Separation and detection method for optical isomer (R)-2-hydroxybutyrate n-butyl ester in (S)-2-hydroxybutyrate n-butyl ester

Abstract

The invention discloses a method for separating and detecting an optical isomer (R)-2-hydroxybutyrate n-butyl ester in (S)-2-hydroxybutyrate n-butyl ester, and belongs to the technical field of analytical chemistry. The method comprises the following steps: firstly, preparing a system applicability solution and a reference substance solution for later use; and then setting gas chromatography detection conditions, and detecting the optical isomer of the (S)-2-n-butyl hydroxybutyrate by adopting a CycSil-B chromatographic column and using a capillary column with the modified dimethylsilyl-beta-cyclodextrin as a stationary phase. The gas chromatography is adopted, the content of the optical isomer (R)-2-n-butyl hydroxybutyrate in (S)-2-n-butyl hydroxybutyrate can be rapidly detected, meanwhile, the method is high in specificity, good in separation degree, high in sensitivity and good in repeatability, the R-configuration enantiomer and the S-configuration enantiomer of 2-n-butyl hydroxybutyrate can be well separated, and the method is suitable for industrial production. Therefore, the quality of the Pinefibrate bulk drug is ensured.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry, and particularly relates to a method for separating and detecting the optical isomer (R)-2-hydroxybutyric acid n-butyl ester in (S)-2-hydroxybutyric acid n-butyl ester. Background Art

[0002] (S)-2-Hydroxybutyric acid n-butyl ester is a colorless to light yellow oily substance, with the molecular formula: C8H 16 O3, molecular weight: 160.21, and the structural formula is shown in Formula I. It is generally formed by the esterification reaction of (S)-2-hydroxybutyric acid and n-butanol under the catalysis of concentrated sulfuric acid. The synthesis route is as Figure 1 shown. Due to the presence of a chiral carbon atom, there is often a small amount of the R-type enantiomer (R)-2-hydroxybutyric acid n-butyl ester in (S)-2-hydroxybutyric acid n-butyl ester, and the structural formula is shown in Formula II.

[0003]

[0004] (S)-2-Hydroxybutyric acid n-butyl ester is an optically active ester compound and has broad application prospects in many fields such as the pharmaceutical field, the fragrance and food industry, chemical engineering and materials science, agriculture and pesticides. In the pharmaceutical field, (S)-2-hydroxybutyric acid n-butyl ester is one of the key chiral starting materials for the raw material drug pemafibrate. Pemafibrate is used to treat hyperlipidemia (including familial hyperlipidemia) and is a new type of highly selective PPARα regulator. It binds to PPARα and regulates the expression of genes involved in lipid metabolism, thereby reducing the plasma triglyceride (TG) level and increasing the high-density lipoprotein cholesterol (HDL-C) level.

[0005] The synthesis route of pemafibrate generally involves the activation of (S)-2-hydroxybutyric acid n-butyl ester with trifluoromethanesulfonic anhydride, and then the SN2 nucleophilic substitution reaction and hydrolysis reaction with compound 1 (3-[[2-benzoxazolyl[3-(4-methoxyphenoxy)propyl]amino]methyl]phenol). The synthesis route is as Figure 2 shown. And a small amount of the R-type enantiomer (R)-2-hydroxybutyric acid n-butyl ester present in (S)-2-hydroxybutyric acid n-butyl ester will also participate in this series of reactions, and the reaction generates the enantiomeric impurity of pemafibrate. The structural formulas of pemafibrate and its enantiomeric impurity are shown in Formulas III and IV.

