WO2021217442A1

WO2021217442A1 - Convergence chromatography analysis method for tocopherol acetate in e-liquid

Info

Publication number: WO2021217442A1
Application number: PCT/CN2020/087564
Authority: WO
Inventors: 袁涛; 蔡佳桐; 徐潇; 李剑政
Original assignee: 深圳波顿香料有限公司
Priority date: 2020-04-27
Filing date: 2020-04-28
Publication date: 2021-11-04
Also published as: CN113640395A

Abstract

A convergence chromatography analysis method for tocopherol acetate in e-liquid, for use in detecting tocopherol acetate in e-liquid. Since the presence of tocopherol acetate in e-liquid may impair human health, the presence or absence of tocopherol acetate in e-liquid is required to be quickly detected during both e-liquid production and e-liquid inspection to implement quality control on the e-liquid. Compared with reported gas chromatography and liquid chromatography, the use of convergence chromatography analysis consumes a shorter time. Optimized chromatography conditions are: an HSS C18 SB chromatography column, 3% of ethanol and 97% of CO₂ used as an elution solvent, a flow rate of 0.8 mL/min, and a back pressure of 2000 psi. In an experiment, a photodiode array (PDA) detector is used, and detection is completed within two minutes when analysis is performed under a wavelength of 220 nm; in addition, the mobile phase comprises 97% of CO₂ and thus is safe and pollution-free to the environment. The analysis method is rapid and sensitive, and thus provides a method basis for security inspection of an electronic cigarette.

Description

Convergence Chromatographic Analysis Method of Tocopherol Acetate in Electronic Cigarette Oil

Technical field

[0001] The present invention relates to the technical field of chemical analysis of electronic cigarette oil, and in particular to a method for analyzing tocopherol acetate in electronic cigarette oil by convergence chromatography.

Background technique

[0002] Recently, the number of deaths from severe pneumonia caused by e-cigarette smoking in the United States has gradually increased, which has triggered widespread concerns about the safety of e-cigarettes. An investigation by the Centers for Disease Control (CDC) showed that tocopherol acetate was detected in the e-liquid consumed by multiple patients; therefore, the rapid and sensitive analysis of the tocopherol acetate in the e-liquid is more effective than the e-liquid. Quality control is very important; conventional tocopherol acetate analysis methods include gas chromatography, gas chromatography-mass spectrometry technology, normal phase liquid chromatography, etc.

[0003] Gas chromatographic analysis methods often have a relatively long analysis time, the analysis time is greater than 30 minutes, the normal phase liquid chromatography analysis time also requires at least 10 minutes, and the normal phase liquid chromatography analysis requires a large amount of organic solvents. Therefore, there is an urgent need to develop an analytical method that can quickly and sensitively analyze tocopherol acetate in order to achieve high-throughput, rapid and sensitive analysis of tocopherol acetate in e-liquid.

technical problem

[0004] There is no efficient and rapid analytical method for the determination of tocopherol acetate in e-liquid.

Technical solutions

[0005] Specifically, a method for analyzing tocopherol acetate in e-liquid by convergence chromatography is used to determine the tocopherol acetate in e-liquid, which is characterized in that it includes the following steps: (1) Electronics E-liquid sample preparation: Weigh the e-liquid, and use a mixed solution of n-heptane and isopropanol as solvent A to dissolve to a constant volume. The volume ratio of n-heptane and isopropanol added is in the range of 8:2-0:10 ; It is also possible to use a mixed solution of n-hexane and isopropanol as solvent A to dissolve to a constant volume. The volume ratio of n-hexane and isopropanol added is in the range of 8:2-0:10; and the volume of e-liquid and solvent A The ratio is 1:10-1000; then the dissolved solution is filtered with a filter membrane to obtain an e-liquid sample.

[0006] (2) Preparation of standard working solution: Weigh the tocopherol acetate standard and dilute it with solvent A to obtain standard series concentrations of 1.0μg/mL, 10.0μg/mL, 20.0μg/mL, 100.0μg/ mL, 500.0μg/mL gradient standard working solution.

