CN114544845A - Method for determining R isomer in dapoxetine hydrochloride - Google Patents
Method for determining R isomer in dapoxetine hydrochloride Download PDFInfo
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- G—PHYSICS
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- 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
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8872—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample impurities
Abstract
The invention discloses a method for determining an R isomer in dapoxetine hydrochloride, which comprises the following steps: determining dapoxetine hydrochloride R isomer in the test solution by high performance liquid chromatography; the solvent used in the test solution is a mixed solution of n-hexane, ethanol and diethylamine; the detection conditions in the high performance liquid chromatography are as follows: the chromatographic column is a cellulose chiral column; the column temperature is 20-30 ℃; the detection wavelength is 228-232 nm or 290-294 nm. The determination method disclosed by the invention can realize effective separation of the R isomer of dapoxetine, dapoxetine and other impurities, and the R isomer of dapoxetine and the dapoxetine have early peak emergence time within 6-8 min, so that the method has the advantages of simplicity, rapidness, high accuracy and the like; the content of the R isomer of the dapoxetine can be accurately determined; the effective control of the R isomer of the dapoxetine hydrochloride in the bulk drugs is realized, and the quality control of the product is facilitated.
Description
Technical Field
The invention belongs to the field of testing, and particularly relates to a method for determining an R isomer in dapoxetine hydrochloride.
Background
Dapoxetine hydrochloride with chemical name (+) -(S) -N, N-dimethyl- (. alpha. -2- (1-naphthoxy) ethyl)]Benzylamine hydrochloride, a selective 5-hydroxytryptamine reuptake inhibitor. The dapoxetine hydrochloride is rapidly absorbed after oral administration, and the maximum plasma concentration (C) is reached after about 1-2 hours after the dapoxetine hydrochloride is rapidly absorbed after oral administrationmax) The absolute bioavailability was 42% (range 15-76%). Peak plasma concentrations were reached after 1.01 and 1.27 hours, respectively, following a single oral administration of 30mg and 60mg dapoxetine in the fasting state. The drug in the blood concentration reaches the bottom 24 hours after the drug administration (less than 5 percent of the peak concentration), and the drug in dapoxetine has little accumulation after the drug is taken every day. The pharmacokinetic characteristic of the dapoxetine hydrochloride can meet the requirement of taking the dapoxetine hydrochloride as required, and the dapoxetine hydrochloride becomes the first 5-hydroxytryptamine reuptake inhibitor approved for treating premature ejaculation. The dapoxetine hydrochloride has a chiral center carbon atom in a molecular structure, has an enantiomer, has an S configuration in a clinical use configuration, and has pharmacological activity 3.5 times of that of an R configuration. According to the existing literature data, the preparation methods of dapoxetine hydrochloride mainly comprise two methods, one method is a method for forming a dapoxetine chiral center by reducing a chiral reagent, the other method is a method for forming the dapoxetine chiral center by a resolution method, and no matter which synthesis method is adopted, the purity and the quality of the medicine are possibly influenced due to incomplete removal of impurities in the synthesis process. At present, the reported dapoxetine hydrochloride detection method has the defects of long peak emergence time of a target compound, trailing phenomenon of a chromatographic peak and influence on the detection efficiency and the production cost of a product, so that the separation and effective determination of the R isomer of dapoxetine hydrochloride have important practical significance on the production and quality control of dapoxetine hydrochloride bulk drugs and preparations.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention aims to provide the method for measuring the R isomer in the dapoxetine hydrochloride, and the method can effectively, quickly, accurately and reliably separate and detect the R isomer in the dapoxetine hydrochloride bulk drug, and the peak shapes of the dapoxetine peak and the R isomer in the dapoxetine peak are symmetrical without tailing, thereby being beneficial to controlling the product quality of the dapoxetine hydrochloride bulk drug and the preparation and ensuring the medication safety of patients.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a method for determining an R isomer in dapoxetine hydrochloride, which comprises the following steps:
determining dapoxetine hydrochloride R isomer in the test solution by high performance liquid chromatography; the solvent used in the test solution is a mixed solution of n-hexane, ethanol and diethylamine; the detection conditions in the high performance liquid chromatography are as follows: the chromatographic column is a cellulose chiral column; the column temperature is 20-30 ℃; the detection wavelength is 228-232 nm or 290-294 nm. The determination method adopted by the invention is suitable for separating and determining the R isomer of the dapoxetine hydrochloride, and compared with the prior art, the determination method is quicker, improves the efficiency, improves the peak pattern and trailing phenomenon, and is more accurate and effective.
