Method for separating and detecting enantiomers in fluralana and intermediate thereof

Abstract

The invention discloses a method for separating and detecting enantiomers in fluralana and intermediates thereof, S-fluralana, R-fluralana, S-intermediates and R-intermediates can be simultaneously detected by adopting a reverse phase chromatography, a test solution, a reference solution and a system applicability solution are prepared by dissolving with a diluent, and the test solution, the reference solution and the system applicability solution are subjected to quantitative analysis. And determining a chromatogram by adopting specific reversed-phase high-performance liquid chromatography conditions, and calculating the content of the enantiomers in the fluralana and the intermediate according to the chromatogram. The separation and detection method can effectively realize complete separation of S-fluralana, R-fluralana, S-intermediate and R-intermediate, has good convenience, specificity, high sensitivity, repeatability and accuracy, can effectively determine the content of enantiomers in fluralana and intermediate, is beneficial to guarantee the quality of fluralana and intermediate, and has a good application prospect. And the method has important significance on splitting and quality control of the fluralana.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis and detection, and in particular to a method for separating and detecting enantiomers of flurellana and its intermediates. Background Art

[0002] Flurellan is an isoxazoline insecticide and acaricide. Its insecticidal mechanism is mainly to control the chloride ion channel by antagonizing the γ-aminobutyric acid receptor and glutamate receptor gates, so that chloride ions cannot penetrate into the postsynaptic membrane, thereby interfering with the transmembrane signal transmission of the nervous system, causing the insect nervous system to be disordered, and then causing the insect to die.

[0003] In recent years, flurellana has been widely used in the clinical treatment of external parasitic diseases in dogs and cats, and has shown good results. The drug ingredients on the market are racemates of R and S configurations. Further research results show that the S configuration is the main insecticidal active ingredient. The process is mainly to synthesize S-configuration flurellana sodium (S-flurellana sodium) from S-configuration intermediates (S-intermediates). Therefore, the quantitative detection of S-intermediates and R-configuration isomers corresponding to S-flurellana sodium plays a very important role in process development. The molecular formulas and structural formulas of S-flurellana, R-flurellana, S-intermediates and R-intermediates are as follows:

[0004]

[0005] At present, there is no method that can simultaneously detect the enantiomers of flurellana and the enantiomers of flurellana's intermediates using the same chromatographic conditions. However, the introduction of the chiral center of flurellana starts from the early intermediates. Therefore, it is urgent to provide a method that can simultaneously determine the enantiomeric content of flurellana and its intermediates, which is of great significance for achieving the separation of the enantiomers of flurellana and its intermediates and the quality control of flurellana. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a method for separating and detecting enantiomers of flurana and its intermediates, which can effectively separate the enantiomers of flurana and its intermediates, has good specificity, sensitivity, repeatability and accuracy, and can more comprehensively realize the quality control of flurana.

[0007] The technical solution adopted by the present invention to solve the technical problem is: a method for separating and detecting enantiomers in flurellana and its intermediates, the method adopts reverse phase chromatography to simultaneously detect and separate S-flurellana and R-flurellana as well as S-intermediate and R-intermediate, the structural formulas of S-flurellana, R-flurellana, S-intermediate and R-intermediate are shown in Formulas I to IV respectively;

[0008]

[0009] The chromatographic conditions of the reverse phase chromatography are: using a mixed solution of phosphoric acid solution and acetonitrile as the mobile phase for isocratic elution, the elution flow rate is 0.8-1.5 mL / min; the column temperature is 30-40° C.; the detection wavelength is 263 nm-265 nm; and the injection volume is 10 μL.

[0010] Furthermore, the volume fraction of the phosphoric acid solution is 0.1%, and the volume ratio of acetonitrile in the mobile phase is 60-70%.

[0011] Preferably, the volume ratio of acetonitrile in the mobile phase is 60%.

[0012] Preferably, the elution flow rate is 0.8-1.2 mL / min, and the column temperature is 35-40°C.

[0013] More preferably, the elution flow rate is 1.0 mL / min and the column temperature is 35°C.

[0014] Preferably, the detection wavelength is 265 nm.

[0015] Furthermore, the chromatographic column used in the reverse phase chromatography is Lux Cell μlose-1, 4.6 mm×250 mm, 3.0 μm.

[0016] Furthermore, the method comprises at least the following steps:

[0017] 1) Prepare the test solution: dissolve and dilute the S-flurana test sample and the S-intermediate test sample with a diluent to prepare an S-flurana test solution and an S-intermediate test solution;

[0018] 2) preparing reference substance solutions: dissolving and diluting the R-flurana reference substance and the R-intermediate reference substance respectively with a diluent to prepare R-flurana reference substance solutions and R-intermediate reference substance solutions;

[0019] 3) Prepare system suitability solution: take appropriate amount of S-flurana reference substance, S-intermediate reference substance, R-flurana reference substance and R-intermediate reference substance, add diluent to dissolve, and prepare system suitability solution;

[0020] 4) Chromatographic test: Inject the test solution, reference solution and system suitability solution into a reverse phase high performance liquid chromatograph for determination, and record the chromatograms; obtain the qualitative detection chromatographic peaks of R-fluerana and R-intermediate in the test solution based on the chromatograms of the system suitability solution and the reference solution; calculate the contents of R-fluerana and R-intermediate in the test solution based on the relationship between the concentration of R-fluerana and R-intermediate in the reference solution and the peak area of ​​their chromatographic peaks and the peak area corresponding to R-fluerana and R-intermediate in the test solution.

