CN115950991B - Su Wo Leisheng intermediate and detection method of enantiomer impurities thereof - Google Patents
Su Wo Leisheng intermediate and detection method of enantiomer impurities thereof Download PDFInfo
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- JYTNQNCOQXFQPK-MRXNPFEDSA-N suvorexant Chemical compound C([C@H]1C)CN(C=2OC3=CC=C(Cl)C=C3N=2)CCN1C(=O)C1=CC(C)=CC=C1N1N=CC=N1 JYTNQNCOQXFQPK-MRXNPFEDSA-N 0.000 claims abstract description 30
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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Abstract
The invention belongs to the technical field of medicine analysis and quality control, and particularly relates to a method for detecting a threo Wo Leisheng intermediate and enantiomer impurities thereof. The invention provides a method for detecting a suvorexant intermediate and enantiomer impurities thereof by HPLC, which can effectively separate the suvorexant intermediate from the enantiomer impurities thereof, accurately measure the content of the enantiomer impurities, has stable and reliable measurement result and strong specificity, can effectively control the quality of the suvorexant intermediate, improve the quality of the suvorexant bulk drug, and can effectively monitor the synthesis process of the suvorexant intermediate and the synthesis process of the suvorexant bulk drug.
Description
Technical Field
The invention belongs to the technical field of medicine analysis and quality control, and particularly relates to a method for detecting a suvorexant intermediate (namely (R) -N-benzyl-N- (3- ((tert-butoxycarbonyl) amino) butyryl) glycine methyl ester) and enantiomer impurities (namely (S) -N-benzyl-N- (3- ((tert-butoxycarbonyl) amino) butyryl) glycine methyl ester) by High Performance Liquid Chromatography (HPLC).
Background
Suvorexant (Suvorexant) is a new drug approved by the FDA in the united states for the treatment of sleep difficulties and its original manufacturer is moxadong. Suvorexant is an orexin receptor antagonist that affects orexin pathway signaling; orexin is a small molecule neuropeptide that is involved in the regulation of the sleep-wake cycle. Suvorexant can be used clinically for treating chronic insomnia.
Suvorexant is known by the chemical name 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 of formula C 23 H 23 ClN 6 O 2 The structural formula is as follows:
the suvorexant synthesis route is shown in fig. 1. As can be seen from the synthetic route of fig. 1, IN3 is a key intermediate for the synthesis of suvorexant API. The chemical name of IN3 is (R) -N-benzyl-N- (3- ((t-butoxycarbonyl) amino) butanoyl) glycine methyl ester. The chemical formula of IN3 is as follows:
IN order to ensure the product quality of suvorexant API or drug substance, strict control of the content of IN3 IN the suvorexant API or drug substance is required IN the drug development and drug registration work. Whereas IN3 contains a number of enantiomers, the chemical name of which is (S) -N-benzyl-N- (3- ((t-butoxycarbonyl) amino) butanoyl) glycine methyl ester (referred to herein as suvorexant intermediate enantiomeric or enantiomeric impurity a or impurity a, these terms being used interchangeably herein).
The suvorexant synthesis process route shows that impurities A and IN3 participate IN subsequent reactions, IN3 is taken as a main reaction material, and finally the suvorexant API is generated; the impurity A IN IN3 will also participate IN the reaction, thereby deriving a series of impurities of the structure of impurity A; these impurities can cause suvorexant API isomers to exceed the limit requirements of the drug substance, so that the quality of suvorexant API is unacceptable and registration for reporting as the drug substance is impossible.
Therefore, it is highly desirable to find a quality control method capable of accurately measuring impurity a so as to control the content and limitation of impurity a IN advance IN 3. The purification process of the Suvorax API is effectively reduced due to the improvement of the purity of the IN3, so that the direct production cost of the Suvorax API or the bulk drug is reduced. Meanwhile, the effective control of the impurity A also indirectly controls the product quality of the Suvorexant API or the bulk drug, so that the product quality of the Suvorexant API is more stable and controllable.
The basic information for impurity a is given in the following table:
by referring to the literature, no detection method related to a suvorexant intermediate IN3 and enantiomer impurity A thereof exists IN the technology disclosed and reported at present; the prior art also does not show the important effect of IN3 and enantiomer impurity a on suvorexant API or quality of bulk drug.
