A method for detecting impurities of suvorexan intermediate and its enantiomeric impurities

Abstract

The present invention belongs to the technical field of drug analysis and quality control, and relates to a method for detecting a suvorexan intermediate and its enantiomeric impurities. More specifically, it relates to a method for detecting a suvorexan intermediate (i.e., (R)-4-benzoazole-7-methyl-1,4-diazepine-2,5-dione) and its enantiomeric impurities (i.e., (S)-4-benzoazole-7-methyl-1,4-diazepine-2,5-dione) by high performance liquid chromatography (HPLC). The detection method of the present invention can effectively separate the suvorexan intermediate from its enantiomeric impurities, accurately determine the enantiomeric impurity content, and the determination result is stable and reliable, with strong specificity, so as to effectively control the quality of the suvorexan intermediate, improve the quality of the suvorexan bulk drug, and effectively monitor the intermediate synthesis process of suvorexan and the synthesis process of the suvorexan bulk drug.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis and quality control, and particularly relates to a method for detecting a suvorexant intermediate (i.e., (R)-4-benzoazole-7-methyl-1,4-diazepine-2,5-dione) and an enantiomeric impurity thereof (i.e., (S)-4-benzoazole-7-methyl-1,4-diazepine-2,5-dione) by high performance liquid chromatography (HPLC). Background Art

[0002] Suvorexant is a new drug for the treatment of sleep difficulties approved by the US FDA in 2014. Its original manufacturer is Merck Pharmaceuticals. Suvorexant is an orexin receptor antagonist that can affect the signal transduction of the orexin pathway; orexin is a small molecule neuropeptide that is related to the regulation of the sleep-wake cycle. Suvorexant can be used clinically to treat chronic insomnia.

[0003] The chemical name of Suvorexant is 5-chloro-2-[(5R)-5-methyl-4-[5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoyl]-1,4-diazepan-1-yl]-1,3-benzoxazole, and its molecular formula is C 23 H 23 ClN6O2, its structural formula is as follows:

[0004]

[0005] The synthetic route of Suvorexant is as follows Figure 1 As shown. Figure 1 It can be seen from the synthetic route that IN4 is a key intermediate for the synthesis of Suvorexan API. The chemical name of IN4 is (R)-4-benzoazole-7-methyl-1,4-diazepine-2,5-dione. The chemical structure of IN4 is as follows:

[0006]

[0007] In order to ensure the product quality of suvorexan API or raw materials, the content of IN4 in suvorexan API or raw materials is required to be strictly controlled during drug research and development and drug registration. IN4 contains a certain number of enantiomers, the chemical name of which is (S)-4-benzoazole-7-methyl-1,4-diazepine-2,5-dione (referred to as suvorexan intermediate enantiomeric impurity or enantiomeric impurity or enantiomeric impurity B or impurity B in this article, and these terms are used interchangeably in this article). Impurity B, as an enantiomer of IN4, has a key impact on the quality of IN4.

[0008] The synthetic process route of svoraxant shows that impurity B and IN4 participate in subsequent reactions, and IN4 will eventually form svoraxant API through multiple steps of reaction; impurity B also reacts chemically with other reactants at the same time, thereby deriving a series of substances with structures similar to impurity B and producing more impurities; if impurity B in IN4 is not subject to limit control, it will eventually cause the isomers of svoraxant API to exceed the limit requirements, resulting in unqualified quality of svoraxant API and even affecting the registration application of svoraxant API.

[0009] Therefore, it is urgent to find a quality control method that can accurately determine impurity B so as to control the content and limit of impurity B in advance in IN4, thereby better controlling the product quality of suvorexan API or raw material drug.

