Method for determining residual solvent 1, 2-ethanedithiol of cetrorelix acetate

Abstract

The invention relates to the technical field of cetrorelix acetate preparation, in particular to a method for determining a residual solvent 1, 2-ethanedithiol of cetrorelix acetate, cetrorelix acetate and a solvent are mixed to prepare a to-be-detected solution containing about 50mg of cetrorelix acetate per 1ml, the method for determining the residual solvent 1, 2-ethanedithiol of cetrorelix acetate is good in specificity, and the method can be used for determining the residual solvent 1, 2-ethanedithiol of cetrorelix acetate. In the range of 10.053-100.530 mu g / ml, the linear relationship between the peak area and the concentration is good, the sample introduction precision, repeatability and intermediate precision are good, the durability is good, and the accuracy of daily detection can be ensured by the determination method of the cetrorelix acetate residual solvent 1, 2-ethanedithiol.

Description

Technical Field

[0001] The present invention relates to the technical field of cetrorelix acetate preparation, in particular to a method for determining 1,2-ethanedithiol, a residual solvent of cetrorelix acetate. Background Art

[0002] Cetrorelix Acetate is a synthetic decapeptide and a gonadotropin-releasing hormone (GnRH) antagonist commonly used in assisted reproductive technology. During the production process, organic solvents may be used, and residual solvents are one of the quality indicators that require strict control. 1,2-Ethanedithiol, an organosulfur compound, may be used as a solvent or reagent during synthesis or purification. Due to its potential toxicity, the residual amount must comply with relevant pharmacopoeias or regulatory requirements.

[0003] The determination of 1,2-ethanedithiol, the residual solvent in cetrorelix acetate, is usually performed based on gas chromatography (GC). During the determination process, the measurement parameters and raw materials need to be set to ensure the measurement results. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for stably determining 1,2-ethanedithiol, a residual solvent in cetrorelix acetate.

[0005] The present invention is achieved through the following technical solution: a method for determining the residual solvent 1,2-ethanedithiol in cetrorelix acetate, comprising the following steps:

[0006] Step 1: Prepare the test solution by mixing cetrorelix acetate with a solvent to prepare a test solution containing approximately 50 mg of cetrorelix acetate per 1 ml;

[0007] Step 2: Detection: The solution to be tested is detected using a chromatograph.

[0008] Furthermore, in step 1, the solvent is dimethyl sulfoxide-anhydrous ethanol solvent, and the volume ratio of dimethyl sulfoxide to anhydrous ethanol is 3:1.

[0009] Furthermore, in step 1, 50 mg of cetrorelix acetate was accurately weighed and placed in a vial, and 700 μl of dimethyl sulfoxide and 300 μl of anhydrous ethanol were accurately added in sequence, the vial was capped, and sonicated to dissolve the vial to prepare a test solution containing approximately 50 mg of cetrorelix acetate per 1 ml.

[0010] Furthermore, in step 2, the chromatograph used is an Agilent DB-624 gas chromatograph.

[0011] Furthermore, in step 2, the temperature is raised to 70°C for 15 minutes, then raised to 240°C at a rate of 30°C per minute and held for 15 minutes.

[0012] Inlet temperature, 220°C; detector temperature, 250°C; injection volume, 1 μl; column flow rate, 1.8 ml / min; split ratio, 1:1; carrier gas, N2.

[0013] Furthermore, in step 2, the peak theoretical plate number of 1,2-ethanedithiol is tested, and the peak theoretical plate number of 1,2-ethanedithiol is not less than 5000.

[0014] Furthermore, in step 2, the average content of 1,2-ethanedithiol is tested, and the RSD value of the average content of 1,2-ethanedithiol is not higher than 25%. Furthermore, in step 2, the RSD value of the peak area of 1,2-ethanedithiol is tested, and the RSD value of the peak area of 1,2-ethanedithiol is not higher than 25%, and the RSD value of the retention time is not higher than 1.0%.

[0015] Furthermore, the quantitative limit and detection limit status were tested, and the quantitative limit concentration was lower than 30% of the limit concentration, and the detection limit was lower than 10% of the limit concentration.

