A detection method for related substances of ethyl 4-bromobutyrate

Abstract

The present invention provides a detection method for related substances of ethyl 4-bromobutyrate, and relates to the field of drug analysis. The method adopts a weak polar chromatographic column; nitrogen is used as a carrier gas, and the flow rate is 0.7-1.5 ml / min; injection is performed in split mode; a temperature control method of programmed heating is adopted, and the detector is a hydrogen flame ionization detector; gas chromatography is used to qualitatively or quantitatively detect related substances in ethyl 4-bromobutyrate. It has the characteristics of stable baseline, good peak shape, high separation, excellent specificity and system suitability, high sensitivity, and good durability, which makes up for the gap in the prior art, and is of great significance to the quality control of ethyl 4-bromobutyrate and the drug safety of elagolid sodium.

Description

Technical Field

[0001] The invention relates to the field of drug analysis, and in particular to a method for detecting 4-bromobutyrate ethyl related substances. Background Art

[0002] Elagolix sodium is an effective, selective, orally active non-peptide antagonist of the gonadotropin-releasing hormone receptor (GnRHR) for the treatment of endometriosis and uterine fibroids. So far, there have been more than 40 clinical trials of Elagolix involving more than 3,000 patients. In addition, Elagolix was launched in the United States on July 23, 2018, and its Phase III clinical trial for the treatment of uterine fibroids is also underway, which has great market prospects.

[0003] Ethyl 4-bromobutyrate (SM4) is the starting material for the synthesis of elagolid sodium. As the production source of the raw material of elagolid sodium, its quality will directly affect the quality and function of elagolid sodium. In the currently published documents or patents, the related substance analysis method of ethyl 4-bromobutyrate is not included, which makes the compound have no reference analysis standard at this stage, thus seriously affecting the quality control of elagolid sodium. Therefore, it is very important to establish a stable and effective related substance analysis method of ethyl 4-bromobutyrate for the quality control of elagolid sodium. Summary of the invention

[0004] The object of the present invention is to provide a method for detecting related substances of ethyl 4-bromobutyrate in view of the above-mentioned problems. The method is suitable for the qualitative and quantitative detection of ethyl 4-bromobutyrate and has the characteristics of stable baseline, good peak shape, high separation, excellent specificity and system applicability, high sensitivity and good durability.

[0005] The technical solution adopted by the present invention is as follows:

[0006] A method for detecting related substances in ethyl 4-bromobutyrate is carried out by gas chromatography, and the chromatographic conditions include: using a weak polarity chromatographic column; using nitrogen as a carrier gas with a flow rate of 0.7 to 1.5 ml / min; injecting in a split mode; using a hydrogen flame ionization detector as a detector; and using a programmed temperature control method, wherein the temperature rise program is:

[0007] Heating rate (℃ / min) Temperature(℃) Holding time (min) — 40～60 2～4 5 80～140 0～5 30 230～245 5～15

[0008] In a preferred embodiment of the present invention, the heating program is:

[0009] Heating rate (℃ / min) Temperature(℃) Holding time (min) — 60 2 5 90 5 30 240 10

[0010] In a preferred embodiment of the present invention, the heating program is:

[0011] Heating rate (℃ / min) Temperature(℃) Holding time (min) — 40 2 5 140 0 30 230 5

[0012] In a preferred embodiment of the present invention, the weak polarity chromatographic column is a capillary column using 5% phenyl-methylpolysiloxane as the stationary liquid or a capillary column with similar polarity.

[0013] In a preferred embodiment of the present invention, the flow rate is 1.0 ml / min.

[0014] In a preferred embodiment of the present invention, the split ratio is 60:1.

[0015] In a preferred embodiment of the present invention, the injection port temperature is 220°C; the detector temperature is 250°C.

[0016] In a preferred embodiment of the present invention, the hydrogen flow rate is 40 ml / min and the air flow rate is 400 ml / min.

