A method for determining benzyl malonate UV absorbers in PBAT products

Abstract

The present invention discloses a method for determining the presence of a benzyl malonate-type ultraviolet absorber in a PBAT product. The method comprises the following steps: (1) preparing a standard working solution and a sample solution to be tested, wherein the sample solution to be tested is obtained by adding a chloroform solution to the PBAT sample to completely dissolve the sample, then adding a precipitation solvent and an ionic salt solution, mixing the mixture evenly, and then centrifuging to obtain an insoluble substance and a supernatant. The supernatant is treated to dryness and then redissolved with a methanol-water solution. NaOH solution is added dropwise to the redissolved solution until no obvious precipitate is formed in the solution. After centrifugation, the supernatant is taken and the volume is fixed with a methanol-water solution to obtain the sample solution to be tested; and (2) performing qualitative and quantitative analysis of the sample solution to be tested and the standard solution using ultraviolet chromatography-tandem mass spectrometry. The method established by the present invention can meet the requirements of relevant laws and regulations and achieve the supervision of the above-mentioned ultraviolet absorbers.

Description

Technical field:

[0001] The present invention relates to the technical field of plastic product detection, and in particular to a method for determining the content of a benzyl malonate ultraviolet absorber in a PBAT product. Background technology:

[0002] Polybutylene adipate terephthalate (PBAT) is a promising polyester biodegradable material. In 2022, my country's annual production reached 400,000 tons. Under the current plastic restriction policy, PBAT is currently used in various film bags, food packaging, disposable tableware, and other products to gradually replace non-degradable plastic products such as polyolefins. However, the aromatic ring-ester conjugated system in the PBAT molecule makes it particularly susceptible to ultraviolet absorption, causing its molecular chain to break, which in turn affects the mechanical and physical properties and service life of PBAT products. Therefore, ultraviolet absorbers (UVAs) are usually added to PBAT products. Their principle is to convert the absorbed ultraviolet light energy into heat energy to reduce the adverse effects of ultraviolet light on the PBAT polymer chain. Currently, common UV absorbers include salicylates, aminobenzoates (such as UV-1), benzophenones (such as UV-531 and UV-9), benzotriazoles (such as UV-326 and UV-327), hydroxyphenyl-s-triazines (such as UV-400 and UV405), oxalic acid anilides (such as UV-312), cyanoacrylates (such as UV-3030 and UV-3039), and nickel-based quenchers (such as UV-1084). All of these have been used in PBAT products (CN106751559A and CN113004665A). However, most of these UV absorbers have been shown to have estrogenic or androgenic activity, and long-term exposure may lead to endocrine disorders (Trend. Anal. Chem., 2009, 28, 708-717). Benzyl malonate UV absorbers, such as UV-988 and UV-1988, offer advantages over traditional UV absorbers in both performance and price (Plast. Addit. Compd., 2007, 9, 32-35), and are also less toxic (Cramer Class I). Commercialized in 2007, they were initially used in plastics such as polycarbonate and, in recent years, have also begun to be used in PBAT products.

[0003] However, for PBAT-based food contact articles, benzyl malonate UV absorbers are not currently included in the positive list of permitted substances (GB 9685-2016), and therefore still require regulation. Unlike traditional UV absorbers for which mature analytical methods have been established, few testing methods have been reported for benzyl malonate UV absorbers. Most importantly, because benzyl malonate and PBAT oligomers (such as dimers and trimers) or fragmentation products all have an aromatic ring-ester structure, their UV absorption wavelengths, mass spectrometry ion fragmentation characteristics, and other characteristics are very similar. This makes benzyl malonate very susceptible to interference from PBAT oligomers or fragmentation products during measurement, increasing the difficulty of establishing an accurate quantitative method. Summary of the invention:

[0004] To address the issue of PBAT oligomer interference in UV-1988 and UV-988 ultraviolet absorber tests, the present invention provides a method for determining benzyl malonate ultraviolet absorbers in PBAT products. The method utilizes a precipitation solvent to precipitate PBAT polymers, and simultaneously utilizes copper acetate as an ionic ligand for PBAT oligomers to efficiently remove PBAT oligomers, thereby avoiding their interference with the determination of the aforementioned ultraviolet absorbers. Furthermore, the method can meet relevant legal and regulatory requirements.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for determining the content of a benzyl malonate UV absorber in a PBAT product, wherein the benzyl malonate UV absorber includes UV-988 and UV-1988. The method comprises the following steps:

[0007] (1) preparing a standard working solution and a sample solution to be tested, wherein the sample solution to be tested is obtained by the following steps: adding a chloroform solution to the PBAT sample to completely dissolve the sample, then adding a precipitation solvent and an ion salt solution to the solution, wherein the precipitation solvent is selected from one or two of methanol, tetrahydrofuran and n-hexane, and the ion salt solution is a salt solution of copper ions or iron ions, mixing the solution uniformly and then centrifuging to obtain an insoluble substance and a supernatant, treating the supernatant to dryness, and then redissolving it with a methanol-water solution, and adding a NaOH solution dropwise to the redissolved solution until no obvious precipitate is generated in the solution, and after centrifugation, taking the supernatant and diluting it with a methanol-water solution to obtain the sample solution to be tested;

[0008] (2) The sample solution and standard solution were taken separately for qualitative and quantitative analysis using ultra-high performance liquid chromatography-tandem mass spectrometry.

[0009] Preferably, the sample solution to be tested is obtained by the following steps: adding a chloroform solution to the PBAT sample to completely dissolve the sample to obtain a PBAT chloroform solution with a mass concentration of 0.05-0.10 g / mL, and then adding a precipitation solvent and an ionic salt solution with a concentration of 0.8-2.0 mol / L to the solution, the volume ratio of the precipitation solvent to the ionic salt solution is 25-35:1, the volume ratio of the precipitation solvent to the chloroform solution is 2:1, the precipitation solvent is selected from one or two of methanol, tetrahydrofuran and n-hexane, and the ionic salt solution is a salt solution of copper ions or iron ions. The solution is mixed evenly and centrifuged to obtain an insoluble matter and a supernatant, the insoluble matter is washed with a precipitation solvent, and the washing solution after washing is mixed with the supernatant to obtain a mixed solution. After the mixed solution is treated to dryness, it is re-dissolved with a methanol-water solution, and NaOH solution is added dropwise to the re-dissolved solution until no obvious precipitate is generated in the solution. After centrifugation, the supernatant is taken and made up to volume with a methanol-water solution to obtain the sample solution to be tested.

[0010] Preferably, in step (1), the precipitation solvent is a mixed solution of methanol and tetrahydrofuran, the volume ratio of methanol to tetrahydrofuran is 2-6:1, and the ionic salt solution is selected from one of copper sulfate, copper nitrate, copper acetate, copper chloride, ferric sulfate, ferric nitrate, ferric acetate and ferric chloride.

[0011] Further preferably, the volume ratio of methanol to tetrahydrofuran in the mixed solution of methanol and tetrahydrofuran in step (1) is 5:1, the concentration of the ionic salt solution is 1.5 mol / L. The mass concentration of PBAT in the chloroform solution of PBAT is 1 / 15 g / mL, and the volume ratio of the precipitation solvent to the ionic salt solution is 30:1.

[0012] Preferably, the volume ratio of methanol to water in the methanol-water solution in step (1) is 1:1.

[0013] Preferably, the standard working solution in step (1) is prepared by dissolving target mass of UV-988 and UV-1988 standards in tetrahydrofuran to obtain a standard stock solution containing UV-988 and UV-1988 at a concentration of 1000 mg / L. Subsequently, the standard stock solution is diluted stepwise with a methanol-water mixture at a volume ratio of 1:1 to obtain standard working solutions with concentrations of 5 μg / L, 10 μg / L, 20 μg / L, 50 μg / L, and 100 μg / L, respectively, of UV-988 and UV-1988.