[0016] The beneficial effects of the present invention are:

[0017] 1. Specificity: The blank solvent does not interfere with the determination of 1,2-ethanedithiol; the determination of 1,2-ethanedithiol peak in the reference solution and the test sample spiked solution is not affected by adjacent chromatographic peaks; the theoretical plate number is greater than 5000;

[0018] 2. Linearity and range: In the range of 10.053 μg / ml to 100.530 μg / ml (0.0201% to 0.2011%), the linear equation between peak area and concentration is y = 1.2268x + 1.4648, the correlation coefficient r = 0.9970, and the residual sum of squares is 59.21, indicating a good linear relationship.

[0019] 3. Detection limit: The quantification limit of 1,2-ethanedithiol is 10.1 ng (0.0201%), and the detection limit is 3.0 ng (0.0060%);

[0020] 4. Precision of the limit of quantification: After 6 injections of the limit of quantification solution, the RSD value of the retention time of the 1,2-ethanedithiol peak was 0.05%, and the RSD value of the peak area was 10.12%, both of which met the requirements. The precision of the limit of quantification of this method is good;

[0021] 5. Repeatability: The average content of 1,2-ethanedithiol measured in 6 samples of spiked solution was 0.093%, and the RSD value was 8.80%, which met the requirements and showed that the method had good repeatability.

[0022] 6. Intermediate precision: For the same batch of samples, different analysts measured 12 sample spiked solutions at different times. The average content of 1,2-ethanedithiol was 0.090%, and the RSD value was 7.76%, which met the requirements. The intermediate precision of this method is good.

[0023] 7. Solution stability: The reference solution was placed at room temperature for 4 hours, and the RSD value of the 1,2-ethanedithiol peak area was 13.84%, and the RSD value of the retention time was 0.05%, which met the requirements, indicating that the reference solution had good stability after being placed at room temperature for 4 hours. The test solution was placed at room temperature for 12 hours, and no 1,2-ethanedithiol peak was detected, indicating that the test solution had good stability after being placed at room temperature for 12 hours;

[0024] 8. Durability: By making minor adjustments to different column flow rates, initial temperatures, split ratios, inlet temperatures, and chromatographic columns, the blank solvent did not interfere with the determination of the 1,2-ethanedithiol peak; the theoretical plate numbers in the spiked solution of the test sample, calculated based on the 1,2-ethanedithiol peak, were all greater than 5000. In summary, this method has good durability;

[0025] 9. Accuracy (recovery rate): The average recovery rate of 1,2-ethanedithiol was 94.4%, and the RSD value was 6.68%, which met the requirements and indicated that the method had good accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a linear relationship graph between peak area and concentration in the range of 10.053 to 100.530 μg / ml. DETAILED DESCRIPTION

[0027] The following will be combined with the embodiments of the invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described 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 efforts are within the scope of protection of the present invention.

[0028] In the following embodiments,

[0029] Instrument name factory model Chromatograph Agilent Technologies Agilent DB-624 Electronic analytical balance Mettler-Toledo Instruments Ltd. MS105DU

[0030] Name of trial drug level factory Dimethyl sulfoxide (DMSO) AR Chengdu Kelong Chemicals Co., Ltd. Diethylamine AR Tianjin Oubok Chemical Co., Ltd. Anhydrous ethanol HPLC Tianjin Saferay Technology Co., Ltd.

[0031] Product Name batch number content source 1,2-Ethanedithiol 20210805 100％ Sinopharm Chemical Reagent Co., Ltd.

[0032] name batch number source Cetrorelix acetate XC220102 Kaifeng Mingren Pharmaceutical Co., Ltd.

[0033] The method for selecting solvents is shown in Appendix 2:

[0034] Schedule 1

[0035]

[0036]

[0037] in,

[0038] In method 1, the blank solvent is dimethyl sulfoxide.

[0039] In method 2, the blank solvent is dimethyl sulfoxide-diethylamine, and the volume ratio of dimethyl sulfoxide to diethylamine is 1000:1.