[0021] Furthermore, the diluents used in step 1) to step 3) are all 60% by volume acetonitrile aqueous solution.

[0022] The invention discloses a method for separating and detecting enantiomers of flurana and its intermediates, which has the following beneficial effects: the invention makes up for the lack of reverse phase chromatography for simultaneously detecting the enantiomer content of flurana and the intermediates, adopts chromatographic conditions different from those in the prior art, and realizes the simultaneous detection of S-flurana and R-flurana as well as S-intermediate and R-intermediate by one chromatographic method; the method can effectively and completely separate the detection peaks of flurana enantiomers (S-flurana, R-flurana) and intermediate enantiomers (S-intermediate and R-intermediate); the method has good convenience, specificity, high sensitivity, repeatability and accuracy, and is of great significance to the separation and quality control of flurana. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 — is the chromatogram of the system suitability solution in Example 1;

[0024] Figure 2 - is the chromatogram of the S-Floranab reference substance solution in Example 1;

[0025] Figure 3 - is the chromatogram of the R-Floranab reference substance solution in Example 1;

[0026] Figure 4 - is the chromatogram of the S-intermediate reference substance solution in Example 1;

[0027] Figure 5 - is the chromatogram of the R-intermediate reference substance solution in Example 1;

[0028] Figure 6 - is the chromatogram of the S-flurana test solution in Example 1;

[0029] Figure 7 - is the chromatogram of the S-intermediate test solution in Example 1;

[0030] Figure 8— is the chromatogram of the system suitability solution in Example 2;

[0031] Fig. 9 — is the chromatogram of the system suitability solution in Example 3;

[0032] Fig.10 — is the chromatogram of the system suitability solution in Example 4;

[0033] Fig.11 - is the chromatogram of the system suitability solution in Example 5;

[0034] Fig.12 — is the chromatogram of the system suitability solution in Example 6;

[0035] Fig.13 — is the chromatogram of the system suitability solution in Comparative Example 1;

[0036] Fig.14 — is the chromatogram of the system suitability solution in Comparative Example 2;

[0037] Fig.15 — is the chromatogram of the system suitability solution in Comparative Example 3;

[0038] Fig.16 — is the chromatogram of the system suitability solution in Comparative Example 4;

[0039] Fig.17 — is the linear relationship diagram of S-Florana;

[0040] Fig.18 — is the linear relationship diagram of R-Florana;

[0041] Fig.19 — is the linear relationship diagram of S-intermediate;

[0042] Fig. 20 — is the linear relationship diagram of R-intermediate. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with the accompanying drawings and examples, but these specific implementation schemes do not limit the protection scope of the present invention in any way.

[0044] Example 1

[0045] A method for separating and detecting enantiomers in flurellana and its intermediates, the method using reverse phase chromatography to simultaneously detect and separate S-flurellana and R-flurellana as well as S-intermediate and R-intermediate, wherein the structural formulas of S-flurellana, R-flurellana, S-intermediate and R-intermediate are shown in Formulas I to IV, respectively;

[0046]

[0047] The separation and detection method comprises the following steps:

[0048] 1) Preparation of test solution: Dissolve and dilute the S-flurana test sample and the S-intermediate test sample respectively with 60% by volume acetonitrile aqueous solution to prepare S-flurana test solution and S-intermediate test solution with a concentration of 1 mg / mL;

[0049] The specific steps are as follows: weigh about 20 mg of S-flurana test sample and S-intermediate test sample, accurately weigh, place in 20 mL volumetric flasks respectively, add 60% acetonitrile aqueous solution to dissolve and dilute to scale, shake well, and obtain 1 mg / ml S-flurana test sample solution and 1 mg / mL S-intermediate test sample solution;

[0050] 2) Preparation of reference substance solution: dissolving and diluting R-flurana reference substance and R-intermediate reference substance respectively with diluent to prepare R-flurana reference substance solution and R-intermediate reference substance solution with a concentration of 0.3 μg to 50 μg / mL; the specific steps are: weighing about 20 mg of R-flurana reference substance and R-intermediate reference substance, accurately weighing, placing them in 20 mL volumetric flasks, adding 60% by volume acetonitrile aqueous solution to dissolve and dilute to the scale, shaking well, then accurately measuring 1 mL, placing it in a 100 mL volumetric flask, adding 60% by volume acetonitrile aqueous solution to dilute to the scale, shaking well, and obtaining R-flurana reference substance solution with a concentration of 0.01 mg / ml and R-intermediate reference substance solution with a concentration of 0.01 mg / mL;

