Method for detecting residual solvent in avibactam sodium

Abstract

The invention provides a method for detecting a residual solvent in avibactam sodium. According to the method, a gas chromatographic method is adopted, a capillary column with 6% cyanopropyl phenyl-94% dimethyl polysiloxane as a stationary phase is adopted, a carrier gas is nitrogen, a detector is a hydrogen flame ionization detector, and a sample introduction mode is headspace sample introduction. The method can be used for simultaneously detecting 12 residual solvents in avibactam sodium. The method has the advantages of strong specificity, high sensitivity, good repeatability, high accuracy and good durability, and is suitable for detecting the content of the residual solvent in avibactam sodium.

Description

Technical Field

[0001] The invention relates to the field of drug analysis, and in particular to a method for detecting residual solvents in avibactam sodium. Background Art

[0002] The chemical name of Avibactam sodium is [(2S,5R)-2-carbamoyl-7-oxo-1,6-diazabicyclo[3.2.1]-octane-6-yl]sodium sulfate, and its chemical structure is as follows.

[0003]

[0004] Avibactam belongs to the diazabicyclooctanone compound and is currently the most promising non-β-lactam inhibitor. It does not have obvious antibacterial activity itself, but can inhibit type A (including ESBL and KPC) and type C β-lactamases. Therefore, when used in combination with various cephalosporins and carbapenem antibiotics, avibactam has broad-spectrum antibacterial activity, especially against Escherichia coli and Klebsiella pneumoniae containing extended-spectrum β-lactamases, Escherichia coli containing excessive AmpC enzymes, and Escherichia coli containing both AmpC and extended-spectrum β-lactamases. However, a variety of solvents are used in the preparation process of avibactam, and there are residual solvents, which brings troubles to the quality inspection of avibactam.

[0005] CN 117871719 A discloses a method for detecting five kinds of residual solvents in avibactam sodium, which uses an external standard method to calculate the content of the residual solvents. The analytical solvent is selected from dimethyl sulfoxide, dimethylformamide or sulfolane, the bonded phase in the gas chromatographic column is 6% cyanopropylphenyl-94% dimethylpolysiloxane; the chromatographic column is Agilent DB-624, 0.53mm×30m, 3.00um. The initial column temperature is 40-70°C, maintained for 3 minutes, then increased to 130°C at a rate of 30°C per minute, maintained for 2 minutes, and then increased to 200°C at a rate of 30°C and maintained for 3 minutes; the injection port temperature is 190-250°C, the detector temperature is 250°C, the carrier gas is nitrogen, and the detector is a hydrogen flame ionization detector; the column flow rate is 2.5-4.0mL per minute, and the split ratio is 10-50:1; the headspace equilibrium temperature is 90-120°C, and the equilibrium time is 20-40 minutes; the injection volume is 1mL.

[0006] CN 118130642 A discloses a method for detecting eight kinds of residual solvents in avibactam sodium, which adopts the standard curve method. With N,N-dimethylformamide aqueous solution as diluent, the program temperature rise is: the initial column temperature is 60℃ and maintained for 2-5 minutes, then the temperature is raised to 140℃ at a rate of 10℃ / min, and maintained for 3-6 minutes. The capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as the stationary liquid is used as the chromatographic column; the injection port temperature is 190℃-230℃; the detector (hydrogen flame ionization detector) temperature is 210℃-50℃; the headspace conditions are: sample equilibrium temperature: 55℃-65℃; equilibrium time: 20-40 minutes; flow rate 1.0ml / min; mobile phase: nitrogen, split ratio: 30:1.

[0007] However, the above methods are only used for the separation and detection of 5 and 8 residual solvents. It is difficult to completely separate and detect the solvents when used for more than 10 residual solvents. In order to ensure the quality of drugs and monitor residual solvents, it is necessary to develop new detection methods.

[0008] This method can detect more types of solvents and uses isopropyl acetate as the internal standard. The results of the internal standard method are more accurate.