[0040] In Methods 1 and 2, for the reference substance stock solution, take an appropriate amount of 1,2-ethanedithiol reference substance, accurately weigh it, dissolve it in a solvent, and quantitatively dilute it to make a solution containing approximately 1.0 mg per 1 ml;

[0041] Reference solution: Accurately measure an appropriate amount of 1,2-ethanedithiol reference stock solution and quantitatively dilute it with solvent to make a solution containing approximately 0.1 mg per 1 ml;

[0042] Test sample spiked solution: Take 100 mg of the test sample, accurately weigh it, and place it in a penicillin bottle. Accurately add 100 μl of 1,2-ethanedithiol reference stock solution and 900 μl of solvent in sequence. Seal the bottle with a cap and sonicate to dissolve it to prepare a mixed solution containing approximately 0.1 mg of 1,2-ethanedithiol and 100 mg of cetrorelix acetate per 1 ml.

[0043] In method 3, the blank solvent is dimethyl sulfoxide-anhydrous ethanol, and the volume ratio of dimethyl sulfoxide to anhydrous ethanol is 1:1.

[0044] In methods 4-6, the blank solvent is dimethyl sulfoxide-anhydrous ethanol, and the volume ratio of dimethyl sulfoxide to anhydrous ethanol is 7:3.

[0045] In methods 3-6, reference substance stock solution: Take an appropriate amount of 1,2-ethanedithiol reference substance, accurately weigh it, dissolve it in anhydrous ethanol, and quantitatively dilute it to make a solution containing approximately 10 mg per 1 ml;

[0046] Reference substance stock solution: Take an appropriate amount of 1,2-ethanedithiol reference substance mother solution, accurately weigh it, dissolve it in anhydrous ethanol and quantitatively dilute it to make a solution containing approximately 0.5 mg per 1 ml;

[0047] Reference solution: Accurately measure an appropriate amount of 1,2-ethanedithiol reference stock solution and quantitatively dilute it with solvent to make a solution containing approximately 0.05 mg per 1 ml;

[0048] Test sample spike solution: Take 50 mg of the test sample, accurately weigh it, and place it in a vial. Accurately add appropriate amounts of dimethyl sulfoxide, anhydrous ethanol, and 1,2-ethanedithiol reference substance stock solution in that order. Seal the bottle with a cap and sonicate to dissolve it. Prepare a mixed solution containing approximately 0.05 mg of 1,2-ethanedithiol and 50 mg of cetrorelix acetate per 1 ml.

[0049] Test solution: Take 50 mg of the test sample, accurately weigh it, and place it in a penicillin bottle. Accurately add 700 μl of dimethyl sulfoxide and 300 μl of anhydrous ethanol in that order. Cover the bottle and sonicate to dissolve it to make a solution containing approximately 50 mg of cetrorelix acetate per 1 ml.

[0050] It can be determined from Appendix 1 that the solvent selected is dimethyl sulfoxide-anhydrous ethanol, and the volume ratio of dimethyl sulfoxide to anhydrous ethanol is 7:3, which can ensure the accuracy of the measurement.

[0051] The specificity test results are shown in Appendix 2.

[0052] Schedule 2

[0053]

[0054] in,

[0055] The blank solvent and solvent were both dimethyl sulfoxide-anhydrous ethanol, and the volume ratio of dimethyl sulfoxide to anhydrous ethanol was 7:3.

[0056] Reference substance stock solution: Take an appropriate amount of 1,2-ethanedithiol reference substance, accurately weigh it, dissolve it in anhydrous ethanol and quantitatively dilute it to make a solution containing about 10 mg per 1 ml;

[0057] Reference substance stock solution: Take an appropriate amount of 1,2-ethanedithiol reference substance mother solution, accurately weigh it, dissolve it in anhydrous ethanol and quantitatively dilute it to make a solution containing approximately 0.5 mg per 1 ml;

[0058] Reference solution: Accurately measure an appropriate amount of 1,2-ethanedithiol reference stock solution and quantitatively dilute it with solvent to make a solution containing approximately 0.05 mg per 1 ml;

[0059] Test sample spike solution: Take 50 mg of the test sample, accurately weigh it, and place it in a vial. Accurately add appropriate amounts of dimethyl sulfoxide, anhydrous ethanol, and 1,2-ethanedithiol reference substance stock solution in that order. Seal the bottle with a cap and sonicate to dissolve it. Prepare a mixed solution containing approximately 0.05 mg of 1,2-ethanedithiol and 50 mg of cetrorelix acetate per 1 ml.

