergence chromatography analysis
[0040] The standard working solution obtained in step (2) and the electronic cigarette oil sample obtained in step (1) are analyzed for concentration using an ultra-high performance convergence chromatograph, and a diode array detector (PDA) is used for detection;
[0041] BEH 2-Ethlpyridine (2.1mm×150mm, 1.7μm) was used; the column temperature was 40°C, the injection volume was 1μL, and the system back pressure was 2000psi; in the selection of the co-solvent of the mobile phase, four control groups were set up for the experiment, namely methanol, acetonitrile, and ethanol; the three co-solvents and the main mobile phase (supercritical fluid CO2) were eluted isocratically at 5%:95%, and the flow rate was 0.6mL / min; the chromatogram is shown in FIG. Figure 2 As shown in the figure, the mobile phase of UPCC is mainly a supercritical fluid with CO2 as the main component. In order to adjust the polarity of the mobile phase and the solubility of the target compound to adapt to the different solubilities of different target compounds, the peak shape and retention time of the target compound can be effectively changed. Methanol, acetonitrile and ethanol are co-solvents with different polarities, which have an impact on the separation optimization of sample solutions with the same concentration. Figure 2 As shown, when acetonitrile is used as the elution solvent, caffeine cannot be eluted within 5 minutes. Using ethanol and methanol as the elution solvent, the retention time is longer than that of methanol. Therefore, methanol is most preferably used as the elution solvent.
[0042] Example 3
[0043] (1) Preparation of e-cigarette oil samples
[0044] Weigh 0.2 g of electronic cigarette oil, dilute to 10 mL volumetric flask with isopropanol solvent, and then filter with a 0.22 μm filter membrane to obtain an electronic cigarette oil sample;
[0045] (2) Preparation of standard working solution
[0046] Weigh 25.4 mg of caffeine standard, dilute to a 25 mL volumetric flask with dichloromethane solvent to obtain a mother solution; take 10, 100, 200, 1000, and 5000 μL of the mother solution, respectively, and dilute to a 10 mL volumetric flask with dichloromethane; obtain standard working solutions with concentrations of 1.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 100.0 μg / mL, and 500.0 μg / mL;
[0047] (3) Convergence chromatography analysis
[0048] The standard working solution obtained in step (2) and the electronic cigarette oil sample obtained in step (1) are analyzed for concentration using an ultra-high performance convergence chromatograph, and a diode array detector (PDA) is used for detection;
[0049] BEH 2-Ethlpyridine (2.1mm×150mm, 1.7μm) was used; the column temperature was 40°C, the injection volume was 1μL, and the system back pressure was 2000psi; methanol and supercritical fluid CO2 were selected for isocratic elution at 5%:95%, and four control groups were set in terms of flow rate selection; they were 0.5mL / min, 0.6mL / min, 0.8mL / min, 1.0mL / min, and 1.5mL / min; the chromatogram is shown in FIG. Figure 3 As shown in the figure, when supercritical CO2 is used as the mobile phase, due to its low viscosity and high diffusion coefficient, it has a higher linear velocity in the separation process, and the analysis speed is 3-10 times faster than that of traditional HPLC, and the analysis time is short. In order to ensure better sensitivity and appropriate chromatographic column pressure, it is particularly important to select a suitable flow rate. Figure 3 It can be seen that as the flow rate increases, the analysis time gradually shortens, but the column pressure will also increase accordingly, so the flow rate of 0.6mL / min is the best.
[0050] Example 4
[0051] (1) Preparation of e-cigarette oil samples
[0052] Weigh 0.2 g of electronic cigarette oil, dilute to 10 mL volumetric flask with isopropanol solvent, and then filter with a 0.22 μm filter membrane to obtain an electronic cigarette oil sample;
[0053] (2) Preparation of standard working solution
[0054] Weigh 25.4 mg of caffeine standard, dilute to a 25 mL volumetric flask with dichloromethane solvent to obtain a mother solution; take 10, 100, 200, 1000, and 5000 μL of the mother solution, respectively, and dilute to a 10 mL volumetric flask with dichloromethane; obtain standard working solutions with concentrations of 1.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 100.0 μg / mL, and 500.0 μg / mL;
[0055] (3) Convergence chromatography analysis
[0056] The standard working solution obtained in step (2) and the electronic cigarette oil sample obtained in step (1) are analyzed for concentration using an ultra-high performance convergence chromatograph, and a diode array detector (PDA) is used for detection;
[0057] BEH 2-Ethlpyridine (2.1 mm × 150 mm, 1.7 μm) was used; the column temperature was 40°C, the injection volume was 1 μL; methanol and supercritical fluid CO2 were used for isocratic elution at 5%:95%, and the flow rate was 0.6 mL / min; in terms of back pressure selection, 5 control groups were set, and their back pressures were 1800 psi, 2000 psi, 2200 psi, 2400 psi, and 2800 psi, respectively. The chromatograms are shown in FIG. Figure 4 As shown in the figure, in ultra-high performance convergence chromatography, dynamic back pressure (ABPR) is one of the important factors affecting the separation process. Its main function is to control CO2 to maintain a supercritical state during the entire operation process, because under different back pressure conditions, the solubility of the sample is different. When the back pressure increases, the density of the supercritical fluid increases, the solubility is enhanced, and the retention time becomes shorter. Figure 4 As can be seen from the figure, as the back pressure increases, the retention time of caffeine gradually shortens, but as the back pressure increases, the consumption rate of CO2 increases greatly, and the column pressure increases significantly. Considering all factors, a back pressure of 2000 psi was selected for the experiment.