[0007] (3) Convergence chromatographic analysis: the standard working solution obtained in step (2) and the e-liquid sample obtained in step (1) are subjected to concentration analysis using an ultra-efficient convergence chromatograph, and the detector is used for detection; Conditions: ACQUITY UPCC BEH (3.0 mm×100 mm, 1.7μm), ACQUITY UPCC BEH 2-Ethlpyridine (2.1mm×150mm, 1.7μm), Viridis HSS C18 SB (2.1mm×150mm, 1.8μm) A chromatographic column; the column temperature is 40℃, the injection volume is 1μL, the system back pressure is 1800-3000psi; the mobile phase is cosolvent and supercritical CO ₂ eluting isocratically in a certain ratio, and the flow rate range is 0.5-1mL/min.

[0008] Preferably, in step (1), a 0.22 μm nylon filter membrane is used for filtration.

[0009] As a preference, in step (1) and step (2), isopropanol, n-heptane, and n-hexane all adopt HPLC purity.

[0010] Preferably, in step (3), the co-solvent is one of methanol, ethanol, isopropanol, and acetonitrile, and its purity is HPLC purity.

[0011] Preferably, in step (3), a diode array detector is used for detection, and the chromatographic peak area is quantified by an external standard method.

[0012] Preferably, the detection wavelength is 220 nm.

[0013] Preferably, in step (3), the mobile phase is methanol and supercritical CO ₂ in a mass ratio of 2%: 98%-7%: 93% isocratic elution.

[0014] Preferably, in step (2), the preparation method of the standard working solution is as follows: weigh 52.0 mg tocopherol acetate standard product into a 50 mL volumetric flask, and dilute the solvent A to 50 mL to obtain a mother liquor; Take 10, 100, 200, 1000, 5000 μL of mother liquor and add it to a 10 mL volumetric flask, and then dilute to 10 mL with the solvent A to obtain standard series concentrations of 1.0 μg/mL, 10.0 μg/mL, 20.0 μg/mL, respectively , 100.0μg/mL, 500.0μg/mL.

Beneficial effect

[0015] The beneficial effect of the present invention is: the method for analyzing tocopherol acetate in electronic cigarette oil proposed by the present invention by convergence chromatography. Because the presence of tocopherol acetate in e-liquid may cause damage to human health, it is necessary to quickly detect the presence of tocopherol acetate in the production and inspection of e-liquid; therefore, the present invention is established for the first time Convergence chromatographic analysis method for the determination of tocopherol acetate in electronic cigarette oil; Convergence chromatographic analysis is adopted, and the analysis time is shorter than that of gas and liquid phases; and the chromatographic conditions are HSS C18 SB chromatographic column, 3% methanol 97 %CO _{2 is} used as the elution solvent, the flow rate is 0.8 mL/min, and the back pressure is 2000 psi. The PDA detector is used in the experiment. When analyzing at a wavelength of 220nm, the retention time is 1.65 minutes, and the detection time is within 2 minutes. The rapid determination of batch samples can be realized, and the mobile phase is 97% CO ₂ , which is safe and pollution-free to the environment. E-cigarette safety testing provides methodological basis; also provides technical support for pharmacy, smoking and health research.

Description of the drawings

[0016] FIG. 1 is a chromatogram of the tocopherol acetate of Example 1 of the present invention on three different convergence chromatographic columns.

[0017] FIG. 2 is a chromatogram of the tocopherol acetate of Example 2 of the present invention under four different elution solvents.

[0018] FIG. 3 is a convergence chromatogram of the tocopherol acetate of Example 3 of the present invention at different flow rates.

[0019] FIG. 4 is a convergence chromatogram of the tocopherol acetate of Example 4 of the present invention under different back pressures.

[0020] FIG. 5 is a combined chromatogram of the tocopherol acetate of Example 5 of the present invention in different ratios of elution solvents.

The best mode of the present invention

[0021] In order to express the present invention more clearly, the present invention will be further described below in conjunction with embodiments.

[0022] First of all, for the original intention and idea of the present invention, it is precisely in recent years that there have been many unidentified electronic pneumonias in the United States. An investigation by the Centers for Disease Control (CDC) has shown that births have been detected in the electronic cigarettes consumed by multiple patients. Phenol acetate; therefore, the rapid and sensitive analysis of tocopherol acetate in e-liquid is essential to the safety of e-liquid. Then, it is the first time to establish the analysis of tocopherol acetate in e-liquid by using the convergence chromatography analysis method; it should be widely used in the quality detection of e-liquid.