[0051] Dissolve and dilute the S-flurana reference substance and the S-intermediate reference substance with a diluent to prepare an S-flurana reference substance solution and an S-intermediate reference substance solution with a concentration of 0.3 μg to 50 μg / mL; the specific steps are: weigh about 20 mg of the S-flurana reference substance and the S-intermediate reference substance, accurately weigh, place them in a 20 mL volumetric flask, add a 60% acetonitrile aqueous solution by volume to dissolve and dilute to the scale, shake well, then accurately measure 1 mL, place it in a 100 mL volumetric flask, add a 60% acetonitrile aqueous solution by volume to dilute to the scale, shake well, and obtain a 0.01 mg / mL S-flurana reference substance solution and a 0.01 mg / ml S-intermediate reference substance solution;

[0052] 3) Preparation of system suitability solution: Take appropriate amounts of S-fluerana reference substance, S-intermediate reference substance, R-fluerana reference substance and R-intermediate reference substance, add 60% by volume acetonitrile aqueous solution to dissolve, and prepare a solution containing 0.25 mg of each of S-fluerana, S-intermediate, R-fluerana and R-intermediate per 1 mL as the system suitability solution;

[0053] 4) Chromatographic test: The test solution, reference solution and system suitability solution were respectively injected into a reverse phase high performance liquid chromatograph for determination. The chromatographic test conditions were as follows: Lux Cell μlose-1, 4.6 mm × 250 mm, 3.0 μm; the mobile phase was 0.1% phosphoric acid solution: acetonitrile, and isocratic elution was performed. The volume ratio of acetonitrile in the mobile phase was 60%, and the elution time was 20 min; flow rate: 1.0 mL / min; column temperature: 35 ° C; UV detector detection wavelength: 265 nm; injection volume: 10 μL;

[0054] Record the chromatogram; obtain the qualitative detection chromatographic peaks of S-fluorolelana, S-intermediate, R-fluorolelana and R-intermediate in the test solution according to the chromatograms of the system suitability solution and the reference solution; calculate the content of R-fluorolelana and R-intermediate in the test solution according to the relationship between the concentration of R-fluorolelana and R-intermediate in the reference solution and the peak area of ​​their chromatographic peaks and the peak area corresponding to R-fluorolelana and R-intermediate in the test solution.

[0055] Figure 1 is the chromatogram of the system suitability solution in this embodiment, Figure 2 is the chromatogram of S-Floranab reference solution, Figure 3 is the chromatogram of R-Floranab reference solution, Figure 4 is the chromatogram of the S-intermediate reference solution, Figure 5 is the chromatogram of the R-intermediate reference solution, Figure 6 is the chromatogram of S-Florana test solution. Figure 7 The chromatogram of the S-intermediate test solution is shown in Table 1.

[0056] Table 1 Liquid chromatography analysis results of system suitability solutions in Example 1

[0057] Component name Retention time (min) Separation R-Florana 8.000 NA S-Florana 8.568 2.47 R-Intermediate 11.729 11.77 S-Intermediate 12.487 2.41

[0058] As shown in Table 1, the peak retention times of R-fluerana, S-fluerana, R-intermediate and S-intermediate are 8.000min, 8.568min, 11.729min and 12.487min respectively. Figure 1It can be seen that the four substances S-Florana, S-intermediate, R-Florana and R-intermediate are completely separated in the same chromatogram, and it can be seen from Table 1 that the separation between any adjacent peaks of the four substances S-Florana, S-intermediate, R-Florana and R-intermediate is greater than 1.5, indicating that the same chromatographic condition of the present invention can not only achieve complete separation of Florana and its enantiomers (S-Florana and R-Florana) and Florana intermediate and its enantiomers (S-intermediate and R-intermediate), but also can achieve complete separation between Florana enantiomers and intermediate enantiomers, so that the method of the present invention can simultaneously detect and separate the four substances S-Florana, S-intermediate, R-Florana and R-intermediate, providing a favorable prerequisite for the simultaneous and accurate determination of S-Florana, S-intermediate, R-Florana and R-intermediate;

[0059] In addition, the content of the corresponding target substance in the test sample can be obtained by the linear relationship between the concentration of each target substance in the reference solution and the peak area of ​​its chromatographic peak and the peak area of ​​the target substance in the test solution. For example, in this embodiment, in order to know the content of R-flurana in the S-flurana test sample and the content of R-intermediate in the S-intermediate test sample, the linear relationship between the concentration and peak area of ​​R-flurana obtained from the R-flurana reference solution and the peak area of ​​R-flurana in the S-flurana test solution were calculated to show that the content of R-flurana in the S-flurana test sample was 0.84%; the linear relationship between the concentration and peak area of ​​R-intermediate obtained from the R-intermediate reference solution and the peak area of ​​R-intermediate in the S-intermediate test solution were calculated to show that the content of R-intermediate in the S-intermediate test sample was 0.62%.

