Analysis method for detection and quality control of (S)-2-n-butyl hydroxybutyrate related substances

Abstract

The invention discloses an analysis method for detection and quality control of (S)-2-n-butyl hydroxybutyrate related substances, and belongs to the technical field of analytical chemistry. The method comprises the following steps: firstly, preparing a system applicability solution and a test solution; setting gas chromatography detection conditions, adopting an HP-1 chromatographic column, and using dimethyl polysiloxane as a capillary column; the system applicability solution and the test solution are taken respectively and injected into a gas chromatograph, a chromatogram is recorded, a standard curve is drawn, the content of impurities in (S)-2-n-butyl hydroxybutyrate is calculated, and the impurities in (S)-2-n-butyl hydroxybutyrate comprise (S)-2-hydroxybutyric acid, n-butyl alcohol, butyl lactate, butyl 2-butanone acid and butyl butyrate. The method disclosed by the invention is rapid, simple, convenient and effective, can separate a plurality of process impurities and degraded impurities from a main peak, fully meets the qualitative and quantitative analysis of each impurity in the product, effectively controls the product quality, and is simple and convenient to operate and good in repeatability.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and particularly relates to an analytical method for detecting and controlling the quality of related substances of (S)-2-hydroxybutyric acid n-butyl ester. Background Art

[0002] (S)-2-Hydroxybutanoic acid n-butyl ester, CAS: 132513-51-0, molecular formula: C8H 16 O3, molecular weight: 160.21, structural formula is shown in Formula I.

[0003]

[0004] (S)-2-Hydroxybutyric acid n-butyl ester is an optically active ester compound with broad application prospects in a variety of fields, including medicine, flavoring and food industries, chemical engineering and materials science, agriculture and pesticides, scientific research, and standard products. (S)-2-Hydroxybutyric acid n-butyl ester is sensitive to acids, bases, temperature, and oxidative conditions, and can participate in reactions such as ester hydrolysis, oxidation, and reduction. As one of the reactants in the Pemabet API, its quality is crucial to the API's purity and other indicators, and has a crucial impact on the safety of Pemabet preparations. To meet relevant regulations and pharmacopoeia requirements and ensure product quality, a detailed analysis and research of the related substances of (S)-2-Hydroxybutyric acid n-butyl ester was conducted.

[0005] according to Figure 1 (S)-2-hydroxybutyric acid n-butyl ester is synthesized by the reaction of (S)-2-hydroxybutyric acid with n-butanol. The product may contain residual reaction materials, reaction byproducts, degradation products, and other substances. Currently, no analysis or detection methods for related impurities of (S)-2-hydroxybutyric acid n-butyl ester have been reported in relevant journals or literature domestically or internationally. The present invention aims to provide an analytical method for the detection and quality control of related substances in (S)-2-hydroxybutyric acid n-butyl ester, further advancing the control level of compound purity monitoring. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an analytical method for the detection and quality control of related substances of (S)-2-hydroxybutyric acid n-butyl ester. The method is rapid, simple and effective, can separate multiple process impurities and degradation impurities from the main peak, fully meets the requirements of qualitative and quantitative analysis of various impurities in the product, effectively controls product quality, is easy to operate and has good repeatability.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] An analytical method for detecting and controlling the quality of (S)-n-butyl 2-hydroxybutyrate-related substances, using gas chromatography, comprises the following steps:

[0009] 1) Prepare system suitability solution and test sample solution and set aside;

[0010] 2) Setting gas chromatography detection conditions: using an HP-1 chromatographic column with 100% dimethylpolysiloxane as a capillary column;

[0011] 3) Take the system suitability solution and the test solution separately, inject them into the gas chromatograph, record the chromatogram, draw the standard curve, and calculate the impurity content of (S)-2-hydroxybutyric acid n-butyl ester;

[0012] The impurities in the (S)-2-hydroxybutyric acid n-butyl ester include (S)-2-hydroxybutyric acid, n-butanol, butyl lactate, butyl 2-ketobutyrate, and butyl butyrate.

[0013] Furthermore, in the step 1), the system suitability solution is prepared as follows: (S)-2-hydroxybutyric acid n-butyl ester and (S)-2-hydroxybutyric acid, n-butanol, butyl lactate, butyl 2-ketobutyrate, and butyl butyrate reference substances are taken in appropriate amounts, accurately weighed, dissolved in dichloromethane, and diluted to prepare a mixed solution containing 10 mg of (S)-2-hydroxybutyric acid n-butyl ester, 5 μg of butyl lactate, 80 μg of butyl 2-ketobutyrate, and 30 μg each of (S)-2-hydroxybutyric acid, n-butanol, and butyl butyrate per 1 mL.