[0023] In the following examples, the instrument used is Waters ACQULITY Ultra High Performance Convergence Chromatograph with ACQULITY UPCC diode array detector (PDA); tocopherol acetate standard sample was purchased from Sigma-Aldrich, HPLC purity. Methanol, ethanol, isopropanol, and acetonitrile were all purchased from Merck with HPLC purity.

[0024] Example 1.

[0025] (1) E-liquid sample preparation.

[0026] Weigh 0.1 g of e-liquid, dilute to a 10 mL volumetric flask with isopropanol solvent, and then filter with a 0.22 μm filter membrane to obtain an e-liquid sample.

[0027] (2) Preparation of standard working solution.

[0028] Weigh 52.0 mg of tocopherol acetate standard, dilute to a 50 mL volumetric flask with isopropanol solvent to obtain mother liquor; take 10, 100, 200, 1000, 5000 μL of mother liquor, and then dilute with isopropanol Into a 10mL volumetric flask; get a standard working solution with a concentration of 1-500μg/mL in the standard series; such as 1.0μg/mL, 10.0μg/mL, 20.0μg/mL, 100.0μg/mL, 500.0μg/mL Standard working solution.

[0029] (3) Convergence chromatographic analysis.

[0030] Concentration analysis of the standard working solution obtained in step (2) and the e-liquid sample obtained in step (1) was carried out using an ultra-efficient convergence chromatograph, and a diode array detector (PDA) was used for detection; Choose, set up three control groups, ACQUITY UPCC BEH (3.0 mm×100 mm, 1.7μm), ACQUITY UPCC BEH 2-Ethlpyridine (2.1mm×150mm, 1.7μm), Viridis HSS C18 SB (2.1mm×150mm, 1.8μm); other chromatographic conditions are the same, as follows: column temperature is 40℃, injection volume is 1μL, system back pressure is 2000psi; mobile phase is methanol and supercritical CO ₂ eluting isocratically at 3%:97%, and the flow rate is 0.8mL/min; the chromatogram comparison is shown in Figure 1. In the convergence chromatographic analysis, the chromatographic column has a greater impact on the resolution and peak shape in a shorter analysis time; then it can be seen from Figure 1 that HSS C18 is used The SB chromatographic column has a short peak time and good separation effect.

[0031] Example 2.

[0032] (1) E-liquid sample preparation.

[0033] Weigh 0.2g of e-liquid, dilute with n-heptane: isopropanol (8:2, V/V) solvent to a 10mL volumetric flask, and then filter with a 0.22μm nylon filter membrane to obtain e-cigarette Oil samples.

[0034] (2) Preparation of standard working solution.

[0035] Weigh 52.0 mg of tocopherol acetate standard, use n-heptane: isopropanol (8:2, V/V) solvent to dilute to a 50 mL volumetric flask to obtain the mother liquor; take 10, 100, and 200 respectively , 1000, 5000μL mother liquor, and then use n-heptane: isopropanol (8:2, V/V) solvent to dilute to a 10mL volumetric flask.

[0036] (3) Convergence chromatographic analysis.

[0037] The concentration analysis of the standard working solution obtained in step (2) and the e-liquid sample obtained in step (1) is carried out by using an ultra-efficient convergence chromatograph, and a diode array detector (PDA) is used for detection.

[0038] Using Viridis HSS C18 SB (2.1 mm×150mm, 1.8 μm); column temperature is 40° C., injection volume is 1 μL, system back pressure is 2000 psi; in the selection of cosolvent for mobile phase, there are four control groups. The experiments were carried out with methanol, acetonitrile, ethanol, and isopropanol; the four co-solvents and the main mobile phase (supercritical fluid CO ₂ ) were eluted isocratically at 3%:97% at a flow rate of 0.8 mL/min; The chromatogram is shown in Figure 2; the mobile phase of UPCC is mainly a _{supercritical fluid with CO 2} as the main body. In order to adjust the polarity of the mobile phase and the solubility of the target, it can adapt to the different solubility of different target compounds. Effectively change the peak shape and retention time of the target compound, and methanol, acetonitrile, ethanol, and isopropanol are used as cosolvents with different polarities to optimize the separation of sample solutions of the same concentration. It can be seen from Figure 2 that when acetonitrile is used as the elution solvent, the chromatographic peak time is the slowest, and the peak width is large, which is not conducive to rapid and accurate quantitative analysis. Using ethanol and isopropanol as elution solvents, the obtained chromatographic peak width is small, but compared with methanol, the retention time is longer. This is because in the convergence chromatography, methanol is the organic solvent with the strongest elution capacity. Therefore, methanol is the best choice as a co-solvent.

