Method for detecting alkylphenol polyoxyethylene ether in microspheres

Abstract

The invention discloses a method for detecting alkylphenol ethoxylates in microspheres, and belongs to the technical field of microsphere detection methods. The method for detecting alkylphenol ethoxylates in microspheres comprises the following steps: S1, grinding to-be-detected microspheres, dissolving the to-be-detected microspheres in water, and filtering to remove water-soluble substances in the to-be-detected microspheres to obtain a filtered substance I; s2, extracting alkylphenol polyoxyethylene ether in the filtrate I by using a first solvent to obtain an extract II; s3, concentrating and drying the extract II, adding a second solvent to separate out impurity components, and filtering to remove the impurity components to obtain filtrate III. S4, adding an alkaline solution into the filtrate III, heating, and adding a third solvent to adjust the pH value, so as to obtain a to-be-detected sample solution. And S5, determining the content of alkylphenol polyoxyethylene ether in the to-be-detected sample solution by a liquid chromatography-mass spectrometry method. The detection method has the characteristics of high accuracy, good repeatability and high sensitivity, and can be used for detecting alkylphenol ethoxylates in microspheres.

Description

Technical Field

[0001] The present application relates to the technical field of microsphere detection methods, and in particular to a method for detecting alkylphenol polyoxyethylene ether in microspheres. Background Art

[0002] Embolic microspheres are widely used in the treatment of malignant tumors and vascular malformations such as liver cancer, cerebral aneurysms, and uterine fibroids. Alkylphenol polyoxyethylene ether (APEO) is an important polyoxyethylene nonionic surfactant that has the characteristics of stable properties, acid and alkali resistance, and low cost. Alkylphenol polyoxyethylene ether is usually added when preparing embolic microspheres.

[0003] APEO metabolites, such as nonylphenol, octylphenol, and APEO with low polymerization degree, have been proven to have estrogenic activity, interfere with human endocrine, and have serious effects on the growth of cancer cells and fertility. Since surfactants such as APEO are usually added when preparing embolic microspheres, and both microspheres and APEO have certain adsorption properties, these APEOs are likely to remain on the microspheres during the subsequent cleaning process, leading to safety issues.

[0004] Therefore, it is necessary to detect the APEO content in the microsphere samples. Summary of the invention

[0005] The purpose of the present application is to provide a method for detecting alkylphenol polyoxyethylene ether in microspheres, so as to detect the content of alkylphenol polyoxyethylene ether in microspheres and improve the detection accuracy.

[0006] The embodiment of the present application is implemented as follows:

[0007] The present application embodiment provides a method for detecting alkylphenol polyoxyethylene ether in microspheres, comprising:

[0008] S1. Grind the microspheres to be tested and dissolve them in water, filter and remove the water-soluble substances in the microspheres to be tested to obtain a filtrate I.

[0009] S2. Using the first solvent to extract the alkylphenol polyoxyethylene ether in the filtrate I to obtain an extract II.

[0010] S3, concentrating and drying the extract II, adding a second solvent to precipitate impurity components, filtering to remove the impurity components, and obtaining a filtrate III.

[0011] S4. Add an alkaline solution to the filtrate III and heat it, add a third solvent to adjust the pH, and obtain a sample solution to be tested.

[0012] S5. Determine the content of alkylphenol polyoxyethylene ether in the sample solution by liquid chromatography-mass spectrometry.

[0013] In the above technical solution, the composition of the microspheres is relatively complex. In addition to APEO, it also contains various salts, solvents, polymers and other substances. These substances will not only interfere with the detection of APEO, but also reduce the sensitivity of the instrument and contaminate the instrument. Therefore, before detection and analysis, the present embodiment of the application performs pre-treatment on the sample.

[0014] First, the microspheres to be tested are ground and dissolved in water. Various water-soluble salts, solvents, etc. will dissolve, while APEO has a low solubility in water and is basically insoluble in water. Therefore, the microspheres to be tested are dissolved in water and then filtered to remove the water-soluble substances in the microspheres and obtain a filtrate containing APEO.