[0060] Example 2

[0061] The difference between this embodiment and embodiment 1 is that the column temperature used in the chromatographic test is 40°C.

[0062] Under the chromatographic conditions of Example 2, it was detected that Figure 8 The chromatogram of the system suitability solution in this embodiment and the specific results of the system suitability solution analysis are shown in Table 2:

[0063] Table 2 Liquid chromatography analysis results of system suitability solutions in Example 2

[0064] Component name Retention time (min) Separation R-Florana 7.702 NA S-Florana 8.247 2.57 R-Intermediate 11.215 11.89 S-Intermediate 11.941 2.49

[0065] As shown in Table 2, the peak retention times of R-Fluorana, S-Fluorana, R-intermediate and S-intermediate are 7.702min, 8.247min, 11.215min and 11.941min respectively, and the separation between any adjacent peaks is greater than 1.5, indicating that when the column temperature is 40°C, the peaks corresponding to S-Fluorana, S-intermediate, R-Fluorana and R-intermediate can also be completely separated, providing a favorable prerequisite for the simultaneous and accurate determination of -Fluorana, S-intermediate, R-Fluorana and R-intermediate. And after calculation, it is known that the content of R-Fluorana in the S-Fluorana sample is 0.82%, and the content of R-intermediate in the S-intermediate sample is 0.61%.

[0066] Example 3

[0067] The difference between this embodiment and embodiment 1 is that the detection wavelength used in the chromatographic test is 263 nm.

[0068] Under the chromatographic conditions of Example 3, it was detected that Fig. 9 The chromatogram of the system suitability solution in this embodiment, the specific results of the system suitability solution analysis are shown in Table 3: Table 3 Liquid chromatography analysis results of the system suitability solution in Example 3

[0069] Component name Retention time (min) Separation R-Florana 7.988 NA S-Florana 8.556 2.47 R-Intermediate 11.714 11.74 S-Intermediate 12.474 2.42

[0070] As shown in Table 3, the peak retention times of R-Fluorana, S-Fluorana, R-intermediate and S-intermediate are 7.988min, 8.556min, 11.714min and 12.474min respectively, and the separation between any adjacent peaks is greater than 1.5, indicating that when the detection wavelength is 263nm, the peaks corresponding to S-Fluorana, S-intermediate, R-Fluorana and R-intermediate can also be completely separated, providing a favorable prerequisite for the simultaneous and accurate determination of -Fluorana, S-intermediate, R-Fluorana and R-intermediate. And after calculation, it is known that the content of R-Fluorana in the S-Fluorana sample is 0.86%, and the content of R-intermediate in the S-intermediate sample is 0.65%.

[0071] Example 4

[0072] The difference between this embodiment and embodiment 1 is that the flow rate used in the chromatographic test is 0.8 mL / min.

[0073] Under the chromatographic conditions of Example 4, it was detected that Fig.10 The chromatogram of the system suitability solution in this embodiment and the specific results of the system suitability solution analysis are shown in Table 4:

[0074] Table 4 Liquid chromatography analysis results of system suitability solutions in Example 4

[0075] Component name Retention time (min) Separation R-Florana 9.990 NA S-Florana 10.700 2.53 R-Intermediate 14.622 11.91 S-Intermediate 15.567 2.45

[0076] As shown in Table 4, the peak retention times of R-fluerana, S-fluerana, R-intermediate and S-intermediate are 9.990min, 10.700min, 14.622min and 15.567min respectively, and the separation between any adjacent peaks is greater than 1.5, indicating that when the flow rate is 0.8mL / min, the peaks corresponding to S-fluerana, S-intermediate, R-fluerana and R-intermediate can also be completely separated, providing a favorable prerequisite for the simultaneous and accurate determination of -fluerana, S-intermediate, R-fluerana and R-intermediate. And after calculation, it is known that the content of R-fluerana in the S-fluerana sample is 0.81%, and the content of R-intermediate in the S-intermediate sample is 0.62%.

[0077] Example 5

[0078] The difference between this embodiment and embodiment 1 is that the flow rate used in the chromatographic test is 1.5 mL / min.

[0079] Under the chromatographic conditions of Example 5, it was detected that Fig.11 Table 5 is the chromatogram of the system suitability solution in this embodiment, and the specific results of the system suitability solution analysis are as follows:

[0080] Table 5 Liquid chromatography analysis results of system suitability solutions in Example 5

[0081] Component name Retention time (min) Separation R-Florana 5.323 NA S-Florana 5.698 2.26 R-Intermediate 7.830 11.00 S-Intermediate 8.338 2.24

[0082] As shown in Table 5, the peak retention times of R-fluerana, S-fluerana, R-intermediate and S-intermediate are 5.323min, 5.698min, 7.830min and 8.338min respectively, and the separation between any adjacent peaks is greater than 1.5, indicating that when the flow rate is 1.5mL / min, the peaks corresponding to S-fluerana, S-intermediate, R-fluerana and R-intermediate can also be completely separated, providing favorable prerequisites for the simultaneous and accurate determination of -fluerana, S-intermediate, R-fluerana and R-intermediate. And after calculation, it is known that the content of R-fluerana in the S-fluerana sample is 0.86%, and the content of R-intermediate in the S-intermediate sample is 0.67%.