The invention aims to provide a high performance liquid chromatography analysis method for a Su Wo Leisheng intermediate IN3 and an enantiomer impurity A thereof, so that the quality of the Su Vol Rainson intermediate IN3 is effectively controlled, and further the quality of a Su Vol Rainson API or a bulk drug is effectively controlled; meanwhile, the method has a certain guiding significance on the research on the synthesis process of the Suvorexant API or the bulk drug.
Disclosure of Invention
In order to effectively control the quality of suvorexant, in one aspect, the present invention provides a method for detecting suvorexant intermediates and enantiomeric impurities thereof by HPLC having chromatographic conditions of:
stationary phase: polysaccharide derivative normal phase coating chiral chromatographic column, silica gel surface coated with cellulose-tri (4-methyl benzoate);
mobile phase: the mixed solution of n-hexane, ethanol and diethylamine, wherein the volume ratio of n-hexane, ethanol and diethylamine is 95% -75%: 5% -25%: 0.01 to 0.2 percent;
elution procedure: isocratic elution is carried out for 5-30 min;
wherein the suvorexant intermediate is (R) -N-benzyl-N- (3- ((t-butoxycarbonyl) amino) butanoyl) glycine methyl ester, and the enantiomeric impurity is (S) -N-benzyl-N- (3- ((t-butoxycarbonyl) amino) butanoyl) glycine methyl ester (referred to herein as suvorexant intermediate enantiomeric impurity or enantiomeric impurity a or impurity a, which terms are used interchangeably herein).
Further, the volume ratio of n-hexane, ethanol and diethylamine in the mobile phase was 90%:10%:0.1%.
Further, the elution procedure is isocratic elution for 5-30 min. More preferably, the elution procedure is isocratic elution for 16min.
Further, the HPLC detection wavelength is full wavelength scanning of 190-450 nm, preferably 220nm; the flow rate is 1.0ml/min; the column temperature is 30 ℃; the sample loading was 20. Mu.l.
Further, the method also includes preparing a test solution that includes dissolving the test with a diluent.
Still further, the test article comprises a suvorexant intermediate to be detected.
Further, the mass volume ratio of the Suvorexant intermediate to be detected and the diluent in the sample solution is 1.0-5.0 mg:1ml, preferably 2.0mg:1ml.
Further, the method also comprises preparing a system applicability solution which comprises respectively taking the Suvorexant intermediate and the enantiomer impurity reference substances thereof, adding a diluent for dissolution and mixing.
Further, the concentration of the suvorexant intermediate in the system applicability solution is 1.0-5.0 mg/ml, and the concentration of the enantiomer impurity reference substance is 100-500 mug/ml. Preferably, the concentration of suvorexant intermediate in the system-applicable solution is 2.0mg/ml and the concentration of enantiomeric impurity reference is 300 μg/ml.
Further, the diluent comprises ethanol.
Further, the method comprises comparing the chromatograms of the sample solution and the system applicability solution, and if the chromatogram of the sample solution shows a chromatographic peak corresponding to the retention time of the chromatographic peak of the enantiomeric impurity A in the chromatogram of the system applicability solution, indicating that the sample contains the enantiomeric impurity A.
Further, the method further comprises calculating the content of the enantiomeric impurity by the formula:
Xi%=Ai/A×100%
wherein ,Xi% is the pair in the test solutionThe content of the enantiomer impurities is calculated,Aiis the peak area of enantiomer impurities in the sample solution,Ais the sum of all peak areas in the sample solution.
In another aspect, the present invention provides a method for detecting the presence of enantiomeric impurities in a suvorexant intermediate by HPLC, comprising the steps of:
a. preparation of test solution: taking a Suvorexant intermediate to be detected, and adding a diluent for dissolution to obtain a sample solution;
b. preparation of a System applicability solution: respectively taking a suvorexant intermediate and an enantiomer impurity A reference substance thereof, adding a diluent for dissolution, and mixing to obtain a system applicability solution;
the suvorexant intermediate is (R) -N-benzyl-N- (3- ((t-butoxycarbonyl) amino) butanoyl) glycine methyl ester;
the enantiomer impurity A is: (S) -N-benzyl-N- (3- ((t-butoxycarbonyl) amino) butanoyl) glycine methyl ester;
c. the following chromatographic conditions are adopted to detect the test sample and the system applicability solution respectively:
stationary phase: polysaccharide derivative normal phase coating chiral chromatographic column, silica gel surface coated with cellulose-tri (4-methyl benzoate);
mobile phase: the mixed solution of n-hexane, ethanol and diethylamine, wherein the volume ratio of n-hexane, ethanol and diethylamine is 95-75%, 5-25%, 0.01-0.2%;
elution procedure: isocratic elution is carried out for 5-30 min;
d. and if the chromatogram of the test solution shows a chromatographic peak corresponding to the retention time of the chromatographic peak of the enantiomer impurity A in the chromatogram of the system applicability solution, indicating that the test solution contains enantiomer impurity A.