[0014] Preferably, the chromatographic conditions in the ultra-liquid chromatography-tandem mass spectrometry described in step (2) are as follows: chromatographic column: C18 stationary phase chromatographic column or phenyl-hexyl stationary phase chromatographic column; mobile phase A: methanol, mobile phase B: water; elution gradient: mobile phase A: mobile phase B starts at 50:50, changes to 0:100 within 5 minutes, maintains for 5.9 minutes, and then switches to 50:50 within 0.1 minutes; flow rate 0.3 mL / min; injection volume: 5 μL; column temperature: 40°C; mass spectrometry conditions in the ultra-liquid chromatography-tandem mass spectrometry are as follows: ESI positive ion mode; ion source: electrospray ionization source ESI; ion source temperature: 350°C-600°C; ionization mode: positive ion mode; spray voltage: 400-500 V; capillary voltage: 3500-4500 V; air flow rate 7 SLM; detection method: multiple reaction monitoring MRM; run time: 10 minutes, post-run 5 minutes.

[0015] Further preferably, the chromatographic column in step (2) is Eclipse Plus C18 100 mm×2.1 mm×1.8 μm, Poroshell EC-C18 100 mm×2.1 mm×2.7 μm, or Eclipse Plus Phenyl-Hexyl 100 mm×2.1 mm×1.8 μm.

[0016] Further preferably, the mass spectrometry conditions in the ultra-liquid chromatography-tandem mass spectrometry described in step (2) are: ESI positive ion mode; ion source: electrospray ionization source ESI; ion source temperature: 450°C; ionization mode: positive ion mode; spray voltage: 470V; capillary voltage: 4000V; air flow rate 7SLM; detection method: multiple reaction monitoring MRM; run time: 10min, post-run 5min.

[0017] The beneficial effects of the present invention are as follows: The present invention adopts a three-step strategy of dissolution precipitation, ion ligand removal and UPLC-MS / MS determination to establish a method for simultaneously determining the content of ultraviolet absorbers UV-1988 and UV-988 in PBAT products. This method has good recovery rate and precision for UV-1988 and UV-988 in PBAT products. At the same time, this method can meet the requirements of relevant laws and regulations and realize the supervision of the above-mentioned ultraviolet absorbers. Description of the drawings:

[0018] Figure 1 The chromatograms are those of UV-1988 and UV-988;

[0019] Figure 2 The secondary mass spectrum of UV-1988 (ESI + , CE=20eV);

[0020] Figure 3 The secondary mass spectrum of UV-988 (ESI + , CE=20eV). Specific implementation method:

[0021] The following is a further description of the present invention, but not a limitation of the present invention.

[0022] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the experimental materials and reagents herein are conventional commercial products in the art.

[0023] Example 1:

[0024] A method for determining the content of a benzyl malonate UV absorber in a PBAT product, wherein the benzyl malonate UV absorber includes UV-988 and UV-1988, comprising the following steps:

[0025] (1) Preparation of standard working solutions: The target mass of UV-988 and UV-1988 standards were dissolved in tetrahydrofuran to obtain standard stock solutions containing 1000 mg / L of UV-988 and UV-1988. Subsequently, the standard stock solutions were diluted stepwise with methanol-water (volume ratio 1:1) to obtain standard working solutions with concentrations of 5 μg / L, 10 μg / L, 20 μg / L, 50 μg / L, and 100 μg / L of UV-988 and UV-1988, respectively.

[0026] (2) Preparation of sample solution to be tested:

[0027] a. Sample pretreatment: Weigh 1.00-2.00 g of PBAT sample and cut the sample into 5 mm × 5 mm pieces with scissors for later use.

[0028] b. Sample Extraction: Weigh 1.0 g (accurate to 0.1 mg) of the PBAT sample obtained in step a and place it into a clean 50 mL centrifuge tube. Add 15 mL of chloroform solution. Once the sample is completely dissolved, add 30 mL of a methanol-tetrahydrofuran mixture and 1 mL of a 1.5 mol / L aqueous copper ion solution (copper acetate). Tighten the cap and vortex the centrifuge tube to thoroughly mix the solution. Afterward, let it sit for 15 minutes. Centrifuge the tube at 5000 g for 15 minutes. Remove the tube and transfer the supernatant to a clean 100 mL flask. Wash the insoluble material from the centrifuge tube with 15 mL of the methanol-tetrahydrofuran solution three times. Repeat this process, adding the wash solution to the 100 mL flask. Purge the flask with liquid nitrogen until nearly dry. Redissolve the solution in 1.5 mL of a methanol-water solution (1:1 by volume). Transfer the entire solution to a 2 mL centrifuge tube and add 1 mol / L NaOH solution dropwise until no significant precipitate forms. Centrifuge at 5000 g for 10 min, and dilute the supernatant to 2 mL with methanol-water (1:1 volume ratio). Filter through a 0.2 μm filter membrane before analysis by UPLC-MS / MS.