Preferably, the column temperature is 22-28 ℃; further preferably, the column temperature is 25-28 ℃; even more preferably, the column temperature is 25 ℃.
Preferably, the detection wavelength is 228-232 nm; further preferably, the detection wavelength is 230-232 nm; still further preferably, the detection wavelength is 230 nm.
Preferably, the detection wavelength is 291-293 nm; further preferably, the detection wavelength is 292-293 nm; still more preferably, the detection wavelength is 292 nm.
Preferably, the mobile phase in the high performance liquid chromatography is a mixed solution of ethanol, n-hexane and diethylamine.
Preferably, in the mobile phase, the volume ratio of ethanol, n-hexane and diethylamine is 1000: (4.9X 10)4～9.9×104): (0.9 to 1.1); further preferably, in the mobile phase, the volume ratio of ethanol, n-hexane and diethylamine is 1000: (6.0X 10)4～9.9×104): (0.9 to 1.1); more preferably, it isIn the mobile phase, the volume ratio of the ethanol to the n-hexane to the diethylamine is 1000: (9.0X 10)4～9.9×104): 1; most preferably, in the mobile phase, the volume ratio of ethanol, n-hexane and diethylamine is 1000: 9.85X 104：1。
Preferably, the flow rate of the mobile phase is 0.9-1.1 mL/min; further preferably, the flow rate of the mobile phase is 1-1.1 mL/min; more preferably, the flow rate of the mobile phase is 1 mL/min.
Preferably, the column length of the chromatographic column is 150-250 mm; further preferably, the column length of the chromatographic column is 200-250 mm; more preferably, the column length of the chromatographic column is 230-250 mm.
Preferably, the cellulose chiral column is a chromatographic column using cellulose-tris (3, 5-dimethylphenyl carbamate) bonded silica gel as a filler.
Preferably, the particle size of the filler is 1.8-5 μm; more preferably, the particle size of the filler is 2-5 μm; more preferably, the particle size of the filler is 3 to 5 μm.
Preferably, the volume ratio of the n-hexane to the ethanol to the diethylamine is 80:20: (0.1-0.5); further preferably, the volume ratio of the n-hexane to the ethanol to the diethylamine is 80:20: (0.3-0.5); more preferably, the volume ratio of n-hexane, ethanol and diethylamine is 80:20: (0.4-0.5); most preferably, the volume ratio of n-hexane, ethanol and diethylamine is 80:20: 0.4.
preferably, the assay method further comprises: and (3) preparing a system applicability solution by using the dapoxetine hydrochloride and the dapoxetine R isomer standard substance, and testing and determining the retention time and the separation degree of the dapoxetine and the dapoxetine R isomer.
Preferably, the concentration of dapoxetine hydrochloride in the system suitability solution is 2 mg/mL.
Preferably, the concentration of dapoxetine R isomer hydrochloride in the system suitability solution is 2. mu.g/mL.
Preferably, the calculation method of the R isomer content of the dapoxetine hydrochloride is a main component self-control method;
preferably, the calculation formula of the R isomer content of the dapoxetine hydrochloride is as follows:
in the formula: a. theRThe peak area of the R isomer of dapoxetine in a chromatogram of a test solution is shown; as is the peak area of dapoxetine in the chromatogram of the control solution; the reference solution is a solution obtained by diluting the test solution by 1000 times.
The invention has the beneficial effects that: (1) the determination method disclosed by the invention can realize effective separation of the R isomer of dapoxetine, dapoxetine and other impurities, and the R isomer of dapoxetine and the dapoxetine have early peak emergence time within 6-8 min, so that the method has the advantages of simplicity, rapidness, high accuracy and the like; (2) the determination method disclosed by the invention not only can realize effective separation of the R isomer of dapoxetine, the main component of dapoxetine and related impurities, but also can accurately determine the content of the R isomer of dapoxetine; (3) the method has the advantages that the diethylamine is added into the solvent adopted by the determination method, so that the peak pattern can be well improved, and the separation degree is improved.