[0017] Beneficial effects of the present invention:

[0018] The invention provides a detection method for 4-bromobutyric acid ethyl ester-related substances, which has the characteristics of stable baseline, good peak shape, high separation, excellent specificity and system applicability, high sensitivity and good durability, is suitable for qualitative and quantitative detection of 4-bromobutyric acid ethyl ester, fills the gap in the prior art, and is of great significance to the quality control of 4-bromobutyric acid ethyl ester and the drug safety of elagolid sodium. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will now be described by way of example with reference to the accompanying drawings, in which:

[0020] Figure 1 is a chromatogram under the chromatographic conditions in Example 1;

[0021] Figure 2 is a chromatogram under the chromatographic conditions in Example 2;

[0022] Figure 3 is the chromatogram under the chromatographic conditions in Example 3;

[0023] Figure 4 is a chromatogram under the chromatographic conditions in Example 4;

[0024] Figure 5 is a chromatogram under the chromatographic conditions in Example 5;

[0025] Figure 6 is a chromatogram under the chromatographic conditions in Example 6;

[0026] Figure 7 It is a chromatogram under the chromatographic conditions in Example 7. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is clearly and completely described below through specific 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 creative work are within the scope of protection of the present invention.

[0028] 1. Instruments and reagents

[0029] Instrument: Agilent 7890B gas chromatograph; 1 / 100,000 electronic balance (XS205DU)

[0030] Reagents: N,N-dimethylformamide (DMF) GC grade.

[0031] 2. Reference substances

[0032] The details of the related substance reference substances used in the present invention are shown in Table 1.

[0033] Table 1 Details of relevant substance reference substances

[0034]

[0035] 3. Test products

[0036] The 4-bromobutyric acid ethyl ester (test sample) used in the present invention is provided by Shandong Linyi Kangaite Chemical Technology Co., Ltd.

[0037] Example 1

[0038] Instrument: Agilent 7890B (with FID detector);

[0039] Chromatographic column: Agilent neutral capillary column (Agilent DB-1701), stationary phase: (14%-cyanopropyl-phenyl)-methylpolysiloxane (30m*0.53mm*1μm);

[0040] Inlet temperature: 220°C;

[0041] Injection method: direct injection;

[0042] Injection mode: split (split ratio: 60:1);

[0043] Column flow rate: 1.4 ml / min (carrier gas: N2);

[0044] Injection volume: 0.4 μl;

[0045] Detector (FID) temperature: 250°C;

[0046] H2 flow rate: 40ml / min;

[0047] Air flow rate: 400ml / min.

[0048] Test solution: Accurately weigh about 1000 mg of ethyl 4-bromobutyrate and place it in a 2 ml volumetric flask, dissolve it in DMF and dilute to the scale as the test solution.

[0049] The heating program is shown in Table 2.

[0050] Table 2 Heating program in Example 1

[0051] Heating rate (℃ / min) Temperature(℃) Holding time (min) — 60 2 20 100 0 10 140 0 30 230 5

[0052] Test results such as Figure 1 As shown. Figure 1 It can be seen that the use of a neutral chromatographic column results in extremely large interference, poor separation between impurities and the main peak, poor separation between impurities, and severe baseline drift. This shows that this method is not suitable for the detection of related substances in 4-bromobutyric acid inhibition.

[0053] Example 2

[0054] The samples tested in this embodiment are the same as those in Example 1, and the chromatographic conditions are the same as those in Example 1, except for the chromatographic column and the temperature program. The chromatographic column used in this embodiment is: Agilent weak polar capillary column (Agilent HP-5), and the stationary liquid is 5% phenyl-methyl polysiloxane (30m*0.32mm*0.25μm). The temperature program is shown in Table 3.

[0055] Table 3 Heating program in Example 2

[0056] Heating rate (℃ / min) Temperature(℃) Holding time (min) — 80 2 10 140 0 30 230 0

[0057] The test results are shown in Table 4 and Figure 2 shown. Figure 2 In the chromatographic peaks 1-7 are related substance peaks, and m is the main peak. Figure 2 It can be seen that this method detected a total of 7 related substances, and the main peak and its subsequent adjacent impurities were not effectively separated. Therefore, this method is not suitable for the detection of related substances in ethyl 4-bromobutyrate.