[0029] Prepare a blank sample: To a clean 50-mL centrifuge tube, add 15 mL of chloroform solution, followed by 30 mL of a methanol-tetrahydrofuran mixture and 1 mL of a 1.5 mol / L copper ion aqueous solution (copper acetate). Tighten the cap and vortex the tube to thoroughly mix the solution. After allowing it to rest for 15 minutes, centrifuge the tube at 5000 g for 15 minutes. Remove the tube and transfer the solution to a clean 100-mL flask. Rinse the tube with 15 mL of the methanol-tetrahydrofuran solution three times, adding the wash solution to the 100-mL flask. Purge the flask nearly dry with liquid nitrogen and reconstitute with 1.5 mL of a methanol-water (1:1 volume ratio). Transfer the entire solution to a 2-mL centrifuge tube and add 1 mol / L NaOH solution dropwise until no visible precipitate forms. Afterwards, the mixture was centrifuged at 5000 g for 10 min, and the supernatant was diluted to 2 mL with methanol-water (volume ratio 1:1), filtered through a 0.2 μm filter membrane, and then subjected to UPLC-MS / MS detection.

[0030] (3) The sample solution and the standard working solution were respectively aspirated for testing, and the chromatographic conditions were as follows: chromatographic column: Poroshell EC-C18 100 mm × 2.1 mm × 2.7 μm; mobile phase A: methanol, mobile phase B: water; elution gradient: mobile phase A: mobile phase B started at 50:50, changed to 0:100 within 5 minutes, maintained for 5.9 minutes, and then switched to 50:50 within 0.1 minutes; flow rate 0.3 mL / min; injection volume: 5 μL; column temperature: 40°C; mass spectrometry conditions in the ultra-liquid chromatography-tandem mass spectrometry method were as follows: ESI positive ion mode; ion source: electrospray ionization source ESI; ion source temperature: 450°C; ionization mode: positive ion mode; spray voltage: 470 V; capillary voltage: 4000 V; air flow rate 7 SLM; detection method: multiple reaction monitoring MRM; run time: 10 minutes, post-run 5 minutes.

[0031] Example 2: UPLC-MS / MS analysis and confirmation of positive results

[0032] The sample and standard working solution were measured using the UPLC-MS / MS conditions described in Example 1. If the sample's chromatographic peak retention time matched that of the standard and all selected ion pairs were present, a positive confirmation was performed based on the type and relative abundance ratio of the selected ion pairs for qualitative analysis. For qualitative analysis, if the relative abundance tolerance did not exceed the range specified in Table 1, the presence of the corresponding analyte in the sample was determined.

[0033] Table 1 Maximum allowable deviation of relative ion abundance ratios for confirmation of positive results

[0034]

[0035] Example 3: Selection of precipitation solvent

[0036] After dissolving PBAT in chloroform, a suitable solvent must be selected to precipitate the PBAT polymers in the chloroform to prevent adverse effects on subsequent UPLC analysis. The precipitation solvent must be miscible with chloroform. Based on the detection conditions in Example 1, this example examined the ability of five solvents—methanol, tetrahydrofuran, n-hexane, methanol-tetrahydrofuran (5:1 volume ratio), and tetrahydrofuran-n-hexane (1:5 volume ratio)—to precipitate the polymer components in chloroform-dissolved PBAT samples. The spiked recoveries of these five solvents were also evaluated. Five 500mL narrow-necked flat-bottom flasks containing 150mL of chloroform were used. 10g of the cut negative PBAT sample was added to each flask. Subsequently, 300mL of each of the five solvents was added. After 15 minutes of simmering, the amount of precipitate produced was measured. As shown in Table 2, the results indicate that methanol-tetrahydrofuran (5:1 volume ratio) produced the most precipitate, so it was selected as the precipitation solvent.