Further, the volume ratio of n-hexane, ethanol and diethylamine in the mobile phase was 90% to 10% to 0.1%.
Further, the elution procedure is isocratic elution for 5-30 min. More preferably, the elution procedure is isocratic elution for 16min.
Further, the HPLC detection wavelength is full wavelength scanning of 190-450 nm, preferably 220nm; the flow rate is 1.0ml/min; the column temperature is 30 ℃; the sample loading was 20. Mu.l.
Further, the mass volume ratio of the Suvorexant intermediate to be detected and the diluent in the sample solution is 1.0-5.0 mg:1ml, preferably 2.0mg:1ml.
Further, the concentration of the suvorexant intermediate in the system applicability solution is 1.0-5.0 mg/ml, and the concentration of the enantiomer impurity reference substance is 100-500 mug/ml. Preferably, the concentration of suvorexant intermediate in the system-applicable solution is 2.0mg/ml and the concentration of enantiomeric impurity reference is 300 μg/ml.
Further, the diluent comprises ethanol.
In another aspect, the present invention provides a method for determining the content of enantiomeric impurity a in a suvorexant intermediate, which is (R) -N-benzyl-N- (3- ((t-butoxycarbonyl) amino) butanoyl) glycine methyl ester, by HPLC, the enantiomeric impurity a being: (S) -N-benzyl-N- (3- ((t-butoxycarbonyl) amino) butanoyl) glycine methyl ester, the method comprising the steps of:
a. preparation of test solution: taking a Suvorexant intermediate to be detected, and adding a diluent for dissolution to obtain a sample solution;
b. the test solution was tested using the following chromatographic conditions:
stationary phase: polysaccharide derivative normal phase coating chiral chromatographic column, silica gel surface coated with cellulose-tri (4-methyl benzoate);
mobile phase: the mixed solution of n-hexane, ethanol and diethylamine, wherein the volume ratio of n-hexane, ethanol and diethylamine is 95-75%, 5-25%, 0.01-0.2%;
elution procedure: isocratic elution is carried out for 5-30 min;
c. the content of enantiomeric impurity A was calculated by the following formula:
Xi%=Ai/A×100%
wherein ,Xi% is the content of enantiomeric impurities in the test solution,Aiis the peak area of enantiomer impurities in the sample solution,Ais a test sampleThe sum of all peak areas in the solution.
Further, the method also comprises the steps of preparing a system applicability solution, which comprises the steps of respectively taking the Suvorexant intermediate and the enantiomer impurity A reference substance thereof, adding a diluent for dissolution and mixing.
Further, the volume ratio of n-hexane, ethanol and diethylamine in the mobile phase was 90%:10%:0.1%.
Further, the elution procedure is isocratic elution for 5-30 min. More preferably, the elution procedure is isocratic elution for 16min.
Further, the HPLC detection wavelength is full wavelength scanning of 190-450 nm, preferably 220nm; the flow rate is 1.0ml/min; the column temperature is 30 ℃; the sample loading was 20. Mu.l.
Further, the mass volume ratio of the Suvorexant intermediate to be detected and the diluent in the sample solution is 1.0-5.0 mg:1ml, preferably 2.0mg:1ml.
Further, the concentration of the suvorexant intermediate in the system applicability solution is 1.0-5.0 mg/ml, and the concentration of the enantiomer impurity reference substance is 100-500 mug/ml. Preferably, the concentration of suvorexant intermediate in the system-applicable solution is 2.0mg/ml and the concentration of enantiomeric impurity reference is 300 μg/ml.
Further, the diluent comprises ethanol.
