CN108872413B - Method for detecting enantiomer content in (S, S) -2, 8-diazabicyclo [4,3,0] nonane - Google Patents
Method for detecting enantiomer content in (S, S) -2, 8-diazabicyclo [4,3,0] nonane Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
Abstract
The invention belongs to the technical field of drug detection, and particularly relates to a method for detecting enantiomer content in (S, S) -2, 8-diazabicyclo [4,3,0] nonane. The invention adopts 1-dimethylamino naphthalene-5-sulfonyl chloride as a derivatization reagent to perform derivatization on (S, S) -2, 8-diazabicyclo [4,3,0] nonane, then adopts HPLC to perform detection, and calculates the content of enantiomer by an area normalization method. The method for detecting the content of the enantiomer in the (S, S) -2, 8-diazabicyclo [4,3,0] nonane has the advantages of low cost, quick reaction, simple operation, complete derivatization reaction, higher accuracy compared with derivatization methods reported in other documents, and great significance for controlling the quality of the (S, S) -2, 8-diazabicyclo [4,3,0] nonane and ensuring the production quality of moxifloxacin hydrochloride.
Description
Technical Field
The invention belongs to the technical field of drug detection, and particularly relates to a method for detecting enantiomer content in (S, S) -2, 8-diazabicyclo [4,3,0] nonane.
Background
Moxifloxacin hydrochloride (Moxifloxacin hydrochloride) belongs to fourth generation fluoroquinolones chemical synthetic antibiotic drugs, and is introduced by Bayer company in Germany in 1999. Since the market, the antibacterial agent has the advantages of wide antibacterial spectrum, strong antibacterial power, good curative effect, small side effect, no cross drug resistance with other antibacterial agents and the like, and is widely applied clinically. As a novel quinolone drug, the azabicyclo structure on the c-7 side chain enhances the antibacterial action on gram-positive bacteria.
In the production process of moxifloxacin hydrochloride, (S, S) -2, 8-diazabicyclo [4,3,0] nonane is widely used as a side chain azabicyclo intermediate, so that the quality control of the moxifloxacin hydrochloride is very important. (S, S) -2, 8-diazabicyclo [4,3,0] nonane has the following structure:
the (S, S) -2, 8-diazabicyclo [4,3,0] nonane has two chiral centers in the structure, one enantiomer and two diastereomers theoretically exist, and the two diastereomers cannot be generated according to the analysis of the chemical synthesis process of (S, S) -2, 8-diazabicyclo [4,3,0] nonane, and thus only the enantiomers thereof are controlled in quality control. The enantiomer (R, R) -2, 8-diazabicyclo [4,3,0] nonane has the following structure:
at present, Chinese patent CN104133019A discloses a method for detecting an enantiomer of diazabicyclononane, which utilizes o-fluoronitrobenzene to carry out pre-column derivatization detection on the enantiomer in (S, S) -2, 8-diazabicyclo [4,3,0] nonane, but the repeatability, precision and accuracy are poor.
The literature (Oriental Journal of chemistry.31(2015)2207-2212) reports a method for measuring isomers by HPLC after derivatization of (S, S) -2, 8-diazabicyclo [4,3,0] nonane with NBD-Cl, but this method has a low recovery rate and cannot accurately quantify the isomer content.
Therefore, it is necessary to further study the method for detecting the enantiomeric content of (S, S) -2, 8-diazabicyclo [4,3,0] nonane.
Disclosure of Invention
In order to solve the above problems, the present invention aims to provide a method for detecting enantiomer content in (S, S) -2, 8-diazabicyclo [4,3,0] nonane, which uses 1-dimethylaminonaphthalene-5-sulfonyl chloride as a derivatization reagent, and has the advantages of low cost, rapid reaction, simple operation, complete derivatization reaction, and high repeatability, precision and accuracy.
The invention adopts 1-dimethylamino naphthalene-5-sulfonyl chloride as a derivatization reagent to perform derivatization on (S, S) -2, 8-diazabicyclo [4,3,0] nonane, then adopts HPLC to perform detection, and calculates the content of enantiomer by an area normalization method.
