Pre-column Derivatization Detection Method for Enantiomers of the Starting Material of Brivaracetam

Abstract

The present invention relates to a detection method for the enantiomer (S)-4-propyl-dihydrofuran-2-one of the starting material (R)-4-propyl-dihydrofuran-2-one of brivaracetam; the detection method includes derivatizing (R)-4-propyl-dihydrofuran-2-one and its enantiomer (S)-4-propyl-dihydrofuran-2-one with trimethylsilyl iodide (TMSI) and ethanol, so as to change the chromatographic peak shape and improve the ultraviolet absorption, and then using reversed-phase high performance liquid chromatography, with water-acetonitrile as the mobile phase and a chiral column as the chromatographic column to detect the enantiomer (S)-4-propyl-dihydrofuran-2-one in the sample of the starting material (R)-4-propyl-dihydrofuran-2-one of brivaracetam. This detection method can completely separate (R)-4-propyl-dihydrofuran-2-one and its enantiomer and can quantitatively detect the isomer with a content higher than 0.09% in (R)-4-propyl-dihydrofuran-2-one, with high sensitivity, strong practicability, and a simple and fast detection process.

Description

Technical Field

[0001] The present invention belongs to the fields of analytical chemistry and pharmaceuticals, and particularly relates to a pre-column derivation detection method for the enantiomers of (R)-4-propyl-dihydrofuran-2-one, which is the starting material of brivaracetam. Background Art

[0002] Brivaracetam is a third-generation anti-epileptic drug developed by UCB Pharma in Belgium. It was approved by the US Food and Drug Administration (FDA) on February 19, 2016, for the adjunctive treatment of partial-onset seizures in patients 16 years of age and older, and has broad anti-epileptic activity and high safety. Compared with the previous-generation drug levetiracetam, brivaracetam has a similar chemical structure and mechanism of action, and it is the first anti-epileptic drug approved by the FDA for the treatment of partial-onset seizures since 2013. The chemical name of brivaracetam is (2S)-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl]butyramide, and its structural formula is:

[0003]

[0004] During the drug R & D process, the content of isomers has strict limit regulations in drug quality. Therefore, it is particularly important to establish chiral analysis methods for starting materials, key intermediates, and active pharmaceutical ingredients. Summary of the Invention

[0005] The chiral center of brivaracetam comes from the starting material, and (R)-4-propyl-dihydrofuran-2-one is an important starting material for the synthesis of brivaracetam and is an optically active compound. According to the regulations of the ICH, to control the isomers of brivaracetam, it is necessary to first control the isomers of the starting material. Currently, there is no liquid chromatography analysis method, and CN109828049A reports a gas chromatography detection method for the enantiomers (S)-furanone of (R)-furanone. This method uses a gas chromatograph to calculate the content of (S)-furanone by the external standard method based on the peak area. To better control the active ingredient of brivaracetam, and considering that (R)-4-propyl-dihydrofuran-2-one and its enantiomers are more stable when measured under liquid chromatography conditions at room temperature, and comprehensively evaluating the synthesis difficulty and purchase cost of enantiomer reference standards, etc., it is necessary to establish a method for quality control of the enantiomers of (R)-4-propyl-dihydrofuran-2-one by liquid chromatography conditions such as area normalization method or external standard method.

[0006] The chemical structural formula of the starting material (R)-4-propyl-dihydrofuran-2-one is:

[0007]

[0008] The chemical structural formula of the enantiomer (S)-4-propyl-dihydrofuran-2-one is as follows:

[0009] .

[0010] The object of the present invention is to provide a method for the liquid-phase separation and determination of the enantiomer (R)-4-propyl-dihydrofuran-2-one of the starting material of brivaracetam. This method can accurately determine the content of the enantiomer of the starting material of brivaracetam, providing a reliable basis for the control of the chiral impurities of brivaracetam and being beneficial to ensuring the quality of brivaracetam raw materials and preparations.

[0011] The present invention provides a method for detecting the enantiomer (S)-4-propyl-dihydrofuran-2-one of the starting material (R)-4-propyl-dihydrofuran-2-one of brivaracetam. This method uses trimethylsilyl iodide (TMSI) to carry out a ring-opening reaction on (R)-4-propyl-dihydrofuran-2-one and its enantiomer (S)-4-propyl-dihydrofuran-2-one, and then uses absolute ethanol to carry out a substitution reaction on the trimethylsilyl group, thereby changing the chromatographic peak shape and improving the ultraviolet absorption. Then, reverse-phase high-performance liquid chromatography is used, with water-acetonitrile as the mobile phase and a chiral column as the chromatographic column to detect the enantiomer (S)-4-propyl-dihydrofuran-2-one in the sample of the starting material (R)-4-propyl-dihydrofuran-2-one of brivaracetam.

[0012] In an embodiment of the present invention, a method for detecting the enantiomer (S)-4-propyl-dihydrofuran-2-one of the starting material (R)-4-propyl-dihydrofuran-2-one of brivaracetam is provided. The operation steps of this detection method are as follows:

[0013] (1) Preparation of the mobile phase

[0014] Mobile phase A: water

[0015] Mobile phase B: acetonitrile

[0016] (2) Preparation of the test solution: Weigh the sample of (R)-4-propyl-dihydrofuran-2-one to be tested, add trimethylsilyl iodide and acetonitrile, and then oscillate and react in a water bath constant temperature oscillator. Then add absolute ethanol and continue to oscillate and react in the water bath constant temperature oscillator. Let it cool to room temperature and prepare a test solution by mixing with an acetonitrile aqueous solution.

[0017] (3) Preparation of the reference solution: Weigh the reference substance of (S)-4-propyl-dihydrofuran-2-one, add trimethylsilyl iodide and acetonitrile, and then oscillate and react in a water bath constant temperature oscillator. Then add absolute ethanol and continue to oscillate and react in the water bath constant temperature oscillator. Let it cool to room temperature and prepare a reference solution by mixing with an acetonitrile aqueous solution.

[0018] (4) Inject the above-mentioned equal amounts of the reference solution and the test solution into a high-performance liquid chromatograph respectively. The chromatographic conditions are as follows: the chromatographic column is a chiral chromatographic column; the detector is an ultraviolet detector; the elution mode is gradient elution;

[0019] (5) Analyze the chromatogram and calculate by the external standard method based on the peak area or by the area normalization method.

[0020] Furthermore, the program of the gradient elution is as follows:

[0021] .

[0022] Furthermore, each 1 ml of the reference solution in step (3) contains 0.5 mg to 5 mg of (S)-4-propyl-dihydrofuran-2-one, preferably 5 mg.

[0023] Furthermore, the addition amount of trimethylsilyl iodide in step (2) and step (3) is 0.35 ml to 0.45 ml, preferably 0.4 ml.

[0024] Furthermore, the addition amount of acetonitrile in step (2) and step (3) is 2.0 ml to 3.0 ml, preferably 2.0 ml.

[0025] Furthermore, the addition amount of absolute ethanol in step (2) and step (3) is 3.5 ml to 4.5 ml, preferably 4.0 ml.

[0026] Furthermore, the water bath temperature in step (2) and step (3) is 50 °C to 70 °C, preferably 57 °C to 63 °C.

[0027] Furthermore, the oscillation mode in step (2) and step (3) is instrument oscillation or manual oscillation.

[0028] Furthermore, the acetonitrile aqueous solution in step (2) and step (3) is a 60% (v / v) acetonitrile aqueous solution.

[0029] Furthermore, the first oscillation time in step (2) and step (3) is 0.45 h to 0.55 h.