[0015] Then, the APEO-containing filtrate is dissolved in the first solvent, and the first solvent can extract the APEO in the filtrate to obtain an extract II containing APEO. Before using the first solvent to extract APEO, the water-soluble substances in the microsphere sample are first removed by using an aqueous solvent, which can reduce the influence of these water-soluble substances on the extraction efficiency and extraction quality of the first solvent to extract APEO, and reduce the content of water-soluble impurity components in the extract II.

[0016] Since the composition of microspheres is relatively complex, they usually contain other emulsifiers with similar molecular weight and polarity to APEO, such as Span 60. These emulsifiers will be extracted by the first solvent to a greater or lesser extent. In the subsequent content detection, if the extraction liquid II is directly used for detection, the impurity components such as Span 60 in the extraction liquid II will interfere and affect the accuracy of the detection results. Therefore, in the embodiment of the present application, the extraction liquid II is concentrated and dried, a second solvent is added to precipitate the impurity components, and the impurity components are filtered to remove the impurity components to obtain a filtrate III containing a small amount of impurity components.

[0017] Then, an alkaline solution is added to the filtrate III and heated, and a third solvent is added to adjust the pH, so that the impurity components can be hydrolyzed, thereby further reducing the content of the impurity components in the sample solution to be tested and improving the detection accuracy.

[0018] Finally, liquid chromatography-mass spectrometry (LC-MS) was used to determine the content of alkylphenol polyoxyethylene ether in the test sample solution. LC-MS has high sensitivity and is suitable for the detection of APEO with low concentration, low volatility, medium polarity and other characteristics in the microsphere test sample solution, which can further improve the detection accuracy.

[0019] The above detection method has good linearity, repeatability and accuracy, and is suitable for the detection of the content of octylphenol polyoxyethylene ether in microspheres. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0021] Figure 1 This is a flow chart of the detection process of alkylphenol polyoxyethylene ether in microspheres provided in the examples of the present application. DETAILED DESCRIPTION

[0022] The embodiments of the present application will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. In the examples, if specific conditions are not specified, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0023] Alkylphenol polyoxyethylene ether (APEO) is an important polyoxyethylene nonionic surfactant with the characteristics of stable properties, acid and alkali resistance and low cost. When preparing embolic microspheres, alkylphenol polyoxyethylene ether is usually added as an emulsifier. APEO metabolites, such as nonylphenol, octylphenol and APEO with low polymerization degree, have been proven to have estrogenic activity, interfere with human endocrine, and have serious effects on the growth of human cancer cells and fertility.

[0024] However, since the microspheres have a certain adsorption capacity and APEO itself also has a strong adsorption capacity, these APEOs are likely to remain on the microspheres during the subsequent cleaning process, thus causing safety issues. Therefore, it is necessary to detect the APEO content in the microsphere samples.

[0025] Based on this, the embodiment of the present application provides a method for detecting alkylphenol polyoxyethylene ether in microspheres to detect the content of alkylphenol polyoxyethylene ether in microspheres and improve the detection accuracy.

[0026] See also Figure 1 The method for detecting alkylphenol polyoxyethylene ether in microspheres provided in the embodiment of the present application includes:

[0027] S1. Grind the microspheres to be tested and dissolve them in water, filter and remove the water-soluble substances in the microspheres to be tested to obtain a filtrate I.

[0028] S2. Using a first solvent to extract the alkylphenol polyoxyethylene ether in the filtrate I to obtain an extract II.

[0029] S3, concentrating and drying the extract II, adding a second solvent to precipitate impurity components, filtering to remove the impurity components, and obtaining a filtrate III.

[0030] S4. Add an alkaline solution to the filtrate III and heat it, add a third solvent to adjust the pH, and obtain a sample solution to be tested.

[0031] S5. Determine the content of the alkylphenol polyoxyethylene ether in the sample solution by liquid chromatography-mass spectrometry.

[0032] Since the composition of the microspheres to be tested is relatively complex, the microspheres to be tested may contain some water-soluble substances. For example, water-soluble salts or water solvents. The presence of these water-soluble substances will interfere with the detection of APEO, reduce the sensitivity of the instrument, and contaminate the instrument. Therefore, in step S1, the microspheres to be tested are ground and then immersed in water, which can increase the contact area between the microspheres to be tested and water, so that the water-soluble substances in the microspheres to be tested can be fully dissolved in water, while APEO is basically insoluble in water, so that these water-soluble substances can be separated from APEO during filtration to obtain the filtrate I containing APEO.