The method for detecting the content of the enantiomer in the (S, S) -2, 8-diazabicyclo [4,3,0] nonane comprises the following steps:
(1) chromatographic conditions are as follows:
a chromatographic column: CHIRALPAK AD-H chiral column, 250 × 4.6mm, particle size 5 μm;
detection wavelength: 246 nm;
mobile phase: a mixed solution of n-hexane and absolute ethyl alcohol;
flow rate: 1.0-1.5 ml/min;
column temperature: 20-40 DEG C
Operating time: at least 25 min;
(2) derivatization:
weighing (S, S) -2, 8-diazabicyclo [4,3,0] nonane, placing the (S, S) -2, 8-diazabicyclo [4,3,0] nonane into a reaction bottle, adding a solvent and 1-dimethylaminonaphthalene-5-sulfonyl chloride, and stirring at 20-30 ℃ for 0.5-1 hour to obtain a (S, S) -2, 8-diazabicyclo [4,3,0] nonane derivative solution;
weighing (R, R) -2, 8-diazabicyclo [4,3,0] nonane, placing the (R, R) -2, 8-diazabicyclo [4,3,0] nonane into a reaction bottle, adding a solvent and 1-dimethylaminonaphthalene-5-sulfonyl chloride, and stirring at 20-30 ℃ for 0.5-1 hour to obtain a (R, R) -2, 8-diazabicyclo [4,3,0] nonane derivative solution;
(3) preparing a test solution:
precisely measuring (S, S) -2, 8-diazabicyclo [4,3,0] nonane derivative solution, placing the solution in a volumetric flask, and adding absolute ethyl alcohol to dilute the solution to a scale to obtain a test solution;
(4) preparation of enantiomer solution:
precisely measuring (R, R) -2, 8-diazabicyclo [4,3,0] nonane derivative solution, placing the solution in a volumetric flask, and adding absolute ethyl alcohol to dilute the solution to a scale to obtain enantiomer solution;
(5) preparing a blank solution:
derivatizing without adding (S, S) -2, 8-diazabicyclo [4,3,0] nonane and (R, R) -2, 8-diazabicyclo [4,3,0] nonane by adopting the method in the step (2), precisely measuring, placing in a volumetric flask, adding absolute ethyl alcohol to dilute to a scale, and taking as a blank solution;
(6) preparation of system applicability solution:
precisely measuring enantiomer solution, placing the enantiomer solution in a volumetric flask, adding absolute ethyl alcohol to dilute the enantiomer solution to a scale, and taking the enantiomer solution as a system applicability solution;
(7) the detection method comprises the following steps:
injecting 10 μ l of blank solution into a liquid chromatograph, and recording the map; injecting 10 μ l of the system applicability solution into a liquid chromatograph, repeating for 6 times, recording the map, requiring that the RSD of the peak area of the enantiomer derivative is determined to be less than or equal to 2.0% for 6 times, the RSD of the retention time of each peak is less than or equal to 1.0%, and the separation degree between the peaks and the number of theoretical plates meet the requirements.
Injecting 10 μ l of the enantiomer solution into a liquid chromatograph, and recording the spectrum; injecting 10 mu l of test solution into a liquid chromatograph, recording a spectrum, recording a peak area, and calculating the content of the enantiomer in the (S, S) -2, 8-diazabicyclo [4,3,0] nonane.
Wherein:
the volume ratio of the normal hexane to the absolute ethyl alcohol is 45-75: 25-55.
Preferably, the volume ratio of the n-hexane to the absolute ethyl alcohol is 55: 45.
Preferably, the column temperature is 25-35 ℃.
In the step (2), the solvent is acetonitrile, tetrahydrofuran or dichloromethane.
The 1-dimethylamino naphthalene-5-sulfonyl chloride is a derivatization reagent and has the following structure:
preferably, in step (2), the solvent is dichloromethane.
In the step (2), the amount of the 1-dimethylaminonaphthalene-5-sulfonyl chloride is 1.0 to 1.2 times of the amount of (S, S) -2, 8-diazabicyclo [4,3,0] nonane or (R, R) -2, 8-diazabicyclo [4,3,0] nonane.
The invention has the following beneficial effects:
the method for detecting the content of the enantiomer in the (S, S) -2, 8-diazabicyclo [4,3,0] nonane has the advantages of low cost, quick reaction, simple operation, complete derivatization reaction, higher accuracy compared with derivatization methods reported in other documents, and great significance for controlling the quality of the (S, S) -2, 8-diazabicyclo [4,3,0] nonane and ensuring the production quality of moxifloxacin hydrochloride.