Drawings
FIG. 1 is a chromatogram of the solvent of example 1.
FIG. 2 is a chromatogram of a system suitability solution in example 1.
Fig. 3 is an enlarged view of fig. 2.
FIG. 4 is a chromatogram of the test solution of example 1.
Fig. 5 is an enlarged view of fig. 4.
FIG. 6 is a chromatogram of the control solution of example 1.
FIG. 7 is a chromatogram of the selective solution in example 3.
Fig. 8 is an enlarged view of fig. 7.
FIG. 9 is a chromatogram of the solvent, system suitability solution and selectivity solution in example 3.
Fig. 10 is an enlarged view of fig. 9.
FIG. 11 is a linear plot of dapoxetine.
FIG. 12 is a linear plot of dapoxetine R isomer.
Detailed Description
Specific embodiments of the present invention are described in further detail below with reference to the figures and examples, but the practice and protection of the present invention is not limited thereto. It is noted that the following processes, if not described in particular detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.
The solvents used in the following examples are all mixed solutions of n-hexane, absolute ethyl alcohol and diethylamine, and the volume ratio of n-hexane, absolute ethyl alcohol and diethylamine is 80:20: 0.04.
Example 1
The method for measuring the content of the R isomer in the dapoxetine hydrochloride in the embodiment adopts high performance liquid chromatography to measure the content of the R isomer in the dapoxetine hydrochloride, and specifically comprises the following steps:
(1) solution preparation
Respectively preparing a test solution, a reference solution and a system applicability solution, wherein the specific preparation method comprises the following steps:
test solution: taking a proper amount of the product, precisely weighing, dissolving by using a solvent and diluting to prepare a solution containing 2.0mg of dapoxetine hydrochloride in 1mL, wherein the solution is used as a test solution.
Control solution: precisely transferring a proper amount of the test solution, and adding a solvent to dilute the test solution into a solution containing 2.0 mu g of dapoxetine hydrochloride in every 1mL, wherein the solution is used as a control solution.
System applicability solution: taking a proper amount of reference substances of the dapoxetine hydrochloride and the R isomer of the dapoxetine hydrochloride, precisely weighing, adding a solvent to dissolve and dilute the reference substances to prepare a mixed solution containing 2.0mg of the dapoxetine hydrochloride and 80 mu g of the R isomer of the dapoxetine hydrochloride in each 1mL of the mixed solution as a system applicability solution.
(2) Setting detection conditions
The chromatographic column is Phenomenex
Cellulose-1 with specification of 4.6mm × 250mm and 5 μm, wherein the mobile phase consists of n-hexane-absolute ethyl alcohol-diethylamine, and the volume ratio of n-hexane to absolute ethyl alcohol is 98.5: 1.5, the volume of the diethylamine is 0.1 percent of the total volume of the absolute ethyl alcohol; the column temperature is 25 ℃, and the flow rate is 1.0 mL/min; the detection wavelength of the high performance liquid chromatograph detector is 292 nm.
(3) System applicability requirement
In the system applicability solution chromatogram, the peak appearance sequence is respectively dapoxetine R isomer, dapoxetine, and the separation degree of the peaks of the dapoxetine and the dapoxetine R isomer is required.
(4) Detection of
Taking 10 μ L of solvent, injecting into high performance liquid chromatograph, detecting, and recording chromatogram, wherein the chromatogram is shown in figure 1.
And (3) injecting 10 mu L of the system applicability solution into a high performance liquid chromatograph for detection, recording a chromatogram, wherein the chromatogram is shown in fig. 2 and fig. 3, the peak emergence time of the dapoxetine R isomer is 6.483min, the peak emergence time of the dapoxetine is 7.157min, the separation degree of the dapoxetine R isomer and the dapoxetine is 2.82 which is more than 1.5, and the system applicability solution meets the requirement.