[0010] The basic information of impurity B is shown in the table below:

[0011]

[0012] After reviewing the literature, no technical reports on the detection method of suvorexan intermediate IN4 and its enantiomeric impurity B have been found, and no public information has been published to explain the important influence of IN4 and its impurity B on the quality of suvorexan API or raw materials. The present invention aims to provide a high performance liquid chromatography analysis method for impurity B of suvorexan, which can effectively control the quality of suvorexan IN4, and then effectively control the quality of suvorexan API or raw materials, and at the same time, it also has certain guiding significance for the research on the synthesis process of suvorexan API or raw materials. Summary of the invention

[0013] In order to effectively control the quality of suvorexan, in one aspect, the present invention provides a method for detecting suvorexan intermediates and enantiomeric impurities thereof by HPLC, wherein the chromatographic conditions of the HPLC are:

[0014] Stationary phase: polysaccharide derivative normal phase coated chiral chromatographic column, the silica surface is coated with cellulose-tri(4-methylbenzoate);

[0015] Mobile phase: a mixed solution of n-hexane, ethanol and diethylamine, wherein the volume ratio of n-hexane, ethanol and diethylamine is 95% to 60%: 5% to 40%: 0.01% to 0.2%;

[0016] Elution procedure: isocratic elution for 5 to 60 minutes;

[0017] The suovoxan intermediate is (R)-4-benzoxazole-7-methyl-1,4-diazepine-2,5-dione, and its enantiomeric impurity is (S)-4-benzoxazole-7-methyl-1,4-diazepine-2,5-dione (referred to herein as suovoxan intermediate enantiomeric impurity or enantiomeric impurity or enantiomeric impurity B or impurity B, and these terms are used interchangeably herein).

[0018] Furthermore, the volume ratio of n-hexane, ethanol and diethylamine in the mobile phase is 90% to 70%: 10% to 30%: 0.01% to 0.2%.

[0019] Furthermore, the volume ratio of n-hexane, ethanol and diethylamine in the mobile phase is 70%:30%:0.1%.

[0020] Further, the elution procedure is isocratic elution for 18 to 60 min. Further, the elution procedure is isocratic elution for 5 to 30 min. Further, the elution procedure is isocratic elution for 18 to 30 min. More preferably, the elution procedure is isocratic elution for 18 min.

[0021] Furthermore, the detection wavelength of the HPLC is a full wavelength scan of 190 to 450 nm, preferably 220 nm; the flow rate is 1.0 ml / min; the column temperature is 30° C.; and the injection volume is 20 μl.

[0022] Furthermore, the method further comprises preparing a test sample solution, which comprises taking the test sample and adding a diluent to dissolve the solution. Furthermore, the test sample comprises a suvorexan intermediate to be tested.

[0023] Furthermore, the mass volume ratio of the suvorexan intermediate to be detected to the diluent in the test solution is 1.0-4.0 mg:1 ml, preferably 2.0 mg:1 ml.

[0024] Furthermore, the method also includes preparing a system suitability solution, which includes taking the suvorexan intermediate and its enantiomeric impurity reference substances respectively, adding a diluent to dissolve and mix.

[0025] Further, the concentration of the suvorexan intermediate in the system suitability solution is 1.0-4.0 mg / ml, and the concentration of the enantiomeric impurity reference substance is 300-800 μg / ml. Preferably, the concentration of the suvorexan intermediate in the system suitability solution is 1.5 mg / ml, and the concentration of the enantiomeric impurity reference substance is 500 μg / ml.

[0026] Furthermore, the diluent includes ethanol.

[0027] Furthermore, the method further comprises comparing the chromatograms of the test solution and the system suitability solution. If a chromatographic peak corresponding to the retention time of the chromatographic peak of the enantiomeric impurity B in the chromatogram of the system suitability solution is presented in the chromatogram of the test solution, it indicates that the test sample contains the enantiomeric impurity B.

[0028] Furthermore, the method further comprises calculating the content of the enantiomeric impurity by the following formula:

[0029] Xi%=Ai / A×100%

[0030] Wherein, Xi% is the content of enantiomeric impurities in the test solution, Ai is the peak area of ​​enantiomeric impurities in the test solution, and A is the sum of all peak areas in the test solution.