[0058] Table 4 Test results in Example 2

[0059] Serial number Retention time min Tailing Factor Separation 1 2.053 1.7 / 2 3.934 1.4 37.5 3 5.190 1.1 19.8 4 6.009 1.2 12.1 m 6.743 0.5 5.3 5 6.902 na na 6 7.017 na na 7 7.240 0.9 4.7

[0060] Note: In Table 2, “ / ” means not applicable, and “na” means cannot be calculated. The same applies below.

[0061] Example 3

[0062] The detection conditions in this embodiment are different from those in embodiment 2 in terms of the elution procedure. The elution procedure in this embodiment is shown in Figure 5.

[0063] Table 5 Heating program of Example 3

[0064] Heating rate (℃ / min) Temperature(℃) Holding time (min) — 80 2 5 140 0 30 230 5

[0065] The test results are shown in Table 6 and Figure 3 shown. Figure 3 In the chromatographic peaks 1-7 are related substance peaks, and m is the main peak. Figure 3 It can be seen that this method detected a total of 7 related substances, the main peak and its adjacent impurities were not completely separated, and there was no effective separation between chromatographic peaks 5 and 6. It can be seen that this method is not suitable for the detection of related substances in ethyl 4-bromobutyrate.

[0066] Table 6 Example 3 test results

[0067] Serial number Retention time min Tailing Factor Separation 1 2.053 1.8 / 2 4.102 1.4 36.3 3 5.758 1.1 21.0 4 6.996 1.2 13.4 m 8.128 0.6 5.4 5 8.369 na na 6 8.464 na na 7 17.287 0.9 na

[0068] Example 4

[0069] The detection conditions in this embodiment are different from those in embodiment 2 in terms of the elution procedure. The elution procedure in this embodiment is shown in Figure 7.

[0070] Table 7 Heating program in Example 4

[0071] Heating rate (℃ / min) Temperature(℃) Holding time (min) — 60 2 30 90 0 10 140 0 30 230 5

[0072] The test results are shown in Table 8 and Figure 4 shown. Figure 4 In the chromatographic peaks 1-8 are related substance peaks, and m is the main peak. Figure 4 It can be seen that this method detected a total of 8 related substances, the peak shapes of related substance peak 1 and the main peak were poor, and related substance peak 6 appeared on the tailing peak of the main peak. It can be seen that this method is not suitable for the detection of related substances in ethyl 4-bromobutyrate.

[0073] Table 8 Test results in Example 4

[0074] Serial number Retention time min Tailing Factor Separation 1 2.177 2.1 / 2 4.666 1.0 59.5 3 6.003 1.1 31.0 4 6.085 1.1 1.7 m 7.125 0.5 6.5 5 7.374 1.0 2.5 6 7.568 1.0 4.6 7 11.509 1.0 104.9 8 12.949 1.0 37.3

[0075] Example 5

[0076] The detection conditions in this embodiment are different from those in embodiment 2 in terms of flow rate and elution procedure. In this embodiment, the flow rate is 1.0 ml / min, and the elution procedure is shown in Figure 9.

[0077] Table 9 Heating program in Example 5

[0078] Heating rate (℃ / min) Temperature(℃) Holding time (min) — 60 2 5 90 5 40 240 5.5

[0079] The test results are shown in Table 10 and Figure 5 shown. Figure 5 In the chromatographic peaks 1-8 are related substance peaks, and m is the main peak. Figure 5 It can be seen that this method detected 8 related substances in total, and the separation between the chromatographic peaks met the requirements, but the baseline drifted and was not stable. Therefore, this method is not suitable for the detection of related substances in ethyl 4-bromobutyrate.

[0080] Table 10 Washing temperature rise sequence in Example 5

[0081] Serial number Retention time min Tailing Factor Separation 1 2.769 1.3 / 2 7.177 1.0 87.5 3 10.722 1.1 44.1 4 10.924 1.2 1.8 5 11.432 1.1 3.0 m 14.047 0.5 12.7 6 14.436 1.0 3.3 7 18.200 1.0 80.9 8 19.768 1.0 40.2

[0082] Example 6

[0083] The detection conditions in this embodiment are different from those in Example 2 in terms of the elution procedure. The elution procedure in this embodiment is shown in Table 11.

