CN111220727B - Method for detecting enantiomer in ivabradine hydrochloride intermediate and application - Google Patents
Method for detecting enantiomer in ivabradine hydrochloride intermediate and application Download PDFInfo
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- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 14
- 125000000250 methylamino group Chemical group [H]N(*)C([H])([H])[H] 0.000 description 14
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 14
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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
- G01N30/02—Column chromatography
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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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
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Abstract
The invention provides a method for detecting enantiomers in an ivabradine hydrochloride intermediate and application thereof, wherein the method comprises the steps of respectively preparing a sample solution to be detected, injecting the sample solution into a high performance liquid chromatograph, and measuring conditions that a chromatographic column is CHIRALPAK IC chromatographic column, the column temperature is 25-40 ℃, and the detection wavelength is 280-290 nm; the mobile phase is a mixture of n-hexane, dichloromethane and diethylamine with the volume ratio of (76-80): 20-24): 0.5, and the flow rate of the mobile phase is 0.8-1.2 mL/min; the method can accurately perform quantitative analysis on the enantiomer in the intermediate of the ivabradine hydrochloride, has symmetrical peak shape and no tailing phenomenon, meets the relevant requirements on the separation degree, the quantitative limit, the detection limit, the repeatability, the precision, the solution stability and the like, can reach 0.0337 mu g/mL, and can detect the enantiomer of which the content is higher than 0.01 percent in the intermediate of the ivabradine hydrochloride.
Description
Technical Field
The invention relates to the technical field of medical chemistry, in particular to a method for detecting an enantiomer in an ivabradine hydrochloride intermediate and application thereof.
Background
Ivabradine hydrochloride (Ivabradine hydrochloride) with the chemical name of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] -methylamino ] propyl ] -7, 8-dimethoxy-1, 3,4, 5-tetrahydro-2H-3-benzazepin-2-one hydrochloride. Ivabradine hydrochloride, which has very valuable pharmacological and therapeutic properties, in particular heart rate-slowing properties, is useful for the treatment or prevention of various clinical manifestations of myocardial ischemia, such as angina pectoris, myocardial infarction and related rhythm disorders, and of various diseases involving rhythm disorders, in particular supraventricular arrhythmias, and for systolic and diastolic heart failure. The chemical structural formula of the ivabradine hydrochloride is as follows:
after the ivabradine is orally taken, the ivabradine can be quickly and completely absorbed, and can reach the blood drug peak concentration after one hour under the fasting condition. In the patient, the plasma protein binding rate of ivabradine is about 70%, and the apparent volume of distribution is close to 100L at steady state. In the recommended 5mg dose each time, twice daily long-term dosing, the maximum plasma concentration was 22ng/mL (CV 29%), and the mean plasma concentration at steady state was 10ng/mL (CV 38%). Within the liver and digestive tract ivabradine is metabolized by oxidation only through the cytochrome P4503 a4, the main active metabolite being the N-demethylated derivative. The elimination half-life of ivabradine in plasma is 2 hours (70% to 75% of the AUC) with an effective half-life of 11 hours. The total clearance rate is 400mL/min, and the renal clearance rate is 70 mL/min. The metabolite is finally excreted through the feces and urine, and 4% of the original drug orally taken can be found in the urine.
The ivabradine hydrochloride is synthesized by using 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepine-2-ketone as a starting material, generating 3- (3-chloropropyl) -7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone through nucleophilic reaction of 3-chloro-1-bromopropane, then generating an intermediate 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1 through condensation of the 3- (3-chloropropyl) -7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone and (1S) -4, 5-dimethoxy-1- [ (methylamino) methyl ] benzocyclobutane under an alkaline condition, 3-dihydro-2H-benzazepin-2-ketone, and finally hydrogenating to prepare the ivabradine hydrochloride, wherein the intermediate 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-ketone has the chemical structural formula:
and the intermediate enantiomer is derived from the enantiomer in the material 4, 5-dimethoxy-1- [ (methylamino) methyl ] benzocyclobutane, which upon further reaction results in the production of the enantiomer of ivabradine hydrochloride key intermediate 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one, 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one.