[0030] Furthermore, the second oscillation time in step (2) and step (3) is 0.9 h to 1.1 h.

[0031] Furthermore, each 1 ml of the test solution in step (2) contains 0.45 mg to 0.55 mg of (R)-4-propyl-dihydrofuran-2-one, preferably 0.5 mg.

[0032] Furthermore, the chiral chromatographic column in step (4) is CHIRALPAK IG-3 (2.1×150 mm, 3 mm).

[0033] Furthermore, the detection wavelength in step (4) is 240 nm to 260 nm, preferably 250 nm.

[0034] Furthermore, the injection volume in step (4) is 5 ml to 100 μl, preferably 20 ml.

[0035] Furthermore, the column temperature in step (4) is 27°C to 33°C, preferably 30°C.

[0036] Furthermore, the flow rate in step (4) is 0.25 ml / min to 0.35 ml / min, preferably 0.3 ml / min.

[0037] Furthermore, in the chromatogram of step (5), the retention time and peak area of the (S)-4-propyl-dihydrofuran-2-one chromatographic peak are determined based on the chromatogram of the reference solution, and the (S)-4-propyl-dihydrofuran-2-one content in the test sample is calculated by the peak area according to the chromatograms of the test solution and the reference solution using the external standard method.

[0038] Furthermore, the (S)-4-propyl-dihydrofuran-2-one content is calculated using the peak area formula according to the external standard method:

[0039] Enantiomer content (%) = C 对照品 ×A 供试品 ×K 供试品 / A 对照品 / M 供试品 ×100%.

[0040] The A 供试品 is the peak area of (S)-4-propyl-dihydrofuran-2-one in the test solution;

[0041] A 对照品 is the peak area of (S)-4-propyl-dihydrofuran-2-one in the reference solution;

[0042] C 对照品 is the concentration of (S)-4-propyl-dihydrofuran-2-one in the reference solution, mg / ml;

[0043] K 供试品 is the dilution factor of (S)-4-propyl-dihydrofuran-2-one in the test solution, ml;

[0044] M 供试品 is the weight of the test sample in the test solution.

[0045] Further, in the chromatogram described in step (5), the retention time and peak area of the chromatographic peak of (S)-4-propyl-dihydrofuran-2-one are determined according to the chromatogram of the reference substance solution, and the content of (S)-4-propyl-dihydrofuran-2-one in the test sample is calculated by the area normalization method without correction factor according to the chromatogram of the test sample solution.

[0046] Furthermore, the calculation formula for the content of (S)-4-propyl-dihydrofuran-2-one by the area normalization method without correction factor is: enantiomer content (%) = A 对映异构体 / A 总 × 100% (only integrating the main peak and the enantiomer peak).

[0047] The A 对映异构体 is the peak area of (S)-4-propyl-dihydrofuran-2-one in the test sample solution;

[0048] A 总 is the sum of the peak areas of (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one in the test sample solution.

[0049] Further, according to the methods for preparing the test sample solution and the reference substance solution described in steps (2) and (3), take the reference substances of (R)-4-propyl-dihydrofuran-2-one and (S)-4-propyl-dihydrofuran-2-one, add an appropriate amount of trimethylsilyl iodide and acetonitrile, then oscillate and react in a water bath thermostatic oscillator for a certain time, add an appropriate amount of absolute ethanol, continue to oscillate and react in the water bath thermostatic oscillator for a certain time, let it stand to room temperature, and prepare a mixed reference substance solution by mixing with an acetonitrile aqueous solution as the system suitability solution.

[0050] Furthermore, each 1 ml of the system suitability solution contains 0.45 mg - 0.55 mg of (R)-4-propyl-dihydrofuran-2-one and 0.5 mg - 5 mg of (S)-4-propyl-dihydrofuran-2-one.

[0051] Furthermore, the system suitability solution is injected into a high performance liquid chromatograph and detected under the chromatographic conditions described in step (4). In the obtained chromatogram, the resolution between the chromatographic peaks of (R)-4-propyl-dihydrofuran-2-one and (S)-4-propyl-dihydrofuran-2-one should be greater than 2.0.

[0052] The present invention provides a pre-column derivation detection method for the enantiomer (S)-4-propyl-dihydrofuran-2-one of the starting material (R)-4-propyl-dihydrofuran-2-one of brexpiprazole. By selecting specific chromatographic conditions and derivation conditions, the enantiomer in (R)-4-propyl-dihydrofuran-2-one can be simply, quickly and accurately separated and detected, thus ensuring the quality control of the starting material of brexpiprazole. It plays a guiding role in the development of the synthesis process and helps to formulate the quality standard of brexpiprazole API.

[0053] Obviously, based on the above content of the present invention, according to the common general technical knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modification, substitution or change can be made.

[0054] The following will further elaborate on the above content of the present invention through specific embodiments. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Description of the Drawings

[0055] Figure 1 shows the HPLC chromatogram of the blank solution in the specificity test for the method validation of the determination of the content of (S)-4-propyl-dihydrofuran-2-one in the starting material (R)-4-propyl-dihydrofuran-2-one of brexpiprazole in Example 1.

[0056] Figure 2 shows the HPLC chromatogram of the system suitability solution in the specificity test for the method validation of the determination of the content of (S)-4-propyl-dihydrofuran-2-one in the starting material (R)-4-propyl-dihydrofuran-2-one of brexpiprazole in Example 1.

[0057] Figure 3 shows the HPLC chromatogram of the reference solution in the specificity test for the method validation of the determination of the content of (S)-4-propyl-dihydrofuran-2-one in the starting material (R)-4-propyl-dihydrofuran-2-one of brexpiprazole in Example 1.

[0058] Figure 4 shows the HPLC chromatogram of the test solution in the specificity test for the method validation of the determination of the content of (S)-4-propyl-dihydrofuran-2-one in the starting material (R)-4-propyl-dihydrofuran-2-one of brexpiprazole in Example 1. Embodiments

[0059] The present invention will be further illustrated by the following examples, but it should be understood that the following examples are not limited to the scope of the present invention. The reagents used in the following examples can be easily obtained from the market.

[0060] (1) Information on reagents, test drugs, samples and reference substances

[0061] (R)-4-propyl-dihydrofuran-2-one raw material [Yikelai (Taizhou) Pharmaceutical Co., Ltd., batch number: D01-SM1-Z03320220301];

[0062] (R)-4-propyl-dihydrofuran-2-one reference substance [Yikelai (Taizhou) Pharmaceutical Co., Ltd., batch number: DZ(D01-SM1-Z03320220301)-S1, content: 99.5%];

[0063] (S)-4-propyl-dihydrofuran-2-one reference substance (Chongqing Liujiang Pharmaceutical Technology Co., Ltd., batch number: DZ(D01-SM1-ZZ1-21110301)-S1, content: 97.7%);

[0064] Acetonitrile (HPLC) [Beijing Merida Technology Co., Ltd., batch number: 0211128];

[0065] Trimethylsilyl iodide (AR) [Shanghai Macklin Biochemical Co., Ltd., batch number: C13078049];

[0066] Absolute ethanol (HPLC) [Chengdu Nuoshi Technology Co., Ltd., batch number: 2021122001].

[0067] (2) Main instruments

[0068] High performance liquid chromatograph: Thermo Fisher Ultimate 3000;

[0069] Electronic balance: XSE105DU;

[0070] Water bath thermostatic oscillator: SHZ-B.