[0033] The content of water-soluble substances in the filter I is low. When the filter I is tested later, the interference of these water-soluble impurities on the detection of APEO content can be reduced, and the detection accuracy can be improved. In addition, the APEO in the microspheres to be tested is basically retained in the filter I and is basically not discharged with the filtration and separation of the aqueous solution. The loss of APEO in the processing process is low. When the filter I is tested later, the detection accuracy can be further improved.

[0034] In order to further reduce the content of water-soluble substances in the filtrate I and promote the full dissolution of the water-soluble substances in the microspheres to be tested in water, in some embodiments, the microspheres to be tested can be ground into powder and then ultrasonicated with water.

[0035] As an example, the microspheres to be tested can be ground into powder, and then ultrasonicated with water for more than 5 minutes.

[0036] The filtrate I obtained in step S1 may contain some other water-insoluble impurities in addition to the APEO to be detected. In step S2, the filtrate I is soaked in a first solvent, which can be used as an extractant to extract the alkylphenol polyoxyethylene ether from the filtrate I to obtain an extract II.

[0037] In some embodiments, the first solvent includes at least one of dichloromethane, methanol, or n-hexane.

[0038] As an example, the first solvent is dichloromethane.

[0039] As an example, the first solvent includes a mixed solution of methanol and dichloromethane.

[0040] As an example, the first solvent includes a mixed solution of methanol and n-hexane.

[0041] As an example, the first solvent includes a mixed solution of methanol, dichloromethane and n-hexane.

[0042] Furthermore, in some embodiments, the mass proportion of methanol in the first solvent is 10% to 30%.

[0043] As an example, the mass proportion of methanol in the first solvent may be in a range of 10%, 10%, 10%, 10%, 10%, or any two thereof.

[0044] Furthermore, in order to improve the extraction quality, increase the APEO content in the extract II, and reduce the loss of APEO during the treatment process, in some embodiments, the first solvent can be poured into the filtrate I, and then the filtrate I can be shaken to accelerate the dissolution of APEO in the filtrate I in the first solvent.

[0045] As an example, the first solvent is poured into the filtrate I and shaken or agitated for more than 5 minutes.

[0046] As an example, a mixture of dichloromethane and methanol is poured into the filtrate I, and the mixture is shaken or vortexed for 5 minutes, and then allowed to stand for extraction, and the lower layer of solution is taken as the filtrate II.

[0047] Furthermore, in some embodiments, in step S2, the volume ratio of the mass of the filtrate I to the first solvent can be 1:1-5.

[0048] The filtrate I obtained in step S1 contains, in addition to the APEO to be detected, some other water-insoluble impurities. These impurities may include some emulsifiers with similar molecular weight and polarity to APEO, such as Span 60. When the filtrate I is soaked in the first solvent, these emulsifiers such as Span 60 with similar molecular weight and polarity to APEO will be extracted into the extraction liquid II together with APEO to a greater or lesser extent. If the extraction liquid II is directly tested, the impurity components such as Span 60 in the extraction liquid II will interfere with the detection of the APEO content and reduce the detection accuracy.

[0049] Therefore, in the detection method provided in the embodiment of the present application, after obtaining the extract II in step S2, the extract II is concentrated and dried, a second solvent is added to precipitate the impurity components, and the impurity components are filtered to remove the impurity components to obtain the filtrate III.

[0050] The solubility of the impurity components in the extract II and APEO in the second solvent is different to a certain extent, and at least part of the impurity components in the extract II can be separated and removed by the second solvent. Compared with the content of the impurity components in the extract II, the content of the impurity components in the filtrate III obtained in step S3 is lower.

[0051] After the extract II is concentrated and dried, it is then immersed in the second solvent, which can remove the first solvent in the extract II and avoid affecting the separation effect of the second solvent on impurity components such as Span 60 due to excessive residues of the first solvent.

[0052] In some embodiments, the second solvent includes at least one of ethanol, isopropanol, or methanol.

[0053] As an example, the second solvent is methanol.

[0054] As an example, the second solvent is isopropyl alcohol.