[0039] Example 3.

[0040] (1) E-liquid sample preparation.

[0041] Weigh 0.5g of e-liquid, dilute to a 10mL volumetric flask with n-hexane: isopropanol (8:2, V/V), and then filter with a 0.22μm filter membrane to obtain a sample of e-liquid .

[0042] (2) Preparation of standard working solution.

[0043] Weigh 52.0 mg of tocopherol acetate standard, use n-hexane: isopropanol (8:2, V/V) solvent to dilute to a 50mL volumetric flask to obtain the mother liquor; take 10, 100, 200, 1000, 5000μL mother liquor, and then use n-hexane: isopropanol (8:2, V/V) to dilute to a 10mL volumetric flask.

[0044] (3) Convergence chromatographic analysis.

[0045] Concentration analysis of the standard working solution obtained in step (2) and the e-liquid sample obtained in step (1) was carried out by using an ultra-efficient convergence chromatograph, and a diode array detector (PDA) was used for detection; using Viridis HSS C18 SB (2.1mm×150mm, 1.8μm); column temperature is 40℃, injection volume is 1μL, system back pressure is 2000psi; methanol and supercritical fluid CO ₂ are eluted at 3%:97% isocratic, and flow rate is selected There are four groups of control groups; respectively 0.5mL/min, 0.6mL/min, 0.8mL/min, 1.0mL/min; the chromatogram is shown in Figure 3; when supercritical CO _{2 is} used as the mobile phase, due to The low viscosity and high diffusion coefficient make it have a higher linear velocity in the separation process, the analysis speed is 3-10 times faster than the traditional high performance liquid chromatography, and the analysis time is short. In order to ensure better sensitivity and suitable chromatography Column pressure: It is particularly important to select an appropriate flow rate. It can be seen from Figure 3 that as the flow rate increases, the analysis time is gradually shortened, but the column pressure will increase accordingly, so the flow rate of 0.8mL/min is the best choice.

[0046] Example 4.

[0047] (1) E-liquid sample preparation.

[0048] Weigh 0.2g of e-liquid, dilute it with n-heptane: isopropanol (5:5, V/V) into a 10mL volumetric flask, and then filter with a 0.22μm filter membrane to obtain e-liquid sample.

[0049] (2) Preparation of standard working solution.

[0050] Weigh 52.0 mg of tocopherol acetate standard, use n-heptane: isopropanol (5:5, V/V) solvent to dilute to a 50 mL volumetric flask to obtain the mother liquor; take 10, 100, and 200 respectively , 1000, 5000μL mother liquor, and then use n-heptane: isopropanol (5:5, V/V) solvent to dilute to a 10mL volumetric flask.

[0051] (3) Convergence chromatographic analysis.

[0052] The concentration analysis of the standard working solution obtained in step (2) and the e-liquid sample obtained in step (1) is performed using an ultra-efficient convergence chromatograph, and a diode array detector (PDA) is used for detection.

[0053] Adopt Viridis HSS C18 SB (2.1mm×150mm, 1.8μm); chromatographic column temperature is 40°C, injection volume is 1μL; methanol and supercritical fluid CO _{2 are selected} according to 3%:97% isocratic elution, flow rate Choose 0.8mL/min; in the selection of back pressure, there are 5 control groups, the back pressures are 1800psi, 2000psi, 2400psi, 2800psi, 3000psi, and the chromatogram is shown in Figure 4; in the 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 CO ₂ to maintain a supercritical state during the entire operation, because the dissolving ability of the sample is different under different back pressure conditions. When the back pressure increases, the density of the supercritical fluid increases, the solvation ability increases, and the column pressure increases. It can be seen from Figure 4 that as the back pressure increases, the retention time gradually shortens, but as the back pressure increases, _{the rate of CO 2} consumption increases greatly. _{The cost of CO 2} consumption needs to be considered, so the back pressure of 2000 psi Most suitable.

[0054] Example 5.

[0055] (1) E-liquid sample preparation.

[0056] Weigh 0.25g of e-liquid, dilute to a 10mL volumetric flask with n-hexane: isopropanol (5:5, V/V), and then filter with a 0.22μm filter membrane to obtain a sample of e-liquid .