[0009] The inventors of the present invention are based on the synthesis process of avibactam sodium. The sample may involve 12 kinds of solvents, including methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, tert-butyl alcohol, methyl tert-butyl ether, ethyl acetate, tetrahydrofuran, toluene and butyl acetate. There are many types of residual solvents, a wide boiling point, and a large difference in polarity. The existing technology cannot meet the demand, and it is necessary to develop an analytical method with high sensitivity, good repeatability, accurate results, and the ability to simultaneously detect multiple residual solvents in avibactam sodium. Summary of the invention

[0010] In order to solve the above technical problems and achieve the purpose of the present invention, the present invention provides a method for detecting residual solvents in avibactam sodium. The detection method of the present invention is for 12 kinds of solvents, including methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, tert-butyl alcohol, methyl tert-butyl ether, ethyl acetate, tetrahydrofuran, toluene and butyl acetate, which may be involved in the synthetic sample of avibactam sodium. There are many kinds of residual solvents, with wide boiling points and large polarity differences. After repeated studies, it was surprisingly found that by changing the heating rate, diluting the solvent and adopting the internal standard method, the above 12 kinds of residual solvents can be accurately separated and detected.

[0011] To achieve the purpose of the present invention, the following embodiments are provided.

[0012] In one embodiment, a method for detecting residual solvents of avibactam sodium of the present invention, wherein the residual solvent is methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, tert-butyl alcohol, methyl tert-butyl ether, ethyl acetate, tetrahydrofuran, toluene and butyl acetate, and the detection method is gas chromatography, characterized in that: the chromatographic conditions are: the column temperature of a capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as a stationary liquid: the initial temperature is 35-45°C, maintained for 4-6 minutes, and then heated to 120°C at a rate of 15-25°C / min, maintained for 1-3 minutes, and then heated to 200-230°C at a rate of 15-25°C / min, maintained for 3-10 minutes, and the diluted solvent is N-methylpyrrolidone (NMP).

[0013] In some embodiments, in the detection method of the present invention, the carrier gas is nitrogen, the split ratio is 20:1, the nitrogen flow rate is 1.0-3.0 ml / min, the hydrogen flow rate is 40 ml / min, and the air flow rate is 400 ml / min; the headspace conditions are: the headspace equilibrium temperature is 75-85°C, the equilibrium time is 25-35 min, the quantitative loop temperature is 100°C, and the transmission line temperature is 110°C.

[0014] Preferably, in the above detection method of the present invention, the starting temperature of the capillary column is 38°C to 42°C.

[0015] Preferably, in the above detection method of the present invention, the gas chromatography column has a specification of 30m×0.32mm 1.8μm.

[0016] In some embodiments, the detection method of the present invention comprises the following steps: 1) Prepare a blank solution; use N-methylpyrrolidone as the blank solution; 2) Preparation of reference solution: Prepare reference solutions containing at least 12 residual solvents with different concentrations; 3) Prepare test solution; 4) Use gas chromatography to detect the blank solution, reference solution and test solution in steps 1), 2) and 3), and calculate the content by peak area using the internal standard method.

[0017] Preferably, in the above detection method of the present invention, the concentrations of the residual solvents in step 2) are different, including 300 μg / ml methanol, 40 μg / ml acetonitrile, 72 μg / ml tetrahydrofuran, 88 μg / ml toluene, 350 μg / ml tert-butanol, 350 μg / ml ethanol, 500 μg / ml acetone, 500 μg / ml isopropanol, 500 μg / ml methyl tert-butyl ether, 500 μg / ml ethyl acetate, and 500 μg / ml butyl acetate, and 2 ml is accurately measured.

[0018] Preferably, in the detection method of the present invention, step 3) of preparing the test solution comprises dissolving the test sample with a blank solution to prepare a test solution with a concentration of 100 mg / ml, accurately adding 2 ml of the internal standard solution, heating in an 80°C water bath to dissolve, and shaking well.

[0019] Preferably, in the detection method of the present invention, the internal standard substance in step 4) is isopropyl acetate, 1 ml of blank solution contains 0.1 mg of isopropyl acetate, and 2 ml of the internal standard solution is accurately measured and placed in a 20 ml empty bottle.

