A method for quantitatively determining trimellitic acid, terephthalic acid and isophthalic acid in electronic cigarette liquid

Abstract

The present application relates to a kind of quantitative determination electronic cigarette liquid trimellitic acid, p-phthalic acid, m-phthalic acid method, belong to electronic cigarette liquid component detection technical field, it is characterized in that: using to electronic cigarette liquid sample adds certain concentration of formic acid aqueous solution extraction and improves the acid-base environment of sample, further purify sample by vortex, filtration, using core-shell (surface porous) reversed-phase chromatographic column, realize the complete separation of three target objects, combined with liquid chromatography mass spectrometer technology, internal standard method quantitative, realize the accurate detection of trimellitic acid, p-phthalic acid, m-phthalic acid in electronic cigarette liquid.The present application has the advantages that simple operation, quantitative accurate, high sensitivity, target can be extracted from complex electronic cigarette liquid matrix and accurately quantified, can satisfy the accurate detection of trimellitic acid, p-phthalic acid, m-phthalic acid in electronic cigarette liquid.

Description

Technical Field

[0001] This invention belongs to the field of electronic cigarette liquid component detection technology, specifically involving a pH adjustment and sample extraction pretreatment method, and an analytical method for determining trimellitic acid (BTCA), terephthalic acid (TPA) and isophthalic acid (IPA) in electronic cigarette liquid using liquid chromatography-mass spectrometry (LC-MS). Background Technology

[0002] Polyethylene terephthalate (PET resin) and the polymer of dimethyl terephthalate with 1,4-cyclohexanediol and 2,2,4,4-tetramethyl-1,3-cyclobutanediol (PCTG resin, trade name Tritan) are commonly used plastic materials in e-cigarettes. BTCA, TPA, and IPA are important monomer raw materials and process aids in the production process. Prolonged contact with e-cigarette liquids (generally between 18 and 24 months) poses a safety risk of migration into the e-cigarette liquid.

[0003] TPA and IPA can disrupt the endocrine system and increase the risk of kidney stones; BTCA compounds can irritate the skin and have suspected reproductive toxicity, and long-term trace intake can accumulate a burden on the liver and kidneys. Currently, both my country and the European Union have regulations and standards to control these three acids and have given clear migration limits of 5 mg / kg, 7.5 mg / kg, and 5 mg / kg, respectively.

[0004] Currently, testing methods for the three acids are only focused on food contact materials, specifying detection methods for food simulants such as water, ethanol, acetic acid, and olive oil. Because e-cigarette liquids are mixtures composed of propylene glycol, glycerol, various natural extracts, and multiple flavor components, their mechanisms are more complex than those of food simulants, and the published methods are insufficient for their testing.

[0005] Therefore, there is an urgent need to establish a simple, accurate, rapid, highly sensitive, and absolutely quantitative method for the separation and analysis of the three acids in e-cigarette liquid. Summary of the Invention

[0006] The purpose of this invention is to find a simple, accurate, and rapid pretreatment method that can extract target substances from the complex e-cigarette liquid matrix. By combining liquid chromatography-mass spectrometry (LC-MS) technology, the optimal chromatographic conditions are sought to achieve simultaneous detection of BTCA, TPA, and IPA in e-cigarette liquid. This method is accurate, sensitive, and easy to operate.

[0007] The objective of this invention is achieved through the following technical solution: a method for quantitatively determining BTCA, TPA, and IPA in e-cigarette liquids. First, a formic acid aqueous solution of a certain concentration is added to the e-cigarette liquid to be tested for extraction and to improve the acid-base environment of the sample. Then, the sample is further purified by vortexing and filtration before entering the mobile phase. Combined with liquid chromatography-mass spectrometry (LC-MS / MS) technology, accurate detection of the three acids BTCA, TPA, and IPA in e-cigarette liquids is achieved. The specific steps are as follows: (1) Sample pretreatment: Take the e-cigarette liquid sample, add formic acid aqueous solution to make the pH of the sample system 2-3, add internal standard working solution and perform vortex extraction, and filter through filter membrane to obtain the test solution; (2) Sample detection: The sample was detected by liquid chromatography-mass spectrometry. A standard curve was prepared using matrix-matched standard working solution. The standard curve was used for quantification to determine trimellitic acid, terephthalic acid and isophthalic acid in e-cigarette liquid. LC-MS analysis conditions: Column: Core-shell (porous surface) reversed-phase column; Stationary phase: (120Å high-purity silica gel, porous surface particles, uncapped C18); Dimensions: 3.0 × 100 mm, 2.7 µm; Column temperature: 40 °C; Injection volume: 5 μL; Mass spectrometry conditions: Equipped with Jet Stream electrospray ionization (ESI); Scan mode: Negative ion scan; Nebulizer pressure: 40 psi; Spray voltage: 1000 V; Capillary voltage: 3500 V; Sheath gas flow rate: 12 L / min; Sheath gas temperature: 350 °C; Gas flow rate: 8 L / min; Gas flow temperature: 330 °C; Data acquisition was performed using multiple reaction monitoring (MRM) mode.