[0083] Example 6

[0084] The difference between this embodiment and embodiment 1 is that in the chromatographic test, the volume ratio of acetonitrile in the mobile phase is 70%.

[0085] Under the chromatographic conditions of Example 6, it was detected that Fig.12 Table 6 is the chromatogram of the system suitability solution in this embodiment, and the specific results of the system suitability solution analysis are shown in Table 6:

[0086] Table 6 Liquid chromatography analysis results of system suitability solutions in Example 6

[0087]

[0088]

[0089] As shown in Table 6, the peak retention times of R-Fluorana, S-Fluorana, R-intermediate and S-intermediate are 4.872min, 5.093min, 6.495min and 6.789min respectively, and the separation between any adjacent peaks is greater than 1.5, indicating that when the volume ratio of acetonitrile in the mobile phase is 70%, the peaks corresponding to S-Fluorana, S-intermediate, R-Fluorana and R-intermediate can also be completely separated, providing a favorable prerequisite for the simultaneous and accurate determination of -Fluorana, S-intermediate, R-Fluorana and R-intermediate. And after calculation, it is known that the content of R-Fluorana in the S-Fluorana sample is 0.79%, and the content of R-intermediate in the S-intermediate sample is 0.68%.

[0090] Comparative Example 1

[0091] The difference between this comparative example and Example 1 is that the mobile phase used in the chromatographic test is 0.1% phosphoric acid solution: methanol, and the elution time is 30 min.

[0092] Under the chromatographic conditions of Comparative Example 1, it was detected that Fig.13 This is the chromatogram of the system suitability solution in this comparative example. It can be seen from the figure that when 0.1% phosphoric acid solution: methanol is used as the mobile phase for elution for 30 minutes, S-flurelana, S-intermediate, R-flurelana and R-intermediate cannot be eluted.

[0093] Comparative Example 2

[0094] The difference between this comparative example and Example 1 is that in the chromatographic test, the volume ratio of acetonitrile in the mobile phase is 75%.

[0095] Under the chromatographic conditions of Comparative Example 2, it was detected that Fig.14 The chromatogram of the system suitability solution in this comparative example and the specific results of the system suitability solution analysis are shown in Table 7:

[0096] Table 7 Liquid chromatography analysis results of system suitability solution in Comparative Example 2

[0097] Component name Retention time (min) Separation R-Florana 3.756 NA S-Florana 3.856 0.93 R-Intermediate 4.549 6.06 S-Intermediate 4.673 1.02

[0098] It can be seen from Table 7 that the peak retention times of R-fluorolane, S-fluorolane, R-intermediate and S-intermediate are 3.756min, 3.856min, 4.549min and 4.673min respectively, and the separation degrees of S-fluorolane and R-fluorolane, S-intermediate and R-intermediate are all less than 1.5, which indicates that when the volume percentage of acetonitrile in the fluidity is 75%, S-fluorolane and R-fluorolane, S-intermediate and R-intermediate cannot be completely separated.

[0099] Comparative Example 3

[0100] The difference between this comparative example and Example 1 is that the flow rate in the chromatographic test is 1.8 mL / min.

[0101] Under the chromatographic conditions of Comparative Example 3, it was detected that Fig.15 The chromatogram of the system suitability solution in this comparative example and the specific results of the system suitability solution analysis are shown in Table 8:

[0102] Table 8 Liquid chromatography analysis results of system suitability solution in Comparative Example 3

[0103] Component name Retention time (min) Separation R-Florana 4.191 NA S-Florana 4.339 1.29 R-Intermediate 5.318 7.83 S-Intermediate 5.508 1.38

[0104] It can be seen from Table 8 that the peak retention times of R-fluororana, S-fluororana, R-intermediate and S-intermediate are 4.191 min, 4.339 min, 5.318 min and 5.508 min, respectively, and the separation degrees of S-fluororana and R-fluororana, S-intermediate and R-intermediate are all less than 1.5, which means that when the flow rate is 1.8 mL / min, S-fluororana and R-fluororana, S-intermediate and R-intermediate cannot be completely separated.

[0105] Comparative Example 4

[0106] The difference between this comparative example and Example 1 is that the chromatographic test column is Excsep C18, 4.6 mm×250 mm, 5.0 μm.

[0107] Under the chromatographic conditions of Comparative Example 4, it was detected that Fig.16 This is the chromatogram of the system suitability solution in this comparative example. It can be seen from the figure that when the chromatographic column is Excsep C18, 4.6mm×250mm, 5.0μm, S-fluerana, S-intermediate, R-fluerana and R-intermediate cannot be eluted.