[0020] Compared with the prior art, the detection method of the present invention has the following technical effects: (1) The detection method for determining residual solvents established in the present invention can simultaneously effectively separate and detect 12 kinds of residual solvents in avibactam sodium, including methanol, acetonitrile, dichloromethane, tetrahydrofuran, toluene, tert-butyl alcohol, ethanol, acetone, isopropanol, methyl tert-butyl ether, ethyl acetate, and butyl acetate. The determination method is fast and simple to operate, which greatly improves work efficiency.

[0021] (2) The present invention adopts programmed temperature rise, which can make components with different boiling points have appropriate retention in the chromatographic column, and the chromatographic peaks are evenly distributed and the peak shape is more symmetrical.

[0022] (3) The method established in the present invention can be used for qualitative or quantitative detection, with high detection sensitivity, good repeatability and low detection limit, and is suitable for routine detection of residual solvents in avibactam sodium. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : HS-GC method for the determination of avibactam sodium residual solvent blank solution chromatogram; Figure 2 : HS-GC method for the determination of avibactam sodium residual solvent reference solution chromatogram; Figure 3 : Chromatogram of the test solution of avibactam sodium residual solvent determined by HS-GC. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with embodiments. It is necessary to point out that the following embodiments are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technical personnel in the field based on the above invention content still fall within the scope of protection of the present invention.

[0025] Example 1 Detection of residual solvents of avibactam sodium: Specific steps: Step 1) Preparation of internal standard solution Take an appropriate amount of isopropyl acetate, weigh it accurately, add N-methylpyrrolidone to dissolve and dilute to make a solution containing about 0.1 mg per 1 ml as the internal standard solution. Accurately measure 2 ml of the internal standard solution, place it in a 20 ml headspace bottle, seal it, and you have it.

[0026] Step 2) Preparation of reference solution (1) Preparation of reference substance stock solution Accurately weigh about 0.75 g of methanol, about 0.1 g of acetonitrile, about 0.15 g of dichloromethane, about 0.18 g of tetrahydrofuran, about 0.22 g of toluene, about 0.875 g of tert-butanol, and about 1.25 g each of ethanol, acetone, isopropanol, methyl tert-butyl ether, ethyl acetate, and butyl acetate, place them in a 50 ml volumetric flask, add internal standard solution to dissolve and dilute to the scale, shake well, and obtain.

[0027] (2) Preparation of reference solution Accurately measure 1 ml of the reference stock solution, place it in a 50 ml volumetric flask, dilute to the mark with the internal standard solution, and shake well. Accurately measure 2 ml, place it in a 20 ml headspace bottle, and seal it.

[0028] Step 3) Preparation of test solution Take about 0.2g of this product, weigh it accurately, put it in a 20ml headspace bottle, accurately add 2ml of internal standard solution, seal it, heat it in a 80℃ water bath to dissolve, shake it well, and you have it.

[0029] Step 4) Use gas chromatography to detect the blank solution, reference solution and test solution, and calculate the content by peak area using the internal standard method.

[0030] The chromatographic conditions are: Chromatographic column: capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as stationary liquid; column temperature: starting temperature is 40℃, maintained for 5min; heated to 120℃ at a rate of 20℃ / min, maintained for 2min, and then heated to 200℃ at a rate of 20℃ / min, maintained for 5min; flow rate: 2.0ml / min: detector: FID: detector temperature: 250℃: injection port temperature: 200℃; carrier gas: nitrogen; split ratio: 20:1; hydrogen flow rate 40ml / min; air flow rate: 400ml / min; Headspace conditions: headspace equilibrium temperature: 80°C, equilibrium time: 30 min; quantitative loop temperature: 100°C; transfer line temperature: 110°C.

[0031] Example 2 Screening of injection methods The solution direct injection method was used instead of the headspace injection method for determination, and the rest was the same as in Example 1.

[0032] Since avibactam sodium is easily degraded under high temperature conditions, after experimental investigation, the direct solution injection method was used. There were many impurity peaks in the test sample, which interfered with the determination of the solvent to be tested, and the peak shape of direct injection of methanol and ethanol was poor. Therefore, the headspace injection method with fewer interfering components and less likely to contaminate the injection port, chromatographic column and detection system was selected for determination.