[0006]

[0007] Therefore, the optical purity of (S)-2-hydroxybutyric acid n-butyl ester will directly affect the optical purity of pemafibrate API, which plays a crucial role in the medication safety of pemafibrate preparations. To ensure the quality of drugs, it is necessary to control the content of a small amount of the optical isomer (R)-2-hydroxybutyric acid n-butyl ester that may exist in (S)-2-hydroxybutyric acid n-butyl ester from the source. At present, there is no report on the optical isomer (R)-2-hydroxybutyric acid n-butyl ester in (S)-2-hydroxybutyric acid n-butyl ester and its detection method. Therefore, through gas chromatography (GC method), this patent aims to provide a detection method with high sensitivity, good resolution, and low matrix interference to monitor the optical purity of (S)-2-hydroxybutyric acid n-butyl ester, providing technical guarantee for the control of enantiomeric impurities of pemafibrate and its preparations from the source. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a separation and detection method for the optical isomer (R)-2-hydroxybutyric acid n-butyl ester in (S)-2-hydroxybutyric acid n-butyl ester, which is fast, simple, effective, highly specific, has good resolution, high sensitivity, and good repeatability.

[0009] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0010] A separation and detection method for the optical isomer (R)-2-hydroxybutyric acid n-butyl ester in (S)-2-hydroxybutyric acid n-butyl ester, characterized in that: gas chromatography is adopted, including the following steps:

[0011] 1) Prepare a system suitability solution and a test solution for standby;

[0012] 2) Set the gas chromatography detection conditions: use a CycloSil-B chromatographic column, a capillary column with modified dimethylsilyl-β-cyclodextrin as the stationary phase;

[0013] 3) Take the system suitability solution and the test solution respectively, inject them into the gas chromatography, and record the chromatogram.

[0014] Further, in the step 1), the solvents used for the system suitability solution and the test solution are dichloromethane.

[0015] Further, in the step 1), the preparation of the system suitability solution: take appropriate amounts of the reference substances of (S)-2-hydroxybutyric acid n-butyl ester and (R)-2-hydroxybutyric acid n-butyl ester, accurately weigh them, dissolve and dilute with dichloromethane to prepare a mixed solution containing about 1 mg of (S)-2-hydroxybutyric acid n-butyl ester and 10 μg of (R)-2-hydroxybutyric acid n-butyl ester per 1 mL, and shake well to obtain.

[0016] Further, in the step 1), preparation of the test solution: Take an appropriate amount of (S)-butyl 2-hydroxybutyrate, weigh it accurately, dissolve it in dichloromethane and dilute to make a solution containing about 0.5 mg per 1 mL, and shake well to obtain.

[0017] Further, in the step 2), the specifications of the CycloSil-B chromatographic column are 30 m × 0.25 mm, and the particle size of the packing is 0.25 μm.

[0018] Further, in the step 2), the programmed temperature rise is as follows: the initial column temperature is 80 °C, maintained for 5 minutes, and then heated to 200 °C at a rate of 5 °C per minute.

[0019] Further, in the step 2), the inlet temperature is 240 °C, and the detector temperature is 250 °C.

[0020] Further, in the step 2), the injection volume is 1 μL.

[0021] Further, in the step 2), the flow rate is 1 mL / min, and the carrier gas is nitrogen.

[0022] Further, in the step 2), the detector is an FID detector, and the split ratio is 5:1.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] The present invention provides a method for separating and detecting the optical isomer (R)-butyl 2-hydroxybutyrate in (S)-butyl 2-hydroxybutyrate. By using gas chromatography, it can not only quickly detect the content of the optical isomer (R)-butyl 2-hydroxybutyrate in (S)-butyl 2-hydroxybutyrate; at the same time, the method of the present invention has strong specificity, good resolution, can well separate the two enantiomers of the R configuration and S configuration of butyl 2-hydroxybutyrate, high sensitivity, good repeatability, and can accurately quantify the trace amount of (R)-butyl 2-hydroxybutyrate in (S)-butyl 2-hydroxybutyrate, thereby ensuring the quality of pemafibrate raw materials. Description of the Drawings

[0025] Figure 1 It is the synthetic route diagram of (S)-butyl 2-hydroxybutyrate in the background technology of this application;