[0071] (3) The detection method is determined by referring to the high performance liquid chromatography method (General Principles 0512, Volume IV, Chinese Pharmacopoeia 2020 Edition). Examples

[0072] 1. Main technical parameters of high performance liquid chromatography

[0073] High performance liquid chromatograph: Thermo Fisher Ultimate 3000; the detector is an ultraviolet detector; the detection wavelength is 250 nm; the column temperature is 30 °C; chromatographic column model: covalently bonded silica gel with amylose tris(3-chloro-5-methylphenylcarbamate) on the surface as the filler [suitable for CHIRALPAK IG-3 (2.1×150 mm, 3 mm)]; the flow rate is 0.3 ml / min; the injection volume is 20 μl;

[0074] Mobile phase: Purified water is used as mobile phase A, and acetonitrile is used as mobile phase B; linear gradient elution is carried out according to the following table, and the elution conditions are as follows:

[0075]

[0076] 2. Assay method

[0077] (1)Solution preparation

[0078] a. Stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance: Weigh accurately 47.63 mg of (S)-4-propyl-dihydrofuran-2-one reference substance (batch number DZ(D01-SM1-ZZ1-21110301)-S1, provided by Chongqing Liujiang Pharmaceutical Technology Co., Ltd., the same below), place it in a 10-ml volumetric flask, dissolve it with acetonitrile (batch number 0211128, provided by Beijing Merck Technology Co., Ltd., the same below) and dilute it to the scale, shake well. Accurately measure 1 ml and place it in a 10-ml volumetric flask, dilute it to the scale with acetonitrile, shake well, and use it as the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance.

[0079] b. Reference substance solution: Accurately measure 1.0 ml of the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance, place it in a 10-ml volumetric flask, add 0.4 ml of trimethylsilyl iodide (batch number C13078049, provided by Shanghai Macklin Biochemical Co., Ltd., the same below), then add 2.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 60 °C for 0.5 h, then add 4 ml of absolute ethanol (batch number 2021122001, provided by Chengdu Nuoshi Technology Co., Ltd., the same below), continue to shake and react in a water bath thermostatic oscillator for 1 h, take it out, let it cool to room temperature, dilute it to the scale with 60% (v / v) acetonitrile aqueous solution, shake well. Accurately measure 1 ml and place it in a 10-ml volumetric flask, dilute it to the scale with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the reference substance solution.

[0080] c. Test sample solution: Weigh accurately 53.95 mg of the test sample of (R)-4-propyl-dihydrofuran-2-one [batch number D01-SM1-Z033202 20301, provided by Yikele (Taizhou) Pharmaceutical Co., Ltd., the same below], place it in a 10-ml volumetric flask, add 0.4 ml of trimethylsilyl iodide, then add 3.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 60 °C for 0.5 h, then add 4 ml of absolute ethanol, continue to shake and react in a water bath thermostatic oscillator for 1 h, take it out, let it cool to room temperature, dilute it to the scale with 60% (v / v) acetonitrile aqueous solution, shake well. Accurately measure 1 ml and place it in a 10-ml volumetric flask, dilute it to the scale with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the test sample solution.

[0081] d. System suitability solution: Accurately weigh 50.94 mg of (R)-4-propyl-dihydrofuran-2-one reference substance [batch number: DZ(D01-SM1-Z03320220301)-S1, provided by Yikele (Taizhou) Pharmaceutical Co., Ltd., the same below], place it in a 10-ml volumetric flask, accurately add 1.0 ml of the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance and 0.4 ml of trimethylsilyl iodide, then add 2.0 ml of acetonitrile. Oscillate and react in a water bath thermostatic oscillator at 60 °C for 0.5 h, then add 4 ml of absolute ethanol, continue to oscillate and react in the water bath thermostatic oscillator for 1 h. Take it out, let it cool to room temperature, dilute it to the mark with 60% (v / v) acetonitrile aqueous solution, shake well. Accurately measure 1 ml and place it in a 10-ml volumetric flask, dilute it to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the system suitability solution.

[0082] e. Blank solution: Accurately measure 0.4 ml of trimethylsilyl iodide, place it in a 10-ml volumetric flask, add 2.0 ml of acetonitrile, oscillate and react in a water bath thermostatic oscillator at 60 °C for 0.5 h, then add 4 ml of absolute ethanol, continue to oscillate and react in the water bath thermostatic oscillator for 1 h. Take it out, let it cool to room temperature, dilute it to the mark with 60% (v / v) acetonitrile aqueous solution, shake well. Accurately measure 1 ml and place it in a 10-ml volumetric flask, dilute it to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the blank solution.

[0083] (2) Detection

[0084] a. Measure 20 ml each of the blank solution and the system suitability solution and inject them into the high-performance liquid chromatograph, record the chromatogram. The chromatograms are shown in Figures 1 to 2, and the results of the system suitability solution are shown in Table 1. It is required that the resolution between (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one should be greater than 2.0;

[0085]

[0086] Conclusion: The resolution between (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one is 3.53, which is greater than 2.0, meeting the standard requirements.

[0087] b. Measure 20 ml each of the reference solution and the test solution and inject them into the high-performance liquid chromatograph, record the chromatogram. The chromatograms are shown in Figures 3 to 4, and the results of the reference solution and the test solution are shown in Table 2:

[0088] .

[0089] Results:

[0090] The content of (S)-4-propyl-dihydrofuran-2-one is calculated by the external standard method with the peak area formula as:

[0091] Enantiomer content (%) = C 对照品 × A 供试品 × K 供试品 / A 对照品 / M 供试品 × 100%.

[0092] C 对照品 —— Concentration of (S)-4-propyl-dihydrofuran-2-one in the reference solution, mg / ml;

[0093] A 供试品 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the test solution; 4]

[0094] K 供试品 —— Dilution factor of (S)-4-propyl-dihydrofuran-2-one in the test solution, ml;

[0095] A 对照品 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the reference solution;

[0096] M 供试品 Is the weighed amount of the test substance in the test solution.

[0097] The calculation result is: 0.079%.[[ID=4)1]]

[0098] (S)-4-propyl-dihydrofuran-2-one content calculation formula by area normalization method without correction factor is:

[0099] Enantiomer content (%) = A 对映异构体 / A 总 × 100% (only integrate the main peak and the enantiomer peak).

[0100] A 对映异构体 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the test solution;

[0101] A 总 —— Sum of the peak areas of (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one in the test solution.

[0102] The calculation result is: 0.084%.

[0103] Conclusion: The content of (S)-4-propyl-dihydrofuran-2-one in (R)-4-propyl-dihydrofuran-2-one obtained by the above two calculation methods is basically the same. Example

[0104] 1. Main technical parameters of high performance liquid chromatography

[0105] High performance liquid chromatograph: Thermo Fisher Ultimate 3000; the detector is an ultraviolet detector; the detection wavelength is 250 nm; the column temperature is 30 °C; the chromatographic column model: covalently bonded silica gel with amylose tris(3-chloro-5-methylphenylcarbamate) on the surface as the filler [CHIRALPAK IG-3 (2.1×150 mm, 3 mm) applicable]; the flow rate is 0.25 ml / min; the injection volume is 20 μl;

[0106] Mobile phase: Purified water is used as mobile phase A, and acetonitrile is used as mobile phase B; linear gradient elution is carried out according to the following table, and the elution conditions are as follows:

[0107] 。

[0108] 2. Assay method

[0109] (1) Solution preparation

[0110] a. Stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance: Weigh accurately 10.42 mg of (S)-4-propyl-dihydrofuran-2-one reference substance, place it in a 20-ml volumetric flask, dissolve it with acetonitrile and dilute to the mark, shake well, and use it as the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance.

