Method for detecting related substances of Alalisgroup

Abstract

The invention discloses a method for detecting related substances of alalisset, which comprises the following steps: carrying out gradient elution by using octadecylsilane chemically bonded silica as a chromatographic column filler, acetonitrile-water mixed liquor as a diluent, mixed solution of acid and alkali containing an organic solvent as a mobile phase A and the organic solvent as a mobile phase B, and detecting the related substances of the alalisset. According to the method, the related substances of the alasset are separated, chromatograms are recorded, calculation is performed according to a main component self-contrast method, the impurities of the alasset are fully revealed, the related substances of the alasset can be rapidly detected, the safety of products is improved, and the method is scientific, reliable and capable of controlling the related substances of the alasset.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical analysis, and relates to the detection of elastomeric related substances, and in particular to a detection method for elastomeric related substances, which can effectively detect the related substances in elastomeric and supplement the detection method in the pharmaceutical research field of elastomeric. Background Art

[0002] Elacestrant is an experimental selective estrogen receptor degrader (SERD) that was approved for marketing by the U.S. Food and Drug Administration (FDA) on January 27, 2023. It is the world's first oral SERD drug on the market. The molecular formula of Elacestrant is: C 30 H 38 N2O2, molecular weight: 458.65, its CAS registration number: 722533-56-4.

[0003] The composition of the elastostat API is clear, mainly containing elastostat and 8 related impurities. It is very important to completely separate these 9 substances. It is of great significance to monitor the quality of elastostat by detecting the related substances of elastostat, and then ensure the safety of the majority of drug users. At present, there are few detection methods for elastostat. There is no pharmacopoeia that includes the analysis method of related substances of elastostat, and there is no literature that provides a high-performance liquid chromatography method for the determination of related substances of elastostat. Summary of the invention

[0004] The technical problem to be solved by the present invention is that there are few methods for detecting elastostat. Currently, no pharmacopoeia has included an analysis method for related substances of elastostat, and no literature provides a high-performance liquid chromatography method for determining related substances of elastostat. Therefore, the present invention provides a method for detecting related substances of elastostat. The present invention can effectively detect related substances in elastostat through system applicability, specificity, detection limit, quantification limit, linearity, precision, accuracy, solution stability, etc., and supplements the detection method in the field of elastostat pharmaceutical research.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for detecting elastomeric related substances, the detection method comprising the following steps:

[0007] 1) Prepare the test solution;

[0008] 2) preparing a control solution;

[0009] 3) Prepare separation solution;

[0010] 4) taking the separation solution, the control solution and the test solution respectively, and detecting them by high performance liquid analysis, using octadecylsilane bonded silica gel as the chromatographic column filler, using a mixed solution of acetonitrile-water as the diluent, using a mixed solution of an acid and a base containing an organic solvent as the mobile phase A and an organic solvent as the mobile phase B, performing gradient elution, separating the elastomeric related substances, recording the chromatogram, and calculating according to the principal component self-reference method;

[0011] The elastostat-related substances include substances represented by formula I, substances represented by formula II, substances represented by formula III, and substances represented by formula IV;

[0012] Among them, the substance shown in formula I:

[0013] The substance shown in formula II:

[0014] The substance shown in formula III:

[0015] The substance shown in formula IV:

[0016] As a preferred embodiment of the present invention, the separation solution is prepared as follows: the substance shown in formula I, the substance shown in formula II, the substance shown in formula III and the substance shown in formula IV are respectively taken to prepare a stock solution containing the substance shown in formula I with a concentration of 1 mg / mL, a stock solution containing the substance shown in formula II with a concentration of 1 mg / mL, a stock solution containing the substance shown in formula III with a concentration of 1 mg / mL and a stock solution containing the substance shown in formula IV with a concentration of 1 mg / mL; the stock solution containing the substance shown in formula I, the stock solution containing the substance shown in formula II, the stock solution containing the substance shown in formula III and the stock solution containing the substance shown in formula IV are respectively taken, mixed and shaken to obtain an impurity mixed stock solution with a concentration of 20 μg / mL; the elastostat reference substance and the impurity mixed stock solution are taken and shaken to obtain a separation solution, wherein the concentration of elastostat is 1 mg / mL and the impurity concentration is 1 μg / mL.