Drawings
FIG. 1 is an HPLC chromatogram of (S, S) -2, 8-diazabicyclo [4,3,0] nonane derivative in example 2;
FIG. 2 is an HPLC chromatogram of (R, R) -2, 8-diazabicyclo [4,3,0] nonane chloro derivative in example 2;
FIG. 3 is an HPLC chromatogram of the first experiment result of the system suitability solution in example 2;
FIG. 4 is an HPLC chromatogram for measuring the enantiomeric content of (S, S) -2, 8-diazabicyclo [4,3,0] nonane in example 4;
FIG. 5 is an HPLC chromatogram for measuring the enantiomeric content of (S, S) -2, 8-diazabicyclo [4,3,0] nonane in example 5;
FIG. 6 is an HPLC chromatogram for measuring the enantiomeric content of (S, S) -2, 8-diazabicyclo [4,3,0] nonane in example 6.
Detailed Description
The present invention is further described below with reference to examples.
The raw materials used in the examples were all commercially available raw materials except for those specifically mentioned.
Example 1
The detection method comprises the following steps:
1. chromatographic conditions are as follows:
a chromatographic column: CHIRALPAK AD-H chiral column, 250 × 4.6mm, particle size 5 μm; the detection wavelength is 246 nm; mobile phase: n-hexane-absolute ethanol (volume ratio 55: 45); column temperature: 30 ℃; flow rate: 1.0 ml/min; operating time: 25 min; sample introduction amount: 10 mu l of the mixture;
2. preparation of the solution
2.1 preparation of (R, R) -2, 8-diazabicyclo [4,3,0] nonane derivative solution:
accurately weighing 50mg of (R, R) -2, 8-diazabicyclo [4,3,0] nonane, placing the weighed mixture into a reaction bottle, adding 20ml of solvent and a derivatization reagent with the amount of 1.2 times of the substance, and stirring for 0.5 hour at the temperature of 20-30 ℃ to obtain a corresponding derivative solution;
precisely measuring 0.15ml of (R, R) -2, 8-diazabicyclo [4,3,0] nonane derivative solution, placing the solution in a 25ml volumetric flask, adding ethanol to dilute the solution to a scale, and shaking up to obtain the (R, R) -2, 8-diazabicyclo [4,3,0] nonane derivative solution;
2.2 preparation of system adaptive solution:
precisely measuring 1.0ml of (R, R) -2, 8-diazabicyclo [4,3,0] nonane derivative solution, placing the solution in a 50ml volumetric flask, and adding absolute ethyl alcohol to dilute the solution to a scale to obtain a system applicability solution.
2.3 preparation of test solution:
weighing 1.0g of (S, S) -2, 8-diazabicyclo [4,3,0] nonane, placing the weighed 1.0g of (S, S) -2, 8-diazabicyclo [4,3,0] nonane into a reaction bottle, adding 20ml of solvent and a derivatization reagent accounting for 1.2 times of the weight of the substance, and stirring at 20-30 ℃ for 0.5 hour to obtain a derivative solution;
precisely measuring 0.15ml of (S, S) -2, 8-diazabicyclo [4,3,0] nonane derivative solution, placing the solution in a 25ml volumetric flask, and adding absolute ethyl alcohol to dilute the solution to a scale to obtain a test solution;
preparing a blank solution:
adding 20ml of solvent and a derivatization reagent with the amount of 1.2 times of the substance into a reaction bottle, and stirring for 1 hour at the temperature of 20-30 ℃ to obtain a blank derivatization solution;
precisely measuring 0.15ml of blank derivative solution, placing the blank derivative solution in a 25ml volumetric flask, and adding absolute ethyl alcohol to dilute the blank derivative solution to a scale to obtain a blank solution;
3. the detection method comprises the following steps:
injecting 10 μ l of blank solution into a liquid chromatograph, and recording the map;
injecting 10 μ l of the system applicability solution into a liquid chromatograph, repeating for 6 times, recording the map, requiring that the RSD of the peak area of the enantiomer derivative is determined to be less than or equal to 2.0% for 6 times, the RSD of the retention time of each peak is less than or equal to 1.0%, and the separation degree between the peaks and the number of theoretical plates meet the requirements. And (R, R) -2, 8-diazabicyclo [4,3,0] nonane derivative solution and 10 mu l of test solution are respectively injected into a liquid chromatograph, the spectrum is recorded, the peak area is recorded, and the content of the enantiomer in the (S, S) -2, 8-diazabicyclo [4,3,0] nonane is calculated.