[0058] Example 5
[0059] (1) Preparation of e-cigarette oil samples
[0060] Weigh 0.2 g of electronic cigarette oil, dilute to 10 mL volumetric flask with isopropanol solvent, and then filter with a 0.22 μm filter membrane to obtain an electronic cigarette oil sample;
[0061] (2) Preparation of standard working solution
[0062] Weigh 25.4 mg of caffeine standard, dilute to a 25 mL volumetric flask with dichloromethane solvent to obtain a mother solution; take 10, 100, 200, 1000, and 5000 μL of the mother solution, respectively, and dilute to a 10 mL volumetric flask with dichloromethane; obtain standard working solutions with concentrations of 1.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 100.0 μg / mL, and 500.0 μg / mL;
[0063] (3) Convergence chromatography analysis
[0064] The standard working solution obtained in step (2) and the electronic cigarette oil sample obtained in step (1) are analyzed for concentration using an ultra-high performance convergence chromatograph, and a diode array detector (PDA) is used for detection;
[0065] BEH 2-Ethlpyridine (2.1mm×150mm, 1.7μm) was used; the column temperature was 40℃, the injection volume was 1μL; the system back pressure was 2000psi; methanol and supercritical fluid CO2 were selected for isocratic elution at 5%:95%, and the flow rate was 0.6mL / min; 6 control groups were set up for the selection of the ratio of elution solvent (methanol and supercritical fluid CO2); they were 2%:98%; 3%:97%; 4%:96%; 5%:95%; 6%:94%; 7%:93%; as the methanol ratio increased from 2% to 7%, the analysis time of caffeine was gradually shortened, and the retention time was shortened from 2.1 minutes to 0.7 minutes. In the experiment, considering the solvent consumption and experimental time, 5% methanol and 95% CO2 were selected as isocratic elution conditions, and the retention time was 1.65 minutes, which could achieve rapid analysis of caffeine within 2 minutes.
[0066] In summary: when the convergence chromatography analysis uses: chromatographic column BEH 2-Ethlpyridine (2.1mm×150mm, 1.7μm); the column temperature is 40°C, the injection volume is 1μL, and the system back pressure is 2000psi; the mobile phase is methanol and supercritical CO2 at a 5%:95% isocratic elution, and the flow rate is 0.6mL / min. The detection effect is the best, and it is analyzed at a wavelength of 273nm. Compared with the existing liquid chromatography and gas chromatography analysis methods, this method greatly improves the analysis speed and greatly reduces the consumption of organic solvents. Since 95% of the components in the mobile phase are CO2, it is safe and pollution-free to the environment. This method can achieve rapid and sensitive analysis of caffeine in electronic cigarettes, providing a methodological basis for the safety detection of electronic cigarettes.
[0067] Methodological investigation
[0068] The above six standard working solutions of gradient were selected and the optimal chromatographic conditions were as follows: the chromatographic column was BEH 2-Ethlpyridine (2.1 mm × 150 mm, 1.7 μm); the chromatographic column temperature was 40°C, the injection volume was 1 μL, and the system back pressure was 2000 psi; the mobile phase was methanol and supercritical CO2 at a 5%:95% isocratic elution rate, and the flow rate was 0.6 mL / min for determination. The peak area of caffeine was used for quantification. The concentration of caffeine and its peak area were linearly related in the range of 1.0 to 500.0 μg / mL. The linear regression equation was y=3212.4x+535.6, R 2 =0.9999, and the detection limit is 0.35 μg / mL (S / N=3). The results of 5 measurements of the standard product show that the relative standard deviation (RSD) of caffeine is 0.12%, indicating that the method of the present invention has good precision and repeatability.