[0026] Figure 2 It is the synthetic route diagram of pemafibrate in the background technology of this application;

[0027] Figure 3 It is the chromatogram of the blank solution detected in Example 1 of this application;

[0028] Figure 4 It is the chromatogram of the system suitability solution detected in Example 1 of this application;

[0029] Figure 5 This is the chromatogram for detecting the test sample solution in Example 1 of this application. Detailed implementation manners

[0030] The following further clarifies the present invention in conjunction with specific embodiments. The embodiments are implemented on the premise of the technical solution of the present invention. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0031] Example 1

[0032] A method for separating and detecting the optical isomers of (S)-2-hydroxybutyric acid n-butyl ester, comprising the following steps:

[0033] 1) Instrument and sample selection: The model of the GC is Agilent 8890 GC System; the batch number of (S)-2-hydroxybutyric acid n-butyl ester is 20240501, sourced from Tianjin Famosi; the batch number of (R)-2-hydroxybutyric acid n-butyl ester is 24-09-080063, sourced from RKD.

[0034] 2) Set the gas chromatography detection conditions: The chromatographic column is a capillary column with modified dimethylsilyl-β-cyclodextrin (i.e., adding 30% hepta-(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin in 14% cyanopropylphenyl, 86% methyl polysiloxane) (CycloSil-B 30m × 0.25mm, 0.25um or similar polarity) as the stationary phase; the temperature programming is that the initial column temperature is 80°C, maintained for 5 minutes, and then heated to 200°C at a rate of 5°C per minute; the injection port temperature is 240°C; the detector temperature is 250°C; the injection volume is 1 μL; the running time is 25 min; the carrier gas is nitrogen; the detector is an FID detector; the split ratio is 5:1; the flow rate is 1 mL / min.

[0035] 3) Preparation of the sample solution

[0036] Blank solution: Dichloromethane;

[0037] System suitability solution: Take appropriate amounts of the reference substances of (S)-2-hydroxybutyric acid n-butyl ester and (R)-2-hydroxybutyric acid n-butyl ester, weigh accurately, dissolve and dilute with dichloromethane to prepare a mixed solution containing about 1 mg of (S)-2-hydroxybutyric acid n-butyl ester and 10 μg of (R)-2-hydroxybutyric acid n-butyl ester per 1 mL, and shake well to obtain;

[0038] Test sample solution: Take an appropriate amount of (S)-2-hydroxybutyric acid n-butyl ester, weigh accurately, dissolve and dilute with dichloromethane to prepare a solution containing about 0.5 mg per 1 mL, and shake well to obtain.

[0039] 4) Sample detection method

[0040] Respectively take the above blank solution, system suitability solution, and test solution, inject them into the gas chromatograph according to the chromatographic conditions described in the method, and record the chromatograms. The chromatograms are as Figures 3 - 5 shown.

[0041] From Figure 3 the chromatogram of the blank solution, it can be seen that the solvent does not peak at the peaks of (S)-butyl 2-hydroxybutyrate and (R)-butyl 2-hydroxybutyrate, and there is no interference in the detection of (S)-butyl 2-hydroxybutyrate and (R)-butyl 2-hydroxybutyrate.

[0042] From Figure 4 the chromatogram of the system suitability solution, it can be seen that the resolution between the peaks of (S)-butyl 2-hydroxybutyrate and (R)-butyl 2-hydroxybutyrate meets the requirements, and the theoretical plate number calculated based on the peak of (S)-butyl 2-hydroxybutyrate is not less than 5000.

[0043] From Figure 5 the chromatogram of the test solution, it can be seen that the peaks of (S)-butyl 2-hydroxybutyrate and (R)-butyl 2-hydroxybutyrate in the sample are consistent with those in the system suitability solution, meeting the requirements for quantitative detection.