[0037] Table 2 Selection of precipitation solvents

[0038]

[0039] Example 4: Selection of ionic ligands

[0040] Based on the detection conditions of Example 1, this example employed metal ion ligands to selectively form macromolecular complexes with PBAT oligomers, thereby preventing interference with UV-1988 and UV-988 assays. The removal ability of PBAT oligomers using salt solutions containing iron and copper ions was investigated. A blank sample was prepared, and a predetermined amount of UV-1988 and UV-988 was added to the sample, resulting in a final concentration of 0.10 mg / L. The sample was treated according to the above method, and the concentrations of UV-1988 and UV-988 were measured, and their recoveries were calculated. As shown in Table 3, the recoveries of UV-1988 and UV-988 exceeded 450% when using the salt solution containing iron ions, indicating that Fe(III) has poor adsorption capacity for PBAT oligomers. When using the salt solution containing copper ions, the recoveries of both ranged from 86.2% to 90.9%, with copper acetate achieving the highest recovery. Therefore, copper acetate was ultimately selected as the ionic ligand for PBAT oligomers.

[0041] Table 3 Selection of ligand ion salt solution

[0042]

[0043]

[0044] Example 5: Selection of chromatographic column

[0045] Based on the detection conditions of Example 1, considering that UV-1988 and UV-988 are both medium-polar compounds, commonly used C18 stationary phase and phenyl-hexyl stationary phase chromatographic columns were selected for their separation, namely, Eclipse Plus C18 (100mm×2.1mm×1.8μm), Poroshell EC-C18 (100mm×2.1mm×2.7μm), and Eclipse Plus Phenyl-Hexyl (100mm×2.1mm×1.8μm). The results showed that the separation effect of UV-1988 and UV-988 on the above C18 columns was better, and the system pressure was the lowest and the mobile phase equilibration time was the shortest when separating on the Poroshell EC-C18 (100mm×2.1mm×2.7μm), so this column was selected for the separation of the above-mentioned ultraviolet absorbers.

[0046] Example 6: Optimization of ion source parameters

[0047] Ion source parameters are crucial for the ionization efficiency of UV-1988 and UV-988, specifically the ionization mode, capillary voltage, spray voltage, and ion source temperature. Both UV absorbers have alkoxy and ester structures, which facilitate the formation of positive ions. Therefore, the positive ionization mode was selected to determine the extraction and separation conditions described above. Based on the detection conditions of Example 1, the capillary voltage (3.5-4.0 kV), spray voltage (400-500 V), and ion source temperature (300°C-500°C) were optimized individually. The instrument response of UV-1988 and UV-988 at the same concentration in methanol-water (50:50 volume ratio) was investigated. Results showed that the instrument response of these UV absorbers was highest when the capillary voltage was set to 4.0 kV, the spray voltage was set to 470 V, and the ion source temperature was set to 450°C.

[0048] Example 7: Selection of Multiple Reaction Monitoring (MRM) Conditions

[0049] Both UV-1988 and UV-988 are easy to form [M+Na] + Because of the presence of peaks, 273 and 441 were selected as parent ions for both products. Full scans of their product ions were performed at collision energies of 10, 20, and 40 eV. The results showed that at a collision energy of 20 eV, both products produced a relatively abundant number of product ions with larger mass-to-charge ratios. For UV1988, the product ions were mainly 121, 159, 161, 189, 191, 193, and 219, while for UV988, the product ions were mainly 215, 301, 303, 327, 329, 345, 349, 351, 373, and 391. Further increasing the collision energy yielded more product ions with smaller mass-to-charge ratios. For UV1988, the product ions were mainly 51, 65, 75, 77, 91, and 93, while for UV988, the product ions were mainly 87, 91, 103, 105, 189, and 203. Considering that multiple reaction monitoring ion pairs established with product ions of larger mass-to-charge ratios have less interference, a collision energy of 20 eV was selected. The product ions with the highest response at this energy were selected to establish the MRM ion pairs for quantitative analysis, while the remaining ion pairs were used for auxiliary qualitative analysis. The optimized parameters are shown in Table 4.