Example 2
Systematic applicability experiment for detecting enantiomer content in (S, S) -2, 8-diazabicyclo [4,3,0] nonane by using 1-dimethylamino naphthalene-5-sulfonyl chloride as a derivatization reagent:
1. derivatization reaction of (S, S) -2, 8-diazabicyclo [4,3,0] nonane
Weighing 1.0g of (S, S) -2, 8-diazabicyclo [4,3,0] nonane, placing the weighed 1.0g of (S, S) -2, 8-diazabicyclo [4,3,0] nonane into a reaction bottle, adding 20ml of dichloromethane and 2.14g (1.2 times of the amount of the substances) of 1-dimethylaminonaphthalene-5-sulfonyl chloride, and stirring at the temperature of 20-30 ℃ for 0.5 hour to obtain a derivative solution;
precisely measuring 0.15ml of (S, S) -2, 8-diazabicyclo [4,3,0] nonane derivative solution, placing the solution in a 25ml volumetric flask, and adding absolute ethyl alcohol to dilute the solution to a scale to obtain a test solution;
2. derivatization reaction of (R, R) -2, 8-diazabicyclo [4,3,0] nonane
Weighing 50mg of (R, R) -2, 8-diazabicyclo [4,3,0] nonane, placing the weighed 50mg into a reaction bottle, adding 20ml of dichloromethane and 1.34g of 1-dimethylaminonaphthalene-5-sulfonyl chloride, and stirring at 20-30 ℃ for 0.5 hour to obtain a derivative solution;
precisely measuring 0.15ml of (S, S) -2, 8-diazabicyclo [4,3,0] nonane derivative solution, placing the solution in a 25ml volumetric flask, and adding absolute ethyl alcohol to dilute the solution to a scale to obtain a reference solution;
3. the detection was carried out according to the detection method in example 1, and the results of the measurement of the system suitability test are shown in Table 1.
TABLE 1 System suitability solution test results
Wherein, FIG. 1 is an HPLC chromatogram of a (S, S) -2, 8-diazabicyclo [4,3,0] nonane derivative; FIG. 2 is an HPLC chromatogram of (R, R) -2, 8-diazabicyclo [4,3,0] nonane derivative.
The results of the detection in fig. 1 and 2 are shown in table 2.
Table 2 test results of fig. 1 and 2
Example 3
An accuracy test for detecting the content of enantiomer in (S, S) -2, 8-diazabicyclo [4,3,0] nonane by using 1-dimethylamino naphthalene-5-sulfonyl chloride as a derivatization reagent.
The accuracy is obtained by adding quantitative limit, 80%, 100% and 120% of three isomers with different concentrations into a test sample. The method is verified to have good accuracy by adding a known amount of isomer, measuring the isomer content in the sample to be added, subtracting the original isomer content in the sample, and expressing the ratio (recovery) between the measurement result and the actual amount added in percentage (%), which is required to be between 90% and 108%.
The test results are shown in Table 3.
TABLE 3 recovery test results
And (4) conclusion: under 4 concentrations, the isomer recovery rate is 96.30-100.11%, the average recovery rate is 98.13%, and the method meets the requirement of a verification scheme (90-108%), and the method is proved to have good accuracy.