[0014] Furthermore, in step 1), the test solution is prepared by taking an appropriate amount of (S)-2-hydroxybutyric acid n-butyl ester, accurately weighing it, adding dichloromethane to dissolve it and diluting it to prepare a solution containing approximately 10 mg per 1 mL.

[0015] Furthermore, in the step 2), the specification of the HP-1 chromatographic column is 25m×0.32mm, and the filler particle size is 0.52um.

[0016] Furthermore, in step 2), the temperature rising program is as follows: the initial column temperature is 70°C, the temperature is raised to 100°C at a rate of 10°C per minute, maintained for 5 minutes, and then the temperature is raised to 280°C at a rate of 30°C per minute, maintained for 3 minutes.

[0017] Furthermore, in step 2), the injection port temperature is 250°C and the detector temperature is 280°C.

[0018] Furthermore, the injection volume is 1 μL.

[0019] Furthermore, in step 2), the gas flow rate is 2 mL / min, and the carrier gas is nitrogen.

[0020] Furthermore, in step 2), the detector is a FID detector.

[0021] Furthermore, in the step 2), the split ratio is 50:1.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] The present invention provides an analytical method for detecting and controlling the quality of (S)-n-butyl 2-hydroxybutyrate-related substances. Gas chromatography is used to detect (S)-2-hydroxybutyric acid, n-butanol, butyl lactate, butyl 2-ketobutyrate, and butyl butyrate impurities in (S)-n-butyl 2-hydroxybutyrate, and good separation can be achieved. The method is simple, rapid, effective, and highly specific, thereby ensuring that the quality of (S)-n-butyl 2-hydroxybutyrate can be controlled. Currently, no method for detecting related impurities in (S)-n-butyl 2-hydroxybutyrate has been reported. The present invention can provide a reliable detection method for detecting related impurities in (S)-n-butyl 2-hydroxybutyrate, has a significant impact in the detection field, and is suitable for comprehensive promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The synthetic route of (S)-2-hydroxybutyric acid n-butyl ester in the background technology of this application;

[0025] Figure 2 This is a chromatogram of the solution for testing the suitability of the system in Example 1 of the present application;

[0026] Figure 3 This is a chromatogram of the test solution detected in Example 1 of the present application. DETAILED DESCRIPTION

[0027] The present invention will be further illustrated below with reference to specific examples. The examples are implemented based on the technical solutions of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0028] Example 1

[0029] An analytical method for detecting and controlling the quality of (S)-n-butyl 2-hydroxybutyrate-related substances comprises the following steps:

[0030] 1) Instrument and sample selection: The gas chromatograph model was Agilent 8890. The sample source information is detailed in Table 1.

[0031] Table 1 Sample information

[0032]

[0033]

[0034] 2) Set gas chromatography detection conditions: the chromatographic column is a capillary column made of 100% dimethylpolysiloxane (HP-1 25m×0.32mm, 0.52um or similar polarity); the temperature program is as follows: the initial column temperature is 70°C, the temperature is increased to 100°C at a rate of 10°C / min, maintained for 5 minutes, and then the temperature is increased to 280°C at a rate of 30°C / min, maintained for 3 minutes; the injection port temperature is 250°C; the detector is an FID detector; the detector temperature is 280°C; the carrier gas is nitrogen; the detector is an FID detector; the split ratio is 50:1; the gas flow rate is 2 mL / min; and the injection volume is 1 μL.

[0035] 3) Sample solution preparation

[0036] System suitability solution: Accurately weigh appropriate amounts of (S)-n-butyl 2-hydroxybutyrate and reference substances (Impurity 15, Impurity 16, Impurity 18, Impurity 19, and Impurity 20). Dissolve and dilute with dichloromethane to prepare a mixed solution containing 10 mg of (S)-n-butyl 2-hydroxybutyrate, 5 μg of Impurity 18, 80 μg of Impurity 19, and 30 μg each of Impurity 15, Impurity 16, and Impurity 20 per 1 mL.

[0037] Test solution: Take an appropriate amount of (S)-2-hydroxybutyric acid n-butyl ester, accurately weigh it, dissolve it in dichloromethane and dilute it to make a solution containing approximately 10 mg per 1 mL.

[0038] 4) Sample testing method

[0039] Take the above system suitability solution and test solution respectively, inject them into the gas chromatograph according to the chromatographic conditions described in the method, and record the chromatogram. Figures 2-3 shown.