[0084] Table 11 Heating program in Example 6

[0085] Heating rate (℃ / min) Temperature(℃) Holding time (min) — 40 2 5 140 0 30 230 5

[0086] The test results are shown in Table 12 and Figure 6 shown. Figure 6 In the chromatographic peaks 1-8 are related substance peaks, and m is the main peak. Figure 6 It can be seen that this method detected a total of 8 related substances, with a stable baseline, good peak shape, and high separation between chromatographic peaks, but the peak of related substance 8 appeared slightly later.

[0087] Table 12 Test results in Example 6

[0088] Serial number Retention time min Tailing Factor Separation 1 2.373 2.0 / 2 2.577 1.4 6.2 3 8.967 2.3 105.9 4 12.119 1.1 37.4 5 14.025 1.4 20.5 m 15.408 0.5 5.7 6 15.940 1.3 2.4 7 16.390 1.0 6.2 8 25.281 1.0 154.9

[0089] Example 7

[0090] The detection conditions in this embodiment are different from those in Example 2 in terms of the elution procedure. The elution procedure in this embodiment is shown in Table 13.

[0091] Table 13 Heating program in Example 7

[0092] Heating rate (℃ / min) Temperature(℃) Holding time (min) — 60 2 5 90 5 30 240 10

[0093] The test results are shown in Table 14 and Figure 7 shown. Figure 7 In the figure, A is the chromatogram of the whole detection process, and B is the enlarged view of the detection process 10-12min. Chromatographic peaks 1-8 are related substance peaks, and m is the main peak. From Table 14 and Figure 7It can be seen that this method detected a total of 8 related substances, with a stable baseline, good peak shapes of each chromatographic peak, appropriate retention time, and high separation between chromatographic peaks. This shows that this method is suitable for the detection of related substances in ethyl 4-bromobutyrate.

[0094] Table 14 Test results in Example 7

[0095] Serial number Retention time min Tailing Factor Separation 1 2.672 1.1 / 2 7.012 1.0 59.5 3 10.479 1.1 37.5 4 10.675 1.2 1.7 m 14.058 0.5 17.1 5 14.488 1.1 2.7 6 14.787 0.9 6.0 7 18.734 1.0 99.5 8 20.071 1.1 38.4

[0096] Example 8

[0097] Accurately weigh appropriate amounts of SM4 and each impurity reference substance, dissolve in DMF and dilute to make a mixed solution containing SM4 500 mg, Z-1 10 mg, Z-2 1 mg, Z-3 1 mg, Z-4 1.5 mg, and ethanol 2.5 mg per 1 ml as the system suitability solution.

[0098] Accurately weigh appropriate amounts of each impurity reference substance of SM4, add DMF to dissolve and dilute to make a solution containing 1 mg of SM4, 10 mg of Z-1, 1 mg of Z-2, 1 mg of Z-3, 1.5 mg of Z-4 and 2.5 mg of ethanol per 1 ml as the reference solution. Prepare two portions in parallel and record them as reference solution 1 and reference solution 2 respectively.

[0099] GC analysis was performed under the chromatographic conditions provided in Example 7. The system suitability investigation results are shown in Tables 15 to 17, wherein Table 15 is the system suitability solution test results, Table 16 is the test results of reference solution 1 after 5 consecutive injections, and Table 17 is the test results of reference solution 2.