[0008] In this invention, the sample pretreatment method involves weighing 2.0 g of e-cigarette liquid into a 15 mL centrifuge tube, adding a certain volume of formic acid aqueous solution, vortexing for 20 min, and then filtering 1 mL of the liquid through a 0.22 μm microporous membrane. The concentration of the formic acid aqueous solution is 1.0%, and the ratio of formic acid aqueous solution to e-cigarette liquid is 1:2, 1:4, or 1:6, preferably 1:4.

[0009] In this invention, the mobile phase consists of an aqueous phase of 0.2% formic acid and an organic phase of acetonitrile, with a flow rate of 0.4 mL / min. The aqueous mobile phase is consistent with the sample pretreatment solvent to eliminate the "solvent effect" and obtain narrower chromatographic peaks, higher mass spectrometry sensitivity, and stable retention time.

[0010] In this invention, the internal standard working solution contains terephthalic acid-d4 for the quantification of terephthalic acid, isophthalic acid-d4 for the quantification of isophthalic acid, and 1,3,5-triphenyltricarboxylic acid for the quantification of trimellitic acid; the concentration of the internal standard working solution is 20 mg / L, and the amount added to each sample is 50 μL. The internal standard trimellitic acid can also be d3-triphenyltricarboxylic acid, 13C3-triphenyltricarboxylic acid, or 1,2,3-triphenyltricarboxylic acid; terephthalic acid can also be ¹³C8-terephthalic acid; and isophthalic acid can also be ¹³C2-isophthalic acid. 。

[0011] In this invention, the gradient elution procedure of the mobile phase in the liquid chromatography is as follows: Table 1. Gradient elution program for mobile phase

[0012] In this invention, the standard curve quantification is achieved by selecting a matrix-matched standard working solution combined with the internal standard method, and calculating the content of the corresponding component based on the detection results and the standard curve of each target analyte.

[0013] In this invention, the MRM parameters in the LC-MS analysis conditions include the determination of retention time, and the selection and optimization of qualitative ion pairs, quantitative ion pairs, and collision energies. First, a full scan analysis is performed on each compound to determine the retention time. Then, a product ion scan is performed on the precursor ion at different collision energies, and two ion pairs and the optimal collision energy are selected for each compound. Finally, the standard solution, matrix extract, and matrix extract with added standards are analyzed using MRM mode. The MRM parameters in the LC-MS analysis conditions are shown in the table below:

[0014] Compared with the prior art, the method of the present invention has the following superior effects: Existing technologies mainly focus on the detection of three target compounds—BTCA, TPA, and IPA—in food simulants because these systems are simple and clean, allowing for separation and quantification. However, existing technologies cannot be applied to complex systems such as e-cigarette liquids. This invention enables the rapid detection and accurate quantification of these three acids in e-cigarette liquid systems.

[0015] (2) The present invention uses a certain volume of 1% formic acid aqueous solution, which not only adjusts the sample pH to 2-3, but also achieves the extraction of the target analyte. Compared with other methods, the pretreatment steps are simple and easy to operate. At the same time, the solvent is consistent with the aqueous mobile phase, which can eliminate the "solvent effect" and obtain a narrower chromatographic peak, higher mass spectrometry sensitivity and stable retention time.

[0016] (3) Current liquid chromatography-mass spectrometry mainly uses the external standard method for quantification. However, the three target substances are highly polar and have weak retention on the reversed-phase C18 column, which will produce a matrix effect and make it impossible to achieve accurate quantification. This invention uses an external matrix-matched standard working solution combined with the internal standard method for quantification, which greatly improves the accuracy and precision of the results and can meet the requirements for accurate quantification of low-content target substances in e-cigarette liquid. Attached Figure Description

[0017] Figure 1 Chromatographic separation of standard solutions on LC-MS; Figure 2 Chromatographic separation diagrams comparing this method with existing techniques: Comparison diagrams of three columns (Poroshell 120SB-C18, Eclipse Plus C18, Synergi Polar-RP); Figure 3 Chromatographic separation diagrams comparing this method with existing techniques: (Poroshell 120SB-C18, EclipsePlus C18) Comparison diagrams of the two columns. Detailed Implementation

[0018] The present invention will be further described below with reference to examples.