[0040] Depend on Figure 2 From the system suitability solution chromatogram, it can be seen that the order of peaks is impurity 16, impurity 15, impurity 20, impurity 18, impurity 19, and (S)-2-hydroxybutyric acid n-butyl ester. The separation between the peaks of each component meets the requirements, and the number of theoretical plates calculated based on the (S)-2-hydroxybutyric acid n-butyl ester peak is not less than 5000.

[0041] Depend on Figure 3 From the chromatogram of the test sample solution, it can be seen that the retention time of the known impurity peaks detected in the sample is consistent with that of the impurity peaks in the system suitability solution. The known impurity peaks, unknown impurity peaks and main peak can be well separated, and the peak shape is good. The baseline in the chromatogram is stable and there is no interference. The method is suitable for the detection of related substances in the test sample.

[0042] Example 2

[0043] The detection method disclosed in Example 1 was verified from the aspects of specificity, limit of quantification, linearity and range, repeatability, intermediate precision, and solution stability, as follows:

[0044] 1. Exclusivity

[0045] Blank solvent: dichloromethane.

[0046] Impurity 15 mother liquor: Take an appropriate amount of Impurity 15 reference substance, accurately weigh it, dissolve it in dichloromethane and dilute it to make a solution containing approximately 3 mg per 1 mL.

[0047] Impurity 16 mother liquor: Take an appropriate amount of impurity 16 reference substance, accurately weigh it, dissolve it in dichloromethane and dilute it to make a solution containing approximately 3 mg per 1 mL.

[0048] Impurity 18 mother liquor: Take an appropriate amount of Impurity 18 reference substance, accurately weigh it, dissolve it in dichloromethane and dilute it to make a solution containing approximately 0.5 mg per 1 mL.

[0049] Impurity 19 mother liquor: Take an appropriate amount of impurity 19 reference substance, accurately weigh it, dissolve it in dichloromethane and dilute it to make a solution containing approximately 0.8 mg per 1 mL.

[0050] Impurity 20 mother liquor: Take an appropriate amount of Impurity 20 reference substance, accurately weigh it, dissolve it in dichloromethane and dilute it to make a solution containing approximately 3 mg per 1 mL.

[0051] Impurity 15 localization solution: Accurately measure an appropriate amount of 15 mother liquor and dilute it with dichloromethane to make a solution containing approximately 30 μg per 1 mL.

[0052] Impurity 16 localization solution: Accurately measure an appropriate amount of 16 mother liquor and dilute it with dichloromethane to make a solution containing approximately 30 μg per 1 mL.

[0053] Impurity 18 localization solution: Accurately measure an appropriate amount of 18 mother liquor and dilute it with dichloromethane to make a solution containing approximately 5 μg per 1 mL.

[0054] Impurity 19 localization solution: Accurately measure an appropriate amount of 19 mother liquor and dilute it with dichloromethane to make a solution containing approximately 80 μg per 1 mL.

[0055] Impurity 20 localization solution: Accurately measure an appropriate amount of 20 mother solution and dilute it with dichloromethane to make a solution containing approximately 30 μg per 1 mL.

[0056] (S)-2-Hydroxybutyric Acid n-Butyl Ester Reference Solution: Take an appropriate amount of (S)-2-Hydroxybutyric Acid n-Butyl Ester reference substance, accurately weigh it, dissolve it in dichloromethane, and dilute it to make a solution containing approximately 10 mg per 1 mL.

[0057] System suitability solution: Accurately weigh appropriate amounts of (S)-n-butyl 2-hydroxybutyrate and reference substances (Impurity 15, Impurity 16, Impurity 18, Impurity 19, and Impurity 20). Dissolve and dilute with dichloromethane to prepare a mixed solution containing approximately 10 mg of (S)-n-butyl 2-hydroxybutyrate, 5 μg of Impurity 18, 80 μg of Impurity 19, and 30 μg each of Impurities 15, 16, and 20 per mL.

[0058] Test solution: Take an appropriate amount of (S)-2-hydroxybutyric acid n-butyl ester, accurately weigh it, and dissolve and dilute it with dichloromethane to make a solution containing approximately 10 mg per 1 mL.

[0059] The above solvent, positioning solution, system suitability solution and test solution were accurately measured respectively, and the test was carried out according to the chromatographic conditions of Example 1. The results are shown in Table 2.