[0044] Example 2

[0045] For the detection method disclosed in Example 1, it is verified from aspects of system suitability, specificity, detection limit and quantitation limit, repeatability, linearity and range, intermediate precision, accuracy, and durability as follows:

[0046] 1. System suitability

[0047] System suitability solution: Take appropriate amounts of reference substances of (S)-butyl 2-hydroxybutyrate and (R)-butyl 2-hydroxybutyrate, weigh accurately, dissolve and dilute with dichloromethane to prepare a mixed solution containing about 1 mg of (S)-butyl 2-hydroxybutyrate and 10 μg of (R)-butyl 2-hydroxybutyrate per 1 mL, and shake well to obtain.

[0048] According to the chromatographic conditions in Example 1 above, after the system is balanced, inject the system suitability solution 6 times and record the chromatograms. The results are shown in Table 1 below.

[0049] Table 1 Results of system suitability test

[0050]

[0051] As can be seen from the results in Table 1, the resolution between the peaks of (S)-butyl 2-hydroxybutyrate and (R)-butyl 2-hydroxybutyrate is greater than 1.5, and the theoretical plate numbers are both much greater than 5000, all meeting the requirements, indicating that the separation of the method of the present invention is good.

[0052] 2. Specificity

[0053] Blank solvent: Dichloromethane;

[0054] (R)-butyl 2-hydroxybutyrate positioning solution: Take an appropriate amount of (R)-butyl 2-hydroxybutyrate reference substance, weigh accurately, dissolve and dilute with dichloromethane to prepare a solution containing about 10 μg of (R)-butyl 2-hydroxybutyrate per 1 mL, and shake well to obtain;

[0055] (S)-butyl 2-hydroxybutyrate positioning solution: Take an appropriate amount of (S)-butyl 2-hydroxybutyrate reference substance, weigh accurately, dissolve and dilute with dichloromethane to prepare a solution containing about 1 mg of (S)-butyl 2-hydroxybutyrate per 1 mL, and shake well to obtain;

[0056] System suitability solution: Take appropriate amounts of (S)-butyl 2-hydroxybutyrate and (R)-butyl 2-hydroxybutyrate reference substances, weigh accurately, dissolve and dilute with dichloromethane to prepare a mixed solution containing about 1 mg of (S)-butyl 2-hydroxybutyrate and 10 μg of (R)-butyl 2-hydroxybutyrate per 1 mL, and shake well to obtain;

[0057] Test solution: Take an appropriate amount of (S)-butyl 2-hydroxybutyrate, weigh accurately, dissolve and dilute with dichloromethane to prepare a solution containing 0.5 mg per 1 mL, and shake well to obtain;

[0058] According to the chromatographic conditions in Example 1 above, after the system suitability is qualified, take the blank solvent, (R)-butyl 2-hydroxybutyrate positioning solution, (S)-butyl 2-hydroxybutyrate positioning solution, system suitability solution, and test solution, inject each 1 injection, record the chromatogram, and the results are shown in Table 2 below.

[0059] Table 2 Results of specificity test

[0060]

[0061] As can be seen from the results in Table 2, the blank solvent has no interference at the elution position of the analyte; the retention times of the analyte in the test solution and the positioning solution are consistent, indicating that the method of the present invention has good specificity.

[0062] 3. Detection limit and quantitation limit

[0063] Mixed reference solution 1: Weigh appropriate amounts of the reference substances of (S)-butyl 2-hydroxybutyrate and (R)-butyl 2-hydroxybutyrate accurately, dissolve in dichloromethane and dilute to prepare a mixed solution containing about 10 μg of (S)-butyl 2-hydroxybutyrate and 10 μg of (R)-butyl 2-hydroxybutyrate per 1 mL. Shake well to obtain.

[0064] Mixed reference solution 2: Pipette 1 mL of mixed reference solution 1 accurately into a 100-mL volumetric flask, dilute to the mark with dichloromethane, and shake well to obtain.