[0019] Example 1: Accurately weigh 2.0 g of e-cigarette liquid into a 15 mL centrifuge tube, add 8 mL of 1.0% formic acid aqueous solution and 50 μL of mixed internal standard solution (TPA-d4, IPA-d4, and JB are all 20 mg / L), vortex for 20 min, take 1 mL of liquid and filter it through a 0.22 μm microporous membrane. The mobile phase gradient elution program is shown in Table 1. Perform LC-MS test.

[0020] The LC-MS analysis conditions are as follows: Chromatographic column: Core-shell (porous surface) reversed-phase column; stationary phase: (120Å high-purity silica gel, porous surface particles, uncapped C18); dimensions: 3.0×100mm, 2.7µm; column temperature: 40℃; injection volume: 5μL.

[0021] Mass spectrometry conditions: Jet Stream electrospray ionization (ESI) source; scanning mode: negative ion scan; nebulizer pressure: 40 psi; spray voltage: 1000 V; capillary voltage: 3500 V; sheath gas flow rate: 12 L / min; sheath gas temperature: 350°C; gas flow rate: 8 L / min; gas flow temperature: 330°C; data acquisition was performed in multiple reaction monitoring (MRM) mode. MRM parameters are shown in Table 2.

[0022] Matrix-matched standard working solutions were prepared with BTCA concentrations of 0.02 mg / kg, 0.05 mg / kg, 0.125 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1 mg / kg, 2.5 mg / kg, and 5 mg / kg, and TPA and IPA concentrations of 0.04 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 5 mg / kg, and 10 mg / kg, respectively. Quantification was performed using the internal standard method. A standard curve was plotted with the ratio of each substance's concentration to the internal standard concentration on the x-axis and the ratio of the peak area of ​​the quantified ion to the peak area of ​​the internal standard on the y-axis. Low-concentration standard solutions were prepared and injected for testing. The method detection limit (S / N=3) and method quantitation limit (S / N=10) were determined. The linear range, linear equation, correlation coefficient, and detection and quantitation limits are shown in Table 3.

[0023] Table 3. Linearity parameters, limit of detection, and limit of quantitation of the method.

[0024] Three commercially available e-cigarette liquids were selected, and mixed standard solutions of low, medium, and high concentrations were added. Six parallel samples were prepared, and the recovery rate and repeatability were analyzed. The specific results are shown in Table 4. The results showed that the recoveries of BTCA, TPA, and IPA were 94.5%–109.8%, 86.7%–91.4%, and 96.0%–108.8%, respectively, with relative standard deviations ranging from 2.69% to 8.44%, 2.04% to 5.36%, and 1.13% to 5.22%, respectively. The recovery rate and precision of the three substances met the detection requirements.

[0025]

[0026] Example 2: Currently, there are three existing technologies for the simultaneous determination of three acids: BTCA, TPA, and IPA. For a detailed comparison, please refer to Table 5.

[0027] Column Comparison. The effects of three liquid chromatography columns—Poroshell 120SB-C18 (3.0 × 100 mm, 2.7 µm), Eclipse Plus C18 (2.1 mm × 150 mm, 3.5 μm), and Synergi Polar-RP (4.6 mm × 250 mm, 4 μm)—on the target compound were compared. The results are shown in [Figure number missing]. Figure 2 .

[0028] like Figure 2As shown, the Synergi Polar-RP column failed to completely separate BTCA, TPA, and IPA. In contrast, both the Poroshell 120SB-C18 and Eclipse Plus C18 columns were able to separate the three compounds, and the peak times and response values ​​of the target compounds on both columns were quite similar. Commercially available e-cigarette liquid was selected as the research object, and a mixed standard solution of BTCA, TPA, and IPA at the same concentration was added. After processing, the target analytes were analyzed using Poroshell 120SB-C18 and Eclipse Plus C18 columns. See details... Figure 3 BTCA, TPA, and IPA exhibit short elution times on the Eclipse Plus C18 column, and TPA fails to achieve complete separation from adjacent interfering peaks. Therefore, the Poroshell 120SB-C18 column can achieve baseline separation of the three acids, eliminate interfering peaks, and produce better peak shapes, meeting the testing requirements for e-cigarette liquids.

[0029] Comparison of limits of quantitation. As shown in Table 5, the limit of quantitation of the present invention is lower than that of the other three methods, which can meet the requirements for low content testing.