[0031] In another aspect, the present invention provides a method for detecting the presence of enantiomeric impurities in a suvorexan intermediate by HPLC, comprising the following steps:

[0032] a. Preparation of test solution: Take the intermediate of suvorexan to be tested, add diluent to dissolve it, and obtain the test solution;

[0033] b. Preparation of system suitability solution: Take the suvorexan intermediate and its enantiomer impurity B reference substance respectively, add a diluent to dissolve them, mix them, and obtain a system suitability solution;

[0034] The suvorexan intermediate is (R)-4-benzoazole-7-methyl-1,4-diazepine-2,5-dione;

[0035] The enantiomeric impurity B is (S)-4-benzoxazole-7-methyl-1,4-diazepine-2,5-dione;

[0036] c. Use the following chromatographic conditions to test the test sample and system suitability solution respectively:

[0037] Stationary phase: polysaccharide derivative normal phase coated chiral chromatographic column, the silica surface is coated with cellulose-tri(4-methylbenzoate);

[0038] Mobile phase: a mixed solution of n-hexane, ethanol and diethylamine, wherein the volume ratio of n-hexane, ethanol and diethylamine is 95% to 60%: 5% to 40%: 0.01% to 0.2%;

[0039] Elution procedure: isocratic elution for 5 to 60 minutes;

[0040] d. If the chromatogram of the test solution shows a chromatographic peak with a retention time corresponding to the chromatographic peak of enantiomeric impurity B in the chromatogram of the system suitability solution, it indicates that the test sample contains enantiomeric impurity B.

[0041] Furthermore, the volume ratio of n-hexane, ethanol and diethylamine in the mobile phase is 90% to 70%: 10% to 30%: 0.01% to 0.2%. Furthermore, the volume ratio of n-hexane, ethanol and diethylamine in the mobile phase is 70%: 30%: 0.1%.

[0042] Further, the elution procedure is isocratic elution for 18 to 60 min. Further, the elution procedure is isocratic elution for 5 to 30 min. Further, the elution procedure is isocratic elution for 18 to 30 min. More preferably, the elution procedure is isocratic elution for 18 min.

[0043] Furthermore, the detection wavelength of the HPLC is a full wavelength scan of 190 to 450 nm, preferably 220 nm; the flow rate is 1.0 ml / min; the column temperature is 30° C.; and the injection volume is 20 μl.

[0044] Furthermore, the mass volume ratio of the suvorexan intermediate to be detected to the diluent in the test solution is 1.0-4.0 mg:1 ml, preferably 2.0 mg:1 ml.

[0045] Further, the concentration of the suvorexan intermediate in the system suitability solution is 1.0-4.0 mg / ml, and the concentration of the enantiomeric impurity reference substance is 300-800 μg / ml. Preferably, the concentration of the suvorexan intermediate in the system suitability solution is 1.5 mg / ml, and the concentration of the enantiomeric impurity reference substance is 500 μg / ml.

[0046] Furthermore, the diluent includes ethanol.

[0047] In another aspect, the present invention provides a method for determining the content of enantiomeric impurity B in a suvorexan intermediate by HPLC, wherein the suvorexan intermediate is (R)-4-benzoazole-7-methyl-1,4-diazepine-2,5-dione, and the enantiomeric impurity B is (S)-4-benzoazole-7-methyl-1,4-diazepine-2,5-dione, and the method comprises the following steps:

[0048] a. Preparation of test solution: Take the intermediate of suvorexan to be tested, add diluent to dissolve it, and obtain the test solution;

[0049] b. Use the following chromatographic conditions to test the test solution:

[0050] Stationary phase: polysaccharide derivative normal phase coated chiral chromatographic column, the silica surface is coated with cellulose-tri(4-methylbenzoate);

[0051] Mobile phase: a mixed solution of n-hexane, ethanol and diethylamine, wherein the volume ratio of n-hexane, ethanol and diethylamine is 95% to 60%: 5% to 40%: 0.01% to 0.2%;

[0052] Elution procedure: isocratic elution for 5 to 60 minutes;

[0053] c. Calculate the content of the enantiomeric impurity B by the following formula:

[0054] Xi%=Ai / A×100%

[0055] Wherein, Xi% is the content of enantiomeric impurities in the test solution, Ai is the peak area of ​​enantiomeric impurities in the test solution, and A is the sum of all peak areas in the test solution.