[0060] Test solution: Take 50 mg of the test sample, accurately weigh it, and place it in a penicillin bottle. Accurately add 700 μl of dimethyl sulfoxide and 300 μl of anhydrous ethanol in that order. Cover the bottle and sonicate to dissolve it to make a solution containing approximately 50 mg of cetrorelix acetate per 1 ml.

[0061] It can be determined from Appendix 2 that the specificity is good and meets the acceptance criteria. The blank solvent has no interference. The determination of 1,2-ethanedithiol peaks in the reference solution and the test sample spiked solution is not affected by adjacent chromatographic peaks. The theoretical plate numbers are all higher than 5000, and the tailing factors are within the acceptable range.

[0062] The results of the linear relationship test are shown in Table 3.

[0063] Schedule 3

[0064]

[0065] In Appendix 3, linear solution: Accurately measure an appropriate amount of linear stock solution (limit of quantitation), 0.3 ml (30%), 0.5 ml (50%), 1 ml (100%), 1.5 ml (150%), and 2 ml (200%) into a 10 ml volumetric flask. Add appropriate amounts of anhydrous ethanol and dimethyl sulfoxide in sequence to dilute to the scale. Shake well to obtain a linear solution with a concentration of the limit of quantitation to 200%.

[0066] It can be determined from Appendix 3 that the injection concentration has a good linear relationship with its peak area within the linear range. The results of the quantitative limit and detection limit tests are shown in Appendix 4.

[0067] Schedule 4

[0068]

[0069] The results of the quantitative limit precision are shown in Appendix 5.

[0070] Schedule 5

[0071]

[0072] In Appendix 4-5, for the detection limit solution 1, the stock solution of the 1,2-ethanedithiol reference substance was diluted and injected, and the chromatogram was recorded. When the signal-to-noise ratio was approximately 10:1, the amount of the sample was the limit of quantification; for the detection limit solution 2, the stock solution of the 1,2-ethanedithiol reference substance was diluted and injected, and the chromatogram was recorded. When the signal-to-noise ratio was approximately 3:1, the amount of the sample was the detection limit.

[0073] Accurately measure the above detection limit solution, inject it into the gas chromatograph, and record the chromatogram. Also, take the quantification limit concentration solution, inject it 6 times in succession, and record the chromatogram.

[0074] It can be seen from Appendix 4-5 that the quantification limit of 1,2-ethanedithiol is 10.1 ng (0.0201%), the detection limit is 3.0 ng (0.0060%), and the quantification limit solution is injected 6 times. The RSD value of the retention time of the 1,2-ethanedithiol peak is 0.05%, and the RSD value of the peak area is 10.12%, both of which meet the requirements. The quantification limit precision of this method is good.

[0075] The repeatability test results are shown in Appendix 6.

[0076] Schedule 6

[0077]

[0078]

[0079] The results of the intermediate precision test are shown in Appendix 7.

[0080] Schedule 7

[0081]

[0082] It can be seen from Appendix 6-7 that the average content of 1,2-ethanedithiol measured in 6 test sample spiked solutions was 0.093%, and the RSD value was 8.80%, which met the requirements, indicating that the method has good repeatability. For the same batch of samples, different analysts measured 12 test sample spiked solutions at different times, and the average content of 1,2-ethanedithiol measured was 0.090%, and the RSD value was 7.76%, which met the requirements. The intermediate precision of this method is good.

[0083] The results of the determination of the recovery rate of 1,2-ethanedithiol are shown in Table 8.