[0033] Example 3 Screening of calculation methods The residual solvent content was calculated using the external standard method, and a recovery test was performed, and the rest was the same as in Example 1.

[0034] Since there are too many residual solvents, a total of 12, the interactions between solvents affect vaporization, and the recovery rate of some solvents (methanol, ethanol, acetonitrile) using the external standard method exceeds 120%. Therefore, a more accurate internal standard method is selected to reduce the system error. According to the type of residual solvent and the boiling point range, isopropyl acetate with similar properties and moderate boiling point is selected as the internal standard.

[0035] The results showed that the recovery rates of 12 residual solvents in avibactam sodium were all in compliance with the requirements when isopropyl acetate was used as the internal standard.

[0036] Example 4 Screening of dilution solvents N,N-dimethylformamide (DMF) and N,N-dimethylacetamide (DMA) were used as the solvent for avibactam sodium instead of NMP, and the rest was the same as in Example 1.

[0037] Avibactam sodium is easily soluble in DMA, DMF, and NMP. In the experimental investigation, it was found that DMF and xylene cannot be completely separated and can interfere with their determination; impurities in DMA interfere with methanol determination; therefore, NMP, which has a higher boiling point and does not interfere with any solvent, was selected as the dilution solvent to meet the detection requirements of the solvent to be tested.

[0038] Example 5 Screening of initial column temperature The initial column temperature was changed, and the rest was the same as in Example 1.

[0039] Take the reference solution and the test solution in Example 1, and except that the starting column temperature is adjusted to 38°C, 42°C, and 45°C, respectively, the rest of the chromatographic column programmed temperature conditions are followed in Example 1 to investigate the separation and interference of the residual solvents to be tested at different starting temperatures.

[0040] The results show that when the initial column temperature is 45°C, the separation between chloroform and tert-butyl alcohol in the reference solution is 1.32, which is less than 1.5. When the initial column temperature is 38°C and 42°C, the separation between the residual solvents meets the requirements, so the optimal initial column temperature is 38°C to 42°C.

[0041] Example 6 Screening of headspace bottle equilibrium temperature The reference solution in Example 1 was taken and measured according to the chromatographic conditions of Example 1 using different headspace bottle equilibrium temperatures (75, 85°C) and equilibrium time of 30 min.

[0042] The results show that the higher the equilibrium temperature of the headspace bottle, the larger the peak area of ​​each component peak in the chromatogram of the reference solution; but it does not affect the test results. Therefore, the present invention selects an equilibrium temperature of 80°C and an equilibrium time of 30 minutes for determination.

[0043] Example 7 System suitability test Take the test solution and the reference solution, inject according to the chromatographic conditions of step 4 in Example 1, and record the chromatogram. Figure 1-3 As shown ( Figure 1 is the blank solution; Figure 2 It is the reference solution; Figure 3 is the test solution; Figure 2 1-methanol; 2-ethanol; 3-acetone; 4-isopropanol; 5-acetonitrile; 6-dichloromethane; 7-tert-butanol; 8-methyl tert-butyl ether; 9-ethyl acetate; 10-tetrahydrofuran; 11-isopropyl acetate (internal standard); 12-toluene; 13-butyl acetate; 14-N-methylpyrrolidone (solvent), determine the separation degree and theoretical plate number between each organic solvent peak.

[0044] Results: By Figure 2 It can be seen that the order of peaks is methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, tert-butanol, methyl tert-butyl ether, ethyl acetate, tetrahydrofuran, isopropyl acetate, toluene, and butyl acetate. The separation degrees between the peaks of each component are 13.69, 7.44, 2.70, 1.93, 4.02, 1.91, 2.80, 16.93, 3.46, 11.51, 30.15, and 13.39, respectively, all greater than 1.5; the theoretical plate numbers based on the above organic solvent peaks are all greater than 5000.

[0045] Example 8 Specificity Test Take blank solution, test solution and reference solution, and measure according to the chromatographic conditions of step 4 in Example 1. The NMP peak has no interference with the determination of methanol, acetonitrile, dichloromethane, tetrahydrofuran, toluene, tert-butanol, ethanol, acetone, isopropanol, methyl tert-butyl ether, ethyl acetate and butyl acetate; in the chromatogram of the test solution, there is no impurity peak that interferes with the determination.