[0055] As an example, the second solvent is ethanol. Compared with methanol or isopropanol, the solubility difference of impurity components such as APEO and Span in ethanol is greater, and the effect of removing impurity components such as Span 60 by using the second solvent is better.

[0056] As an example, the second solvent includes a mixed solution of methanol and ethanol.

[0057] As an example, the second solvent includes a mixed solution of ethanol and isopropanol.

[0058] As an example, the second solvent includes a mixture of methanol and isopropanol.

[0059] As an example, the second solvent includes a mixed solution of methanol, ethanol and isopropanol.

[0060] In step S3, by utilizing the difference in solubility of APEO and impurity components such as Span 60 in the second solvent, a portion of the impurity components can be removed, but it is still possible that some impurity components such as Span 60 will dissolve in the second solvent, and the filtrate III obtained in step S3 will still inevitably contain a small amount of impurity components such as Span 60. If filtrate III is directly used for APEO detection, impurity components such as Span 60 in filtrate III may still interfere with the APEO content detection, which will reduce the detection accuracy.

[0061] Therefore, in the detection method provided in the embodiment of the present application, after re-dissolving with the second solvent in step S3, a certain alkaline solution is added to the filtrate III obtained in step S3, and a heating treatment is performed to hydrolyze the impurity components such as Span 60 in the filtrate III, and then a third solvent is added to adjust the pH to obtain a liquid to be tested. The hydrolysis products of the impurity components such as Span 60 have a low degree of interference with APEO in the subsequent detection process, and basically do not affect the detection accuracy of APEO.

[0062] In some possible embodiments, in step S4, the alkaline solution includes a sodium hydroxide solution.

[0063] Furthermore, in some embodiments, the concentration of the alkaline solution is 0.1-1 mol / L.

[0064] If the concentration of the alkaline solution is too low, the degradation degree of impurity components such as Span 60 is low, which will cause the subsequent measured APEO content results to be too high.

[0065] If the concentration of the alkaline solution is too high, APEO may degrade, resulting in a lower APEO content result in the subsequent measurement.

[0066] As an example, the concentration of the alkaline solution is in the range of one or between any two of 0.1 mol / L, 0.2 mol / L, 0.4 mol / L, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L or 1 mol / L.

[0067] Furthermore, in some embodiments, in step S4, an alkaline solution can be added to the filtrate III and heated to 55°C to 65°C.

[0068] As an example, the heating time may be one of 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C or 65°C, or a range between any two thereof.

[0069] Furthermore, in some embodiments, an alkaline solution can be added to the filtrate III, heated to 55° C. to 65° C., and then stopped after heating for 1 to 3 hours, and a third solvent can be added.

[0070] As an example, the heating time may be one of 1 h, 1.5 h, 2 h, 2.5 h, 3 h or 3.5 h, or a range between any two thereof.

[0071] In some embodiments, in step S4, the third solvent may include a formic acid solution.

[0072] Furthermore, in some embodiments, in step S4, a third solvent is added to adjust the pH to 6-8.

[0073] As an example, a third solvent is added to adjust the pH to one of 6, 6.5, 7, 7.5 or 8, or a range between any two thereof.

[0074] Before testing, the microspheres to be tested are pre-treated by the above method, and the obtained filtrate to be tested contains only a low content of impurity components such as Span 60, or even no impurity components such as Span 60, and the loss of APEO during the pretreatment process is low, which can improve the detection accuracy of APEO.

[0075] In some embodiments, the content of impurity components such as Span 60 in the sample solution can be determined by gas chromatography (GC).

[0076] As an example, the detection conditions for determining the content of impurity components by gas chromatography include:

[0077] The chromatographic column HP-5 was used, the flow rate was set to 1-1.5 ml / min, the gradient heating mode was used, and the FID detector was used for quantitative testing.

[0078] Since APEO is a medium polar, low volatile compound, and the APEO content in the microspheres is low, only a few micrograms or even a few tenths of a microgram, if high performance liquid chromatography (HPLC) or gas chromatography-mass spectrometry (GC-MS) is used for detection, it will lead to large deviations in the test results.

[0079] In step S5, the content of alkylphenol polyoxyethylene ether in the sample solution is determined by liquid chromatography-mass spectrometry (LC-MS), which can further improve the detection accuracy.