[0069] And the chromatographic analysis conditions are: the chromatographic column is BEH 2-Ethlpyridine (2.1mm×150mm, 1.7μm); the chromatographic column temperature is 40°C, the injection volume is 1μL, the system back pressure is 2000psi; the mobile phase is methanol and supercritical CO2 according to 5%:95% isocratic elution, the flow rate is 0.6mL / min, and 5 batches of electronic cigarette oils of different brands are randomly purchased on the market. Each sample is measured in parallel 3 times according to the above method, and no caffeine component is detected. After adding the standard solution, the spiked recovery rate is measured to be 94.3-104.3%, and the relative standard deviation (RSD) is 1.32-5.51%. The method can realize sensitive analysis of caffeine in cigarette oil samples, the method is fast, and the results are reliable.
[0070] The advantages of the present invention are:
[0071] 1) The present invention establishes a method for simultaneously and rapidly detecting caffeine in electronic cigarette oil by using ultra-high performance convergence chromatography in series with a diode array detector, which can accurately perform qualitative and quantitative analysis on the caffeine in the electronic cigarette oil, and provides a scientific basis for the accurate determination and rapid detection of caffeine in the electronic cigarette oil;
[0072] 2) The BEH 2-Ethlpyridine (2.1 mm×150 mm, 1.7 μm) of the present invention and CO2 and methanol at a ratio of 5%:95% as the mobile phase achieved excellent separation effect on caffeine in electronic cigarette oil;
[0073] 3) Rapid and sensitive analysis of samples can be achieved within 2 minutes, with a detection limit of 0.35 μg / mL; since 95% of the components in the mobile phase are CO2, it is safe and pollution-free to the environment.
[0074] The above disclosures are only several specific embodiments of the present invention, but the present invention is not limited thereto, and any changes that can be conceived by those skilled in the art should fall within the protection scope of the present invention.
Claims
1. A convergence chromatography analysis method for caffeine in electronic cigarette oil, used for determining caffeine in electronic cigarette oil, characterized in that: The following steps are involved: (1) Preparation of e-cigarette oil samples Weigh the electronic cigarette oil, dissolve it in a 10 mL volumetric flask using isopropanol as solvent A, and then filter the dissolved solution using a filter membrane to obtain an electronic cigarette oil sample; (2) Preparation of standard working solution Weigh the caffeine standard and dilute it to a 25 mL volumetric flask using dichloromethane as a solvent to obtain a mother solution; and dilute the mother solution with dichloromethane to obtain a gradient standard working solution of 1.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 100.0 μg / mL, and 500.0 μg / mL; (3) Convergence chromatography analysis Using an ultra-high performance convergence chromatograph to analyze the concentration of the standard working solution obtained in step (2) and the electronic cigarette oil sample obtained in step (1), and using a PDA detector for detection; Chromatographic conditions: Use any one of the chromatographic columns ACQUITY UPCC BEH, 3.0mm×100mm, 1.7μm, ACQUITY UPCC BEH 2-Ethlpyridine, 2.1mm×150mm, 1.7μm, and Viridis HSS C18 SB, 2.1mm×150mm, 1.8μm; the chromatographic column temperature is 40°C, the injection volume is 1μL, and the system back pressure is 1800-3000psi; the mobile phase is isocratic elution of co-solvent and supercritical CO2 in a certain proportion, and the flow rate range is 0.5-1.5mL / min.
2. The convergence chromatography analysis method for caffeine in electronic cigarette oil according to claim 1, characterized in that: In step (1), filtration is performed using a 0.2 μm nylon filter membrane.
3. The convergence chromatography analysis method for caffeine in electronic cigarette oil according to claim 1, characterized in that: In step (1) and step (2), isopropanol and dichloromethane are both of HPLC purity.
4. The convergence chromatography analysis method for caffeine in electronic cigarette oil according to claim 1, characterized in that: In step (3), the co-solvent is one of methanol, ethanol and acetonitrile, and its purity is HPLC purity.
5. The convergence chromatography analysis method for caffeine in electronic cigarette oil according to claim 1, characterized in that: In step (3), a PDA detector is used for detection and the chromatographic peak area is quantified using the external standard method.
6. The method for convergence chromatography analysis of caffeine in electronic cigarette oil according to claim 4, characterized in that: The detection wavelength is 273 nm.
7. The method for convergence chromatography analysis of caffeine in electronic cigarette oil according to claim 1, characterized in that: In step (3), the mobile phase is isocratic elution with methanol and supercritical CO2 in a mass ratio of 2%:98% to 7%:93%.