In order to ensure the safety and effectiveness of clinical medication, according to the guiding principle of chiral drugs, the effective control of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one in the key intermediate 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one of ivabradine hydrochloride is necessary. However, in the prior art, only aiming at the effective control method and detection of the chirality of the ivabradine hydrochloride finished product, the content detection method of an ivabradine hydrochloride optical isomer in patent CN102478560A refers to the adoption of an OD-RH (chiralcel, 150mm × 4.6mm, 5 μm) chiral column, a mobile phase (n-hexane-absolute ethyl alcohol-triethylamine ═ 85:15:0.5), a column temperature of 35 ℃, a flow rate of 1.0ml/min, a detection wavelength of 287nm, and the detection of only the ivabradine hydrochloride finished product. Therefore, in order to continuously improve the safety and the effectiveness of the ivabradine hydrochloride, a quantitative detection method of an enantiomer of a key intermediate 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] octyl-1, 3, 5-triene-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one in the ivabradine hydrochloride, which has the advantages of simple operation, high sensitivity and good reproducibility, is urgently needed to be established.
Disclosure of Invention
The detection method can realize effective separation and quantification of 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octyl-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-ketone in the key intermediate of the ivabramine hydrochloride, the separation degree, the specificity, the quantification limit, the detection limit, the linearity, the repeatability, the accuracy, the precision, the solution stability, the durability and other aspects are verified in detail, and each verification result meets the requirements of relevant regulations and guidance principles, and the actual detection effect is good.
The technical scheme of the invention is realized as follows:
a method for detecting enantiomer in ivabradine hydrochloride intermediate, wherein the ivabradine hydrochloride intermediate is 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one, and the enantiomer thereof is 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one;
respectively preparing a sample solution to be detected: injecting a reference substance solution of enantiomer, a test substance solution of ivabradine hydrochloride intermediate and a mixed solution of the ivabradine hydrochloride intermediate and enantiomer into a high performance chromatograph for measurement, wherein the measurement conditions comprise that: the chromatographic column is CHIRALPAK IC chromatographic column, the column temperature is 25-40 ℃, and the detection wavelength is 280-290 nm; the mobile phase is a mixture of n-hexane, dichloromethane and diethylamine, wherein the n-hexane: dichloromethane: the volume ratio of the diethylamine is (76-80): 20-24): 0.5, and the flow rate of the mobile phase is 0.8-1.2 mL/min.
Further illustrated, the n-hexane: dichloromethane: the volume ratio of diethylamine is 78: 22: 0.5.
further, the column temperature of the chromatographic column is 35 ℃, and the detection wavelength is 287 nm.
Further, the flow rate of the mobile phase was 1 mL/min.
Further, the method for preparing the sample solution to be tested comprises the following steps:
(1) test solution for preparing ivabradine hydrochloride intermediate
Weighing an ivabradine hydrochloride intermediate sample, mixing the ivabradine hydrochloride intermediate sample with a mobile phase, and preparing a test solution of the ivabradine hydrochloride intermediate for later use;
(2) enantiomeric control stock solutions
Weighing an enantiomer sample, mixing the enantiomer sample with a mobile phase, and preparing a reference substance stock solution of the enantiomer for later use;
(3) control solutions for enantiomeric preparations
Weighing the prepared enantiomer reference substance stock solution, mixing with a mobile phase, and preparing into enantiomer reference substance solution for later use;
(4) preparing a mixed solution of ivabradine hydrochloride intermediate and enantiomer
Weighing an ivabradine hydrochloride intermediate sample, weighing a reference substance stock solution of an enantiomer, adding into a volumetric flask, diluting by adopting a mobile phase, uniformly mixing, and preparing into a mixed solution for later use;
(5) respectively sucking equivalent enantiomer reference substance solution, ivabradine hydrochloride intermediate sample solution and ivabradine hydrochloride intermediate and enantiomer mixed solution, and injecting into a high performance chromatograph for determination.