[0080] In some embodiments, the detection conditions of liquid chromatography-mass spectrometry (LC-MS) include:

[0081] Column: Waters XBridges C18;

[0082] Column temperature: 30-40°C;

[0083] Mobile phase: a mixture of ammonium formate aqueous solution and acetonitrile, the volume ratio of ammonium formate aqueous solution to acetonitrile is 1:3-5, the ammonium formate aqueous solution contains 0.1%-1% formic acid and the concentration is 5-10mmol / L; the flow rate is 0.5-1mL / min, isocratic elution; the injection volume is 2-5μL;

[0084] Mass spectrometry conditions: electrospray ion source, selective scanning mode.

[0085] As an example, the column temperature may be one of 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, or a range between any two thereof.

[0086] As an example, the volume ratio of the aqueous ammonium formate solution to acetonitrile may be 1:3, 1:4, 1:5, or a range between any two of them.

[0087] As an example, the concentration of the aqueous ammonium formate solution may be in the range of 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L or 10 mmol / L, or any two thereof.

[0088] The aqueous solution of ammonium formate contains 0.1% to 1% by mass of formic acid. The aqueous solution of ammonium formate usually contains formic acid because ammonium formate partially ionizes into formate ions (HCOO-) and ammonium ions (NH+) in water, and the formate ions partially hydrolyze to form formic acid (HCOOH). Specifically, ammonium formate partially ionizes into formate ions and ammonium ions in water, and the formate ions undergo a weaker hydrolysis reaction to form formic acid.

[0089] As an example, the flow rate of the mobile phase may be one of 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min or 1 mL / min, or a range between any two thereof.

[0090] As an example, when performing LC-MS detection, the injection volume is in the range of one or any two of 2 μL, 3 μL, 4 μL or 5 μL.

[0091] Furthermore, when the content of APEO in the sample solution to be tested is determined by liquid chromatography-mass spectrometry (LC-MS), standard linear solutions of APEO with different concentrations can be prepared first, and then the APEO content in the standard solution can be determined according to the above LC-MS test method, and a standard curve can be drawn based on the corresponding results of the measured peak area and concentration. When the sample solution to be tested is subsequently determined by LC-MS, the result of the measured peak area is substituted into the standard curve to calculate the content of APEO in the sample solution to be tested.

[0092] According to the above detection method, since the composition of the microspheres is relatively complex, before the APEO content in the microspheres is detected by the LC-MS method, the microspheres are first pre-treated by immersion in an aqueous solution, extraction with a first solvent, redissolution with a second solvent, and hydrolysis with an alkaline solution, etc., which can reduce the content of impurities in the sample solution to be tested, reduce the interference of impurities on the detection of APEO content, and improve the detection accuracy. In addition, according to the detection method provided in the embodiment of the present application, the detection content of APEO in the microspheres is detected with high detection repeatability and relatively stable detection results.

[0093] The following is a further detailed description of the method for detecting the content of alkylphenol polyoxyethylene ether in the microspheres of the present application in conjunction with the examples.

[0094] Example 1

[0095] Example 1 provides a method for testing the APEO content in microspheres, comprising:

[0096] (1) Obtaining the sample solution to be tested

[0097] Take 1g of microsphere sample and grind it into powder, add water and ultrasonic for 5min to dissolve salts, solvents and other water-soluble substances, and filter to obtain filtrate I. Then add 2mL of the first solvent (the first solvent is a mixture of dichloromethane and methanol, with a methanol content of 10%) to the filtrate I and shake it for 5min, let it stand for extraction and take the lower layer solution to obtain extract II. Concentrate and dry the extract II, then add 2mL of the second solvent (the second solvent is ethanol) to dissolve the precipitated Span 60 and filter to obtain filtrate III. The filtrate is dried and weighed. Take filtrate III and add 1mL of alkaline solution (the alkaline solution is 0.5mol / L NaOH solution) and heat it for 1h, then add the third solvent (the third solvent is formic acid solution) to adjust the pH to 6-8, and filter to obtain the sample solution to be tested.