Example 4
1-dimethylaminonaphthalene-5-sulfonyl chloride is used as a derivatization reagent to detect the content of enantiomer in (S, S) -2, 8-diazabicyclo [4,3,0] nonane (batch number: MO-1-20160821):
1. derivatization reaction of (S, S) -2, 8-diazabicyclo [4,3,0] nonane
Weighing 1.0g of (S, S) -2, 8-diazabicyclo [4,3,0] nonane, placing the weighed 1.0g of (S, S) -2, 8-diazabicyclo [4,3,0] nonane into a reaction bottle, adding 20ml of dichloromethane and 1.34g (1.2 times of the amount of the substances) of 1-dimethylaminonaphthalene-5-sulfonyl chloride, and stirring at the temperature of 20-30 ℃ for 0.5 hour to obtain a derivative solution;
precisely measuring 0.15ml of (S, S) -2, 8-diazabicyclo [4,3,0] nonane derivative solution, placing the solution in a 25ml volumetric flask, and adding absolute ethyl alcohol to dilute the solution to a scale to obtain a test solution;
2. derivatization reaction of (R, R) -2, 8-diazabicyclo [4,3,0] nonane
Weighing 50mg of (R, R) -2, 8-diazabicyclo [4,3,0] nonane, placing the weighed 50mg into a reaction bottle, adding 20ml of dichloromethane and 1.34g of 1-dimethylaminonaphthalene-5-sulfonyl chloride, and stirring at 20-30 ℃ for 0.5 hour to obtain a derivative solution;
precisely measuring 0.15ml of (R, R) -2, 8-diazabicyclo [4,3,0] nonane derivative solution, placing the solution in a 25ml volumetric flask, and adding absolute ethyl alcohol to dilute the solution to a scale to obtain a reference solution;
3. the enantiomeric content of a sample of (S, S) -2, 8-diazabicyclo [4,3,0] nonane, as measured by the detection method described in example 1, was 0.045 wt%, and the results are shown in Table 4 and the spectrum is shown in FIG. 4.
TABLE 4 isomer sample detection results
Example 5
1-dimethylaminonaphthalene-5-sulfonyl chloride is used as a derivatization reagent to detect the content of enantiomer in (S, S) -2, 8-diazabicyclo [4,3,0] nonane (batch number: MO-1-20170303):
the enantiomeric content of a sample of (S, S) -2, 8-diazabicyclo [4,3,0] nonane, as measured by the detection method described in example 4, was 0.045 wt%, and the results are shown in Table 5 and the spectrum is shown in FIG. 5.
TABLE 5 isomer sample detection results
Example 6
1-dimethylamino naphthalene-5-sulfonyl chloride is used as a derivatization reagent to detect the content of the enantiomer in (S, S) -2, 8-diazabicyclo [4,3,0] nonane (batch number: MO-1-20170701):
the enantiomeric content of the (S, S) -2, 8-diazabicyclo [4,3,0] nonane sample was 0.047% by weight as determined by the detection method in example 4, and the results are shown in Table 6 and in FIG. 6.
TABLE 6 isomer sample test results
Comparative example 1
An enantiomer content accuracy experiment in (S, S) -2, 8-diazabicyclo [4,3,0] nonane is carried out by taking o-fluoronitrobenzene as a derivatization reagent and referring to CN 104133019A.
The detection method comprises the following steps:
chromatographic conditions are as follows:
a chromatographic column: CHIRALPAK AD-H chiral column, 250 × 4.6mm, particle size 5 μm; the detection wavelength is 246 nm; mobile phase: n-hexane-anhydrous ethanol-triethylamine (volume ratio 65:35: 0.1); column temperature: 30 ℃; flow rate: 0.5 ml/min; operating time: 60 min; sample introduction amount: 20 μ l.
The experimental steps are as follows:
taking 1.0ml of o-fluoronitrobenzene, putting the o-fluoronitrobenzene into a 25ml volumetric flask, dissolving the o-fluoronitrobenzene with 10ml of dichloromethane, and precisely adding 1.65ml of triethylamine to prepare a solvent A; taking 0.5g of (S, S) -2, 8-diazabicyclo [4,3,0] nonane to be placed in a 25ml volumetric flask, and dissolving the (S, S) -2, 8-diazabicyclo [4,3,0] nonane by using 10ml of dichloromethane to prepare a solvent B; adding the solution B into the solution A, fixing the volume to the scale with dichloromethane, and heating in a water bath at 25 ℃ for 60min to obtain a solution C. Precisely measuring 0.2ml of the solution C, placing the solution C in a 25ml volumetric flask, adding an ethanol solution, and diluting to a scale to obtain a test solution.
The method is proved to have good accuracy by adding four isomers with different concentrations of a quantitative limit and a limit concentration of 80%, 100% and 120%, measuring the content of the isomer in a sample to be added, subtracting the original isomer content in the sample, and expressing the ratio (recovery rate) between the obtained measurement result and the actual addition amount in percentage (%).
The test results are shown in Table 7.