[0030]

[0031] It is evident that existing food simulant methods for e-cigarette liquids suffer from matrix interference, insufficient separation of the target peak from adjacent peaks, and high limits of quantitation. This invention solves these problems by combining acid extraction, a homogeneous mobile phase, core-shell reversed-phase C18, and matrix-matched internal standard quantification. Furthermore, the limit of quantitation of this invention is significantly lower than that of the compared food simulant detection methods, indicating that this invention is more suitable for detecting low-content target substances in e-cigarette liquids.

Claims

1. A method for quantitatively determining trimellitic acid, terephthalic acid, and isophthalic acid in e-cigarette liquid, characterized in that, Extraction was performed using a formic acid aqueous solution of a certain concentration to improve the acid-base environment of the sample. The sample was then further purified by vortexing and filtration before being introduced into the mobile phase. Combined with liquid chromatography-mass spectrometry (LC-MS / MS) technology, accurate detection of BTCA, TPA, and IPA in e-cigarette liquid was achieved. The specific steps are as follows: (1) Sample pretreatment: Take the e-cigarette liquid sample, add formic acid aqueous solution to make the pH of the sample system 2-3, add internal standard working solution and perform vortex extraction, and filter through filter membrane to obtain the test solution; (2) Sample detection: The sample was detected by liquid chromatography-mass spectrometry. A standard curve was prepared using matrix-matched standard working solution. The standard curve was used for quantification to determine trimellitic acid, terephthalic acid and isophthalic acid in e-cigarette liquid. LC-MS analysis conditions: Column: Core-shell (porous surface) reversed-phase column; Stationary phase: (120Å high-purity silica gel, porous surface particles, uncapped C18); Dimensions: 3.0 × 100 mm, 2.7 µm; Column temperature: 40 °C; Injection volume: 5 μL; Mass spectrometry conditions: Equipped with JetStream electrospray ionization source (ESI); Scan mode: Negative ion scan; Nebulizer pressure: 40 psi; Spray voltage: 1000 V; Capillary voltage: 3500 V; Sheath gas flow rate: 12 L / min; Sheath gas temperature: 350 °C; Gas flow rate: 8 L / min; Gas flow temperature: 330 °C; Data acquisition was performed using multiple reaction monitoring (MRM) mode.

2. The method according to claim 1, characterized in that: In the sample pretreatment, 2.0 g of e-cigarette liquid is placed in a 15 mL centrifuge tube, and a 1.0% formic acid aqueous solution is added. The ratio of formic acid aqueous solution to e-cigarette liquid is 1:2, 1:4, or 1:6, preferably 1:

4.

3. The method according to claim 1, characterized in that: In the sample pretreatment, after adding the internal standard working solution, the sample was vortexed for 20 min, and 1 mL of the liquid was filtered through a 0.22 μm microporous membrane.

4. The method according to claim 1, characterized in that: In liquid chromatography-mass spectrometry analysis, the aqueous mobile phase is formic acid aqueous solution, the organic mobile phase is acetonitrile, and the flow rate is 0.4 mL / min. The aqueous mobile phase is consistent with the sample pretreatment solvent to eliminate the "solvent effect" and obtain narrower chromatographic peaks, higher mass spectrometry sensitivity, and stable retention time.

5. The method according to claim 5, characterized in that: The gradient elution procedure for the mobile phase in the liquid chromatography is as follows:

6. The method according to claim 1, characterized in that: The internal standard working solution contains terephthalic acid-d4 for the quantification of terephthalic acid, isophthalic acid-d4 for the quantification of isophthalic acid, and 1,3,5-triphenyltricarboxylic acid for the quantification of trimellitic acid; the concentration of the internal standard working solution is 20 mg / L, and the amount added to each sample is 50 μL.

7. The method according to claim 1 or 7, characterized in that: The internal standard trimellitic acid mentioned above can also be d3-trimethadiene acid. 13 C3-triphenyltricarboxylic acid, 1,2,3-triphenyltricarboxylic acid, and terephthalic acid can also be replaced by ¹³C8-terephthalic acid, and isophthalic acid can also be replaced by ¹³C2-isophthalic acid.

8. The method according to claim 1, characterized in that: The MRM parameters in the LC-MS analysis conditions include the determination of retention time, and the selection and optimization of qualitative ion pairs, quantitative ion pairs, and collision energies. First, a full scan analysis was performed on each compound to determine the retention time. Then, a product ion scan was performed on the precursor ion at different collision energies, and two ion pairs and the optimal collision energy were selected for each compound. Finally, the standard solution, matrix extract, and matrix extract with added standards were analyzed using MRM mode. The MRM parameters in the LC-MS analysis conditions are shown in the table below. 。

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