[0111] b. Reference substance solution: Accurately measure 1.0 ml of the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance, place it in a 10-ml volumetric flask, add 0.4 ml of trimethylsilyl iodide, then add 2.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 60 °C for 0.5 h, then add 4 ml of absolute ethanol, continue to shake and react in a water bath thermostatic oscillator for 1 h, take it out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, and dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the reference substance solution.

[0112] c. System suitability solution: Weigh accurately 47.78 mg of (R)-4-propyl-dihydrofuran-2-one reference substance, place it in a 10-ml volumetric flask, accurately add 1.0 ml of the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance and 0.4 ml of trimethylsilyl iodide, then add 2.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 60 °C for 0.5 h, then add 4 ml of absolute ethanol, continue to shake and react in a water bath thermostatic oscillator for 1 h, take it out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, and dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the system suitability solution.

[0113] d. Test solution: Accurately weigh 48.47 mg of the (R)-4-propyl-dihydrofuran-2-one sample to be tested, place it in a 10-ml volumetric flask, add 0.4 ml of trimethylsilyl iodide, then add 3.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 60 °C for 0.5 h, then add 4 ml of absolute ethanol, continue to shake and react in the water bath thermostatic oscillator for 1 h, take out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the test solution.

[0114] e. Blank solution: Accurately measure 0.4 ml of trimethylsilyl iodide, place it in a 10-ml volumetric flask, add 2.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 60 °C for 0.5 h, then add 4 ml of absolute ethanol, continue to shake and react in the water bath thermostatic oscillator for 1 h, take out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the blank solution.

[0115] (2) Detection

[0116] a. Measure 20 ml each of the blank solution and the system suitability solution and inject them into the high-performance liquid chromatograph, and record the chromatogram. The results of the system suitability solution are shown in Table 3. It is required that the resolution between (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one should be greater than 2.0.

[0117] Conclusion: The resolution between (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one is 3.49, which is greater than 2.0, meeting the standard requirements.

[0118] b. Measure 20 ml each of the reference solution and the test solution and inject them into the high-performance liquid chromatograph, and record the chromatogram. The results of the reference solution and the test solution are shown in Table 4:

[0119] .

[0120] Results:

[0121] The content of (S)-4-propyl-dihydrofuran-2-one is calculated by the external standard method with the peak area formula as:

[0122] Enantiomer content (%) = C 对照品 ×A 供试品 ×K 供试品 / A 对照品 / M 供试品 ×100%.

[0123] C 对照品 —— Concentration of (S)-4-propyl-dihydrofuran-2-one in the reference solution, mg / ml;

[0124] A 供试品 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the test solution;

[0125] K 供试品 —— Dilution factor of (S)-4-propyl-dihydrofuran-2-one in the test solution, ml;

[0126] A 对照品 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the reference solution;

[0127] M 供试品 is the weighed amount of the test substance in the test solution.

[0128] The calculation result is: 0.081%.

[0129] The formula for calculating the content of (S)-4-propyl-dihydrofuran-2-one by area normalization method without correction factor is:

[0130] Enantiomer content (%) = A 对映异构体 / A 总 × 100% (only integrate the main peak and the enantiomer peak).

[0131] A 对映异构体 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the test solution;

[0132] A 总 —— Sum of the peak areas of (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one in the test solution.

[0133] The calculation result is: 0.086%.

[0134] Conclusion: The content of (S)-4-propyl-dihydrofuran-2-one in (R)-4-propyl-dihydrofuran-2-one obtained by the above two calculation methods is basically the same. Example

[0135] 1. Main technical parameters of high performance liquid chromatography

[0136] High-performance liquid chromatograph: Thermo Fisher Ultimate 3000; the detector is an ultraviolet detector; the detection wavelength is 250 nm; the column temperature is 30 °C; chromatographic column model: covalently bonded silica gel with amylose tris(3-chloro-5-methylphenylcarbamate) on the surface as the filler [suitable for CHIRALPAK IG-3 (2.1×150 mm, 3 mm)]; the flow rate is 0.35 ml / min; the injection volume is 20 μl;

[0137] Mobile phase: Purified water is used as mobile phase A, and acetonitrile is used as mobile phase B; linear gradient elution is carried out according to the following table, and the elution conditions are as follows:

[0138] 。

[0139] 2. Determination method

[0140] (l) Solution preparation

[0141] a. Stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance: Accurately weigh 10.42 mg of (S)-4-propyl-dihydrofuran-2-one reference substance, place it in a 20-ml volumetric flask, dissolve it with acetonitrile and dilute it to the mark, shake well, and use it as the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance.

[0142] b. Reference substance solution: Accurately measure 1.0 ml of the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance, place it in a 10-ml volumetric flask, add 0.4 ml of trimethylsilyl iodide, then add 2.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 60 °C for 0.5 h, then add 4 ml of absolute ethanol, continue to shake and react in a water bath thermostatic oscillator for 1 h, take it out, let it cool to room temperature, dilute it to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, and dilute it to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the reference substance solution.

[0143] c. System suitability solution: Accurately weigh 47.78 mg of (R)-4-propyl-dihydrofuran-2-one reference substance, place it in a 10-ml volumetric flask, accurately add 1.0 ml of the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance and 0.4 ml of trimethylsilyl iodide, then add 2.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 60 °C for 0.5 h, then add 4 ml of absolute ethanol, continue to shake and react in a water bath thermostatic oscillator for 1 h, take it out, let it cool to room temperature, dilute it to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, and dilute it to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the system suitability solution.

[0144] d. Test solution: Accurately weigh 48.47 mg of the (R)-4-propyl-dihydrofuran-2-one sample to be tested, place it in a 10-ml volumetric flask, add 0.4 ml of trimethylsilyl iodide, then add 3.0 ml of acetonitrile, shake and react in a water bath thermostatic shaker at 60 °C for 0.5 h, then add 4 ml of absolute ethanol, continue to shake and react in the water bath thermostatic shaker for 1 h, take out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the test solution.

[0145] e. Blank solution: Accurately measure 0.4 ml of trimethylsilyl iodide, place it in a 10-ml volumetric flask, add 2.0 ml of acetonitrile, shake and react in a water bath thermostatic shaker at 60 °C for 0.5 h, then add 4 ml of absolute ethanol, continue to shake and react in the water bath thermostatic shaker for 1 h, take out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the blank solution.

[0146] (2) Detection

[0147] a. Measure 20 ml each of the blank solution and the system suitability solution and inject them into the high-performance liquid chromatograph, and record the chromatogram. The results of the system suitability solution are shown in Table 5. It is required that the resolution between (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one should be greater than 2.0;

[0148]

[0149] Conclusion: The resolution between (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one is 3.56, which is greater than 2.0, meeting the standard requirements.

[0150] b. Measure 20 ml each of the reference solution and the test solution and inject them into the high-performance liquid chromatograph, and record the chromatogram. The results of the reference solution and the test solution are shown in Table 6:

[0151] .

[0152] Results:

[0153] The content of (S)-4-propyl-dihydrofuran-2-one is calculated by the external standard method using the peak area formula as:

[0154] Enantiomer content (%) = C 对照品 × A 供试品 × K 供试品 / A 对照品 / M 供试品×100%.

[0155] C 对照品 —— Concentration of (S)-4-propyl-dihydrofuran-2-one in the reference solution, mg / ml;

[0156] A 供试品 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the test solution;

[0157] K 供试品 —— Dilution factor of (S)-4-propyl-dihydrofuran-2-one in the test solution, ml;

[0158] A 对照品 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the reference solution;

[0159] M 供试品 is the weighed amount of the test substance in the test solution.