[0056] Furthermore, the method also includes preparing a system suitability solution, which includes taking the suvorexan intermediate and its enantiomer impurity B reference substance respectively, adding a diluent to dissolve and mix.

[0057] Furthermore, the volume ratio of n-hexane, ethanol and diethylamine in the mobile phase is 90% to 70%: 10% to 30%: 0.01% to 0.2%. Furthermore, the volume ratio of n-hexane, ethanol and diethylamine in the mobile phase is 70%: 30%: 0.1%.

[0058] Further, the elution procedure is isocratic elution for 18 to 60 min. Further, the elution procedure is isocratic elution for 5 to 30 min. Further, the elution procedure is isocratic elution for 18 to 30 min. More preferably, the elution procedure is isocratic elution for 18 min.

[0059] Furthermore, the detection wavelength of the HPLC is a full wavelength scan of 190 to 450 nm, preferably 220 nm; the flow rate is 1.0 ml / min; the column temperature is 30° C.; and the injection volume is 20 μl.

[0060] Furthermore, the mass volume ratio of the suvorexan intermediate to be detected to the diluent in the test solution is 1.0-4.0 mg:1 ml, preferably 2.0 mg:1 ml.

[0061] Further, the concentration of the suvorexan intermediate in the system suitability solution is 1.0-4.0 mg / ml, and the concentration of the enantiomeric impurity reference substance is 300-800 μg / ml. Preferably, the concentration of the suvorexan intermediate in the system suitability solution is 1.5 mg / ml, and the concentration of the enantiomeric impurity reference substance is 500 μg / ml.

[0062] Furthermore, the diluent includes ethanol.

[0063] Beneficial Effects of the Invention

[0064] 1. The present invention can effectively control the quality of the suvorexan intermediate IN4 and can qualitatively and quantitatively detect its impurity B.

[0065] 2. While effectively controlling the quality of IN4, the present invention can indirectly control the quality of suvorexan API or raw material medicine, so that its quality meets the relevant requirements of chemical raw materials.

[0066] 3. The present invention controls the content limit of impurity B in IN4, thereby reducing other impurities with a structure similar to impurity B that impurity B subsequently brings to suvorexan API or raw materials; thereby simplifying the purification process of suvorexan API or raw materials, reducing the production cost, and partially alleviating the economic burden of medication for patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 It is the synthetic route map of Suvorexant.

[0068] Figure 2 It is the HPLC chart of the test solution in Example 1.

[0069] Figure 3 It is the HPLC chart of the test solution in Example 2.

[0070] Figure 4 is a typical HPLC profile of a system suitability solution.

[0071] Figure 5 is a typical HPLC profile of the system suitability solution of Example 6. DETAILED DESCRIPTION

[0072] It should be understood that based on the above contents of the present invention, according to common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0073] The above contents of the present invention are further described in detail below by the specific implementation of the embodiment form. However, this should not be interpreted as the scope of the above theme of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0074] (1) Materials

[0075] IN4 (provided by Chengdu Weibang Pharmaceutical Co., Ltd., batch number IN4-221201, 221202);

[0076] Impurity B reference substance (provided by Chengdu Weibang Pharmaceutical Co., Ltd., batch number MS203421-221001).

[0077] (2) Main instruments

[0078] High performance liquid chromatograph: Shimadzu LC-20AT, chromatographic column: CHIRALCEL OJ-H, 4.6×250 mm, 5 μm.