[0046] Example 9 Investigation of linear relationship Accurately weigh 0.7507g of methanol, 0.1022g of acetonitrile, 0.1450g of dichloromethane, about 0.1848g of tetrahydrofuran, about 0.2135g of toluene, 1.2424g of ethanol, 1.2530g of acetone, 1.2452g of isopropanol, 1.2450g of tert-butyl alcohol, 1.2410g of methyl tert-butyl ether, 1.2473g of ethyl acetate, and 1.2504g of butyl acetate, and place them in a 50ml volumetric flask, dissolve and dilute to the mark with the internal standard solution under Example 1, shake well, and use it as a reference stock solution. Accurately measure 10ml of the reference stock solution and place it in a 50ml volumetric flask, dilute to the mark with the internal standard solution under the "Specificity" item, shake well, and use it as a linear stock solution. Accurately measure 1 ml, 2 ml, 2.5 ml, 3 ml and 5 ml of the linear stock solution respectively, place them in 25 ml volumetric flasks, dilute to the scale with the internal standard solution under the "Specificity" item, shake well, and use them as linear solutions 2 to 6. The quantitative limit solution is used as linear solution 1.

[0047] Linear regression was performed on the peak area (A) of each organic solvent against the mass concentration (C, ug / ml), and the standard curve equations and correlation coefficients of 12 residual solvents were obtained. The results showed that methanol, acetonitrile, dichloromethane, tetrahydrofuran, toluene, tert-butyl alcohol, ethanol, acetone, isopropanol, methyl tert-butyl ether, ethyl acetate and butyl acetate all showed good linear relationships with the peak area within the concentration range of their standard curves (the correlation coefficients were between 0.999 and 1.000), as shown in Table 1.

[0049]

[0050] Example 10 Precision test Accurately weigh about 0.2g of the test sample, place it in an empty bottle, accurately add 2ml of the internal standard solution, seal it, heat it in a water bath at 80℃ to dissolve it, and shake it well. Prepare 6 copies in parallel; Take 5 portions of the test solution and the reference solution prepared in Example 1, and perform continuous headspace injection according to the chromatographic conditions under Example 1 to calculate the RSD values ​​of the peak areas of 5 residual solvents. The results are as follows: in the test solution, methanol, isopropanol, acetonitrile, dichloromethane, tert-butyl alcohol, methyl tert-butyl ether, ethyl acetate, tetrahydrofuran, toluene, and butyl acetate were not detected; the detection results of ethanol and acetone had extreme differences of 0.02% and 0.001% (n=6); in the reference solution, the RSD values ​​of the peak areas of each residual solvent were less than 5.0% (n=5); the above shows that the method for determining 12 residual solvents in avibactam sodium has good precision.

[0051] Example 11 Recovery Test Weigh 0.2g of the test sample accurately, a total of 10 portions, and place them in headspace bottles. Accurately add 2ml of the recovery stock solution with a limit concentration of 80%, 100%, and 120%, seal, heat in a water bath at 80°C to dissolve, shake well, and use as the test solution with a recovery rate of 80%, 100%, and 120%. Prepare 3 portions for each concentration. Accurately add 2ml of the internal standard solution under Example 1, seal, heat in a water bath at 80°C to dissolve, shake well, and use as the background test solution. According to the chromatographic conditions under Example 1, calculate the recovery rates of methanol, acetonitrile, dichloromethane, tetrahydrofuran, toluene, tert-butyl alcohol, ethanol, acetone, isopropanol, methyl tert-butyl ether, ethyl acetate, and butyl acetate. Results: The average recovery rates of the 12 residual solvents ranged from 91.8% to 112.9% (n=9) (Table 2), all of which met the recovery rate limit requirements specified in the guidance for validation of analytical methods for drug quality standards.

[0052]

[0053] Example 12 Solution Stability Take 4 portions of the reference solution and the test solution in Example 1, place them at room temperature for 0, 10, 22, and 38 hours, and then measure them according to the chromatographic conditions in Example 1. Results: The ratio of the peak area of ​​each residual solvent in the reference solution to the peak area at 0 hour is between 98.0% and 102.0%; the detection results of each residual solvent content in the test solution are less than the limit of 10% compared with the peak area at 0 hour; indicating that the reference solution and the test solution are relatively stable within 32 hours.