[0160] The calculation result is: 0.078%.

[0161] The calculation formula for the content of (S)-4-propyl-dihydrofuran-2-one by area normalization method without correction factor is:

[0162] Content of enantiomer (%) = A 对映异构体 / A 总 ×100% (only integrate the main peak and the enantiomer peak).

[0163] A 对映异构体 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the test solution;

[0164] A 总 —— Sum of the peak areas of (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one in the test solution.

[0165] The calculation result is: 0.083%.

[0166] Conclusion: The content of (S)-4-propyl-dihydrofuran-2-one in (R)-4-propyl-dihydrofuran-2-one obtained by the above two calculation methods is basically the same. Example

[0167] 1. Main technical parameters of high performance liquid chromatography

[0168] High-performance liquid chromatograph: Thermo Fisher Ultimate 3000; the detector is an ultraviolet detector; the detection wavelength is 250 nm; the column temperature is 27 °C; chromatographic column model: covalently bonded silica gel with amylose tris(3-chloro-5-methylphenylcarbamate) on the surface as the filler [suitable for CHIRALPAK IG-3 (2.1×150 mm, 3 mm)]; the flow rate is 0.3 ml / min; the injection volume is 20 μl;

[0169] Mobile phase: Purified water is used as mobile phase A, and acetonitrile is used as mobile phase B; linear gradient elution is carried out according to the following table, and the elution conditions are as follows:

[0170] .

[0171] 2. Assay method

[0172] (1) Solution preparation

[0173] a. Stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance: Accurately weigh 10.42 mg of (S)-4-propyl-dihydrofuran-2-one reference substance, place it in a 20-ml volumetric flask, dissolve it with acetonitrile and dilute to the mark, shake well, and use it as the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance.

[0174] b. Reference substance solution: Accurately measure 1.0 ml of the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance, place it in a 10-ml volumetric flask, add 0.4 ml of trimethylsilyl iodide, then add 2.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 60 °C for 0.5 h, then add 4 ml of absolute ethanol, continue to shake and react in a water bath thermostatic oscillator for 1 h, take it out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, and dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the reference substance solution.

[0175] c. System suitability solution: Accurately weigh 47.78 mg of (R)-4-propyl-dihydrofuran-2-one reference substance, place it in a 10-ml volumetric flask, accurately add 1.0 ml of the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance and 0.4 ml of trimethylsilyl iodide, then add 2.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 60 °C for 0.5 h, then add 4 ml of absolute ethanol, continue to shake and react in a water bath thermostatic oscillator for 1 h, take it out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, and dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the system suitability solution.

[0176] d. Test solution: Weigh accurately 48.47 mg of the sample of (R)-4-propyl-dihydrofuran-2-one to be tested, place it in a 10-ml volumetric flask, add 0.4 ml of trimethylsilyl iodide, then add 3.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 60 °C for 0.5 h, then add 4 ml of absolute ethanol, continue to shake and react in the water bath thermostatic oscillator for 1 h, take out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the test solution.

[0177] e. Blank solution: Accurately measure 0.4 ml of trimethylsilyl iodide, place it in a 10-ml volumetric flask, add 2.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 60 °C for 0.5 h, then add 4 ml of absolute ethanol, continue to shake and react in the water bath thermostatic oscillator for 1 h, take out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the blank solution.

[0178] (2) Detection

[0179] a. Measure 20 ml each of the blank solution and the system suitability solution and inject them into the high performance liquid chromatograph, and record the chromatogram. The results of the system suitability solution are shown in Table 7. It is required that the resolution between (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one should be greater than 2.0;

[0180]

[0181] Conclusion: The resolution between (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one is 3.73, which is greater than 2.0 and meets the standard requirements.

[0182] b. Measure 20 ml each of the reference solution and the test solution and inject them into the high performance liquid chromatograph, and record the chromatogram. The results of the reference solution and the test solution are shown in Table 8:

[0183] .

[0184] Results:

[0185] The content of (S)-4-propyl-dihydrofuran-2-one is calculated by the external standard method with the peak area formula as:

[0186] The content of enantiomer (%) = C 对照品 ×A 供试品 ×K 供试品 / A 对照品 / M 供试品×100%.

[0187] C 对照品 ——Concentration of (S)-4-propyl-dihydrofuran-2-one in the reference solution, mg / ml;

[0188] A 供试品 ——The peak area of (S)-4-propyl-dihydrofuran-2-one in the test solution;

[0189] K 供试品 ——Dilution factor of (S)-4-propyl-dihydrofuran-2-one in the test solution, ml;

[0190] A 对照品 ——Peak area of (S)-4-propyl-dihydrofuran-2-one in the reference solution;

[0191] M 供试品 is the weight of the test sample in the test solution.

[0192] The calculation result is: 0.074%.

[0193] The content of (S)-4-propyl-dihydrofuran-2-one was calculated by area normalization method without adding correction factor:

[0194] Enantiomer content (%) = A 对映异构体 / A 总 ×100% (integrate only the main peak and enantiomer peaks).

[0195] A 对映异构体 ——The peak area of (S)-4-propyl-dihydrofuran-2-one in the test solution;

[0196] A 总 ——The sum of the peak areas of (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one in the test solution.

[0197] The calculation result is: 0.078%.

[0198] Conclusion: The contents of (S)-4-propyl-dihydrofuran-2-one in (R)-4-propyl-dihydrofuran-2-one obtained by the above two calculation methods are basically the same. Example

[0199] 1. Main technical parameters of HPLC

[0200] High-performance liquid chromatograph: Thermo Fisher Ultimate 3000; the detector is an ultraviolet detector; the detection wavelength is 250 nm; the column temperature is 33 °C; the chromatographic column model: covalently bonded silica gel with amylose tris(3-chloro-5-methylphenylcarbamate) on the surface as the filler [suitable for CHIRALPAK IG-3 (2.1×150 mm, 3 mm)]; the flow rate is 0.3 ml / min; the injection volume is 20 μl;

[0201] Mobile phase: Purified water is used as mobile phase A, and acetonitrile is used as mobile phase B; linear gradient elution is carried out according to the following table, and the elution conditions are as follows:

[0202] 。

[0203] 2. Determination method

[0204] (1) Solution preparation

[0205] a. Stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance: Accurately weigh 10.42 mg of (S)-4-propyl-dihydrofuran-2-one reference substance, place it in a 20-ml volumetric flask, dissolve it with acetonitrile and dilute to the mark, shake well, and use it as the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance.

[0206] b. Reference substance solution: Accurately measure 1.0 ml of the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance, place it in a 10-ml volumetric flask, add 0.4 ml of trimethylsilyl iodide, then add 2.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 60 °C for 0.5 h, then add 4 ml of absolute ethanol, continue to shake and react in a water bath thermostatic oscillator for 1 h, take it out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, and dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the reference substance solution.

[0207] c. System suitability solution: Accurately weigh 47.78 mg of (R)-4-propyl-dihydrofuran-2-one reference substance, place it in a 10-ml volumetric flask, accurately add 1.0 ml of the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance and 0.4 ml of trimethylsilyl iodide, then add 2.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 60 °C for 0.5 h, then add 4 ml of absolute ethanol, continue to shake and react in a water bath thermostatic oscillator for 1 h, take it out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, and dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the system suitability solution.