[0084] Schedule 8

[0085]

[0086]

[0087] It can be seen from Appendix 8 that the average recovery rate of 1,2-ethanedithiol is 94.4% and the RSD value is 6.68%, which meets the requirements and indicates that the method has good accuracy.

[0088] The results of the stability test of the reference solution are shown in Table 9.

[0089] Schedule 9

[0090]

[0091] The results of the stability test of the test solution are shown in Table 10.

[0092] Schedule 10

[0093]

[0094] As shown in Tables 9-10, after the reference solution was placed at room temperature for 4 hours, the RSD value of the 1,2-ethanedithiol peak area was 13.84%, and the RSD value of the retention time was 0.05%, which met the requirements. This shows that the reference solution has good stability after being placed at room temperature for 4 hours.

[0095] The test solution was placed at room temperature for 12 hours, and no 1,2-ethanedithiol peak was detected, indicating that the test solution had good stability after being placed at room temperature for 12 hours.

[0096] The results of the column flow rate change are shown in Appendix 11.

[0097] Schedule 11

[0098]

[0099] The results of the measurement of the changed initial temperature are shown in Table 12.

[0100] Schedule 12

[0101]

[0102] The results of the split ratio test are shown in Table 13.

[0103] Schedule 13

[0104]

[0105] The results of the measurement by changing the injection port temperature are shown in Table 14.

[0106] Schedule 14

[0107]

[0108] The results of the determination by changing the chromatographic column are shown in Appendix 15.

[0109] Schedule 15

[0110]

[0111] Tables 11-15 show that, with minor adjustments to column flow rate, initial temperature, split ratio, inlet temperature, and chromatographic column, the blank solvent did not interfere with the determination of the 1,2-ethanedithiol peak. The theoretical plate numbers calculated based on the 1,2-ethanedithiol peak in the spiked sample solutions were all greater than 5000. Overall, this method demonstrates excellent robustness.

[0112] Example 1

[0113] A method for determining 1,2-ethanedithiol, a residual solvent of cetrorelix acetate, comprises the following steps:

[0114] Step 1: Prepare the test solution. Accurately weigh 50 mg of cetrorelix acetate and place it in a vial. Accurately add 700 μl of dimethyl sulfoxide and 300 μl of anhydrous ethanol in sequence. Cap the vial and sonicate to dissolve the solution to be tested, with a concentration of approximately 50 mg of cetrorelix acetate per ml.

[0115] Step 2: Detection: The test solution was detected using a chromatograph. The heating program was as follows: the starting temperature was 70°C, maintained for 15 minutes, then heated to 240°C at a rate of 30°C per minute, and maintained for 15 minutes; the injection port temperature was 220°C; the detector temperature was 250°C; the injection volume was 1 μl; the column flow rate was 1.8 ml / min; the split ratio was 1:1; and the carrier gas was N2. The test results are shown in Appendix 16.

[0116] Comparative Example 1

[0117] A method for determining 1,2-ethanedithiol, a residual solvent of cetrorelix acetate, comprises the following steps:

[0118] Step 1: Prepare the test solution. Accurately weigh 100 mg of cetrorelix acetate, place it in a vial, dissolve it in dimethyl sulfoxide, and quantitatively dilute it to make a reference stock solution containing approximately 1.0 mg of 1,2-ethanedithiol per 1 ml.

[0119] Accurately add 100 μl of the reference substance stock solution and 900 μl of dimethyl sulfoxide in sequence, cap the tube, and sonicate to dissolve to prepare a test solution containing approximately 0.1 mg of 1,2-ethanedithiol and 100 mg of cetrorelix acetate per 1 ml.

[0120] Step 2: Detection: The test solution was detected using a chromatograph. The heating program was as follows: the starting temperature was 100°C, maintained for 15 minutes, then heated to 240°C at a rate of 30°C per minute, and maintained for 15 minutes; the injection port temperature was 220°C; the detector temperature was 250°C; the injection volume was 0.4 μl; the column flow rate was 2.0 ml / min; the split ratio was 3:1; and the carrier gas was N2. The test results are shown in Appendix 16.