[0065] Quantitation limit solution: Pipette 2 mL of mixed reference solution 2 into a 10-mL volumetric flask, dilute to the mark with dichloromethane, and shake well to obtain.

[0066] Detection limit solution: Pipette 1 mL of mixed reference solution 2 into a 10-mL volumetric flask, dilute to the mark with dichloromethane, and shake well to obtain.

[0067] After the system suitability is qualified, operate according to the chromatographic conditions in Example 1 above. Inject the quantitation limit solution and the detection limit solution respectively, record the chromatogram, and the results are shown in Tables 3 to 4 below.

[0068] Table 3 Results of quantitation limit test

[0069] compound sample weight (mg) peak area S / N concentration (μg / mL) (R)-butyl 2-hydroxybutyrate 10.33 0.058 173.8 0.0206 (S)-butyl 2-hydroxybutyrate 10.02 0.046 159.7 0.0200

[0070] Table 4 Results of detection limit test

[0071] compound sample weight (mg) peak area S / N concentration (μg / mL) (R)-butyl 2-hydroxybutyrate 10.33 0.029 52.3 0.0103 (S)-butyl 2-hydroxybutyrate 10.02 0.023 49.5 0.0100

[0072] It can be seen from the results in Table 3 and Table 4 above that the signal-to-noise ratio of the analyte in the detection limit solution is ≥ 3, and the signal-to-noise ratio of the analyte in the quantitation limit solution is ≥ 10. Both the detection limit and the quantitation limit meet the detection requirements.

[0073] 4. Repeatability

[0074] Test solution: Weigh appropriate amount of (S)-butyl 2-hydroxybutyrate accurately, dissolve in dichloromethane and dilute to prepare a solution containing about 0.5 mg per 1 mL. Shake well to obtain, and prepare 6 portions in parallel.

[0075] After the system suitability is qualified, operate according to the chromatographic conditions in Example 1 above. Inject 6 portions of the test solution, one injection for each, record the chromatogram, and the results are shown in Table 5 below.

[0076] Table 5 Results of repeatability test

[0077]

[0078]

[0079] As can be seen from the results in Table 5, the RSD of the analyte content in the 6 repeatability solutions was 1.80%, RSD ≤ 15.0%, indicating good repeatability.

[0080] 5. Linearity and Range

[0081] Linear stock solution: Weigh an appropriate amount of the reference substance of (R)-2-hydroxybutyric acid n-butyl ester accurately, dissolve it in dichloromethane and dilute to make a mixed solution containing about 100 μg of (R)-2-hydroxybutyric acid n-butyl ester per 1 mL, and shake well to obtain;

[0082] 150% linear solution: Pipette 1.5 mL of the linear stock solution into a 10 mL volumetric flask, dilute to the mark with dichloromethane, and shake well to obtain;

[0083] 100% linear solution: Pipette 1.0 mL of the linear stock solution into a 10 mL volumetric flask, dilute to the mark with dichloromethane, and shake well to obtain;

[0084] 50% linear solution: Pipette 0.5 mL of the linear stock solution into a 10 mL volumetric flask, dilute to the mark with dichloromethane, and shake well to obtain;

[0085] 10% linear solution: Pipette 0.1 mL of the linear stock solution into a 10 mL volumetric flask, dilute to the mark with dichloromethane, and shake well to obtain;

[0086] 1% linear solution: Pipette 1.0 mL of the 10% linear solution into a 10 mL volumetric flask, dilute to the mark with dichloromethane, and shake well to obtain.

[0087] After the system suitability is qualified, operate according to the chromatographic conditions in Example 1 above. Inject 1 injection of each linear solution respectively, record the chromatogram, and the results are shown in Table 6 below.

[0088] Table 6 Results of linearity test

[0089]

[0090] As can be seen from the results in Table 6, the concentration of (R)-2-hydroxybutyric acid n-butyl ester showed a linear relationship in the range of 0.1062 μg / mL - 15.93 μg / mL. The linear equation was y = 1.9706x - 0.0698, and the r value was 0.9998, meeting the requirements.