[0079] Example 1: Determination of the content of impurity B in the intermediate IN4 of Suvorexan

[0080] (1) Preparation of test solution: Accurately weigh IN4 (batch number IN4-221201), dissolve it in diluent ethanol, and prepare a 2.0 mg / ml test solution.

[0081] (2) Preparation of system suitability solution: Accurately weigh the impurity B reference substance, dissolve it with diluent ethanol, and prepare a solution with a concentration of 1.0 mg / ml as the impurity B reference substance stock solution. Accurately weigh 15.00 mg of IN4 and accurately measure 5.0 ml of the impurity B stock solution, place them in a 10 ml volumetric flask, dissolve it with diluent ethanol, shake well, and make up to volume to prepare the system suitability solution.

[0082] (3) Chromatographic condition setting

[0083] Chromatographic column: CHIRALCEL OJ-H, specification 4.6×250mm, 5μm;

[0084] Detection wavelength: 220nm;

[0085] Flow rate: 1.0 ml / min;

[0086] Column temperature: 30°C;

[0087] Injection volume: 20 μl;

[0088] Mobile phase: n-hexane-ethanol-diethylamine = (70%:30%:0.1%, v / v / v);

[0089] Gradient elution program: isocratic elution for 18 min.

[0090] (4) Determination

[0091] Pipette 20 μl of the test solution and system suitability solution into the chromatograph, record the chromatogram, and record the peak area A of impurity B in the test sample. i The content of impurity B in the test sample was calculated by area normalization method based on the sum of all peak areas A in the test sample solution.

[0092] The calculation formula is as follows:

[0093] Xi%=Ai / A×100%

[0094] Among them, X i %: the content of impurity B in the test sample,

[0095] A i : Peak area of ​​impurity B in the test sample,

[0096] A: The sum of all peak areas in the test solution. The HPLC spectrum of the test solution is shown in Figure 2 .

[0097] Calculation results: The impurity B content of IN4 sample batch number 221201 is: impurity B = 0.038%.

[0098] Example 2: Determination of the content of impurity B in the intermediate IN4 of Suvorexan

[0099] (1) Preparation of test solution: Accurately weigh IN4 (batch number IN4-221202), dissolve it in diluent ethanol, and prepare a 2.0 mg / ml test solution.

[0100] (2) Preparation of system suitability solution: Accurately weigh the impurity B reference substance, dissolve it with diluent ethanol, and prepare a solution with a concentration of 1.0 mg / ml as the impurity B reference substance stock solution. Accurately weigh 15.00 mg of IN4 and accurately measure 5.0 ml of the impurity B stock solution, place them in a 10 ml volumetric flask, dissolve it with diluent ethanol, shake well, and make up to volume to prepare the system suitability solution.

[0101] (3) Chromatographic condition setting

[0102] Chromatographic column: CHIRALCEL OJ-H, specification 4.6×250mm, 5μm;

[0103] Detection wavelength: 220nm;

[0104] Flow rate: 1.0 ml / min;

[0105] Column temperature: 30°C;

[0106] Injection volume: 20 μl;

[0107] Mobile phase: n-hexane-ethanol-diethylamine = (70%:30%:0.1%, v / v / v);

[0108] Gradient elution program: isocratic elution for 18 min.

[0109] (4) Determination

[0110] Pipette 20 μl of the test solution and system suitability solution into the chromatograph, record the chromatogram, and record the peak area A of impurity B in the test sample. i The content of impurity B in the test sample was calculated by area normalization method based on the sum of all peak areas A in the test sample solution.

[0111] The calculation formula is as follows:

[0112] Xi%=Ai / A×100%

[0113] Among them, X i %: the content of impurity B in the test sample,

[0114] A i : Peak area of ​​impurity B in the test sample,

[0115] A: The sum of all peak areas in the test solution. The HPLC spectrum of the test solution is shown in Figure 3 .