Injecting 10 mu L of the control solution into a high performance liquid chromatograph, adjusting the detection sensitivity to ensure that the peak height of the main component peak is about 20-50% of the full range, the chromatogram is shown in figure 6, the peak emergence time of dapoxetine is 7.307min, and the blank solvent has no interference; then, 10. mu.L of the test solution is precisely measured and injected into a high performance liquid chromatograph, and the chromatogram is recorded, as shown in fig. 4 and 5. As can be seen from the graphs in FIGS. 4 and 5, the peak-off time of the R isomer of dapoxetine is 6.567min, the peak-off time of dapoxetine is 7.263min, the R isomer of dapoxetine can be well separated from dapoxetine and other impurities, the separation degree is 2.85 and is greater than 1.5, the requirement is met, no interference exists in a blank solvent, and the detection method is suitable for use.
Example 2
In the method for measuring the content of the R isomer in the dapoxetine hydrochloride, the content of the R isomer in the dapoxetine hydrochloride is measured by adopting a high performance liquid chromatography, and the method specifically comprises the following steps:
(1) solution preparation
Respectively preparing a test solution, a reference solution and a system applicability solution, wherein the specific preparation method comprises the following steps:
test solution: taking a proper amount of the product, precisely weighing, dissolving by using a solvent and diluting to prepare a solution containing 2.0mg of dapoxetine hydrochloride in 1mL, wherein the solution is used as a test solution.
Control solution: precisely transferring a proper amount of the test solution, and adding a solvent to dilute the test solution into a solution containing 2.0 mu g of dapoxetine hydrochloride in every 1mL, wherein the solution is used as a control solution.
System applicability solution: taking a proper amount of reference substances of dapoxetine hydrochloride and dapoxetine hydrochloride R isomer, precisely weighing, adding a solvent to dissolve and dilute to prepare a mixed solution containing 2.0mg of dapoxetine hydrochloride and 80 mu g of dapoxetine hydrochloride R isomer in each 1mL, and taking the mixed solution as a system applicability solution.
(2) Setting detection conditions
The chromatographic column is Phenomenex
Cellulose-1 with specification of 4.6mm × 250mm and 5 μm, wherein the mobile phase is prepared by mixing n-hexane, absolute ethyl alcohol and diethylamine, and the volume ratio of n-hexane to absolute ethyl alcohol is 98.5: 1.5, the volume of diethylamine is 0.1% of the total amount of absolute ethyl alcohol: the column temperature is 25 ℃, and the flow rate is 1.0 mL/min; the detection wavelength of the high performance liquid chromatograph detector is 230 nm.
(3) System applicability requirement
In the system applicability solution chromatogram, the peak appearance sequence is respectively dapoxetine R isomer, dapoxetine, and the separation degree of the peaks of the dapoxetine and the dapoxetine R isomer is required.
(4) And (3) taking 10 mu L of the control solution, injecting into a high performance liquid chromatograph, adjusting the detection sensitivity to enable the peak height of the main component peak to be about 20-50% of the full range, precisely measuring 10 mu L of the sample solution, injecting into the high performance liquid chromatograph, and recording the chromatogram. Under the detection conditions, the R isomer of the dapoxetine can be well separated from the dapoxetine and other impurities, the separation degree is more than 1.5, and no interference exists in a blank solvent.
Example 3
The detection method of example 1 was verified in terms of system applicability, specificity, linear relationship, quantitation limit, detection limit, precision, accuracy, solution stability, durability, and the like, and will be described in detail below.
(1) Applicability of the system:
taking a proper amount of reference substances of dapoxetine hydrochloride and dapoxetine hydrochloride R isomer, precisely weighing, adding a solvent to dissolve and dilute to prepare a mixed solution containing 2.0mg of dapoxetine hydrochloride and 80 mu g of dapoxetine hydrochloride R isomer in each 1mL, and taking the mixed solution as a system applicability solution.
Injecting 10 μ L of the system applicability solution into a high performance liquid chromatograph, detecting the wavelength at 292nm, recording the chromatogram, and separating the components as shown in Table 1.