[0121] Comparative Example 2

[0122] A method for determining 1,2-ethanedithiol, a residual solvent of cetrorelix acetate, comprises the following steps:

[0123] Step 1: Preparation of test solution: Accurately weigh 100 mg of cetrorelix acetate and place it in a vial. Dissolve it in dimethyl sulfoxide-diethylamine and quantitatively dilute it to prepare a reference stock solution containing approximately 1.0 mg of 1,2-ethanedithiol per 1 ml. Accurately add 100 μl of the reference stock solution and 900 μl of dimethyl sulfoxide in sequence, cap the vial, and sonicate to dissolve it to prepare a test solution containing approximately 0.1 mg of 1,2-ethanedithiol and 100 mg of cetrorelix acetate per 1 ml, wherein the volume ratio of dimethyl sulfoxide to diethylamine is 1000:1;

[0124] Step 2: Detection: The test solution was detected using a chromatograph. The heating program was as follows: the starting temperature was 100°C, maintained for 15 minutes, then heated to 240°C at a rate of 30°C per minute, and maintained for 15 minutes; the injection port temperature was 220°C; the detector temperature was 250°C; the injection volume was 0.4 μl; the column flow rate was 2.0 ml / min; the split ratio was 3:1; and the carrier gas was N2. The test results are shown in Appendix 16.

[0125] Comparative Example 3

[0126] A method for determining 1,2-ethanedithiol, a residual solvent of cetrorelix acetate, comprises the following steps:

[0127] Step 1: Preparation of test solution: Accurately weigh 100 mg of cetrorelix acetate and place it in a vial. Dissolve it in dimethyl sulfoxide-diethylamine and quantitatively dilute it to prepare a reference stock solution containing approximately 1.0 mg of 1,2-ethanedithiol per 1 ml. Accurately add 100 μl of the reference stock solution and 900 μl of dimethyl sulfoxide in sequence, cap the vial, and sonicate to dissolve it to prepare a test solution containing approximately 0.1 mg of 1,2-ethanedithiol and 100 mg of cetrorelix acetate per 1 ml, wherein the volume ratio of dimethyl sulfoxide to diethylamine is 1000:1;

[0128] Step 2: Detection: The test solution was detected using a chromatograph. The heating program was as follows: the starting temperature was 100°C, maintained for 15 minutes, then heated to 240°C at a rate of 30°C per minute, and maintained for 15 minutes; the injection port temperature was 220°C; the detector temperature was 250°C; the injection volume was 0.4 μl; the column flow rate was 2.0 ml / min; the split ratio was 3:1; and the carrier gas was N2. The test results are shown in Appendix 16.

[0129] Comparative Example 3

[0130] A method for determining 1,2-ethanedithiol, a residual solvent of cetrorelix acetate, comprises the following steps:

[0131] Step 1: Prepare the test solution. Accurately weigh 50 mg of cetrorelix acetate and place it in a vial. Accurately add 500 μl of dimethyl sulfoxide and 500 μl of anhydrous ethanol in sequence. Cap the vial and sonicate to dissolve the solution to be tested, to prepare a solution containing approximately 50 mg of cetrorelix acetate per 1 ml.

[0132] Step 2: Detection: The test solution was detected using a chromatograph. The heating program was as follows: the starting temperature was 100°C, maintained for 15 minutes, then heated to 240°C at a rate of 30°C per minute, and maintained for 15 minutes; the injection port temperature was 220°C; the detector temperature was 250°C; the injection volume was 0.4 μl; the column flow rate was 2.0 ml / min; the split ratio was 3:1; and the carrier gas was N2. The test results are shown in Appendix 16.

[0133] Comparative Example 4

[0134] A method for determining 1,2-ethanedithiol, a residual solvent of cetrorelix acetate, comprises the following steps:

[0135] Step 1: Prepare the test solution. Accurately weigh 50 mg of cetrorelix acetate and place it in a vial. Accurately add 700 μl of dimethyl sulfoxide and 300 μl of anhydrous ethanol in sequence. Cap the vial and sonicate to dissolve the solution to be tested, with a concentration of approximately 50 mg of cetrorelix acetate per ml.