[0017] As a preferred embodiment of the present invention, in the mixed solution containing acid and alkali, the acid solution is a polyacidic solution, and the alkali solution is a polyacidic alkaline solution.

[0018] As a preferred embodiment of the present invention, the acid solution is a phosphoric acid solution with a volume concentration of 0.09%-0.11%, the alkali solution is a triethylamine solution, and the pH value of the mixed solution is 2.0-4.0.

[0019] As a preferred embodiment of the present invention, the organic solvent is a cyanide solvent or an alcohol solvent.

[0020] As a preferred embodiment of the present invention, the cyanide solvent is acetonitrile, and the alcohol solvent is methanol.

[0021] As a preferred embodiment of the present invention, the concentration of the test solution is 0.5-2.0 mg / mL.

[0022] As a preferred embodiment of the present invention, the separation solution is obtained by mixing and diluting different reference substance solutions.

[0023] As a preferred embodiment of the present invention, during detection, the detection wavelength is 210-230 nm, the flow rate is 0.9-1.1 mL / min, and the column temperature is 20-40°C.

[0024] As a preferred embodiment of the present invention, the gradient elution is: 0 min, mobile phase A 100%, mobile phase B 0%; 1 min, mobile phase A 100%, mobile phase B 0%; 15 min, mobile phase A 30%, mobile phase B 70%; 20 min, mobile phase A 30%, mobile phase B 70%; 22 min, mobile phase A 100%, mobile phase B 0%; 30 min, mobile phase A 100%, mobile phase B 0%.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1) The present invention can effectively detect related substances in elastomeric via system applicability, specificity, detection limit, quantification limit, linearity, precision, accuracy, solution stability, etc., and supplements the detection method in the field of elastomeric pharmaceutical research.

[0027] 2) The method for determining elastostat related substances of the present invention selects octadecylsilane bonded silica gel and a mixed solution of acid and base containing an organic solvent as mobile phase A and an organic solvent as mobile phase B to facilitate the separation and elution of elastostat related substances, fully reveals the impurities of elastostat, can quickly detect elastostat related substances and improve product safety. The method is scientific and reliable, and can control elastostat related substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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 will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0029] Figure 1 This is the HPLC spectrum of the resolution of elastostat.

[0030] Figure 2 This is the HPLC spectrum of the test results of elastomeric samples.

[0031] Figure 3 This is the HPLC spectrum of the limit of quantitation of elastostat.

[0032] Figure 4 This is the HPLC profile of the detection limit of elastostat.

[0033] In the figure, 1 is a substance represented by formula I; 2 is a substance represented by formula II; 3 is elastomeric; 4 is a substance represented by formula III; and 5 is a substance represented by formula IV. DETAILED DESCRIPTION

[0034] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0035] The instruments (see Table 1), reagents (see Table 2), samples and reference substances (see Table 3) used in the present invention are as follows:

[0036] Table 1. Instruments

[0037]

[0038] Table 2. Reagents

[0039] name level Manufacturer Acetonitrile HPLC Shanghai Xingke High Purity Solvent Co., Ltd. water / Hangzhou Wahaha Group Co., Ltd. Triethylamine AR Sinopharm Chemical Reagent Co., Ltd. Phosphoric acid AR Sinopharm Chemical Reagent Co., Ltd.

[0040] Table 3. Samples and controls

[0041]

[0042] Example 1

[0043] Detection of elastomeric related substances:

[0044] 1) Chromatographic conditions:

[0045] Chromatographic column: MicroPuilte Perfect T3 (4.6mm×250mm, 5μm) or chromatographic column with equivalent performance.

[0046] Ghost peak trapping column: Welch Ghost-Buster Column (4.6mm×50mm) or chromatographic column with equivalent performance.

[0047] Wavelength: 220 nm, flow rate: 1.0 mL / min, injection volume: 5 μL, column temperature: 35°C, mobile phase A: 0.1% triethylamine solution (adjust pH to 3.0 with phosphoric acid)-acetonitrile (90:10) (v / v), mobile phase B: acetonitrile, diluent: acetonitrile-water (10:90) (v / v).

[0048] Gradient elution was performed as shown in Table 4.

[0049] Table 4. Elution program

[0050] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 1 100 0 15 30 70 20 30 70 22 100 0 30 100 0

[0051] Solution preparation

[0052] Blank / diluent: acetonitrile-water (10:90).