Further, in the step (1), the ratio of the ivabradine hydrochloride intermediate to the mobile phase is (0.1-5) in terms of mass-to-volume ratio g/L: 1.
more preferably, in the step (1), the ratio of the ivabradine hydrochloride intermediate to the mobile phase is 1: 1.
further, in the step (2), the ratio of the enantiomer to the mobile phase is (0.001-0.1): 1.
more preferably, in step (2), the ratio of the enantiomer to the mobile phase is 0.01: 1.
further, in the step (3), the ratio of the enantiomer to the mobile phase is (0.0001-0.01) in terms of mass-to-volume ratio g/L: 1.
more preferably, in step (3), the ratio of the enantiomer to the mobile phase is 0.001: 1.
further explaining, in the step (4), the mass volume ratio of the ivabradine hydrochloride intermediate to the mobile phase is (0.1-5): 1; the ratio of the enantiomer to the mobile phase is (0.0001-0.01): 1.
more preferably, in the step (4), the ratio of the ivabradine hydrochloride intermediate to the mobile phase is 1: 1; the ratio of enantiomer to mobile phase was 0.001: 1.
further, in the step (5), in the determination conditions of the high performance liquid chromatography, the model of the CHIRALPAK IC chromatographic column is: the length is 150mm, the inner diameter is 4.6mm, the cellulose surface is covalently bonded with a silica gel filler, and the particle size of the filler is 5 μm.
An application of a method for detecting an enantiomer in an ivabradine hydrochloride intermediate in drug detection and analysis.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a method for controlling chiral molecules of ivabradine hydrochloride intermediate, namely a method for separating and detecting 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-ketone and 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-ketone, which adopts chromatographic conditions different from the prior art, and can see that a certain distance exists between the two from an HPLC (high performance liquid chromatography) chart, the method can effectively separate and detect the enantiomer in the ivabradine hydrochloride key intermediate, and has symmetrical peak type and no tailing phenomenon.
By adopting the detection method of impurity reference substance contrast, the aspects of separation degree, specificity, quantitative limit and detection limit, linearity, precision, repeatability, accuracy, solution stability, durability and the like are verified in detail, and each verification result meets the requirements of relevant regulations and guiding principles, so that the actual detection effect is better.
The method has strong practicability, in the actual detection process, the detection limit of 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepine-2-ketone can reach 0.0337 mu g/mL, and 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepine-2-ketone with the content of 3- [3- [ [ [ (7R) -3 higher than 0.01 percent can be detected, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one, and has the advantages of strong practicability and simple and rapid detection process.
The high performance liquid chromatography provided by the invention has effective values in the range contained in the determination conditions, can accurately detect the enantiomer in the key intermediate after taking any value in each parameter range, can effectively separate the enantiomer, is convenient to detect, and is suitable for popularization and use. Among them, the optimum measurement conditions are: the chromatographic column is CHIRALPAK IC chromatographic column, the column temperature is 35 ℃, and the detection wavelength is 287 nm; n-hexane of the mobile phase: dichloromethane: the volume ratio of diethylamine is 78: 22: 0.5, the flow rate of the mobile phase was 1 mL/min.
Drawings
FIG. 1 is an HPLC chromatogram of a test sample of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one of example 3 of the present invention.
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
Example 1-for quality control of key intermediate of ivabradine hydrochloride and for increasing the safety and effectiveness of ivabradine hydrochloride pharmaceutical products, the invention proposes a method for detecting the enantiomer of the ivabradine hydrochloride intermediate, which is 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one, the enantiomer of which is 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, the detection method of the 3-dihydro-2H-benzazepin-2-ketone comprises the following steps:
respectively preparing a sample solution to be detected: injecting a reference substance solution of enantiomer, a test substance solution of ivabradine hydrochloride intermediate and a mixed solution of the ivabradine hydrochloride intermediate and enantiomer into a high performance chromatograph for measurement, wherein the measurement conditions comprise that: the chromatographic column is CHIRALPAK IC chromatographic column, the column temperature is 25-40 ℃, and the detection wavelength is 280-290 nm; the mobile phase is a mixture of n-hexane, dichloromethane and diethylamine, wherein the n-hexane: dichloromethane: the volume ratio of the diethylamine is (76-80): 20-24): 0.5, and the flow rate of the mobile phase is 0.8-1.2 mL/min.