[0050] Table 4 MRM data acquisition information of UV988 and UV1988 (* indicates quantitative ion)

[0051]

[0052] Example 8: Linear range, detection limit and quantification limit

[0053] 1. According to the determination conditions of Example 1, the standard working solution (0.02-0.5 mg / L) was tested, with the concentration of UV-1988 and UV-988 in the silicone rubber sample as the abscissa, expressed in mg / L, and the corresponding peak area average as the ordinate. The standard working curve was drawn to obtain the linear equation and correlation coefficient. The test results showed that there was a good linear relationship between the concentration and the response value. The linear equation and correlation coefficient are shown in Table 4.

[0054] As can be seen from Table 5, UV-1988 and UV-988 have a wide linear range, and the linear correlation coefficients are both greater than 0.9990, which can well meet the needs of the test work.

[0055] Table 5 Linear equations, detection limits, and quantification limits of UV988 and UV1988

[0056]

[0057] 2. Detection limit and quantification limit

[0058] The detection limit and lower limit of determination of this method are determined based on the sensitivity of UV-1988 and UV-988 in UPLC-MS / MS detection. The test was performed using a sample with a blank sample matrix according to the optimized measurement conditions. The methanol-water solution with the blank sample matrix was used to prepare a series of standard solutions containing 0.02-0.50 mg / L (UV-1988) and 0.18-0.50 mg / L (UV-988). The signal-to-noise ratio (S / N) of 3 times was used as the lowest detection limit, and the detection limit concentration obtained was calculated by the standard deviation S of 7 repeated tests. MDL × coefficient T (n-1，1-α＝0.99) To validate the detection limit, T was 3.143 when the number of replicates was 7 and the confidence level was 99%. This was established when the calculated value was ≤ the detection limit derived from the signal-to-noise ratio. Using a 10-fold signal-to-noise ratio (S / N) as the limit of quantification, the detection and quantification limits for UV-1988 and UV-988 are shown in Table 5.

[0059] 3. Recovery and precision of the method

[0060] Recovery tests for UV-1988 and UV-988 were conducted using a blank sample spike method. Blank samples containing no UV absorbers were extracted using the two methods described above. Spiked solutions containing 0.020 mg / L, 0.10 mg / L, and 0.50 mg / L UV-1988 and 0.018 mg / L, 0.10 mg / L, and 0.50 mg / L UV-1988 were prepared. Six separate measurements were performed at each concentration, and recovery and precision tests were conducted. The spiked recoveries of UV-1988 and UV-988 in silicone rubber products determined by this method ranged from 85.9% to 91.2%, with relative standard deviations ranging from 2.1% to 7.2%, demonstrating good recovery and precision.

[0061] Comparative Example 1:

[0062] CN115350502A discloses a method for rapidly extracting UV absorbers from biodegradable films. The biodegradable film is dissolved, a precipitant is added, and the mixture is centrifuged to allow for sufficient precipitation. The supernatant is then aspirated, concentrated, and dried. The precipitant comprises n-hexane, methanol, or a mixture thereof. The UV988 and UV1988 levels in PBAT-positive products supplemented with specific levels of UV988 and UV1988 were determined using the method of Example 1. The results were compared with those obtained using the detection method of Example 1. The results are shown in Table 6.

[0063] Table 6 Comparison of the results of Example 1 and Comparative Example 1

[0064]

[0065] As shown in Table 6, when the existing method (Comparative Example 1) is used, the recoveries of UV1988 and UV988 are higher than 280% and 160%, respectively, that is, the determination results are significantly higher, indicating that their quantification is severely interfered with by PBAT oligomers or fragmentation products. When the present method (Example 1) is used, the recoveries can be maintained at 91% and 103%, respectively, indicating that the method proposed in the present invention can better avoid the above-mentioned interference and achieve accurate determination of UV988 and UV1988 in PBAT.