[0053] Test sample solution: Take about 20 mg of the test sample and place it in a 20 mL volumetric flask. Add diluent to dissolve and dilute to the scale, and shake well (concentration: 1 mg / mL).

[0054] Control solution: Accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, dilute it to the mark with diluent, and shake well; then accurately transfer 1 mL of the solution to a 10 mL volumetric flask, dilute it to the mark with diluent, and shake well (concentration: 1 μg / mL).

[0055] Resolution solution:

[0056] (1) HNA001-CPZ1 stock solution: Take about 20 mg of HNA001-CPZ1 reference substance, accurately weigh it, place it in a 20 mL volumetric flask, add about 2 / 3 of the volume of the flask with diluent to dissolve it, dilute it to the scale with diluent, and shake well (concentration: 1 mg / mL).

[0057] (2) HNA001-207Z2 stock solution: Take about 20 mg of HNA001-207Z2 reference substance, accurately weigh it, place it in a 20 mL volumetric flask, add about 2 mL of acetonitrile to dissolve it, dilute it to the scale with diluent, and shake well (concentration: 1 mg / mL).

[0058] (3) HNA001-207Z4 stock solution: Take about 20 mg of HNA001-207Z4 reference substance, accurately weigh it, place it in a 20 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, and shake well (concentration: 1 mg / mL).

[0059] (4) D-(+)-DBTA stock solution: Take about 20 mg of D-(+)-DBTA reference substance, accurately weigh it, place it in a 20 mL volumetric flask, add about 2 / 3 of the volume of the flask with diluent to dissolve it, dilute it to the scale with diluent, and shake well (concentration: 1 mg / mL).

[0060] (5) Impurity mixed stock solution: Accurately measure 2 mL of each of the above stock solutions (1)-(4) and place it in a 100 mL volumetric flask. Dilute to the mark with diluent and shake well (concentration: 20 μg / mL, equivalent to 2% of the concentration of the test solution).

[0061] (6) Separation solution: Take about 20 mg of HNA001 reference substance, accurately weigh it, place it in a 20 mL volumetric flask, add about 2 / 3 of the volume of the flask with diluent to dissolve it, accurately measure 1 mL of (5) impurity mixed stock solution, place it in the same volumetric flask, dilute to the scale with diluent, and shake well (HNA001 concentration: 1 mg / mL, impurity concentration: 1 μg / mL, equivalent to 0.1% of the test solution concentration).

[0062] Detection

[0063] Take the separation solution, control solution and test solution respectively, inject them under the above chromatographic conditions, record the chromatogram, and calculate according to the principal component self-reference method.

[0064] Experimental results: Separation solution liquid chromatogram see Figure 1 And Table 6, the liquid chromatogram of the test solution is shown in Figure 2 and Table 7. The detection results of related substances of elastomeric are shown in Table 5.

[0065] Table 5. Detection results of related substances of elastostat

[0066]

[0067] The experimental results show that in the separation solution chromatogram, the theoretical plate number of the main peak is not less than 5000, the separation degree between the main peak and the nearest known impurity is not less than 1.5, and the content of each impurity can be effectively detected in the two batches of test solution.

[0068] Table 6. Separation results

[0069]

[0070] Table 7. Test results of the test products

[0071]

[0072]

[0073] Example 2

[0074] Methodological validation of related substances of elastostat:

[0075] System suitability and specificity

[0076] The separation solution and the reference solution were prepared according to the solution of Example 1, and the sample was injected using the chromatographic conditions of Example 1. The chromatogram was recorded. The results are shown in Table 8 and the separation diagram is shown in Table 8. Figure 2 .

[0077] Table 8. Results of system suitability and specificity test of elastomeric

[0078]

[0079] The experimental results show that: in the reference solution, the RSD of the peak area of ​​the main peak for 6 consecutive injections is 1.84%, not more than 5.0%; the RSD of the retention time is 0.03%, not more than 1.0%; the blank solution has no interference on the main peak and the impurity peaks; the retention time of each impurity positioning solution in the separation test is basically consistent with the retention time of the main peak and each known impurity peak in the separation solution, and in the separation solution, the separation between the main peak and the adjacent impurity peak, and the separation between each known impurity peak and the nearest peak are not less than 1.5, and the theoretical plate number of the main peak is not less than 5000.