The beneficial effects of the invention are that
The detection method can effectively separate the Suvorexant intermediate IN3 from the impurity A, accurately measure the content of the impurity A, has stable and reliable measurement results and strong specificity, and the method verification results show that the method meets the requirements of analysis methodologies IN the 2020 edition of Chinese pharmacopoeia, thereby effectively controlling the quality of the Suvorexant intermediate, improving the quality of the Suvorexant bulk drug, and simultaneously effectively monitoring the synthesis process of the Suvorexant intermediate and the synthesis process of the Suvorexant bulk drug.
Drawings
Fig. 1 is a suvorexant synthetic route diagram.
FIG. 2 is an HPLC chart of a sample solution in example 1.
FIG. 3 is an HPLC chart of the sample solution in example 2.
Fig. 4 is a typical HPLC diagram of a system applicability solution.
FIG. 5 is a typical HPLC plot of a system applicability solution of comparative example 1.
Description of the embodiments
It should be understood that, based on the above description of the present invention, various other modifications, substitutions, and alterations can be made hereto without departing from the basic technical spirit of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
(1) Material
IN3 (supplied by genu bang pharmaceutical company, lot numbers 221201, 221202);
impurity A control (lot number MS203421-221001, available from Chengdu brand pharmaceutical Co., ltd.).
(2) Main instrument
High performance liquid chromatograph: shimadzu LC-20AT, chromatographic column: CHIRALCEL OJ-H, 4.6X250 mm,5 μm.
Example 1: determination of impurity A content IN Suvorexant intermediate IN3
(1) Preparation of test solution: accurately weighing IN3 (batch No. 221201), adding diluent ethanol, and dissolving to obtain 2.0mg/ml sample solution.
(2) Preparation of a System applicability solution: precisely weighing the impurity A reference substance, dissolving with ethanol as diluent to prepare a solution with the concentration of 1.0mg/ml, and taking the solution as the impurity A reference substance stock solution. Accurately weighing IN 3.00 mg, accurately weighing 3mL of impurity A stock solution, placing into a 10mL volumetric flask, dissolving with diluent, shaking, and fixing volume to obtain system applicability solution.
(3) Chromatographic condition setting
Chromatographic column: CHIRALCEL OJ-H, specification 4.6X105 mm,5 μm;
detection wavelength: 220nm;
the flow rate is: 1.0ml/min;
the column temperature is: 30 ℃;
sample injection volume: 20 μl;
mobile phase: n-hexane-ethanol-diethylamine= (90%: 10%:0.1%, v/v/v);
gradient elution procedure: isocratic elution was carried out for 16min.
(4) Measurement
Respectively sucking 10 μl sample injection amount of sample solution and system applicability solution, injecting into chromatograph, recording chromatogram, and respectively recording peak area of impurity A in sampleA i And the sum of all peak areas in the test solutionAAnd calculating the content of the impurity A in the sample by an area normalization method. The calculation formula is as follows:
Xi%=Ai/A×100%
wherein ,X i %: the content of the impurity A in the test sample,
A i : the area of the peak A of the impurity in the test sample,
A: the sum of all peak areas in the test solution is shown in figure 2.
Calculation results: the impurity a content of IN3 sample lot 221201 is: impurity a=0.01%.
Example 2: determination of impurity A content IN Suvorexant intermediate IN3
(1) Preparation of test solution: accurately weighing IN3 (batch No. 221202), adding diluent ethanol, and dissolving to obtain 2.0mg/ml sample solution.
(2) Preparation of a System applicability solution: precisely weighing the impurity A reference substance, dissolving with ethanol as diluent to prepare a solution with the concentration of 1.0mg/ml, and taking the solution as the impurity A reference substance stock solution. Accurately weighing IN 3.00 mg, accurately weighing 3mL of impurity A stock solution, placing into a 10mL volumetric flask, dissolving with diluent, shaking, and fixing volume to obtain system applicability solution.
(3) Chromatographic condition setting
Chromatographic column: CHIRALCEL OJ-H, specification 4.6X105 mm,5 μm;
detection wavelength: 220nm;
the flow rate is: 1.0ml/min;
the column temperature is: 30 ℃;
sample injection volume: 20 μl;
mobile phase: n-hexane-ethanol-diethylamine= (90%: 10%:0.1%, v/v/v);
gradient elution procedure: isocratic elution was carried out for 16min.