[0091] 6. Intermediate precision

[0092] Test solution: Weigh an appropriate amount of (S)-2-hydroxybutyric acid n-butyl ester accurately, dissolve it in dichloromethane and dilute to make a solution containing about 0.5 mg per 1 mL, and shake well to obtain. Prepare 6 portions in parallel.

[0093] The test was carried out by different people on different days with different instruments. After the system suitability was qualified, the chromatographic conditions of Example 1 were followed. Six portions of the test solution were taken and injected into each portion. The chromatograms were recorded. The results are shown in Table 7 below. The RSD values of a total of 12 samples were calculated together with the repeatability test results, which should meet the requirements.

[0094] Table 7 Intermediate precision test results

[0095]

[0096] From the results in Table 7, it can be seen that the RSD of the analyte content in the 12 intermediate precision samples is 2.78%, RSD≤15.0%, and the intermediate precision is good.

[0097] 8. Accuracy

[0098] Reference solution: Take an appropriate amount of (R)-2-hydroxybutyric acid n-butyl ester reference substance, weigh accurately, add dichloromethane to dissolve and dilute to make a mixed solution containing about 100 μg (R)-2-hydroxybutyric acid n-butyl ester per 1 mL, and shake well to obtain;

[0099] Test solution: Take an appropriate amount of (S)-2-hydroxybutyric acid n-butyl ester, weigh accurately, dissolve in dichloromethane and dilute to make a solution containing about 0.5 mg per 1 mL, shake well to obtain;

[0100] Accuracy solution: Weigh 10 mg of (S)-2-hydroxybutyric acid n-butyl ester, accurately weigh it, place it in a 20 mL volumetric flask, add 2 mL of the reference solution, then add dichloromethane to dissolve and dilute to the scale, shake well, and prepare 6 copies in parallel;

[0101] After the system suitability is qualified, operate according to the chromatographic conditions of Example 1 above, take the test sample and 6 portions of accuracy solution into one needle each, record the chromatogram, and the results are shown in Table 8 below.

[0102] Table 8 Accuracy test results

[0103]

[0104]

[0105] From the results in Table 8, it can be seen that the individual recoveries of the analytes in the six accuracy solutions ranged from 90.0% to 110.0%, and the RSD of the overall recovery was 4.02%, which met the requirements, indicating that the method had good accuracy.

[0106] 8. Durability

[0107] In Comparative Examples 1 to 8, except for the following conditions, other parameters and operations are the same as the chromatographic conditions of Example 1. The specific test conditions and test results are shown in Table 9.

[0108] Blank solvent: dichloromethane;

[0109] System suitability solution: Weigh appropriate amounts of reference substances of (S)-butyl 2-hydroxybutyrate and (R)-butyl 2-hydroxybutyrate accurately, dissolve them in dichloromethane, and prepare a mixed solution containing about 1 mg of (S)-butyl 2-hydroxybutyrate and 10 μg of (R)-butyl 2-hydroxybutyrate per 1 mL. Shake well to obtain the solution.

[0110] Test solution: Weigh an appropriate amount of (S)-butyl 2-hydroxybutyrate accurately, dissolve it in dichloromethane and dilute to prepare a solution containing about 0.5 mg per 1 mL. Shake well to obtain the solution.

[0111] Operate according to the chromatographic conditions in Comparative Examples 1 to 8. Inject the system suitability solution and the test solution, record the chromatogram, and the results are shown in Table 9.

[0112] Table 9 Test results of Comparative Examples 1 to 8

[0113]

[0114] It can be seen from the results in Table 9 that under the chromatographic conditions of the present invention, by slightly adjusting chromatographic parameters such as the initial column temperature, the heating rate, the injector temperature, and the detector temperature, and using the adjusted detection conditions to detect the content of the optical isomer (R)-butyl 2-hydroxybutyrate in (S)-butyl 2-hydroxybutyrate, it can be accurately detected. Therefore, the method of the present invention has good durability.