[0080] Repeatability test

[0081] 1) Solution preparation:

[0082] Test sample spiked solution: Take about 20 mg of the test sample, accurately weigh it, place it in a 20 mL volumetric flask, add about 2 / 3 of the volume of the flask with diluent to dissolve it, add 1 mL of the impurity mixed stock solution (5) in Example 1, dilute to the scale with diluent, shake well, and prepare 6 portions in parallel (HNA001 concentration is about 1 mg / mL, impurity concentration is about 1 μg / mL, and the 6 sample weights are: 20.54 mg, 20.76 mg, 20.95 mg, 20.89 mg, 20.57 mg, 20.87 mg, respectively).

[0083] Control solution: Accurately measure 1 mL of the test sample spiked solution with a sample weight of 20.54 mg, place it in a 100 mL volumetric flask, dilute to the scale with diluent, shake well, and then accurately measure 1 mL of the solution, place it in a 10 mL volumetric flask, dilute to the scale with diluent, and shake well (HNA001 concentration: 1 μg / mL, equivalent to 0.1% of the test sample solution concentration).

[0084] 2) Detection

[0085] Take the control solution and the test sample spiked solution respectively, inject the samples using the chromatographic conditions of Example 1, record the chromatogram, and calculate according to the principal component self-reference method.

[0086] Experimental results: The detection results of elastomeric related substances are shown in Table 9.

[0087] Table 9. Results of the Elastranosequential Test

[0088]

[0089] The experimental results show that the RSD of each impurity in the 6 test sample spiked solutions (n=6) is no more than 15%, and the RSD of the total impurity (n=6) is no more than 15%, indicating that the repeatability of this method meets the requirements.

[0090] Limit of Detection and Limit of Quantitation

[0091] 1) Solution preparation:

[0092] Mixed stock solutions: Accurately measure 2 mL of the test solution and each of the stock solutions (2) to (6) in Example 1 above, place them in the same 100 mL volumetric flask, dilute to the mark with diluent, and shake well (concentration is about 20 μg / mL, equivalent to 2% of the concentration of the test solution).

[0093] Detection limit solution: Accurately measure 1 mL of the above mixed stock solution, place it in a 100 mL volumetric flask, dilute to the scale with diluent, and shake well (concentration is about 0.2 μg / mL, equivalent to 0.02% of the test solution concentration).

[0094] Quantitation limit solution: Accurately measure 0.5 mL of the above mixed stock solution, place it in a 20 mL volumetric flask, dilute to the scale with diluent, and shake well (concentration is about 0.5 μg / mL, equivalent to 0.05% of the test solution concentration).

[0095] Detection

[0096] The detection limit solution and the quantitative limit solution were taken separately, and the chromatographic conditions of Example 1 were used for injection, and the chromatogram was recorded. The detection results are shown in Table 10, and the detection limit spectrum is shown in Figure 3 See Table 11 for the quantitative limit graph. Figure 4 With Table 12.

[0097] Table 10. Results of the ELASTRAN LODA and LOQ test

[0098]

[0099] The experimental results show that when the concentration of HNA001 and each impurity is equivalent to 0.02% of the test solution, the S / N is greater than 3; when the concentration of HNA001 and each impurity is equivalent to 0.05% of the test solution, the S / N is greater than 10.

[0100] Table 11. Detection limit results

[0101]

[0102]

[0103] Table 12. Quantitation limit results

[0104]

[0105] Linear

[0106] Solution preparation:

[0107] Mixed stock solutions: Accurately measure 2 mL of the test solution and each of the stock solutions (2)-(6) in Example 1 above, place them in the same 100 mL volumetric flask, dilute to the mark with diluent, and shake well (concentration is about 20 μg / mL, equivalent to 2% of the concentration of the test solution).

[0108] Linear solution: Accurately measure the mixed stock solution according to Table 13, place it in the corresponding volumetric bottle, dilute it to the scale with diluent, and shake well. See Table 13 for specific preparation.

[0109] Table 13. Linearity solutions

[0110]

[0111] 2) Detection

[0112] Take each linear solution respectively, adopt the chromatographic conditions of Example 1 for sample injection, record the chromatogram, and the test results are shown in Table 14.