(4) Measurement
Respectively sucking 10 μl sample injection amount of sample solution and system applicability solution, injecting into chromatograph, recording chromatogram, and respectively recording peak area of impurity A in sampleA i And the sum of all peak areas in the test solutionAAnd calculating the content of the impurity A in the sample by an area normalization method. The calculation formula is as follows:
Xi%=Ai/A×100%
wherein ,X i %: the content of the impurity A in the test sample,
A i : the area of the peak A of the impurity in the test sample,
A: the sum of all peak areas in the test solution is shown in the HPLC chart of FIG. 3.
Calculation results: the impurity a content of IN3 sample lot 221202 was: impurity a=n.d.
The following examples are presented to evaluate the beneficial effects of the detection method of the present invention.
Example 3: system applicability and specificity
Instrument: high performance liquid chromatograph: shimadzu LC-20AT, chromatographic column: CHIRALCEL OJ-H column, 4.6X250 mm,5 μm flow rate: 1.0ml/min, column temperature: 30 ℃, 20ul sample injection amount, detector wavelength: 220nm, the mobile phase is n-hexane-ethanol-diethylamine= (90%: 10%:0.1%, v/v/v), and isocratic elution is 16min.
(1) Blank solution (following dilutions): ethanol;
(2) Impurity a stock: precisely weighing 10mg of impurity A in a 10ml measuring flask, diluting the solution and fixing the volume (impurity A:1.0 mg/ml);
(3) System applicability solution: accurately weighing IN3 20mg IN a 10ml measuring flask, accurately adding 3ml of impurity A stock solution, adding diluent, diluting to constant volume (IN 3.0 mg/ml, impurity A300 ug/ml);
(4) Impurity a localization solution: the impurity A3 mg was weighed and placed in 10ml measuring flask, respectively, and diluted solution was added to dissolve the impurity A to a constant volume (impurity A:300 ug/ml).
(5) Test solution: accurately weighing IN 3-20 mg IN a 10ml measuring flask, and adding the diluent solution to fix the volume.
Blank solution, system applicability solution and sample solution are injected according to chromatographic conditions, chromatographic processes are recorded, and the results are shown in Table 1. The system applicability solution HPLC diagram is shown in FIG. 4.
Table 1 System applicability and specificity
Conclusion: the separation degree of the system applicability solution of the method is 2.92, the separation degree is good, the impurity A has no interference in peak, and the system applicability and the specificity of the method meet the analysis requirement.
Example 4: limit of detection and limit of quantification
Experimental conditions, liquid chromatography methods and solution formulations were as in example 3.
For known impurity A, the detection Limit (LOD) and the quantification Limit (LOQ) are determined from the signal to noise ratio. Diluting the stock solution of the impurity A with known concentration to low concentration for sample injection, determining the detection signal to noise ratio S/N to be more than or equal to 10 as a quantitative limit, and determining S/N=2-8 as a detection limit. The test results are shown in Table 2.
TABLE 2 quantitative limits and detection limits
Results: the quantitative limit of the impurity A is 50ug/ml, which corresponds to 0.125% of the sample concentration, the detection limit is 10ug/ml, which corresponds to 0.025% of the sample concentration, and the method fully proves that the sensitivity of the method is good and meets the requirements of medicine related analysis.
Example 5: solution stability
Experimental conditions, liquid chromatography methods and solution formulations were as in example 3.
And taking a system applicability solution, respectively placing the solution at room temperature (25 ℃) for 0, 2, 6, 12, 18 and 24 hours, and sequentially injecting samples, wherein the solution stability result is shown in Table 3.
TABLE 3 stability
Conclusion: the RSD of the solution impurity A peak area% of the solution in 24 hours at room temperature (25 ℃) is 0.62%, which proves that the solution has good stability and meets the requirements of medicine related analysis.
The research results of examples 3-5 show that the method for measuring the IN3 impurity A provided by the invention has good specificity, sensitivity and durability (good solution stability IN 24 hours at room temperature of 25 ℃), and can be suitable for quality control of intermediate impurity A IN raw material medicine synthesis. The quality analysis and summarization of the suvorexant intermediate by the method of the invention ensure the quality of the suvorexant API prepared by taking the suvorexant intermediate as the raw material, and the content of the impurity A in the suvorexant intermediate is controlled to be 0.5 percent.