[0208] d. Test solution: Accurately weigh 48.47 mg of the (R)-4-propyl-dihydrofuran-2-one sample to be tested, place it in a 10-ml volumetric flask, add 0.4 ml of trimethylsilyl iodide, then add 3.0 ml of acetonitrile, shake and react in a water bath thermostatic shaker at 60 °C for 0.5 h, then add 4 ml of absolute ethanol, continue to shake and react in the water bath thermostatic shaker for 1 h, take out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the test solution.

[0209] e. Blank solution: Accurately measure 0.4 ml of trimethylsilyl iodide, place it in a 10-ml volumetric flask, add 2.0 ml of acetonitrile, shake and react in a water bath thermostatic shaker at 60 °C for 0.5 h, then add 4 ml of absolute ethanol, continue to shake and react in the water bath thermostatic shaker for 1 h, take out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the blank solution.

[0210] (2)Detection

[0211] a. Measure 20 ml each of the blank solution and the system suitability solution and inject them into the high-performance liquid chromatograph, and record the chromatogram. The results of the system suitability solution are shown in Table 9. It is required that the resolution between (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one should be greater than 2.0;

[0212] 。

[0213] Conclusion: The resolution between (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one is 3.42, which is greater than 2.0, meeting the standard requirements.

[0214] b. Measure 20 ml each of the reference solution and the test solution and inject them into the high-performance liquid chromatograph, and record the chromatogram. The results of the reference solution and the test solution are shown in Table 10:

[0215] 。

[0216] Results:

[0217] (S)-4-propyl-dihydrofuran-2-one content is calculated by the external standard method with the peak area formula as: <{

[0218] Enantiomer content (%) = C 对照品 ×A 供试品 ×K 供试品 / A 对照品 / M 供试品×100%.

[0219] C 对照品 —— Concentration of (S)-4-propyl-dihydrofuran-2-one in the reference solution, mg / ml;

[0220] A 供试品 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the test solution;

[0221] K 供试品 —— Dilution factor of (S)-4-propyl-dihydrofuran-2-one in the test solution, ml;

[0222] A 对照品 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the reference solution;

[0223] M 供试品 is the weighed amount of the test substance in the test solution.

[0224] The calculation result is: 0.068%.

[0225] The calculation formula for the content of (S)-4-propyl-dihydrofuran-2-one by area normalization method without correction factor is:

[0226] Enantiomer content (%) = A 对映异构体 / A 总 × 100% (only integrate the main peak and the enantiomer peak).

[0227] A 对映异构体 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the test solution;

[0228] A 总 —— Sum of the peak areas of (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one in the test solution.

[0229] The calculation result is: 0.073%.

[0230] Conclusion: The content of (S)-4-propyl-dihydrofuran-2-one in (R)-4-propyl-dihydrofuran-2-one obtained by the above two calculation methods is basically the same. Example

[0231] 1. Main technical parameters of high performance liquid chromatography

[0232] High-performance liquid chromatograph: Thermo Fisher Ultimate 3000; the detector is an ultraviolet detector; the detection wavelength is 250 nm; the column temperature is 30 °C; the chromatographic column model: covalently bonded silica gel with amylose tris(3-chloro-5-methylphenylcarbamate) on the surface as the filler [suitable for CHIRALPAK IG-3 (2.1×150 mm, 3 mm)]; the flow rate is 0.3 ml / min; the injection volume is 20 μl;

[0233] Mobile phase: Purified water is used as mobile phase A, and acetonitrile is used as mobile phase B; linear gradient elution is carried out according to the following table, and the elution conditions are as follows:

[0234]

[0235] 2. Determination method

[0236] (1) Solution preparation

[0237] a. Stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance: Accurately weigh 10.67 mg of (S)-4-propyl-dihydrofuran-2-one reference substance, place it in a 20-ml volumetric flask, dissolve it with acetonitrile and dilute to the mark, shake well, and use it as the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance.

[0238] b. Reference substance solution: Accurately measure 1.0 ml of the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance, place it in a 10-ml volumetric flask, add 0.35 ml of trimethylsilyl iodide, then add 2.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 57 °C for 0.45 h, then add 4 ml of absolute ethanol, continue to shake and react in a water bath thermostatic oscillator for 0.9 h, take it out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, and dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the reference substance solution.

[0239] c. System suitability solution: Accurately weigh 57.68 mg of (R)-4-propyl-dihydrofuran-2-one reference substance, place it in a 10-ml volumetric flask, accurately add 1.0 ml of the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance and 0.35 ml of trimethylsilyl iodide, then add 2.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 57 °C for 0.45 h, then add 4 ml of absolute ethanol, continue to shake and react in a water bath thermostatic oscillator for 0.9 h, take it out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, and dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the system suitability solution.

[0240] d. Test solution: Accurately weigh 59.12 mg of the sample to be tested, (R)-4-propyl-dihydrofuran-2-one, place it in a 10-ml volumetric flask, add 0.35 ml of trimethylsilyl iodide, then add 3.0 ml of acetonitrile. React with shaking in a water bath thermostatic oscillator at 57 °C for 0.45 h, then add 4 ml of absolute ethanol, continue to react with shaking in the water bath thermostatic oscillator for 0.9 h. Take it out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well. Accurately measure 1 ml, place it in a 10-ml volumetric flask, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the test solution.

[0241] e. Blank solution: Accurately measure 0.35 ml of trimethylsilyl iodide, place it in a 10-ml volumetric flask, add 2.0 ml of acetonitrile. React with shaking in a water bath thermostatic oscillator at 57 °C for 0.45 h, then add 4 ml of absolute ethanol, continue to react with shaking in the water bath thermostatic oscillator for 0.9 h. Take it out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well. Accurately measure 1 ml, place it in a 10-ml volumetric flask, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the blank solution.

[0242] (2) Detection

[0243] a. Measure 20 ml each of the blank solution and the system suitability solution and inject them into the high-performance liquid chromatograph, and record the chromatogram. The results of the system suitability solution are shown in Table 11. It is required that the resolution between (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one should be greater than 2.0;

[0244] .

[0245] Conclusion: The resolution between (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one is 3.54, which is greater than 2.0, meeting the standard requirements.

[0246] b. Measure 20 ml each of the reference solution and the test solution and inject them into the high-performance liquid chromatograph, and record the chromatogram. The results of the reference solution and the test solution are shown in Table 12:

[0247] .

[0248] Results:

[0249] The content of (S)-4-propyl-dihydrofuran-2-one is calculated by the external standard method using the peak area formula as follows:

[0250] Enantiomer content (%) = C 对照品 × A 供试品 × K 供试品 / A 对照品 / M供试品 × 100%。

[0251] C 对照品 —— Concentration of (S)-4-propyl-dihydrofuran-2-one in the reference solution, mg / ml;

[0252] A 供试品 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the test solution;

[0253] K 供试品 —— Dilution factor of (S)-4-propyl-dihydrofuran-2-one in the test solution, ml;

[0254] A 对照品 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the reference solution;

[0255] M 供试品 is the weighed amount of the test substance in the test solution.

[0256] The calculation result is: 0.080%.

[0257] The calculation formula for the content of (S)-4-propyl-dihydrofuran-2-one by area normalization method without correction factor is:

[0258] Enantiomer content (%) = A 对映异构体 / A 总 × 100% (only integrate the main peak and the enantiomer peak).

[0259] A 对映异构体 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the test solution;

[0260] A 总 —— Sum of the peak areas of (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one in the test solution.

[0261] The calculation result is: 0.086%.