[0113] Table 14. Elastranose linearity test results

[0114] name Linear range (μg / mL) Linear equations Correlation coefficient HNA001-CPZ1 0.5047～2.019 A=0.1732C-0.0047 0.9997 D-(+)-DBTA 0.5049～2.020 A=0.1745C-0.0010 0.9995 HNA001 0.4468～1.787 A=0.2724C-0.0026 0.9982 HNA001-207Z2 0.5027～2.011 A=0.2102C+0.0007 1.000 HNA001-207Z4 0.5023～2.009 A=0.5083C+0.0066 0.9999

[0115] The experimental results show that HNA001 and its impurities show good linearity within the specified concentration range; the correlation coefficients r are all greater than 0.990.

[0116] Accuracy

[0117] 1) Solution preparation:

[0118] Impurity mixed stock solution: Accurately measure 2 mL of each stock solution of (1)-(4) in Example 1, place in a 100 mL volumetric flask, dilute to scale with diluent, and shake well (concentration: 20 μg / mL, equivalent to 2% of the concentration of the test solution).

[0119] Impurity reference solution: Accurately measure 1 mL of the above impurity mixed stock solution, place it in a 20 mL volumetric flask, add about 2 / 3 of the volume of the flask with diluent to dissolve, dilute to the scale with diluent, and shake well (impurity concentration is about 1 μg / mL, equivalent to 0.1% of the test solution concentration).

[0120] Accuracy solution: Accurately weigh the test sample according to the table below and place it in the corresponding volumetric bottle, a total of 10 parts, 1 part as the matrix sample, 9 parts accurately transferred into the impurity mixed stock solution, add diluent to dissolve and dilute to the scale, shake well, as the accuracy solution. See Table 15 for specific preparation.

[0121] Table 15. Accuracy Solutions

[0122]

[0123]

[0124] 2) Detection

[0125] Take the impurity reference solution and each accuracy solution respectively, use the chromatographic conditions of Example 1 for sample injection, record the chromatogram, calculate according to the following formula, and the results are shown in Table 16.

[0126]

[0127] Where: W SS : Weight of impurity reference substance, mg;

[0128] D SS : Dilution multiple of impurity reference substance;

[0129] V SS : Volume of impurity reference substance stock solution added, mL;

[0130] A 样杂 : Accuracy Peak area of ​​impurities in solution;

[0131] A 基杂 : Peak area of ​​impurities in matrix solution;

[0132] A R : Peak area of ​​impurity reference substance;

[0133] W R : The weight of the impurity reference substance, mg;

[0134] Ws: weighing amount of accuracy sample, mg;

[0135] W 基 : The weight of the matrix sample, mg;

[0136] D R : Dilution multiple of impurity reference solution;

[0137] D S : Dilution multiple of accuracy solution;

[0138] D 基 : dilution factor of matrix solution;

[0139] P: The content of impurity reference substance.

[0140] Table 16. Results of accuracy determination of each impurity of elastostat

[0141]

[0142]

[0143] The experimental results show that the recoveries at the quantitative limit concentration are all in the range of 70% to 130%, and the recoveries at other concentrations are all in the range of 80% to 120%. The recoveries were calculated using the external standard method, and the RSD (n=9) of the recoveries of each impurity was no more than 10%.

[0144] Solution stability

[0145] 1) Solution preparation:

[0146] Test sample spiked solution: Take about 20.40 mg of the test sample, accurately weigh it, place it in a 20 mL volumetric flask, add about 2 / 3 of the volume of the flask with diluent to dissolve it, add 1 mL of the impurity mixed stock solution (5) in Example 1, dilute to the scale with diluent, and shake well (HNA001 concentration is about 1 mg / mL, impurity concentration is about 1 μg / mL).

[0147] Reference solution: Accurately measure 1 mL of the above-mentioned test sample spiked solution, place it in a 100 mL volumetric flask, dilute it to the mark with diluent, shake well, then accurately measure 1 mL of the solution, place it in a 10 mL volumetric flask, dilute it to the mark with diluent, shake well (HNA001 concentration: 1 μg / mL, equivalent to 0.1% of the test sample solution concentration).

[0148] 2) Detection

[0149] The reference solution and the test sample spiked solution were placed at room temperature for different time periods and injected separately. The chromatograms were recorded. The results are shown in Table 17.