[0136] Step 2: Detection: The test solution was detected using a chromatograph. The heating program was as follows: the starting temperature was 100°C, maintained for 15 minutes, then heated to 240°C at a rate of 30°C per minute, and maintained for 15 minutes; the injection port temperature was 220°C; the detector temperature was 250°C; the injection volume was 0.4 μl; the column flow rate was 2.0 ml / min; the split ratio was 3:1; and the carrier gas was N2. The test results are shown in Appendix 16.

[0137] Comparative Example 5

[0138] A method for determining 1,2-ethanedithiol, a residual solvent of cetrorelix acetate, comprises the following steps:

[0139] Step 1: Prepare the test solution. Accurately weigh 50 mg of cetrorelix acetate and place it in a vial. Accurately add 700 μl of dimethyl sulfoxide and 300 μl of anhydrous ethanol in sequence. Cap the vial and sonicate to dissolve the solution to be tested, with a concentration of approximately 50 mg of cetrorelix acetate per ml.

[0140] Step 2: Detection: The test solution was detected using a chromatograph. The heating program was as follows: the starting temperature was 70°C, maintained for 15 minutes, then heated to 240°C at a rate of 30°C per minute, and maintained for 15 minutes; the injection port temperature was 220°C; the detector temperature was 250°C; the injection volume was 1 μl; the column flow rate was 2.0 ml / min; the split ratio was 3:1; and the carrier gas was N2. The test results are shown in Appendix 16.

[0141] Schedule 16

[0142]

[0143]

[0144] In Comparative Example 1, the blank solvent was dimethyl sulfoxide.

[0145] In Comparative Example 2, the blank solvent was dimethyl sulfoxide-diethylamine, and the volume ratio of dimethyl sulfoxide to diethylamine was 1000:1.

[0146] In Comparative Examples 1 and 2,

[0147] Reference substance stock solution: Take an appropriate amount of 1,2-ethanedithiol reference substance, accurately weigh it, dissolve it in solvent and quantitatively dilute it to make a solution containing approximately 1.0 mg per 1 ml;

[0148] Reference solution: Accurately measure an appropriate amount of 1,2-ethanedithiol reference stock solution and quantitatively dilute it with solvent to make a solution containing approximately 0.1 mg per 1 ml;

[0149] Test sample spiked solution: Take 100 mg of the test sample, accurately weigh it, and place it in a penicillin bottle. Accurately add 100 μl of 1,2-ethanedithiol reference stock solution and 900 μl of solvent in sequence. Seal the bottle with a cap and sonicate to dissolve it to prepare a mixed solution containing approximately 0.1 mg of 1,2-ethanedithiol and 100 mg of cetrorelix acetate per 1 ml.

[0150] In Comparative Example 3, the blank solvent was dimethyl sulfoxide-anhydrous ethanol, and the volume ratio of dimethyl sulfoxide to anhydrous ethanol was 1:1.

[0151] In Comparative Examples 4-5 and Example 1,

[0152] The blank solvent was dimethyl sulfoxide-anhydrous ethanol, and the volume ratio of dimethyl sulfoxide to anhydrous ethanol was 7:3.

[0153] In Comparative Examples 3-5 and Example 1,

[0154] Reference substance stock solution: Take an appropriate amount of 1,2-ethanedithiol reference substance, accurately weigh it, dissolve it in anhydrous ethanol and quantitatively dilute it to make a solution containing about 10 mg per 1 ml;

[0155] Reference substance stock solution: Take an appropriate amount of 1,2-ethanedithiol reference substance mother solution, accurately weigh it, dissolve it in anhydrous ethanol and quantitatively dilute it to make a solution containing approximately 0.5 mg per 1 ml;

[0156] Reference solution: Accurately measure an appropriate amount of 1,2-ethanedithiol reference stock solution and quantitatively dilute it with solvent to make a solution containing approximately 0.05 mg per 1 ml;

[0157] Test sample spike solution: Take 50 mg of the test sample, accurately weigh it, and place it in a vial. Accurately add appropriate amounts of dimethyl sulfoxide, anhydrous ethanol, and 1,2-ethanedithiol reference substance stock solution in that order. Seal the bottle with a cap and sonicate to dissolve it. Prepare a mixed solution containing approximately 0.05 mg of 1,2-ethanedithiol and 50 mg of cetrorelix acetate per 1 ml.