[0115] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for separating and detecting the optical isomer (R)-butyl 2-hydroxybutyrate in (S)-butyl 2-hydroxybutyrate, characterized in that: Using gas chromatography, it includes the following steps: 1) Prepare the system suitability solution and the test solution for standby; 2) Set the gas chromatography detection conditions: Use a CycloSil-B chromatographic column, a capillary column with modified dimethylsilyl-β-cyclodextrin as the stationary phase; 3) Respectively take the system suitability solution and the test solution, inject them into the gas chromatography, and record the chromatogram.

2. The method for separating and detecting the optical isomer (R)-2-hydroxybutyric acid n-butyl ester in (S)-2-hydroxybutyric acid n-butyl ester according to claim 1, wherein: In step 1), the solvent used for the system suitability solution and the test solution is dichloromethane.

3. The method for separating and detecting the optical isomer (R)-butyl 2-hydroxybutyrate in (S)-butyl 2-hydroxybutyrate according to claim 1, characterized in that: In step 1), the preparation of the system suitability solution: Take appropriate amounts of the reference substances of (S)-2-hydroxybutyric acid n-butyl ester and (R)-2-hydroxybutyric acid n-butyl ester, accurately weigh them, dissolve them in dichloromethane and dilute to make a mixed solution containing about 1 mg of (S)-2-hydroxybutyric acid n-butyl ester and 10 μg of (R)-2-hydroxybutyric acid n-butyl ester per 1 mL, and shake well to obtain.

4. The method for separating and detecting the optical isomer (R)-2-hydroxybutyric acid n-butyl ester in (S)-2-hydroxybutyric acid n-butyl ester according to claim 2, characterized in that: In step 1), the preparation of the test solution: Take an appropriate amount of (S)-2-hydroxybutyric acid n-butyl ester, accurately weigh it, dissolve it in dichloromethane and dilute to make a solution containing about 0.5 mg per 1 mL, and shake well to obtain.

5. The method for separating and detecting the optical isomer (R)-butyl 2-hydroxybutyrate in (S)-butyl 2-hydroxybutyrate according to claim 2, characterized in that: In step 2), the specification of the CycloSil-B chromatographic column is 30 m × 0.25 mm, and the particle size of the packing is 0.25 μm.

6. The method for separating and detecting the optical isomer (R)-2-hydroxybutyric acid n-butyl ester in (S)-2-hydroxybutyric acid n-butyl ester according to claim 1, characterized in that: In step 2), the programmed temperature rise is that the initial column temperature is 80 °C, maintained for 5 minutes, and then heated to 200 °C at a rate of 5 °C per minute.

7. The method for separating and detecting the optical isomer (R)-2-hydroxybutyric acid n-butyl ester in (S)-2-hydroxybutyric acid n-butyl ester according to claim 1, characterized in that: In step 2), the inlet temperature is 240 °C, and the detector temperature is 250 °C.

8. The method for separating and detecting the optical isomer (R)-2-hydroxybutyric acid n-butyl ester in (S)-2-hydroxybutyric acid n-butyl ester according to claim 1, characterized in that: In step 2), the injection volume is 1 μL.

9. The method for separating and detecting the optical isomer (R)-2-hydroxybutyric acid n-butyl ester in (S)-2-hydroxybutyric acid n-butyl ester according to claim 1, characterized in that: In step 2), the flow rate is 1 mL / min, and the carrier gas is nitrogen.

10. The method for separating and detecting the optical isomer (R)-2-hydroxybutyric acid n-butyl ester in (S)-2-hydroxybutyric acid n-butyl ester according to claim 1, characterized in that: In step 2), the detector is an FID detector, and the split ratio is 5:1.

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