[0060] Table 2 Impurity location and separation test results

[0061]

[0062]

[0063] As can be seen from Table 2, the order of the peaks of each component is impurity 16, impurity 15, impurity 20, impurity 18, impurity 19, and (S)-2-hydroxybutyric acid n-butyl ester. The separation between the peaks of each component is greater than 1.5, and the theoretical plate number of the main peak is greater than 5000, which meets the requirements; the blank solvent does not affect the detection of related substances in the sample; in the test solution, the theoretical plate number of the impurity peaks detected is high and has good separation, indicating that the specificity of this method is good.

[0064] 2. Limit of Quantitation

[0065] Take appropriate amounts of (S)-2-hydroxybutyric acid n-butyl ester and impurity 16, impurity 15, impurity 20, impurity 18, and impurity 19 reference substances, accurately weigh them, dissolve them in dichloromethane and dilute them to make solutions of certain concentrations. Dilute them stepwise and inject them. The limit of quantification is when the signal-to-noise ratio (S / N) is ≈10. The results are shown in Table 3.

[0066] Table 3 Quantitation limit test results

[0067]

[0068] As shown in Table 3, the limits of quantification for (S)-n-butyl 2-hydroxybutyrate, impurity 16, impurity 15, impurity 20, impurity 18, and impurity 19 were 0.4188 μg / mL, 1.887 μg / mL, 17.54 μg / mL, 3.501 μg / mL, 0.2289 μg / mL, and 0.3045 μg / mL, respectively. These concentrations were all less than 0.05% of the test sample concentration (10 mg / mL). This indicates that the test sample concentration of 10 mg / mL selected for this method is appropriate, the method has high sensitivity, and meets the validation requirements.

[0069] 3. Linearity and range

[0070] Mother liquors of impurities 15, 16, 18, and 20: Prepare in the same manner as the mother liquors of each impurity under the “Specificity” item.

[0071] Linearization stock solution: Take approximately 1000 mg of (S)-2-hydroxybutyric acid n-butyl ester reference substance and approximately 8 mg of impurity 19 reference substance, accurately weigh, place in a 10 mL volumetric flask, add appropriate amount of dichloromethane to dissolve, then accurately add 1 mL each of the above impurity 15, 16, 18, and 20 stock solutions, dilute to the mark with dichloromethane, and shake well. (100 mg / mL (S)-2-hydroxybutyric acid n-butyl ester, 0.05 mg / mL impurity 18, 0.8 mg / mL impurity 19, 0.3 mg / mL impurity 15, 0.3 mg / mL impurity 16, and 0.3 mg / mL impurity 20)

[0072] Linear series solutions: Take appropriate amounts of the linear mother solution and dilute with dichloromethane to prepare a series of mixed solutions with concentrations ranging from the quantitative limit of each component to 120% of the impurity limit.

[0073] Take the linear series solution, inject it into the gas chromatograph, record the peak area, and perform linear regression with concentration as the horizontal axis and peak area as the vertical axis. The results are shown in Table 4.

[0074] Table 4 Linearity and correction factor test results

[0075]

[0076] As shown in Table 4, the linear correlation coefficients of (S)-2-hydroxybutyric acid n-butyl ester, impurity 16, impurity 15, impurity 20, impurity 18, and impurity 19 were all greater than 0.998 in the concentration range from the limit of quantification to 120% of the limit concentration of the test sample, and the intercept was less than 20% of the y value at the limit concentration, indicating that the components had a good linear relationship under the detection conditions.

[0077] 4. Repeatability

[0078] Test solution: Take an appropriate amount of (S)-2-hydroxybutyric acid n-butyl ester, accurately weigh it, dissolve it in dichloromethane and dilute it to make a solution containing approximately 10 mg per 1 mL.

[0079] Six test solutions were prepared in parallel and tested according to the chromatographic conditions of Example 1. The results are shown in Table 5.

[0080] Table 5 Repeatability test results

[0081]

[0082] As shown in Table 5, the number of impurities in the 6 parallel preparations of the test solution was basically the same, and there was no significant difference in the sample purity, the known impurities, and the maximum unknown single impurity content, indicating that this detection method has good repeatability.

[0083] 5. Intermediate precision

[0084] Test solution: Prepare the same as the test solution in “Repeatability”.

[0085] The test was conducted by different test personnel at different test times on different test instruments according to the chromatographic conditions of Example 1, and the impurity content in the test solution was calculated as the RSD value. The results are shown in Table 6.

[0086] Table 6 Intermediate precision test results

[0087]

[0088]

[0089] As shown in Table 6, the 12 test sample solutions tested by different test personnel on different test dates using different test instruments had basically the same number of impurities, and there was no significant difference in sample purity, known impurities, and maximum unknown single impurity content, indicating that the intermediate precision of this test method is good.