[0108] Experimental Example 1 System Applicability Experiment

[0109] 1) Preparation of reference solution: Weigh about 20 mg of R-flurana reference substance and R-intermediate reference substance respectively, accurately weigh, place in a 20 mL volumetric flask, add 60% by volume acetonitrile aqueous solution to dissolve and dilute to scale, shake well, then accurately measure 1 mL, place in a 100 mL volumetric flask, add 60% by volume acetonitrile aqueous solution to dilute to scale, shake well, and obtain reference solution with R-flurana and R-intermediate concentration of 0.01 mg / mL;

[0110] 2) Preparation of sensitivity solution: accurately measure 1 mL of the reference solution and place it in a 10 mL volumetric flask, add 60% acetonitrile aqueous solution to dilute to the mark, shake well, and obtain a sensitivity solution with a concentration of 1 μg / mL of R-flurana and R-intermediate;

[0111] 3) Preparation of system suitability solution: Weigh appropriate amounts of R-flurana reference substance, S-flurana reference substance, R-intermediate reference substance, and S-intermediate reference substance, and dissolve them in 60% by volume acetonitrile aqueous solution to prepare system suitability solutions containing 1 mg each of S-flurana and S-intermediate, and 0.01 mg each of R-flurana and R-intermediate.

[0112] 4) High performance liquid chromatography determination: The chromatographic test conditions are as follows: Lux Cell μlose-1, 4.6 mm × 250 mm, 3.0 μm; the mobile phase is 0.1% phosphoric acid solution: acetonitrile, isocratic elution is performed, the volume ratio of the mobile phase acetonitrile is 60%, and the elution time is 20 min; flow rate: 1.0 mL / min; column temperature: 40°C; UV detector detection wavelength: 265 nm; injection volume: 10 μL;

[0113] The reference solution was injected 6 times, the system suitability solution was injected once, and the sensitivity solution was injected once.

[0114] Table 9 System suitability test results

[0115]

[0116] As shown in Table 9, the reference solution was injected 6 times, and the RSDs of the peak areas of R-flurana and R-intermediate were less than 2.0%, the signal-to-noise ratios of each component in the sensitivity solution were greater than 100, and the separations of each component from adjacent peaks in the system suitability solution were greater than 1.5, indicating that the method of the present invention has good applicability.

[0117] Experimental Example 2: Linear Relationship Investigation Experiment

[0118] 1) Preparation of linear solutions: Weigh appropriate amounts of R-flurana reference substance, S-flurana reference substance, R-intermediate reference substance and S-intermediate reference substance, accurately weigh them, and dilute them with 60% by volume acetonitrile aqueous solution to obtain linear solutions of different mass concentrations, which correspond to solution concentrations of 0.3 μg / mL, 1 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL and 50 μg / mL, respectively.

[0119] 2) High performance liquid chromatography determination: Take each concentration linear solution and inject it once. The chromatographic test conditions are as follows: LuxCellμlose-1, 4.6mm×250mm, 3.0μm; the mobile phase is 0.1% phosphoric acid solution: acetonitrile, isocratic elution, the volume ratio of the mobile phase acetonitrile is 60%, and the elution time is 20min; flow rate: 1.0mL / min; column temperature: 40℃; UV detector detection wavelength: 265nm; injection volume: 10μL.

[0120] Linear regression was performed on the peak area (y) versus the concentration (x), and the linear regression equation of R-fluerana was y=441475x+1251.1, and the correlation coefficient (R) was 0.9998; the linear regression equation of R-intermediate was y=569179x-462.68, and the correlation coefficient (R) was 0.9998; the linear regression equation of S-fluerana was y=440839x-691.1, and the correlation coefficient (R) was 0.9999; the linear regression equation of S-intermediate was y=588490x-2099.7, and the correlation coefficient (R) was 0.9999. This shows that R-fluerana, S-fluerana, R-intermediate and S-intermediate have a good linear relationship in the range of 0.3μg / ml to 50μg / ml, and the linear relationship diagram of S-fluerana is shown in FIG. Fig.17 The linear relationship diagram of S-intermediate is shown in Fig.18 As shown, the linear relationship diagram of R-Fluorana is as follows Fig.19 The linear relationship diagram of R-intermediate is shown in Fig. 20 shown.

[0121] Experimental Example 3: Precision Investigation

[0122] 1) Preparation of reference solution: Weigh about 20 mg of R-flurana reference substance and R-intermediate reference substance respectively, accurately weigh, place in a 20 mL volumetric flask, add 60% by volume acetonitrile aqueous solution to dissolve and dilute to scale, shake well, then accurately measure 1 mL, place in a 100 mL volumetric flask, add 60% by volume acetonitrile aqueous solution to dilute to scale, shake well, and obtain reference solution with R-flurana and R-intermediate concentration of 0.01 mg / mL;

[0123] 2) Preparation of S-Florana test solution: Weigh about 20 mg of S-Florana test sample, accurately weigh, place in a 20 mL volumetric flask, add 60% acetonitrile aqueous solution to dissolve and dilute to scale, shake well, and obtain a S-Florana test solution with a concentration of 1 mg / mL; (6 portions prepared by different personnel)