[0150] Table 17. Results of stability test of elastomeric solution

[0151]

[0152]

[0153] The experimental results show that when the reference solution and the spiked test solution are placed at room temperature for 31 hours, the RSD (n=8) of the main peak area of ​​the reference solution is 3.41%, which is no more than 10%; the maximum RSD (n=8) of the single impurity and total impurity content in the spiked test solution is 2.32%, which is less than 20%, indicating that the reference solution and the spiked test solution are stable within 31 hours at room temperature.

[0154] In summary, the method for determining elastostat related substances of the present invention selects octadecylsilane bonded silica gel and a mixed solution of acid and base containing an organic solvent as mobile phase A and an organic solvent as mobile phase B to facilitate the separation and elution of elastostat related substances, fully reveals the impurities of elastostat, can quickly detect elastostat related substances and improve the safety of the product. The method is scientific and reliable, and elastostat related substances can be controlled.

[0155] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for detecting elastomeric related substances, characterized in that: The detection method comprises the following steps: 1) Prepare the test solution; 2) preparing a control solution; 3) preparing separation solution; 4) taking the separation solution, the control solution and the test solution respectively, and performing detection by high performance liquid analysis, using octadecylsilane bonded silica gel as the chromatographic column filler, using a mixed solution of acetonitrile-water as the diluent, using a mixed solution of an acid and a base containing an organic solvent as the mobile phase A and an organic solvent as the mobile phase B, performing gradient elution, separating the elastomeric related substances, recording the chromatogram, and calculating; The elastomeric related substances include substances represented by formula I, substances represented by formula II, substances represented by formula III, and substances represented by formula IV; Among them, the substance shown in formula I: The substance shown in formula II: The substance shown in formula III: The substance shown in formula IV:

2. The method for detecting elastomeric related substances according to claim 1, characterized in that: The preparation of the separation solution is as follows: taking the substance shown in formula I, the substance shown in formula II, the substance shown in formula III and the substance shown in formula IV respectively, preparing a stock solution containing the substance shown in formula I with a concentration of 1 mg / mL, a stock solution containing the substance shown in formula II with a concentration of 1 mg / mL, a stock solution containing the substance shown in formula III with a concentration of 1 mg / mL and a stock solution containing the substance shown in formula IV with a concentration of 1 mg / mL; taking the stock solution containing the substance shown in formula I, the stock solution containing the substance shown in formula II, the stock solution containing the substance shown in formula III and the stock solution containing the substance shown in formula IV respectively, mixing and shaking to obtain an impurity mixed stock solution with a concentration of 20 μg / mL; taking the elastomeric reference substance and the impurity mixed stock solution, shaking and obtaining a separation solution, wherein the concentration of elastomeric is 1 mg / mL and the impurity concentration is 1 μg / mL.

3. The method for detecting elastomeric related substances according to claim 1, characterized in that: In the mixed solution containing acid and alkali, the acid solution is a polyacidic solution, and the alkali solution is a polyacidic alkaline solution.

4. The method for detecting elastomeric related substances according to claim 3, characterized in that: The acid solution is a phosphoric acid solution with a volume concentration of 0.09%-0.11%, the alkali solution is a triethylamine solution, and the pH value of the mixed solution is 2.0-4.

0.

5. The method for detecting elastomeric related substances according to claim 1, characterized in that: The organic solvent is a cyanide solvent or an alcohol solvent.

6. The method for detecting elastomeric related substances according to claim 5, characterized in that: The cyanide solvent is acetonitrile, and the alcohol solvent is methanol.

7. The method for detecting elastomeric related substances according to claim 1, characterized in that: The concentration of the test solution is 0.5-2.0 mg / mL.

8. The method for detecting elastomeric related substances according to claim 1, characterized in that: The separation solution is obtained by mixing and diluting different reference substance solutions.

9. The method for detecting elastomeric related substances according to claim 1, characterized in that: During detection, the detection wavelength is 210-230 nm, the flow rate is 0.9-1.1 ml / min, and the column temperature is 20-40°C.

10. The method for detecting elastomeric related substances according to claim 1, characterized in that: The gradient elution is: 0 min, mobile phase A 100%, mobile phase B 0%; 1 min, mobile phase A 100%, mobile phase B 0%; 15 min, mobile phase A 30%, mobile phase B 70%; 20 min, mobile phase A 30%, mobile phase B 70%; 22 min, mobile phase A 100%, mobile phase B 0%; 30 min, mobile phase A 100%, mobile phase B 0%.

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