[0100] Table 15 System suitability solution test results

[0101] name Retention time min Peak area Separation Theoretical plates Ethanol 2.702 182 / 24002 Z-1 7.095 694 51.6 60478 Z-2 9.896 65 28.0 175374 Z-3 10.535 84 6.4 156875 Z-4 12.400 82 14.3 99427 SM4 14.083 23211 8.2 30797

[0102] Table 16 Test results of reference solution 1

[0103]

[0104]

[0105] Table 17 Test results of reference solution 2

[0106]

[0107] It can be seen from Table 15 that in the system suitability solution, the separation degree between each known impurity and the adjacent peak is greater than 1.5, which meets the analysis requirements. It can be seen from Table 16 that in the reference solution 1, the separation degree between each impurity and the adjacent peak is greater than 1.5, and the theoretical plate number of the main peak and each related substance peak is greater than 5000. It can be seen from Table 17 that in the reference solution 2, the recovery rate of the response factor of each impurity relative to the average response factor of each impurity in the reference solution 1 is between 90.0 and 110.0%. It can be seen that the method specificity and system applicability provided in Example 7 are excellent.

[0108] Example 9

[0109] Accurately weigh an appropriate amount of each impurity reference substance, add a diluent to dissolve, and continuously dilute each impurity reference substance solution until the S / N is between 9 and 30 under the detection method provided in Example 7, which is the quantitative limit of each impurity. The quantitative limit investigation results are shown in Table 18.

[0110] Table 18 Sensitivity test results

[0111]

[0112] From the results in Table 18, it can be seen that the quantitative limit concentration of each impurity is less than 0.01%, and the quantitative limit repeatability is good, indicating that the method has high sensitivity.

[0113] Example 10

[0114] In this example, based on the method provided in Example 7, the flow rate was changed by 0.1 ml / min, the starting temperature was changed by 2° C., and different instruments and chromatographic columns were used to examine their durability.

[0115] Test solution: Same as Example 1.

[0116] The results of the durability test are shown in Table 19 (the chromatogram can be referred to Figure 7 ). It can be seen from Table 19 that under various conditions of the test solution, the RSD of the content of each related substance and the total content of related substances were all <10.0%, and the absolute difference of the content of related substances less than 0.2% was within ±0.02%, proving that the method has good durability.

[0117] Table 19 Chromatographic conditions durability test results (%)

[0118]

[0119] Note: In Table 19, the data under flow rate, starting temperature, original condition change instrument, and chromatographic column are the percentage content of each relevant substance in the test solution (calculated using the external standard method); each component is Figure 7 The chromatographic peaks represent the components, and “ / ” means not applicable.

[0120] In summary, the detection method of 4-bromobutyric acid ethyl ester-related substances provided by the present invention has the characteristics of stable baseline, good peak shape, high separation, excellent specificity and system applicability, high sensitivity and good durability, is suitable for the qualitative and quantitative detection of 4-bromobutyric acid ethyl ester, fills the gap in the prior art, and is of great significance to the quality control of 4-bromobutyric acid ethyl ester and the drug safety of elagolid sodium.

Claims

1. A method for detecting related substances in ethyl 4-bromobutyrate, characterized in that: Gas chromatography was used for detection, and the chromatographic conditions included: A weak polar chromatographic column is used; the weak polar chromatographic column is a capillary column with 5% phenyl-methyl polysiloxane as the stationary liquid or a capillary column with similar polarity; Use nitrogen as carrier gas with a flow rate of 0.7-1.5 ml / min; inject in split flow mode; The detector is a hydrogen flame ionization detector; The temperature control method of programmed heating is adopted, and the heating program is: The related substances include: cyclobutyrolactone, ethyl-4-ethoxybutyric acid, ethyl 2-bromobutyrate, ethyl 4-chlorobutyrate and ethanol.

2. The detection method according to claim 1, characterized in that: The heating program is:

3. The detection method according to claim 1, characterized in that: The heating program is:

4. The detection method according to any one of claims 1 to 3, characterized in that: The flow rate is 1.0 ml / min.

5. The detection method according to any one of claims 1 to 3, characterized in that: The split ratio is 60:

1.

6. The detection method according to any one of claims 1 to 3, characterized in that: The injection port temperature was 220°C; the detector temperature was 250°C.

7. The detection method according to any one of claims 1 to 3, characterized in that: The hydrogen flow rate was 40 ml / min, and the air flow rate was 400 ml / min.

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