[0158] Test solution: Take 50 mg of the test sample, accurately weigh it, and place it in a penicillin bottle. Accurately add 700 μl of dimethyl sulfoxide and 300 μl of anhydrous ethanol in that order. Cover the bottle and sonicate to dissolve it to make a solution containing approximately 50 mg of cetrorelix acetate per 1 ml.

[0159] The determination method for 1,2-ethanedithiol, a residual solvent in cetrorelix acetate, has good specificity. In the range of 10.053 to 100.530 μg / ml, the peak area has a good linear relationship with the concentration. The injection precision, repeatability, and intermediate precision are good. The durability and accuracy are good. In summary, the determination method for 1,2-ethanedithiol, a residual solvent in cetrorelix acetate, can ensure the accuracy of routine testing.

[0160] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for determining the residual solvent 1,2-ethanedithiol in cetrorelix acetate, characterized in that: The following steps are involved: Step 1: Prepare the test solution by mixing cetrorelix acetate with a solvent to prepare a test solution containing approximately 50 mg of cetrorelix acetate per 1 ml; Step 2: Detection: Use a chromatograph to detect the solution to be tested. In step 1, the solvent is dimethyl sulfoxide-anhydrous ethanol solvent, and the volume ratio of dimethyl sulfoxide to anhydrous ethanol is 3:

1.

2. The method for determining the residual solvent 1,2-ethanedithiol in cetrorelix acetate according to claim 1, wherein In step 1, 50 mg of cetrorelix acetate was accurately weighed and placed in a vial. 700 μl of dimethyl sulfoxide and 300 μl of anhydrous ethanol were accurately added in sequence. The vial was capped and dissolved by ultrasonication to prepare a test solution containing approximately 50 mg of cetrorelix acetate per 1 ml.

3. The method for determining the residual solvent 1,2-ethanedithiol in cetrorelix acetate according to claim 1, characterized in that: In step 2, the chromatograph used is an Agilent DB-624 gas chromatograph.

4. The method for determining the residual solvent 1,2-ethanedithiol in cetrorelix acetate according to claim 1, characterized in that: In step 2, the temperature was raised from 70°C for 15 minutes, then raised to 240°C at a rate of 30°C per minute and held for 15 minutes. Inlet temperature, 220°C; detector temperature, 250°C; injection volume, 1 μl; column flow rate, 1.8 ml / min; split ratio, 1:1; carrier gas, N2.

5. The method for determining the residual solvent 1,2-ethanedithiol in cetrorelix acetate according to claim 1, characterized in that: In step 2, the peak theoretical plate number of 1,2-ethanedithiol is tested, and the peak theoretical plate number of 1,2-ethanedithiol is not less than 5000.

6. The method for determining the residual solvent 1,2-ethanedithiol in cetrorelix acetate according to claim 1, characterized in that: In step 2, the average content of 1,2-ethanedithiol is tested, and the RSD value of the average content of 1,2-ethanedithiol is not higher than 25%.

7. The method for determining the residual solvent 1,2-ethanedithiol in cetrorelix acetate according to claim 1, characterized in that: In step 2, the RSD value of the peak area of 1,2-ethanedithiol is tested. The RSD value of the peak area of 1,2-ethanedithiol is not higher than 25%, and the RSD value of the retention time is not higher than 1.0%.

8. The method for determining the residual solvent 1,2-ethanedithiol in cetrorelix acetate according to claim 1, characterized in that: The limit of quantification and limit of detection status of the test were lower than 30% of the limit concentration, and the limit of detection was lower than 10% of the limit concentration.

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