TABLE 7 recovery test results with o-fluoronitrobenzene as derivatizing reagent
And (4) conclusion: under 4 concentrations, the recovery rate of the enantiomer is between 90.31% and 93.83%, the average recovery rate is 91.37%, and compared with the average recovery rate of 98.13% in example 3, the method using 1-dimethylamino naphthalene-5-sulfonyl chloride as a derivatization reagent is proved to be superior to the method using o-fluoronitrobenzene as a derivatization reagent.
Claims (4)
1. A method for detecting the enantiomeric content of (S, S) -2, 8-diazabicyclo [4,3,0] nonane, characterized in that: the method comprises the following steps:
(1) chromatographic conditions are as follows:
a chromatographic column: CHIRALPAK AD-H chiral column, 250 × 4.6mm, particle size 5 μm;
detection wavelength: 246 nm;
mobile phase: a mixed solution of n-hexane and absolute ethyl alcohol;
flow rate: 1.0-1.5 ml/min;
column temperature: 20-40 DEG C
Operating time: at least 25 min;
(2) derivatization:
weighing (S, S) -2, 8-diazabicyclo [4,3,0] nonane, placing the (S, S) -2, 8-diazabicyclo [4,3,0] nonane into a reaction bottle, adding a solvent and 1-dimethylaminonaphthalene-5-sulfonyl chloride, and stirring at 20-30 ℃ for 0.5-1 hour to obtain a (S, S) -2, 8-diazabicyclo [4,3,0] nonane derivative solution;
weighing (R, R) -2, 8-diazabicyclo [4,3,0] nonane, placing the (R, R) -2, 8-diazabicyclo [4,3,0] nonane into a reaction bottle, adding a solvent and 1-dimethylaminonaphthalene-5-sulfonyl chloride, and stirring at 20-30 ℃ for 0.5-1 hour to obtain a (R, R) -2, 8-diazabicyclo [4,3,0] nonane derivative solution;
(3) preparing a test solution:
precisely measuring (S, S) -2, 8-diazabicyclo [4,3,0] nonane derivative solution, placing the solution in a volumetric flask, and adding absolute ethyl alcohol to dilute the solution to a scale to obtain a test solution;
(4) preparation of enantiomer solution:
precisely measuring (R, R) -2, 8-diazabicyclo [4,3,0] nonane derivative solution, placing the solution in a volumetric flask, and adding absolute ethyl alcohol to dilute the solution to a scale to obtain enantiomer solution;
(5) preparing a blank solution:
derivatizing without adding (S, S) -2, 8-diazabicyclo [4,3,0] nonane and (R, R) -2, 8-diazabicyclo [4,3,0] nonane by adopting the method in the step (2), precisely measuring, placing in a volumetric flask, adding absolute ethyl alcohol to dilute to a scale, and taking as a blank solution;
(6) preparation of system applicability solution:
precisely measuring enantiomer solution, placing the enantiomer solution in a volumetric flask, and adding absolute ethyl alcohol to dilute the enantiomer solution to a scale to obtain a system applicability solution;
(7) the detection method comprises the following steps:
injecting 10 μ l of blank solution into a liquid chromatograph, and recording the map; injecting 10 μ l of the enantiomer solution into a liquid chromatograph, and recording the spectrum; injecting 10 mu l of test solution into a liquid chromatograph, recording a spectrum, recording a peak area, and calculating the content of enantiomer in (S, S) -2, 8-diazabicyclo [4,3,0] nonane;
wherein:
in the step (2), the solvent is dichloromethane;
in the step (2), the amount of the 1-dimethylaminonaphthalene-5-sulfonyl chloride is 1.0 to 1.2 times of the amount of (S, S) -2, 8-diazabicyclo [4,3,0] nonane or (R, R) -2, 8-diazabicyclo [4,3,0] nonane.
2. The method for detecting the enantiomeric content of (S, S) -2, 8-diazabicyclo [4,3,0] nonane according to claim 1, wherein: the volume ratio of the normal hexane to the absolute ethyl alcohol is 45-75: 25-55.
3. The method for detecting the enantiomeric content of (S, S) -2, 8-diazabicyclo [4,3,0] nonane according to claim 2, wherein: the volume ratio of the n-hexane to the absolute ethyl alcohol is 55: 45.
4. The method for detecting the enantiomeric content of (S, S) -2, 8-diazabicyclo [4,3,0] nonane according to claim 1, wherein: the column temperature is 25-35 ℃.
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