[0124] 3) Preparation of S-intermediate test solution: Weigh about 20 mg of S-intermediate test sample, accurately weigh, place in a 20 mL volumetric flask, add 60% acetonitrile aqueous solution to dissolve and dilute to scale, shake well, and obtain a test solution with a S-intermediate concentration of 1 mg / ml; (6 portions prepared by different personnel)

[0125] 4) High performance liquid chromatography determination: The chromatographic test conditions are as follows: Lux Cell μlose-1, 4.6 mm × 250 mm, 3.0 μm; the mobile phase is 0.1% phosphoric acid solution: acetonitrile, isocratic elution is performed, the volume ratio of the mobile phase acetonitrile is 60%, and the elution time is 20 min; flow rate: 1.0 mL / min; column temperature: 40°C; UV detector detection wavelength: 265 nm; injection volume: 10 μL;

[0126] The above test was performed by different analysts at different times using different instruments as repeatability and intermediate precision. 10 μl of each of the reference solution, S-flurana test solution, and S-intermediate test solution was injected into the liquid chromatograph, the chromatogram was recorded, and the contents of R-flurana and R-intermediate enantiomers were calculated.

[0127] Table 10 Precision results

[0128]

[0129] From the above results, it can be seen that in Experiment 1, the average detection amount of R-fluerana and R-intermediate was 0.82% and 0.63%, and the RSD values ​​were 3.23% and 2.99%, respectively; in Experiment 2, the average detection amount of R-fluerana and R-intermediate enantiomer was 0.81% and 0.63%, and the RSD values ​​were 1.59% and 3.37%, respectively;

[0130] The average detection amounts of R-flurana and R-intermediate enantiomer in a total of 12 test solutions in Experiments 1 and 2 were 0.81% and 0.63%, respectively, and the RSD values ​​were 2.76% and 3.08%, respectively, which were no more than 5.0%, indicating that the repeatability and intermediate precision of this method met the detection requirements and had good precision.

[0131] Experimental Example 4: Accuracy Experiment

[0132] Diluent: 60% acetonitrile aqueous solution;

[0133] 1) Mixed stock solution: Accurately weigh appropriate amount of R-flurana and R-intermediate reference substances and prepare to 1 mg / mL with diluent;

[0134] 2) Mixed stock solution: Accurately pipette 5.0 mL of the mixed stock solution into a 50 mL volumetric flask, dilute with diluent and make up to volume;

[0135] 3) Control solution: Accurately pipette 1.0 mL of the mixed stock solution into a 100 mL volumetric flask, dilute with diluent and make up to volume;

[0136] 4) Test solution (matrix solution): Accurately weigh an appropriate amount of S-fluranabol test sample and prepare it to 1 mg / mL with diluent.

[0137] Make two parallel portions;

[0138] 5) Test solution (matrix solution): Accurately weigh an appropriate amount of S-intermediate test sample and prepare it to 1 mg / mL with diluent.

[0139] Make two parallel portions;

[0140] 6) Spiked test solution 1: Take about 20 mg of each of S-flurana and S-intermediate test samples, accurately weigh, place in a 20 ml volumetric flask, accurately add 1.0 ml of mixed stock solution, add diluent to dissolve to the scale, shake well, and use as the spiked solution; prepare 3 copies in parallel;

[0141] 7) Spiked test solution 2: Take about 20 mg of each of S-Florana and S-intermediate test samples, weigh accurately, place in a 20 ml volumetric flask, accurately add 2.0 ml of mixed stock solution, add diluent to dissolve to the scale, shake well, and use as spiked solution. Prepare 3 copies in parallel.

[0142] 8) Spiked test solution 3: Take about 20 mg of each of S-Florana and S-intermediate test samples, weigh accurately, place in a 20 ml volumetric flask, accurately add 3.0 ml of mixed stock solution, add diluent to dissolve to the scale, shake well, and use as spiked solution. Prepare 3 portions in parallel.

[0143] 9) High performance liquid chromatography test: Take the blank solution (diluent), the control solution and the spiked test solution and inject them into the liquid chromatograph respectively;

[0144] The chromatographic test conditions are as follows: Lux Cell μlose-1, 4.6 mm × 250 mm, 3.0 μm; the mobile phase is 0.1% phosphoric acid solution: acetonitrile, isocratic elution is performed, the volume ratio of the mobile phase acetonitrile is 60%, and the elution time is 20 min; flow rate: 1.0 mL / min; column temperature: 40°C; UV detector detection wavelength: 265 nm; injection volume: 10 μL; record the chromatogram and calculate the content of R-flurana and R-intermediate enantiomers. The results are shown in the following table:

[0145] Table 11 Accuracy experimental results

[0146]

[0147] As can be seen from the above table, the recoveries of the nine accuracy solutions at three concentration levels were all between 90% and 108%, and the RSD values ​​were all less than 10%, indicating that the method of the present invention is accurate and feasible.