[0262] Conclusion: The content of (S)-4-propyl-dihydrofuran-2-one in (R)-4-propyl-dihydrofuran-2-one obtained by the above two calculation methods is basically the same. Example

[0263] 1. Main technical parameters of high performance liquid chromatography

[0264] High-performance liquid chromatograph: Thermo Fisher Ultimate 3000; the detector is an ultraviolet detector; the detection wavelength is 250 nm; the column temperature is 30 °C; chromatographic column model: covalently bonded silica gel with amylose tris(3-chloro-5-methylphenylcarbamate) on the surface as the filler [suitable for CHIRALPAK IG-3 (2.1×150 mm, 3 μm)]; the flow rate is 0.3 ml / min; the injection volume is 20 μl;

[0265] Mobile phase: Purified water is used as mobile phase A, and acetonitrile is used as mobile phase B; linear gradient elution is carried out according to the following table, and the elution conditions are as follows:

[0266] 。

[0267] 2. Determination method

[0268] (1) Solution preparation

[0269] a. Stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance: Weigh accurately 10.67 mg of (S)-4-propyl-dihydrofuran-2-one reference substance, place it in a 20-ml volumetric flask, dissolve it with acetonitrile and dilute to the mark, shake well, and use it as the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance.

[0270] b. Reference substance solution: Accurately measure 1.0 ml of the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance, place it in a 10-ml volumetric flask, add 0.45 ml of trimethylsilyl iodide, then add 2.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 63 °C for 0.55 h, then add 4 ml of absolute ethanol, continue to shake and react in a water bath thermostatic oscillator for 1.1 h, take it out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, and dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the reference substance solution.

[0271] c. System suitability solution: Weigh accurately 43.84 mg of (R)-4-propyl-dihydrofuran-2-one reference substance, place it in a 10-ml volumetric flask, accurately add 1.0 ml of the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance and 0.45 ml of trimethylsilyl iodide, then add 2.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 63 °C for 0.55 h, then add 4 ml of absolute ethanol, continue to shake and react in a water bath thermostatic oscillator for 1.1 h, take it out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, and dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the system suitability solution.

[0272] d. Test solution: Accurately weigh 46.32 mg of the (R)-4-propyl-dihydrofuran-2-one sample to be tested, place it in a 10-ml volumetric flask, add 0.45 ml of trimethylsilyl iodide, then add 3.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 63 °C for 0.55 h, then add 4 ml of absolute ethanol, continue to shake and react in the water bath thermostatic oscillator for 1.1 h, take out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the test solution.

[0273] e. Blank solution: Accurately measure 0.45 ml of trimethylsilyl iodide, place it in a 10-ml volumetric flask, add 2.0 ml of acetonitrile, shake and react in a water bath thermostatic oscillator at 63 °C for 0.55 h, then add 4 ml of absolute ethanol, continue to shake and react in the water bath thermostatic oscillator for 1.1 h, take out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the blank solution.

[0274] (2) Detection

[0275] a. Measure 20 ml each of the blank solution and the system suitability solution and inject them into the high-performance liquid chromatograph, and record the chromatogram. The results of the system suitability solution are shown in Table 13. It is required that the resolution between (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one should be greater than 2.0;

[0276] .

[0277] Conclusion: The resolution between (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one is 3.63, which is greater than 2.0, meeting the standard requirements.

[0278] b. Measure 20 ml each of the reference solution and the test solution and inject them into the high-performance liquid chromatograph, and record the chromatogram. The results of the reference solution and the test solution are shown in Table 14:

[0279] .

[0280] Results:

[0281] The content of (S)-4-propyl-dihydrofuran-2-one is calculated by the external standard method using the peak area formula as:

[0282] Enantiomer content (%) = C 对照品 × A 供试品 × K 供试品 / A 对照品 / M供试品 × 100%。

[0283] C 对照品 —— Concentration of (S)-4-propyl-dihydrofuran-2-one in the reference solution, mg / ml;

[0284] A 供试品 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the test solution;

[0285] K 供试品 —— Dilution factor of (S)-4-propyl-dihydrofuran-2-one in the test solution, ml;

[0286] A 对照品 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the reference solution;

[0287] M 供试品 is the weighed amount of the test substance in the test solution.

[0288] The calculated result is: 0.085%.

[0289] (S)-4-propyl-dihydrofuran-2-one content is calculated by the area normalization method without correction factor as follows:

[0290] Enantiomer content (%) = A 对映异构体 / A 总 × 100% (only integrate the main peak and the enantiomer peak).

[0291] A 对映异构体 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the test solution;

[0292] A 总 —— Sum of the peak areas of (S)-4-propyl-dihydrofuran-zo-one and (R)-4-propyl-dihydrofuran-2-one in the test solution.

[0293] The calculated result is: 0.087%.

[0294] Conclusion: The content of (S)-4-propyl-dihydrofuran-2-one in (R)-4-propyl-dihydrofuran-2-one obtained by the above two calculation methods is basically the same. Example

[0295] 1. Main technical parameters of high performance liquid chromatography

[0296] High-performance liquid chromatograph: Thermo Fisher Ultimate 3000; the detector is an ultraviolet detector; the detection wavelength is 250 nm; the column temperature is 30 °C; the chromatographic column model: covalently bonded silica gel with amylose tris(3-chloro-5-methylphenylcarbamate) on the surface is used as the filler [CHIRALPAK IG-3 (2.1×150 mm, 3 mm) is applicable]; the flow rate is 0.3 ml / min; the injection volume is 20 μl;

[0297] Mobile phase: Purified water is used as mobile phase A, and acetonitrile is used as mobile phase B; linear gradient elution is carried out according to the following table, and the elution conditions are as follows:

[0298] 。

[0299] 2. Determination method

[0300] (1) Solution preparation

[0301] a. Stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance: Accurately weigh 12.27 mg of (S)-4-propyl-dihydrofuran-2-one reference substance, place it in a 25-ml volumetric flask, dissolve it with acetonitrile and dilute to the mark, shake well, and use it as the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance.

[0302] b. Reference substance solution: Accurately measure 1.0 ml of the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance, place it in a 10-ml volumetric flask, add 0.4 ml of trimethylsilyl iodide, then add 2.0 ml of acetonitrile, heat it in a water bath at 60 °C, and manually shake it once every 5 minutes. After reacting for 0.5 h, take it out, then add 4 ml of absolute ethanol, continue to manually shake and react for 1 h at intervals of 5 minutes. Take it out, let it cool to room temperature, dilute it to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, and dilute it to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the reference substance solution.

[0303] c. System suitability solution: Accurately weigh 50.45 mg of (R)-4-propyl-dihydrofuran-2-one reference substance, place it in a 10-ml volumetric flask, accurately add 1.0 ml of the stock solution of (S)-4-propyl-dihydrofuran-2-one reference substance and 0.4 ml of trimethylsilyl iodide, then add 2.0 ml of acetonitrile, heat it in a water bath at 60 °C, and manually shake it once every 5 minutes. After reacting for 0.5 h, take it out, then add 4 ml of absolute ethanol, continue to manually shake and react for 1 h at intervals of 5 minutes. Take it out, let it cool to room temperature, dilute it to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, and dilute it to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, and use it as the system suitability solution.

[0304] d. Test solution: Accurately weigh 50.91 mg of the (R)-4-propyl-dihydrofuran-2-one sample to be tested, place it in a 10-ml volumetric flask, add 0.4 ml of trimethylsilyl iodide, then add 3.0 ml of acetonitrile, heat in a water bath at 60 °C, and shake manually every 5 minutes. After reacting for 0.5 h, take it out, add 4 ml of absolute ethanol, continue to shake manually every 5 minutes and react for 1 h, take it out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, and dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, as the test solution.