TABLE 1 detection results of dapoxetine and its R isomer
As can be seen from Table 1, at a detection wavelength of 292nm, the appearance sequence of the system applicability solution is dapoxetine R isomer and dapoxetine, the separation degree between the dapoxetine and each impurity is 2.82, and the solvent peak does not interfere with the measurement of the dapoxetine R isomer and the main component.
(2) Specificity
Selective solution: weighing 100mg of dapoxetine hydrochloride raw material precisely, placing the raw material into a 50mL volumetric flask, weighing 1mL of R isomer reference substance stock solution precisely, placing the R isomer reference substance stock solution into the volumetric flask, adding a solvent to dissolve and dilute the R isomer reference substance stock solution to a scale, and shaking up the R isomer reference substance stock solution.
Precisely measuring 10 μ l of each of the solvent, the system applicability solution and the selective solution, injecting into a high performance liquid chromatograph, and recording a chromatogram, specifically shown in fig. 7-10, wherein fig. 7 and 8 are chromatograms of the selective solution; fig. 9 and 10 are chromatograms of a solvent, a system suitability solution, and a selective solution. As can be seen from figures 7-10, the solvent has no interference at the peak of dapoxetine and the peak of R isomer of the peak of dapoxetine; the dapoxetine peak and the R isomer peak of the dapoxetine peak in the system applicability solution and the selective solution have definite attributions, and the separation degrees of the dapoxetine peak and the R isomer peak of the dapoxetine peak are both larger than 1.5.
(3) Linear relationship and correction factor
Respectively and precisely weighing 10mg of dapoxetine hydrochloride reference substance and 10mg of dapoxetine hydrochloride R isomer reference substance, placing in a 100ml volumetric flask, adding a solvent to dissolve and dilute to a scale, and shaking up to obtain a linear stock solution. Precisely measuring appropriate amount of linear stock solution, diluting according to Table 2, preparing linear solutions with series concentrations, respectively introducing sample, and recording chromatogram. Taking the peak area y as the ordinate and the concentration x as the abscissa to carry out linear regression, the result shows that the concentration is 0.20-6.2 mu g/mL-1The concentration of dapoxetine hydrochloride in the range has a good linear relation with the peak area, wherein the linear equation y is 0.1835x-0.0025, and r is 1.0000, which is shown in a specific figure 11; in the range of 0.20 to 5.8. mu.g/mL-1The concentration of dapoxetine hydrochloride R isomer in the range has a good linear relation with the peak area, the regression equation y is 0.1903x-0.0024, and R is 1.0000, and the specific figure is shown in figure 12. The correction factor of the R isomer of dapoxetine hydrochloride relative to dapoxetine hydrochloride is 0.96, and the specific results are shown in Table 3.
Calculation of R isomer correction factor K:
in the formula: sDapoxetine hydrochloride: the slope of dapoxetine hydrochloride; sR isomer: slope of the R isomer.
TABLE 2 Linear solution preparation Table
TABLE 3 table of linearity and range results
(4) Detection limit and quantification limit
The linear stock solution was taken and the detection limit and the quantitation limit were determined by stepwise dilution, the test results for the detection limit are shown in Table 4 and the test results for the quantitation limit are shown in Table 5.
TABLE 4 detection Limit results
TABLE 5 quantitative limit results
Limit of quantitation, i.e. linear L1 solution
As can be seen from tables 4 and 5, according to the signal-to-noise ratio method, the detection limit of dapoxetine hydrochloride is 0.041 mu g/mL with a signal-to-noise ratio (S/N) of 3:1-1(corresponding to 0.002%), the detection limit of the R isomer of dapoxetine hydrochloride is 0.040 mu g.mL-1(corresponding to 0.002%); the quantitative limit of obtaining the dapoxetine hydrochloride by using the signal-to-noise ratio (S/N) of 10:1 is 0.205 mu g.mL-1(corresponding to 0.01%) and the limit of quantitation of the R isomer of dapoxetine hydrochloride is 0.195. mu.g.mL-1(corresponding to 0.01%). And (3) continuously feeding the quantitative limiting solution for 6 times, wherein the RSD of the peak area of the dapoxetine hydrochloride is 3.8%, and the RSD of the peak area of the R isomer of the dapoxetine hydrochloride is 3.1%, so that the precision of the quantitative limiting solution meets the requirement.