[0057] (2) Preparation of standard working solution.

[0058] Weigh 52.0 mg of tocopherol acetate standard, use n-hexane: isopropanol (5:5, V/V) solvent to dilute to a 50 mL volumetric flask to obtain the mother liquor; take 10, 100, 200, 1000, 5000μL mother liquor, then use n-hexane: isopropanol (5:5, V/V) solvent to dilute to a 10mL volumetric flask.

[0059] (3) Convergence chromatographic analysis.

[0060] Concentration analysis is performed on the standard working solution obtained in step (2) and the e-liquid sample obtained in step (1) using an ultra-efficient convergence chromatograph, and a diode array detector (PDA) is used for detection.

[0061] Adopt Viridis HSS C18 SB (2.1mm×150mm, 1.8μm); column temperature is 40°C, injection volume is 1μL; system back pressure is 2000psi; methanol and supercritical fluid CO _{2 are selected} according to 3%:97%, etc. For high-degree elution, the flow rate is 0.8mL/min; _{there are 6 control groups in the selection of the ratio of elution solvent (methanol and supercritical fluid CO 2} ); 2%:98%; 3%:97%; 4 %:96%; 5%:95%; 6%:94%; 7%:93%; the chromatogram is shown in Figure 5; it can be seen from the figure that as the proportion of methanol increases from 2% to 7% , The analysis speed of tocopherol acetate is gradually shortened, and the retention time is shortened from 2.1 minutes to 1.2 minutes. In the experiment, considering solvent consumption and experiment time, 3% methanol and 97% CO _{2 were selected} as isocratic elution conditions, and the retention time was 1.65 minutes, which can achieve rapid treatment of tocopherol acetate within 2 minutes. analyze.

[0062] In summary: When the convergence chromatographic analysis is selected as: chromatographic column Viridis HSS C18 SB (2.1mm×150mm, 1.8μm); chromatographic column temperature is 40°C, sample injection volume is 1μL, system back pressure is 2000psi; mobile phase is Methanol and supercritical CO ₂ are eluted isocratically at 3%:97%, and the detection effect is best when the flow rate is 8mL/min. Compared with the existing liquid chromatography and gas chromatography analysis methods, the analysis speed of this method is greatly improved, and the consumption of organic solvents is greatly reduced. Since 97% of the mobile phase is CO ₂ , it is safe and pollution-free to the environment. This method can realize the rapid and sensitive analysis of tocopherol acetate in electronic cigarettes, and provide a method basis for the safety detection of electronic cigarettes.

[0063] Methodological investigation.

[0064] Select the above-mentioned six gradient standard working solutions, according to the optimal chromatographic conditions-the chromatographic column is Viridis HSS C18 SB (2.1mm×150mm, 1.8μm): the column temperature is 40°C, the sample injection volume is 1μL, the system Back pressure is 2000psi; the mobile phase is methanol and supercritical CO ₂ are eluted isocratically at 3%:97%, and the flow rate is 0.8mL/min. The peak area of tocopherol acetate is used for quantification. The concentration and its peak area have a linear relationship in the range of 0-500.0μg/mL. When the signal-to-noise ratio is S/N=3, the linear regression equation is y=1351.0x-934.2, R ² =0.9999; the detection limit is 0.2 μg/mL.

[0065] And the chromatographic analysis conditions are: the chromatographic column is Viridis HSS C18 SB (2.1mm×150mm, 1.8μm): the column temperature is 40°C, the sample volume is 1μL, the system back pressure is 2000psi; the mobile phase is methanol and ultra Critical CO ₂ is eluted isocratically according to 3%:97%, and when the flow rate is 0.8mL/min, 6 batches of different brands of e-liquid are randomly purchased on the market, and each sample is measured in parallel for 3 times according to the above method. No tocopherol acetate component was detected. After adding the standard solution, the recovery rate of standard addition was 96.3-102.2%.