[0098] (2) Content detection

[0099] ①LC-MS (liquid chromatography-mass spectrometry) detection conditions are:

[0100] Chromatographic column: Waters XBridges C18 (4.6×150 mm, 3.5 μm);

[0101] Column temperature: 35°C;

[0102] Mobile phase: isocratic elution of ammonium formate aqueous solution containing 0.1% formic acid and a concentration of 10 mmol / L-acetonitrile, the volume ratio of ammonium formate aqueous solution to acetonitrile is 1:4. The flow rate is 0.5 mL / min, and the injection volume is 2 μL;

[0103] The mass spectrometry conditions were: electrospray ionization source (ESI source), selective scanning mode (SIM).

[0104] ②Standard curve preparation:

[0105] Different concentrations of APEO linear solutions were prepared, with the concentrations being 0.1 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 2 μg / mL, and 5 μg / mL. The test was performed according to the above LC-MS determination conditions, and a standard curve was drawn. The results are shown in Table 1.

[0106] Table 1

[0107]

[0108]

[0109] ③ Detection method of Span 60 content in the sample solution:

[0110] GC (gas chromatography) detection conditions: using a chromatographic column HP-5 (30m×0.32mm×0.25μm), setting the flow rate to 1-1.5ml / min, gradient heating mode, and FID detector for quantitative testing.

[0111] ④ Detection of APEO and Span 60 content in the sample liquid:

[0112] Take 1 mL of the sample solution treated in step (1) and inject it, and perform APEO content test according to the detection conditions of step ① and step ② above. Perform Span 60 content test according to the GC method of step ③.

[0113] The test results are shown in Table 2.

[0114] Example 2

[0115] Example 2 provides a method for testing the APEO content in microspheres, which differs from Example 1 in that:

[0116] In step (1), the first solvent was replaced with 2 mL of a mixture of n-hexane and methanol (methanol content was 10%). The test results are shown in Table 2.

[0117] Example 3

[0118] Example 3 provides a method for testing the APEO content in microspheres, which differs from Example 1 in that:

[0119] In step (1), the first solvent was replaced with 2 mL of dichloromethane. The test results are shown in Table 2.

[0120] Example 4

[0121] Example 4 provides a method for testing the APEO content in microspheres, which differs from Example 1 in that:

[0122] In step (1), the second solvent was replaced with isopropanol. The test results are shown in Table 2.

[0123] Example 5

[0124] Example 5 provides a method for testing the APEO content in microspheres, which differs from Example 1 in that:

[0125] In step (1), the second solvent was replaced with methanol. The test results are shown in Table 2.

[0126] Example 6

[0127] Example 6 provides a method for testing the APEO content in microspheres, which differs from Example 1 in that:

[0128] In step (1), the alkaline solution was replaced with a 0.1 mol / L NaOH solution. The test results are shown in Table 2.

[0129] Example 7

[0130] Example 7 provides a method for testing the APEO content in microspheres, which differs from Example 1 in that:

[0131] In step (1), the alkaline solution was replaced with a 1.0 mol / L NaOH solution. The test results are shown in Table 2.

[0132] Example 8

[0133] Example 8 provides a method for testing the APEO content in microspheres, which differs from Example 1 in that:

[0134] In step (1), the alkaline solution was replaced with a 0.01 mol / L NaOH solution. The test results are shown in Table 2.

[0135] Example 9

[0136] Example 9 provides a method for testing the APEO content in microspheres, which differs from Example 1 in that:

[0137] In step (1), the alkaline solution was replaced with a 10 mol / L NaOH solution. The test results are shown in Table 2.

[0138] Table 2

[0139]

[0140] Result analysis:

[0141] In Table 2, it can be seen from the comparison of Examples 1 to 3 that the APEO detection content and the weight of the filtrate in Example 1 are slightly greater than those in Examples 2 and 3, indicating that the extraction effect of the dichloromethane and methanol mixture as the first solvent is better, and more APEO and Span 60 can be extracted from the microspheres. The subsequent concentration data detected has a smaller deviation from the actual concentration, which can more truly reflect the APEO concentration in the microspheres. As the first solvent, the extraction effect of the dichloromethane and methanol mixture is better than the extraction effect of the n-hexane and methanol mixture, and better than the extraction effect of the simple dichloromethane solution, and can more completely extract the residual APEO in the microspheres.