(5) Precision degree
1) Repeatability of
R isomer control stock solutions: weighing 10mg of dapoxetine hydrochloride R-isomer reference substance precisely, placing the reference substance in a 100mL volumetric flask, adding a solvent to dissolve and dilute the reference substance to a scale, and shaking up to obtain the R isomer reference substance stock solution.
Weighing 100mg of dapoxetine hydrochloride raw material precisely, placing the raw material into a 50mL volumetric flask, adding 1mL of R isomer reference stock solution, dissolving and diluting the solution to a scale with a solvent, and shaking up. 6 parts are prepared in parallel, 10 mu l of the mixture is precisely measured and injected into a high performance liquid chromatograph, a chromatogram is recorded, and the RSD value of the R isomer is calculated by an external standard method, and specific results are shown in Table 6.
TABLE 6 repeatability results table
As can be seen from table 6, the RSD of the measured content of dapoxetine hydrochloride R isomer is 0.5% (n ═ 6), indicating that the detection method of the present invention has good repeatability and good stability.
2) Intermediate precision
Different analysts adopt different instruments on different dates to prepare 6 parts of test solution in parallel according to a repeated sample preparation method by taking the same batch of raw materials, precisely measuring 10 mu l of the test solution, injecting the test solution into a high performance liquid chromatograph, and recording a chromatogram. The RSD value of the R isomer of dapoxetine hydrochloride is calculated by an external standard method, and the specific test results are shown in Table 7.
TABLE 7 intermediate precision results table
As can be seen from table 7, different analysts measure that RSD of the content of dapoxetine hydrochloride R isomer in 6 test sample solutions is 1.0% (n ═ 6), and compared with the results of parallel determination of 6 test sample solutions in the repeatability test, RSD of the content of dapoxetine hydrochloride R isomer in 12 test sample solutions in two persons is 2.6%, and the results show that the intermediate precision of the detection method of the present invention is good.
(6) Accuracy of
Weighing 100mg of dapoxetine hydrochloride raw material precisely, placing the raw material into a 50mL volumetric flask, adding a solvent to dissolve and dilute the raw material to a scale, shaking up the solution to serve as a blank test solution, and preparing 2 parts in parallel.
Weighing 100mg of dapoxetine hydrochloride raw material precisely, placing the raw material into a 50mL volumetric flask, respectively weighing 0.5mL, 1.0mL and 1.5mL of R isomer reference substance stock solution precisely into the volumetric flask, adding a solvent to dissolve and dilute the solution to a scale, shaking up, and respectively taking the solution as a 50% limit solution, a 100% limit solution and a 150% limit accuracy solution. 3 parts are prepared in parallel for each limiting concentration.
Precisely measuring 10 μ l of the above solution, injecting into high performance liquid chromatograph, and recording chromatogram. The recovery was calculated by the external standard method and the specific results are shown in Table 8.
Table 8R isomer accuracy results table
As can be seen from Table 8, the recovery rate of the dapoxetine hydrochloride R isomer is 98.1-104.2%, the average recovery rate is 100.8%, and the RSD is 2.2%, which indicates that the detection method has good accuracy.
(7) Stability of solution
The linear L3 solution was sampled and tested at 0, 2, 6, 22, 36, and 42 hours, and the 100% limit accuracy solution was sampled and tested at 0, 4, 8, 24, 38, and 45 hours, respectively, and the specific test results are shown in Table 9.
TABLE 9 stability results Table
As can be seen from Table 9, RSD of the peak area of dapoxetine hydrochloride and the peak area of R isomer of dapoxetine hydrochloride in the linear L3 solution are 2.2% and 0.8%, respectively, indicating that the control solution is stable after standing for 42 hours. The RSD of the R isomer content of the dapoxetine hydrochloride is 1.0% by taking a 100% limit accuracy solution as a test solution, and the result shows that the R isomer content of the dapoxetine hydrochloride of the 100% limit accuracy solution has no obvious change and is basically stable in 45 hours.