Comparative example 1: system applicability and specificity of analysis of suvorexant intermediate IN3 and enantiomeric impurity a thereof under different chromatographic conditions
(1) Blank solution (following dilutions): ethanol;
(2) Impurity a stock: precisely weighing 10mg of impurity A in a 20ml measuring flask, diluting the solution and fixing the volume (impurity A:1.0 mg/ml);
(3) System applicability solution: accurately weighing IN3, 20mg, and accurately adding 3ml of impurity A stock solution into a 10ml measuring flask, and diluting with diluent to constant volume (IN 3.0 mg/ml, impurity A300 ug/ml).
The blank solution and the system applicability solution are respectively absorbed and injected into the chromatograph, and the chromatographic conditions are as follows:
chromatographic column: CHIRALPAK IA, specification 4.6X105 mm,5 μm;
mobile phase: n-hexane-ethanol-diethylamine= (70%: 30%:0.1%, v/v/v); detection wavelength: 200nm;
column temperature: 30 ℃, chromatographic column flow: 1.0ml/min, and the sample injection amount is 10 μl;
gradient elution conditions are isocratic elution for 30min.
Results: the HPLC diagram of the system applicability solution is shown in figure 5; IN the same analysis time, the method has the separation degree of 0.79, no obvious baseline separation (insufficient selectivity) and no subsequent possibility of optimization, and IN3 and impurity A can not meet the analysis requirements, which indicates that the system applicability and specificity of the analysis of the Suvorexant intermediate IN3 and the enantiomer impurity A thereof under the chromatographic condition are not IN accordance with the analysis requirements.
IN conclusion, the invention meets the requirements of analysis methodology IN the 2020 edition of Chinese pharmacopoeia, improves the quality of the Suvorexant raw material medicine by effectively controlling the quality of the Suvorexant intermediate IN3, and simultaneously can effectively monitor the synthesis process of the Suvorexant intermediate and the synthesis process of the Suvorexant raw material medicine, thereby having a certain guiding effect on the drug research and development work of the Suvorexant.
Claims (5)
1. A method for detecting suvorexant intermediates and enantiomeric impurities thereof by HPLC, wherein the HPLC is performed under chromatographic conditions of:
stationary phase: polysaccharide derivative normal phase coating chiral chromatographic column, silica gel surface coated with cellulose-tri (4-methyl benzoate);
mobile phase: the mixed solution of n-hexane, ethanol and diethylamine, wherein the volume ratio of n-hexane, ethanol and diethylamine is 95% -75%: 5% -25%: 0.01 to 0.2 percent;
elution procedure: isocratic elution is carried out for 5-30 min;
wherein the suvorexant intermediate is (R) -N-benzyl-N- (3- ((tert-butoxycarbonyl) amino) butanoyl) glycine methyl ester, and the enantiomer impurity is (S) -N-benzyl-N- (3- ((tert-butoxycarbonyl) amino) butanoyl) glycine methyl ester.
2. The method according to claim 1, wherein the volume ratio of n-hexane, ethanol and diethylamine in the mobile phase is 90%:10%:0.1%.
3. The method according to claim 1, wherein the elution procedure is isocratic elution for 16-30 min; the detection wavelength of the HPLC is 220nm; the flow rate is 1.0ml/min; the column temperature is 30 ℃; the sample loading was 20. Mu.l.
4. A method according to any one of claims 1-3, characterized in that the method comprises the steps of:
a. preparation of test solution: taking a Suvorexant intermediate to be detected, and adding a diluent for dissolution to obtain a sample solution;
b. preparation of a System applicability solution: respectively taking a suvorexant intermediate and an enantiomer impurity reference substance thereof, adding a diluent for dissolution, and mixing to obtain a system applicability solution;
c. detecting the sample to be tested and the system applicability solution by adopting the chromatographic conditions;
d. and if the chromatogram of the test solution shows a chromatographic peak corresponding to the retention time of the chromatographic peak of the enantiomer impurity in the chromatogram of the system applicability solution, indicating that the test solution contains the enantiomer impurity.
5. The method of claim 4, further comprising calculating the content of enantiomeric impurities by the formula:
Xi%=Ai/A×100%
wherein ,Xi% is the content of enantiomeric impurities in the test solution,Aiis enantiomer in the test solutionThe peak area of the impurity is defined as the area of the peak,Ais the sum of all peak areas in the sample solution.
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