[0116] Calculation results: The impurity B content of IN4 sample batch number 221202 is: Impurity B = ND.

[0117] The beneficial effects of the detection method of the present invention are evaluated through the following examples.

[0118] Example 3: System Applicability and Specificity

[0119] Instrument: High performance liquid chromatograph: Shimadzu LC-20AT;

[0120] Chromatographic column: CHIRALCEL OJ-H, specification 4.6×250mm, 5μm;

[0121] Detection wavelength: 220nm;

[0122] Flow rate: 1.0 ml / min;

[0123] Column temperature: 30°C;

[0124] Injection volume: 20 μl;

[0125] Mobile phase: n-hexane-ethanol-diethylamine = (70%:30%:0.1%, v / v / v);

[0126] Gradient elution program: isocratic elution for 18 min.

[0127] Solution preparation:

[0128] (1) Blank solution (the following dilutions): ethanol;

[0129] (2) Impurity B stock solution: Accurately weigh 10 mg of impurity B into a 10 ml volumetric flask, dissolve it with diluent and make up to volume (impurity B: 1.0 mg / ml);

[0130] (3) System suitability solution: Accurately weigh 15 mg of IN4 and 5 mg of impurity B into a 10 ml volumetric flask, and dilute to volume with diluent (IN4 1.5 mg / ml, impurity B 0.5 mg / ml);

[0131] (4) Impurity B localization solution: Weigh 5 mg of impurity B and place it in a 10 ml volumetric flask, then add diluent to dissolve and make up the volume (impurity B: 0.5 mg / ml).

[0132] (5) Test solution: Accurately weigh 20 mg of IN4 into a 10 ml volumetric flask and add diluent solution to make up to volume.

[0133] Inject blank solution, system suitability solution, and test solution according to the chromatographic conditions, record the chromatographic process, and the results are shown in Table 1. The HPLC spectrum of the system suitability solution is shown in Figure 4 .

[0134] Table 1 System applicability and specificity

[0135]

[0136] Conclusion: The system applicability of this method has a solution separation degree of 13.26, which is good, and the impurity B peak has no interference, indicating that the system applicability and specificity of the present invention meet the requirements of quality analysis.

[0137] Example 4: Limit of Detection and Limit of Quantitation

[0138] The experimental conditions, liquid chromatography method and solution preparation were the same as those in Example 3.

[0139] For known impurity B, the limit of detection (LOD) and limit of quantification (LOQ) are determined based on the signal-to-noise ratio. The impurity B stock solution with known concentration is diluted to a low concentration for injection. The detection signal-to-noise ratio S / N ≥ 10 is determined as the limit of quantification, and S / N = 2 to 8 is determined as the limit of detection. The test results are shown in Table 2.

[0140] Table 2 Limit of quantitation and detection limit

[0141]

[0142] Results: The quantitative limit of impurity B was 10ug / ml, equivalent to 0.10% of the sample concentration, and the detection limit was 5ug / ml, equivalent to 0.05% of the sample concentration, indicating that the method of the present invention has good sensitivity and meets the requirements of drug-related analysis methods.

[0143] Example 5: Solution Stability

[0144] The experimental conditions, liquid chromatography method and solution preparation were the same as those in Example 3.

[0145] The system suitability solution was placed at room temperature (25°C) for 0, 2, 6, 12, 18, and 24 hours, and then injected in sequence. The solution stability results are shown in Table 3.

[0146] Table 3 Stability

[0147] time Impurity B% 0 25.171 2 25.427 6 25.057 12 25.159 18 25.118 24 25.240 RSD% 0.51％

[0148] Conclusion: The peak area %RSD of impurity B in the solution at room temperature (25°C) within 24 hours is 0.51%, indicating that the solution of the present invention has good stability and meets the requirements of drug-related analytical methods.