[0054] Example 13 Limit of Quantitation and Limit of Detection Take the reference substance stock solution in Example 1 and make a solution of a certain concentration by stepwise dilution. Accurately measure 2 ml and place it in a 20 ml headspace bottle, seal it, and analyze it by headspace injection according to the chromatographic conditions in Example 1, and record the chromatogram. When the signal-to-noise ratio is not less than 10 and 3 (and the sensitivity meets the requirements), calculate the quantitative limit and detection limit of methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, tert-butyl alcohol, methyl tert-butyl ether, ethyl acetate, tetrahydrofuran, isopropyl acetate, toluene, and butyl acetate respectively. The results are shown in Table 3.

[0055] In summary, the detection method of the present invention can simultaneously determine 12 residual solvents in avibactam sodium by headspace-gas chromatography. The method is simple, accurate, reliable, and sensitive, can meet the detection requirements, and is suitable for the daily detection of residual solvents in avibactam sodium, thereby improving the quality of human pharmaceutical preparations and reducing the risk of clinical use of preparations.

Claims

1. A method for detecting residual solvents of avibactam sodium, wherein the residual solvent is methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, tert-butyl alcohol, methyl tert-butyl ether, ethyl acetate, tetrahydrofuran, toluene and butyl acetate, and the detection method is gas chromatography, characterized in that: The chromatographic conditions are as follows: a capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as the stationary phase; the column temperature is: the initial temperature is 35-45°C, maintained for 4-6 minutes, then increased to 120°C at a rate of 15-25°C / min, maintained for 1-3 minutes, then increased to 200-230°C at a rate of 15-25°C / min, maintained for 3-10 minutes.

2. The detection method as described in claim 1, wherein the carrier gas is nitrogen, the split ratio is 20:1, the nitrogen flow rate is 1.0-3.0 ml / min, the hydrogen flow rate is 40 ml / min, and the air flow rate is 400 ml / min.

3. The detection method as claimed in claim 1, wherein the headspace conditions are: headspace equilibrium temperature is 75-85°C, equilibrium time is 25-35 min, quantitative loop temperature is 100°C, and transmission line temperature is 110°C.

4. The detection method according to claim 1, wherein the initial temperature of the capillary column is 38°C to 42°C.

5. The detection method according to claim 1, wherein the capillary column has a specification of 30m×0.32mm 1.8μm.

6. The detection method according to claim 1, comprising the following steps: 1) Prepare a blank solution; use N-methylpyrrolidone as the blank solution; 2) Preparation of reference solution: Prepare reference solutions containing at least 12 residual solvents with different concentrations; 3) Prepare test solution; 4) Use gas chromatography to detect the blank solution, reference solution and test solution in steps 1), 2) and 3), and calculate the content by peak area using the internal standard method.

7. The detection method as claimed in claim 6, wherein the internal standard substance in step 4) is isopropyl acetate, 1 ml of blank solution contains 0.1 mg of isopropyl acetate, and 2 ml of the internal standard solution is accurately measured and placed in a 20 ml empty bottle.

8. The detection method as described in claim 6, wherein the concentrations of the residual solvents in step 2) are different, including 300 μg / ml methanol, 40 μg / ml acetonitrile, 72 μg / ml tetrahydrofuran, 88 μg / ml toluene, 350 μg / ml tert-butyl alcohol, 350 μg / ml ethanol, 500 μg / ml acetone, 500 μg / ml isopropanol, 500 μg / ml methyl tert-butyl ether, 500 μg / ml ethyl acetate, and 500 μg / ml butyl acetate, and 2 ml is accurately measured.

9. The detection method according to claim 6, wherein the step 3) preparing the test solution comprises dissolving the test sample in a blank solution to prepare a test solution with a concentration of 100 mg / ml, accurately adding 2 ml of an internal standard solution, heating in an 80°C water bath to dissolve, and shaking well.

10. The detection method according to claim 1, wherein the dilution solvent is N-methylpyrrolidone.

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