Industrial applicability

[0066] The advantages of the present invention are: 1) The present invention establishes a method for rapid detection of tocopherol acetate in e-cigarette oil by using ultra-efficient convergence chromatography in series with a diode array detector, which can accurately detect e-cigarettes The tocopherol acetate in the oil is qualitatively and quantified to provide a scientific basis for the accurate determination and rapid detection of the tocopherol acetate in the electronic cigarette oil; 2) The HSS C18 SB chromatographic column of the present invention (2.1mm×150mm, 1.8μm) ) The choice of 3%:97% with CO ₂ and methanol as the mobile phase achieves excellent separation of tocopherol acetate in e-liquid; 3) Fast and sensitive analysis of samples within 2 minutes, method The detection limit is up to 0.2μg/mL; since 97% of the components in the mobile phase are CO ₂ , it is safe and non-polluting to the environment.

Sequence Listing Free Content

[0067] The above-disclosed are only a few specific embodiments of the present invention, but the present invention is not limited thereto, and any changes that can be thought of by those skilled in the art should fall into the protection scope of the present invention.

Claims

A convergence chromatographic analysis method for tocopherol acetate in e-liquid is used for the determination of tocopherol acetate in e-liquid, which is characterized in that it comprises the following steps:

(1) E-liquid sample preparation

Weigh the e-liquid, use the mixed solution of n-heptane and isopropanol as solvent A to dissolve to a constant volume. The volume ratio of n-heptane and isopropanol added is in the range of 8:2-0:10; n-hexane can also be used The mixed solution of alkane and isopropanol is used as solvent A to dissolve to a constant volume. The volume ratio of n-hexane and isopropanol added is in the range of 8:2-0:10; and the volume ratio of e-liquid to solvent A is 1:10 -1000; Then use a filter membrane to filter the dissolved solution to obtain an e-liquid sample;

(2) Preparation of standard working solution

Weigh the tocopherol acetate standard and dilute it with solvent A to obtain a standard series of concentrations of 1.0μg/mL, 10.0μg/mL, 20.0μg/mL, 100.0μg/mL, 500.0μg/mL gradient standard working solution ；

(3) Convergence chromatographic analysis

The concentration analysis of the standard working solution obtained in step (2) and the e-liquid sample obtained in step (1) is carried out with an ultra-high performance convergence chromatograph, and the detector is used for detection;

Chromatographic conditions: ACQUITY UPCC BEH (3.0mm×100mm, 1.7μm), ACQUITY UPCC BEH 2-Ethlpyridine (2.1mm×150mm, 1.7μm), Viridis HSS C18 SB (2.1mm×150mm, 1.8μm) A chromatographic column; the column temperature is 40℃, the injection volume is 1μL, the system back pressure is 1800-3000psi; the mobile phase is cosolvent and supercritical CO 2 eluting isocratically in a certain ratio, and the flow rate range is 0.5-1mL/min.
The method for analyzing tocopherol acetate in electronic cigarette oil according to claim 1, characterized in that, in step (1), a 0.22 μm nylon filter membrane is used for filtration.

3. The method for analyzing tocopherol acetate in e-liquid according to claim 1, characterized in that, in step (1) and step (2), isopropanol, n-heptane, All n-hexane adopts HPLC purity.

4. The method for analyzing tocopherol acetate in e-liquid according to claim 1, characterized in that, in step (3), the co-solvent is methanol, ethanol, isopropanol, One of acetonitrile, and its purity is HPLC purity.

5. The method for analyzing tocopherol acetate in electronic cigarette oil according to claim 1, characterized in that, in step (3), a diode array detector is used for detection, and the external standard method chromatographic peak Quantitative area.

6. The method for analyzing tocopherol acetate in electronic cigarette oil according to claim 4, wherein the detection wavelength is 220 nm.

7. The method for analyzing tocopherol acetate in e-liquid according to claim 1, characterized in that, in step (3), the mobile phase is methanol and supercritical CO 2 according to the mass ratio of 2%: 98%-7%: 93% isocratic elution

8. The method for analyzing tocopherol acetate in electronic cigarette oil according to claim 1, characterized in that, in step (2), the preparation method of the standard working solution is: weighing 52.0 mg of tocopherol acetate The phenol acetate standard was added to a 50mL volumetric flask, and the solvent A was used to dilute the volume to 50mL to obtain the mother liquor; 10, 100, 200, 1000, 5000 μL of the mother liquor were respectively added to the 10mL volumetric flask, and then the solvent A was used to determine The volume is 10mL, and the standard series concentrations are 1.0μg/mL, 10.0μg/mL, 20.0μg/mL, 100.0μg/mL, 500.0μg/mL, respectively.