(8) Durability
Weighing 100mg of dapoxetine hydrochloride raw material precisely, placing the raw material into a 50mL volumetric flask, adding 1mL of R isomer reference substance stock solution, dissolving and diluting the R isomer reference substance stock solution to a scale mark by using a solvent, shaking up, and taking the solution as a 100% limit concentration added standard sample solution.
Taking the system applicability solution and the 100% limit concentration standard sample solution, injecting sample under various durability conditions (column temperature + -2 deg.C, flow rate + -0.1 ml/min, mobile phase proportion + -0.5%, wavelength + -2 nm), analyzing, recording chromatogram, and specific test results are shown in Table 10.
TABLE 10 durability results table (durability Condition examination)
As can be seen from Table 10, the separation degrees between the dapoxetine hydrochloride peak and the dapoxetine hydrochloride R isomer peak in the system applicability solution are both greater than 1.5 within the range of column temperature +/-2 ℃, flow rate +/-0.1 ml/min, mobile phase proportion +/-0.5% and wavelength +/-2 nm; RSD of the content of the R isomer of the dapoxetine hydrochloride in the 100% limit standard sample solution is less than 2.0%, and results show that small changes of column temperature, flow rate, wavelength and mobile phase proportion have no significant influence on the measurement result of the R isomer of the dapoxetine hydrochloride, which shows that the method has good durability and repeatability.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the embodiments, and various changes can be made without departing from the gist of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A method for determining an R isomer in dapoxetine hydrochloride is characterized by comprising the following steps: the method comprises the following steps:
determining dapoxetine hydrochloride R isomer in a test solution by adopting a high performance liquid chromatography, wherein a solvent used in the test solution is a mixed solution of n-hexane, ethanol and diethylamine; the detection conditions in the high performance liquid chromatography are as follows: the chromatographic column is a cellulose chiral column; the column temperature is 20-30 ℃; the detection wavelength is 228-232 nm or 290-294 nm.
2. The method for determining the R isomer of dapoxetine hydrochloride according to claim 1, wherein: the mobile phase in the high performance liquid chromatography is a mixed solution of ethanol, n-hexane and diethylamine.
3. The method for determining the R isomer of dapoxetine hydrochloride according to claim 2, wherein: the volume ratio of the ethanol to the n-hexane to the diethylamine is 1000: (4.9X 10)4～9.9×104)：(0.9～1.1)。
4. The method for determining the R isomer of dapoxetine hydrochloride according to claim 2 or 3, wherein: the flow rate of the mobile phase is 0.9-1.1 mL/min.
5. The method for determining the R isomer of dapoxetine hydrochloride according to claim 1, wherein: the length of the chromatographic column is 150-250 mm.
6. The method for determining the R isomer of dapoxetine hydrochloride according to claim 1, wherein: the cellulose chiral column is a chromatographic column which takes cellulose-tri (3, 5-dimethyl phenyl carbamate) bonded silica gel as a filling agent.
7. The method for determining the R isomer in dapoxetine hydrochloride, according to claim 6, wherein: the particle size of the filler is 1.8-5 μm.
8. The method for determining the R isomer of dapoxetine hydrochloride according to claim 1, wherein: the volume ratio of the n-hexane to the ethanol to the diethylamine is 80:20: (0.1-0.5).
9. The method for determining the R isomer in dapoxetine hydrochloride, according to claim 1, wherein: the assay method further comprises: and (3) preparing a system applicability solution by using the dapoxetine hydrochloride and the dapoxetine R isomer standard substance, and testing and determining the retention time and the separation degree of the dapoxetine and the dapoxetine R isomer.
10. The method for determining the R isomer of dapoxetine hydrochloride according to claim 1, wherein: the calculation method of the R isomer content of the dapoxetine hydrochloride is a main component self-control method; the calculation formula of the R isomer content of the dapoxetine hydrochloride is as follows:
in the formula: a. theRThe peak area of the R isomer of dapoxetine in a chromatogram of a test solution is shown; as is the peak area of dapoxetine in the chromatogram of the control solution; the reference solution is a solution obtained by diluting the test solution by 1000 times.
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