[0149] The results of Examples 3-5 show that the method for determining impurity B in IN4 provided by the present invention has good specificity, sensitivity and durability (good solution stability within 24 hours at room temperature of 25°C), and can be applied to the quality control of impurity B in the intermediate IN4 of suvorexan API. The quality analysis of suvorexan intermediates by the method of the present invention is summarized. In order to ensure the quality of suvorexan API prepared with suvorexan intermediates as raw materials, the content of enantiomers in suvorexan intermediates should be controlled at 0.5%.

[0150] Example 6: Analysis of Suvorexan Intermediate IN4 and Its Enantiomeric Impurity B under Different Chromatographic Conditions

[0151] (1) Blank solution (the following dilutions): ethanol;

[0152] (2) Impurity B stock solution: Accurately weigh 10 mg of impurity B into a 10 ml volumetric flask, dissolve it with diluent and make up to volume (impurity B: 1.0 mg / ml);

[0153] (3) System suitability solution: Accurately weigh 15 mg of IN4 and 5 mg of impurity B into a 10 ml volumetric flask, and dilute to volume with diluent (IN4 1.5 mg / ml, impurity B 0.5 mg / ml);

[0154] (4) Determination.

[0155] Pipette 20 μl of the system suitability solution and inject it into the chromatograph. The chromatographic conditions are as follows:

[0156] Chromatographic column: CHIRALCEL OJ-H, specification 4.6×250mm, 5μm;

[0157] Mobile phase: n-hexane-ethanol-diethylamine = (90%:10%:0.1%, v / v / v);

[0158] Detection wavelength: 220nm;

[0159] Flow rate: 1.0 ml / min;

[0160] Column temperature: 30°C;

[0161] Injection volume: 20 μl;

[0162] Gradient elution program: isocratic elution for 60 min.

[0163] Record the chromatogram and record the peak area A of impurity B in the system suitability solution. i The content of impurity B in the system suitability solution was calculated by the area normalization method based on the sum of all peak areas A in the system suitability solution.

[0164] The calculation formula is as follows:

[0165] Xi%=Ai / A×100%

[0166] Among them, X i %: Content of impurity B in the system suitability solution,

[0167] A i : Impurity B peak area in system suitability solution,

[0168] A: The sum of all peak areas in the system suitability solution;

[0169] Calculation results: System suitability solution HPLC spectrum see Figure 5 ; Under different mobile phase ratios, the separation degree of this method is 15.39, which meets the analysis requirements, but the retention time of impurity B is 40.446min, the analysis time is too long, and the analysis cost increases. While meeting the analysis requirements, it should be economical and efficient. Therefore, this method can be further optimized as the method in Examples 1-5 of the present invention.

[0170] In summary, the present invention meets the requirements of the 2020 edition of the "Chinese Pharmacopoeia" for analytical methodology, and effectively controls the quality of the svoraxant intermediate IN4, thereby improving the quality of the svoraxant raw material. At the same time, it can effectively monitor the synthesis process of the intermediates of svoraxant and the synthesis process of the svoraxant raw material, which has a certain guiding role in the drug research and development of svoraxant.

Claims

1. A method for detecting impurities of suvorexan intermediates and enantiomers thereof by HPLC, characterized in that: The chromatographic conditions of the HPLC are: Stationary phase: polysaccharide derivative normal phase coated chiral chromatographic column, the silica surface is coated with cellulose-tri(4-methylbenzoate); Mobile phase: a mixed solution of n-hexane, ethanol and diethylamine, wherein the volume ratio of n-hexane, ethanol and diethylamine is 95%-60%: 5%-40%: 0.01%-0.2%; Elution procedure: isocratic elution for 5 to 60 minutes; The suvorexan intermediate is (R)-4-benzoazole-7-methyl-1,4-diazepine-2,5-dione, and its enantiomeric impurity is (S)-4-benzoazole-7-methyl-1,4-diazepine-2,5-dione.

2. The method according to claim 1, characterized in that The volume ratio of n-hexane, ethanol and diethylamine in the mobile phase is 90%-70%: 10%-30%: 0.01%-0.2%.