[0148] Experimental Example 5 Quasi-durability Test

[0149] Diluent: 60% acetonitrile aqueous solution;

[0150] 1) S-Fluorellanal stock solution: Take an appropriate amount of S-Fluorellanal reference substance and prepare it to 1 mg / mL with diluent;

[0151] 2) R-Fluorana stock solution: Take an appropriate amount of R-Fluorana reference substance and prepare it to 1 mg / mL with diluent;

[0152] 3) S-intermediate stock solution: Take an appropriate amount of S-intermediate reference substance and prepare it to 1 mg / mL with diluent;

[0153] 4) R-intermediate stock solution: Take an appropriate amount of R-intermediate reference substance and prepare it to 1 mg / mL with diluent;

[0154] 5) High performance liquid chromatography detection: Take the stock solutions of S-flurelana, R-flurelana, S-intermediate and R-intermediate which have been placed at room temperature (25°C) for 0 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 30 hours and 36 hours respectively, and inject them into the liquid chromatograph;

[0155] The chromatographic test conditions are as follows: Lux Cellμlose-1, 4.6mm×250mm, 3.0μm; the mobile phase is 0.1% phosphoric acid solution: acetonitrile, isocratic elution is performed, the volume ratio of the mobile phase acetonitrile is 60%, and the elution time is 20min; flow rate: 1.0mL / min; column temperature: 40℃; detection wavelength of the ultraviolet detector: 265nm; injection volume: 10μL; record the chromatogram.

[0156] The results are shown in Table 12. It can be seen from Table 12 that within 36 hours, the RSD of the peak area percentage content of S-flurana, R-flurana, S-intermediate and R-intermediate was less than 2.0%, indicating that each stock solution was stable within 36 hours under room temperature conditions, and the solution did not need to be prepared before use and had good durability.

[0157] Table 12 Durability test results

[0158]

[0159] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for separating and detecting enantiomers of flurellana and its intermediates, characterized in that: The method adopts reverse phase chromatography to simultaneously detect and separate S-flurana and R-flurana as well as S-intermediate and R-intermediate. The structural formulas of S-flurana, R-flurana, S-intermediate and R-intermediate are shown in Formulas I to IV respectively; The chromatographic conditions of the reverse phase chromatography are: using a mixed solution of phosphoric acid solution and acetonitrile as the mobile phase for isocratic elution, the elution flow rate is 0.8-1.5 mL / min; the column temperature is 30-40° C.; the detection wavelength is 263 nm-265 nm; and the injection volume is 10 μL.

2. The method for separating and detecting enantiomers of flurellana and its intermediates according to claim 1, characterized in that: The volume fraction of the phosphoric acid solution is 0.1%, and the volume ratio of acetonitrile in the mobile phase is 60-70%.

3. The method for separating and detecting enantiomers of flurellana and its intermediates according to claim 2, characterized in that: The volume ratio of acetonitrile in the mobile phase is 60%.

4. The method for separating and detecting enantiomers of flurellana and its intermediates according to claim 1, characterized in that: The elution flow rate is 0.8-1.2 mL / min, and the column temperature is 35-40°C.

5. The method for separating and detecting enantiomers of flurellana and its intermediates according to claim 1, characterized in that: The elution flow rate was 1.0 mL / min, and the column temperature was 35°C.

6. The method for separating and detecting enantiomers of flurellana and its intermediates according to claim 1, characterized in that: The detection wavelength is 265 nm.

7. The method for separating and detecting enantiomers of flurellana and its intermediates according to claim 1, characterized in that: The chromatographic column used in the reverse phase chromatography is Lux Cell μlose-1, 4.6 mm×250 mm, 3.0 μm.

8. The method for separation and detection of enantiomers in flurellana and its intermediates according to any one of claims 1 to 7, characterized in that: The method comprises at least the following steps: 1) Prepare the test solution: dissolve and dilute the S-flurana test sample and the S-intermediate test sample with a diluent to prepare an S-flurana test solution and an S-intermediate test solution; 2) preparing reference substance solutions: dissolving and diluting the R-flurana reference substance and the R-intermediate reference substance respectively with a diluent to prepare R-flurana reference substance solutions and R-intermediate reference substance solutions; 3) Prepare system suitability solution: take appropriate amount of S-flurana reference substance, S-intermediate reference substance, R-flurana reference substance and R-intermediate reference substance, add diluent to dissolve, and prepare system suitability solution; 4) Chromatographic test: Inject the test solution, reference solution and system suitability solution into a reverse phase high performance liquid chromatograph for determination, and record the chromatograms; obtain the qualitative detection chromatographic peaks of R-fluerana and R-intermediate in the test solution based on the chromatograms of the system suitability solution and the reference solution; calculate the contents of R-fluerana and R-intermediate in the test solution based on the relationship between the concentration of R-fluerana and R-intermediate in the reference solution and the peak area of ​​their chromatographic peaks and the peak area corresponding to R-fluerana and R-intermediate in the test solution.

9. The method for separating and detecting enantiomers of flurellana and its intermediates according to claim 8, characterized in that: The diluents used in the steps 1) to 3) are all 60% by volume acetonitrile aqueous solution.

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