[0090] 6. Solution stability

[0091] System suitability solution: Prepare the same as the system suitability solution in “Specificity”.

[0092] Test solution: Prepare the same as the test solution in “Specificity”.

[0093] The system suitability solution and the test solution were taken separately, and the test was carried out according to the chromatographic conditions of Example 1. The results were observed at different time points. The results are shown in Tables 7 and 8.

[0094] Table 7 System suitability solution stability test results

[0095]

[0096]

[0097] Table 8 Test solution stability test results

[0098]

[0099] As shown in Table 7, when the system suitability solution was placed at room temperature for 24 hours, the peak areas of (S)-2-hydroxybutyric acid n-butyl ester and various impurities did not change significantly, and the solution had good stability.

[0100] As shown in Table 8, when the test solution was placed at room temperature for 24 hours, the main peak purity, the content of known impurities, unknown impurities and the number of impurities did not change significantly, and the solution stability was good.

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An analytical method for the detection and quality control of related substances of (S)-n-butyl 2-hydroxybutyrate, characterized by: The gas chromatography method includes the following steps: 1) Prepare system suitability solution and test sample solution and set aside; 2) Setting gas chromatography detection conditions: using an HP-1 chromatographic column with dimethylpolysiloxane as the capillary column; 3) Take the system suitability solution and the test solution separately, inject them into the gas chromatograph, record the chromatogram, draw the standard curve, and calculate the impurity content of (S)-2-hydroxybutyric acid n-butyl ester; The impurities in the (S)-2-hydroxybutyric acid n-butyl ester include (S)-2-hydroxybutyric acid, n-butanol, butyl lactate, butyl 2-ketobutyrate, and butyl butyrate.

2. The analytical method for related substance detection and quality control of (S)-2-hydroxybutyric acid n-butyl ester according to claim 1, characterized in that: In the step 1), the system suitability solution is prepared by taking appropriate amounts of (S)-n-butyl 2-hydroxybutyrate and (S)-2-hydroxybutyric acid, n-butanol, butyl lactate, butyl 2-ketobutyrate, and butyl butyrate reference substances, accurately weighing them, adding dichloromethane to dissolve and dilute them to prepare a mixed solution containing 10 mg of (S)-n-butyl 2-hydroxybutyrate, 5 μg of butyl lactate, 80 μg of butyl 2-ketobutyrate, and 30 μg each of (S)-2-hydroxybutyric acid, n-butanol, and butyl butyrate per 1 mL.

3. The analytical method for detection and quality control of related substances of (S)-2-hydroxybutyric acid n-butyl ester according to claim 1, characterized in that: In step 1), the test solution is prepared by taking an appropriate amount of (S)-2-hydroxybutyric acid n-butyl ester, accurately weighing it, adding dichloromethane to dissolve it and diluting it to make a solution containing approximately 10 mg per 1 mL.

4. The analytical method for detection and quality control of related substances of (S)-2-hydroxybutyric acid n-butyl ester according to claim 1, characterized in that: In the step 2), the specification of the HP-1 chromatographic column is 25m×0.32mm, and the filler particle size is 0.52um.

5. The analytical method for detection and quality control of related substances of (S)-2-hydroxybutyric acid n-butyl ester according to claim 1, characterized in that: In step 2), the temperature program is as follows: the initial column temperature is 70°C, the temperature is increased to 100°C at a rate of 10°C per minute, maintained for 5 minutes, and then the temperature is increased to 280°C at a rate of 30°C per minute, and maintained for 3 minutes.

6. The analytical method for detection and quality control of related substances of (S)-2-hydroxybutyric acid n-butyl ester according to claim 1, characterized in that: In the step 2), the injection port temperature is 250°C and the detector temperature is 280°C.

7. The analytical method for detection and quality control of related substances of (S)-n-butyl 2-hydroxybutyrate according to claim 1, characterized in that: The injection volume was 1 μL.

8. The analytical method for detection and quality control of related substances of (S)-2-hydroxybutyric acid n-butyl ester according to claim 1, characterized in that: In the step 2), the gas flow rate is 2 mL / min, and the carrier gas is nitrogen.

9. The analytical method for detection and quality control of related substances of (S)-2-hydroxybutyric acid n-butyl ester according to claim 1, characterized in that: In the step 2), the detector is a FID detector.

10. The analytical method for detection and quality control of related substances of (S)-2-hydroxybutyric acid n-butyl ester according to claim 1, characterized in that: In the step 2), the split ratio is 50:1.

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