[0142] In Table 2, by comparing Example 1, Example 4 and Example 5, it can be seen that Example 1 uses ethanol as the second solvent for redissolution and precipitation, and the weight of the precipitated filtrate Span 60 after drying is the heaviest, indicating that the effect of using ethanol to precipitate Span 60 is better, Span 60 can be better removed, the influence of Span 60 on the test results can be reduced, the concentration of APEO in the microspheres can be more realistically reflected, and the detection accuracy can be improved. As the second solvent, ethanol has a better precipitation effect on Span 60 than isopropanol and methanol.

[0143] In Table 2, comparing Example 1 with Examples 6-7, Span 60 was not detected in the test sample solutions provided by Example 1, Example 6 and Example 7, and the concentrations of APEO detected in Example 1, Example 6 and Example 7 were not much different and were basically consistent.

[0144] In Table 2, by comparing Example 1 and Example 8, it can be seen that the detection concentration of APEO in Example 8 is higher than that in Example 1, and the weight of the filtrate is consistent with that in Example 1. In Example 8, 72.5 μg / kg of Span 60 is detected, while Span 60 is not detected in the test sample solution of Example 1. This indicates that when the concentration of the alkaline solution is 0.01 mol / L in Example 8, Span 60 is not fully degraded, resulting in a certain amount of Span 60 in the test sample solution. The residual amount of Span 60 in the test sample solution is too high, which will lead to a high detection concentration of APEO.

[0145] In Table 2, it can be seen from the comparison between Example 1 and Example 9 that the test concentration of APEO in Example 9 is much lower than that in Example 1, and Span 60 is not detected in the test sample solutions provided in Example 9 and Example 1, and the weights of the filtrates obtained by filtration in Example 1 and Example 9 are consistent. This indicates that when the concentration of the alkaline solution is 1 mol / L in Example 9, APEO will degrade, resulting in a lower concentration of APEO measured in Example 9.

[0146] From a comprehensive comparison of Example 1 and Examples 6 to 9, it can be seen that the embodiment of the present application uses an alkaline solution with a concentration of 0.1 mol / L to 1 mol / L to redissolve the extract concentrate after the first solvent extraction, which can further remove Span 60 in the extract, reduce the content of Span 60 in the test liquid, and improve the detection accuracy of APEO. If the concentration of the alkaline solution is lower than 0.1 mol / L, Span 60 in the extract concentrate cannot be fully hydrolyzed, resulting in a high detection concentration of APEO. If the concentration of the alkaline solution is higher than 1 mol / L, APEO will degrade, resulting in a low detection concentration of APEO.

[0147] Verification experiment

[0148] 1. Reference substance: Take 1.0 μg / mL APEO linear solution from the linear solution.

[0149] 2. Repeatability: Take 1.0g microsphere sample (APEO concentration in the microsphere sample is about 0.051μg / mL) and add 1.0mL reference solution, and prepare 6 parallel copies.

[0150] 3. Accuracy: Take 1.0g of microsphere sample (APEO concentration in the microsphere sample is about 0.051μg / mL) and add 1.0mL of APEO linear solution with concentrations of 0.1μg / mL, 1.0μg / mL, and 5μg / mL, and prepare 3 copies of each concentration in parallel.

[0151] 4. Sample processing: The above microsphere samples were processed according to the processing method of step (1) in Example 1, and the APEO content was tested according to the LC-MS method of step (2) in Example 1.

[0152] The repeatability test results are shown in Table 3. The accuracy test results are shown in Table 4.

[0153] Table 3

[0154]

[0155] Table 4

[0156]

[0157]

[0158] Result analysis:

[0159] In Table 3, six tests were performed, and the APEO concentrations in each test were not much different, indicating that the detection method provided in the embodiment of the present application has good detection repeatability for detecting the content of alkylphenol polyoxyethylene ether in microspheres, and the detection results are relatively stable.

[0160] As can be seen from Table 4, according to the detection method provided in the embodiment of the present application, the APEO content of microspheres with various APEO concentrations was detected multiple times, and the detection results of the microspheres of each concentration had good stability, indicating that according to the detection method provided in the embodiment of the present application, the content of alkylphenol polyoxyethylene ether in the microspheres is detected, and the detection result is consistent with the actual concentration, with high accuracy, high detection accuracy and high sensitivity.