3. The method according to claim 2, characterized in that The volume ratio of n-hexane, ethanol and diethylamine in the mobile phase is 70%:30%:0.1%.

4. The method according to claim 1, characterized in that: The elution procedure is isocratic elution for 18 to 30 minutes; the detection wavelength of the HPLC is 220 nm; the flow rate is 1.0 ml / min; the column temperature is 30° C.; and the injection volume is 20 μl.

5. A method for detecting the presence of enantiomeric impurities in a suvorexan intermediate by HPLC, characterized in that: The following steps are involved: a. Preparation of test solution: Take the intermediate of suvorexan to be tested, add diluent to dissolve it, and obtain the test solution; b. Preparation of system suitability solution: Take the suvorexan intermediate and its enantiomeric impurity reference substance respectively, add diluent to dissolve, mix, and obtain system suitability solution; The suvorexan intermediate is (R)-4-benzoazole-7-methyl-1,4-diazepine-2,5-dione; The enantiomeric impurity is (S)-4-benzoxazole-7-methyl-1,4-diazepine-2,5-dione; c. Use the following chromatographic conditions to test the test sample and system suitability solution respectively: Stationary phase: polysaccharide derivative normal phase coated chiral chromatographic column, the silica surface is coated with cellulose-tri(4-methylbenzoate); Mobile phase: a mixed solution of n-hexane, ethanol and diethylamine, wherein the volume ratio of n-hexane, ethanol and diethylamine is 95%-60%: 5%-40%: 0.01%-0.2%; Elution procedure: isocratic elution for 5 to 60 minutes; d. If the chromatogram of the test solution shows a chromatographic peak with a retention time corresponding to the chromatographic peak of the enantiomeric impurity in the chromatogram of the system suitability solution, it indicates that the test sample contains enantiomeric impurities.

6. The method according to claim 5, characterized in that The volume ratio of n-hexane, ethanol and diethylamine in the mobile phase is 70%:30%:0.1%.

7. The method according to claim 5, characterized in that The elution procedure is isocratic elution for 18 to 30 minutes; the detection wavelength of the HPLC is 220 nm; the flow rate is 1.0 ml / min; the column temperature is 30° C.; and the injection volume is 20 μl.

8. A method for determining the content of enantiomeric impurities in a suvorexan intermediate by HPLC, wherein the suvorexan intermediate is (R)-4-benzoxazole-7-methyl-1,4-diazepine-2,5-dione, and the enantiomeric impurity is (S)-4-benzoxazole-7-methyl-1,4-diazepine-2,5-dione, characterized in that: The method comprises the following steps: a. Preparation of test solution: Take the intermediate of suvorexan to be tested, add diluent to dissolve it, and obtain the test solution; b. Use the following chromatographic conditions to test the test solution: Stationary phase: polysaccharide derivative normal phase coated chiral chromatographic column, the silica surface is coated with cellulose-tri(4-methylbenzoate); Mobile phase: a mixed solution of n-hexane, ethanol and diethylamine, wherein the volume ratio of n-hexane, ethanol and diethylamine is 95% to 60%: 5% to 40%: 0.01% to 0.2%; Elution procedure: isocratic elution for 5 to 60 minutes; c. Calculate the content of the enantiomeric impurity by the following formula: Xi%=Ai / A×100% Wherein, Xi% is the content of enantiomeric impurities in the test solution, Ai is the peak area of ​​enantiomeric impurities in the test solution, and A is the sum of all peak areas in the test solution.

9. The method according to claim 7, characterized in that: The volume ratio of n-hexane, ethanol and diethylamine in the mobile phase is 70%:30%:0.1%.

10. The method according to claim 7, characterized in that The elution procedure is isocratic elution for 18 to 30 minutes; the detection wavelength of the HPLC is 220 nm; the flow rate is 1.0 ml / min; the column temperature is 30° C.; and the injection volume is 20 μl.

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