Claims

1. A method for determining the content of benzyl malonate UV absorbers in PBAT products, characterized in that: The benzyl malonate UV absorbers include UV-988 and UV-1988. The determination method comprises the following steps: (1) Prepare a standard working solution and a sample solution to be tested, wherein the sample solution to be tested is obtained by the following steps: a. Sample pretreatment: Weigh 1.00~2.00 g of PBAT sample and cut the sample into 5 mm × 5 mm pieces with scissors for later use; b. Sample extraction: Weigh 1.0 g of the PBAT sample obtained in step a and place it in a clean 50 mL centrifuge tube, add 15 mL of chloroform solution, and after the sample is completely dissolved, add 30 mL of methanol-tetrahydrofuran mixed solution and 1 mL of 1.5 mol / L copper ion aqueous solution, wherein the copper ion aqueous solution is an aqueous solution of copper sulfate, copper nitrate, copper acetate or copper chloride. After tightening the bottle cap, shake the centrifuge tube to fully mix the solution in the tube. Then, let it stand for 15 minutes, place the centrifuge tube in a 5000 g centrifuge for 15 minutes, remove the centrifuge tube, transfer the supernatant therein to a 100 mL clean flask, wash the insoluble matter in the centrifuge tube with 15 mL of methanol-tetrahydrofuran solution, repeat three times, and combine the washing solution into the above 100 mL flask. Blow the liquid nitrogen in the flask to almost dryness, and redissolve it with 1.5 mL of methanol-water solution. The volume ratio of methanol to water in the methanol-water solution is 1:

1. Transfer all the solution to a 2 mL centrifuge tube and add 1 dropwise mol / L NaOH solution until no obvious precipitate is formed in the solution. Then, centrifuge at 5000 g for 10 min, and take the supernatant and dilute it to 2 mL with methanol-water (the volume ratio of methanol to water is 1:1). After filtering through a 0.2 µm filter membrane, it is used for UPLC-MS / MS detection. (2) The sample solution and standard solution were taken separately and qualitative and quantitative analysis was performed using ultra-performance liquid chromatography-tandem mass spectrometry.

2. The measuring method according to claim 1, wherein The standard working solution in step (1) is prepared by the following steps: taking the target mass of UV-988 and UV-1988 standards and dissolving them in tetrahydrofuran to obtain a standard stock solution containing UV-988 and UV-1988 at a concentration of 1000 mg / L, and then gradually diluting the above standard stock solution with a methanol-water mixed solution with a volume ratio of 1:1 to obtain standard working solutions with UV-988 and UV-1988 at concentrations of 5 μg / L, 10 μg / L, 20 μg / L, 50 μg / L and 100 μg / L.

3. The measuring method according to claim 1, wherein The chromatographic conditions in the ultra-high performance liquid chromatography-tandem mass spectrometry described in step (2) are as follows: chromatographic column: C18 stationary phase chromatographic column or phenyl-hexyl stationary phase chromatographic column; mobile phase A: methanol, mobile phase B: water; elution gradient: mobile phase A: mobile phase B starts at 50:50, changes to 0:100 within 5 minutes, maintains for 5.9 minutes, and then switches to 50:50 within 0.1 minutes; flow rate 0.3 mL / min; injection volume: 5 μL; column temperature: 40°C; mass spectrometry conditions in the ultra-high performance liquid chromatography-tandem mass spectrometry are as follows: ESI positive ion mode; ion source: electrospray ionization source ESI; ion source temperature: 350°C-600°C; ionization mode: positive ion mode; spray voltage: 400-500 V; capillary voltage: 3500~4500 V; gas flow rate 7 SLM; detection method: multiple reaction monitoring MRM; run time: 10 minutes, post-run 5 minutes.

4. The measuring method according to claim 3, wherein The chromatographic column described in step (2) is EclipsePlus C18 100 mm × 2.1 mm × 1.8 µm, Poroshell EC-C18 100 mm × 2.1 mm × 2.7 µm, or Eclipse Plus Phenyl-Hexyl 100 mm × 2.1 mm × 1.8 µm.

5. The measuring method according to claim 3, wherein The mass spectrometry conditions in the ultra-high performance liquid chromatography-tandem mass spectrometry described in step (2) are as follows: ESI positive ion mode; ion source: electrospray ionization source ESI; ion source temperature: 450°C; ionization mode: positive ion mode; spray voltage: 470 V; capillary voltage: 4000 V; gas flow rate 7 SLM; detection method: multiple reaction monitoring MRM; run time: 10 min, post-run 5 min.

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