[0161] In summary, since the composition of the microspheres is relatively complex, according to the detection method provided in the embodiment of the present application, before the APEO content in the microspheres is detected by the LC-MS method, the microspheres to be tested are sequentially soaked in an aqueous solution to remove water-soluble substances, the first solvent is used to extract APEO, the second solvent is used to dissolve and precipitate impurity components such as Span 60, and the alkaline solution is used to hydrolyze impurity components such as Span 60. This can reduce the content of impurities in the sample solution to be tested, reduce the interference of impurities on the detection of APEO content, and improve the detection accuracy. In addition, according to the detection method provided in the embodiment of the present application, the detection repeatability is high when detecting the content of APEO in the microspheres.

[0162] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for detecting alkylphenol polyoxyethylene ether in microspheres, characterized in that: The detection method comprises: S1, grinding the microspheres to be tested and dissolving them in water, filtering to remove water-soluble substances in the microspheres to be tested, and obtaining a filtrate I; S2, extracting the alkylphenol polyoxyethylene ether in the filtrate I with a first solvent to obtain an extract II; S3, concentrating and drying the extract II, adding a second solvent to precipitate impurity components, filtering to remove the impurity components, and obtaining a filtrate III; S4, adding an alkaline solution to the filtrate III and heating it, adding a third solvent to adjust the pH, and obtaining a sample solution to be tested; S5. Determine the content of the alkylphenol polyoxyethylene ether in the test sample solution by liquid chromatography-mass spectrometry.

2. The detection method according to claim 1, characterized in that: In step S2, the first solvent includes at least one of dichloromethane, methanol or n-hexane; Optionally, the first solvent includes the methanol and the dichloromethane, or the methanol and the n-hexane, or a mixture of the methanol, the dichloromethane and the n-hexane; optionally, the mass proportion of the methanol in the first solvent is 10% to 30%.

3. The detection method according to claim 1, characterized in that: In step S3, the second solvent includes at least one of ethanol, isopropanol or methanol; Optionally, the second solvent is ethanol.

4. The detection method according to claim 1, characterized in that: In step S4, the alkaline solution includes a sodium hydroxide solution; and / or, the concentration of the alkaline solution is 0.1 to 1 mol / L; And / or, the heating temperature in step S4 is 55°C to 65°C.

5. The detection method according to claim 4, characterized in that: In step S4, the third solvent includes a formic acid solution; And / or, adding the third solvent to adjust the pH to 6-8.

6. The detection method according to claim 1, characterized in that: In step S5, the detection conditions of the liquid chromatography-mass spectrometry method include: Column: Waters XBridges C18; Column temperature: 30-40°C; Mobile phase: a mixture of ammonium formate aqueous solution and acetonitrile, wherein the volume ratio of the ammonium formate aqueous solution to the acetonitrile is 1:3-5, and the ammonium formate aqueous solution contains 0.1%-1% formic acid and the concentration is 5-10 mmol / L; the flow rate is 0.5-1 mL / min, isocratic elution; the injection volume is 2-5 μL; Mass spectrometry conditions: electrospray ion source, selective scanning mode.

7. The detection method according to claim 6, characterized in that: In step S5, after the standard solutions of alkylphenol polyoxyethylene ether with different concentrations are measured by the liquid chromatography-mass spectrometry method, a standard curve of peak area and concentration is drawn; after the sample solution is measured by the liquid chromatography-mass spectrometry method, the content of the alkylphenol polyoxyethylene ether is calculated using the standard curve.

8. The detection method according to claim 1, characterized in that: The impurity components include Span 60; Optionally, in step S5, the content of the impurity component in the sample liquid is determined by gas chromatography.

9. The detection method according to claim 8, characterized in that: The detection conditions for determining the content of the impurity components by gas chromatography include: The chromatographic column HP-5 was used, the flow rate was set to 1-1.5 ml / min, the gradient heating mode was used, and the FID detector was used for quantitative testing.

10. The detection method according to claim 1, characterized in that: In step S1, the microspheres to be tested are ground and dissolved in water, and ultrasonicated for more than 5 minutes.

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