[0305] e. Blank solution: Accurately measure 0.4 ml of trimethylsilyl iodide, place it in a 10-ml volumetric flask, add 2.0 ml of acetonitrile, heat in a water bath at 60 °C, and shake manually every 5 minutes. After reacting for 0.5 h, take it out, add 4 ml of absolute ethanol, continue to shake manually every 5 minutes and react for 1 h, take it out, let it cool to room temperature, dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, accurately measure 1 ml, place it in a 10-ml volumetric flask, and dilute to the mark with 60% (v / v) acetonitrile aqueous solution, shake well, as the blank solution.

[0306] (2)Detection

[0307] a. Measure 20 ml each of the blank solution and the system suitability solution and inject them into the high-performance liquid chromatograph, and record the chromatogram. The results of the system suitability solution are shown in Table 15. It is required that the resolution between (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one should be greater than 2.0;

[0308]

[0309] Conclusion: The resolution between (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one is 3.21, which is greater than 2.0, meeting the standard requirements.

[0310] b. Measure 20 ml each of the reference solution and the test solution and inject them into the high-performance liquid chromatograph, and record the chromatogram. The results of the reference solution and the test solution are shown in Table 16:

[0311] 。

[0312] Results:

[0313] The content of (S)-4-propyl-dihydrofuran-2-one is calculated by the external standard method with the peak area formula as:

[0314] The enantiomer content (%) = C 对照品 ×A 供试品 ×K 供试品 / A 对照品 / M供试品 × 100%.

[0315] C 对照品 —— Concentration of (S)-4-propyl-dihydrofuran-2-one in the reference solution, mg / ml;

[0316] A 供试品 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the test solution;

[0317] K 供试品 —— Dilution factor of (S)-4-propyl-dihydrofuran-2-one in the test solution, ml;

[0318] A 对照品 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the reference solution;

[0319] M 供试品 is the weighed amount of the test substance in the test solution.

[0320] The calculation result is: 0.105%.

[0321] The formula for calculating the content of (S)-4-propyl-dihydrofuran-2-one by area normalization method without correction factor is:

[0322] Enantiomer content (%) = A 对映异构体 / A 总 × 100% (only integrate the main peak and the enantiomer peak).

[0323] A 对映异构体 —— Peak area of (S)-4-propyl-dihydrofuran-2-one in the test solution;

[0324] A 总 —— Sum of the peak areas of (S)-4-propyl-dihydrofuran-2-one and (R)-4-propyl-dihydrofuran-2-one in the test solution.

[0325] The calculation result is: 0.107%.

[0326] Conclusion: The contents of (S)-4-propyl-dihydrofuran-2-one in (R)-4-propyl-dihydrofuran-2-one obtained by the above two calculation methods are basically the same.

Claims

1. A method for detecting the enantiomers of the starting material of brivaracetam, wherein, The starting material of brivaracetam is (R)-4-propyl-dihydrofuran-2-one, and the (R)-4-propyl-dihydrofuran-2-one is the raw material of (R)-4-propyl-dihydrofuran-2-one from Yikele Taizhou Pharmaceutical Co., Ltd., with batch number D01-SM1-Z03320220301; the enantiomer of the starting material of brivaracetam is (S)-4-propyl-dihydrofuran-2-one; it is characterized in that the detection method includes the following steps: (1) Prepare the mobile phase: Mobile phase A: water Mobile phase B: acetonitrile (2) Prepare the test solution: Weigh the sample of (R)-4-propyl-dihydrofuran-2-one, add trimethylsilyl iodide and acetonitrile, and react by shaking in a water bath thermostatic oscillator. Then add anhydrous ethanol and continue to react by shaking in a water bath thermostatic oscillator. Let it cool to room temperature and prepare the test solution by mixing with an acetonitrile aqueous solution for later use; (3) Prepare the reference solution: Weigh the reference substance of (S)-4-propyl-dihydrofuran-2-one, add trimethylsilyl iodide and acetonitrile, and react by shaking in a water bath thermostatic oscillator. Then add anhydrous ethanol and continue to react by shaking in a water bath thermostatic oscillator. Let it cool to room temperature and prepare the reference solution by mixing with an acetonitrile aqueous solution for later use; (4) Respectively draw equal amounts of the above test solution and reference solution and inject them into a high performance liquid chromatograph for determination. The chromatographic conditions include: Chromatographic column: chiral column; the chromatographic column is: CHIRALPAK IG-3; length 150 mm, inner diameter 2.1 mm, packing particle size 3 μm; Detector: UV detector; Detection wavelength: 240 nm - 260 nm; Column temperature: 27 °C - 33 °C; Flow rate: 0.25 ml / min - 0.35 ml / min; Using the above chromatographic conditions, perform linear gradient elution with mobile phase A and mobile phase B; the program of the gradient elution is: ； (5) Calculation method: Calculate by the external standard method with peak area or by the area normalization method.

2. The detection method according to claim 1, characterized in that: In step (4) described above, the column temperature is 30 °C.

3. The detection method according to claim 1, wherein: In step (4) described above, the flow rate is 0.3 ml / min.

4. The detection method according to claim 1, wherein: In steps (2) and (3) described above, the shaking method is shaking with an instrument or manual shaking.

5. The detection method according to claim 1, characterized in that: In steps (2) and (3) described above, the temperature of the shaking reaction is 50 °C - 70 °C.

6. The detection method according to claim 5, wherein: In steps (2) and (3) described above, the temperature of the shaking reaction is 57 °C - 63 °C.

7. The detection method according to claim 1, wherein: In steps (2) and (3) described above, in steps (2) and (3) described above, the concentration of the acetonitrile aqueous solution is 60% by volume.

8. The detection method according to claim 1, characterized in that: In steps (2) and (3) described above, the first shaking time is 0.45 h - 0.55 h.

9. The detection method according to claim 1, wherein: In steps (2) and (3) described above, the second shaking time is 0.9 h - 1.1 h.

10. The detection method according to any one of claims 1 to 9, characterized in that: In steps (2) and (3) described above, the volume of trimethylsilyl iodide added to the test solution and reference solution is 0.35 - 0.45 ml, the volume of acetonitrile added is 2.0 ml - 3.0 ml, and the volume of anhydrous ethanol added is 3.5 ml - 4.5 ml.

11. The detection method according to claim 10, characterized in that: In the said step (2) and step (3), the volume of trimethylsilyl iodide added to the test solution and the reference solution is 0.4 ml, the volume of acetonitrile added is 2.0 ml, and the volume of absolute ethanol added is 4 ml.

12. The detection method according to any one of claims 1 to 9, characterized in that: In the said step (2), the concentration of the test solution is 0.4 mg / ml to 0.6 mg / ml.

13. The detection method according to claim 12, wherein: In the said step (2), the concentration of the test solution is 0.45 mg / ml to 0.55 mg / ml.

14. The detection method according to claim 13, wherein: In the said step (2), the concentration of the test solution is 0.5 mg / ml.

15. The detection method according to any one of claims 1 to 9, characterized in that: In the said step (3), the concentration of the reference solution is 0.1 μg / ml to 5 μg / ml.

16. The detection method according to claim 15, wherein: In the said step (3), the concentration of the reference solution is 5 μg / ml.

17. The detection method according to any one of claims 1 to 9, characterized in that: In the said step (4), 5 μl to 100 μl of the test solution and the reference solution respectively; inject into the liquid chromatograph.

18. The detection method according to claim 17, characterized in that: In the said step (4), 20 μl of the test solution and the reference solution respectively; inject into the liquid chromatograph.

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