Example 2-a method for the detection of enantiomers of an intermediate in ivabradine hydrochloride comprising the following steps:
(1) preparing a sample solution to be detected:
s1, preparation of a test solution of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one:
weighing an ivabradine hydrochloride intermediate sample, mixing the ivabradine hydrochloride intermediate sample with a mobile phase to prepare a test solution for later use, wherein the mass-to-volume ratio (g/L) of the ivabradine hydrochloride intermediate sample is as follows: the ratio of the mobile phase is (0.1-5): 1.
s2, preparation of a control stock solution of 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one:
weighing enantiomer, adding the enantiomer into a volumetric flask, mixing the enantiomer with a mobile phase to prepare a reference stock solution for later use, wherein the enantiomer is obtained by the following steps in terms of mass-to-volume ratio (g/L): the ratio of the mobile phase is (0.001-0.1): 1.
s3, preparation of a control solution of 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one:
measuring a reference substance stock solution of the enantiomer, adding the reference substance stock solution into a volumetric flask, mixing the reference substance stock solution with a mobile phase to prepare a reference substance solution of the enantiomer, and measuring the enantiomer according to a mass-to-volume ratio (g/L): the ratio of the mobile phase is (0.0001-0.01): 1.
s4, preparation of a mixed solution:
weighing an ivabradine hydrochloride intermediate sample, weighing a reference substance stock solution of an enantiomer, adding into a volumetric flask, diluting with a mobile phase, and uniformly mixing to prepare a mixed solution for later use; according to the mass-to-volume ratio (g/L), the ivabradine hydrochloride intermediate sample: the ratio of the mobile phase is (0.1-5): 1; according to the mass-to-volume ratio (g/L), the enantiomer: the ratio of the mobile phase is (0.0001-0.01): 1.
(2) and (3) determination of high performance liquid chromatography:
respectively sucking the reference substance solution of equal amount of enantiomer, the test substance solution of the ivabradine hydrochloride intermediate and the mixed solution of the ivabradine hydrochloride intermediate and the enantiomer, and injecting the mixture into a high performance chromatograph for measurement.
Wherein, the measuring conditions of the high performance liquid chromatography comprise:
the checking method comprises the following steps: high performance liquid chromatography (0512) of the four general rules of Chinese pharmacopoeia 2015 edition;
a chromatographic column: CHIRALPAK IC (type: 150mm long, 4.6mm inner diameter, cellulose surface covalently bonded silica gel filler, filler particle size 5 μm);
a detector: a UV detector;
detection wavelength: 287 nm;
column temperature: 25-40 ℃, preferably 35 ℃;
flow rate: 0.8-1.2 mL/min, preferably 1 mL/min;
mobile phase: calculated by volume ratio, n-hexane: dichloromethane: diethylamine (76-80): (20-24): 0.5, the mobile phases used above are all the same.
In the actual detection process, for the high performance liquid chromatography method adopted by the invention, after the appropriate adjustment in each parameter range in the determination conditions, the 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepine-2-ketone can be accurately detected, and the 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepine-2-ketone and 3- [3- [ [ [ (7R) -3 can be detected, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one is effectively separated and detected.
The following 14 typical examples illustrate embodiments of the invention, and samples of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one from the same lot (191104 lots) were taken for testing.
Example 3
The content of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one of lot number 191104 was selected for detection, including the following steps:
s1, preparing a test solution of the ivabradine hydrochloride intermediate:
weighing 10mg of ivabradine hydrochloride intermediate sample, adding 10mL of mobile phase, uniformly mixing, and preparing 1mL of solution containing 1.0mg of ivabradine hydrochloride intermediate as a test solution for later use.
S2, preparation of enantiomeric control stock solutions:
weighing appropriate amount of enantiomer control, precisely weighing, dissolving with mobile phase, and quantitatively diluting to obtain solution containing about 10 μ g of enantiomer control stock solution per 1 mL.
S3, preparation of control solutions of enantiomers:
an appropriate amount of the enantiomer control stock solution was measured, dissolved in a mobile phase, and quantitatively diluted to give a solution containing about 1. mu.g of enantiomer per 1 mL.
S4, preparation of a mixed solution:
weighing 10mg of the ivabradine hydrochloride intermediate sample, adding the sample into a 10mL volumetric flask, precisely weighing 1mL of the enantiomer reference stock solution obtained in the step S2, adding the enantiomer reference stock solution into the volumetric flask, diluting the enantiomer reference stock solution with a mobile phase to a scale, mixing, shaking up to obtain a mixed solution for later use.
S5, measuring 20 mu L of enantiomer control solution, injecting into a high performance liquid chromatograph, adjusting detection sensitivity to enable the peak height of the main component chromatographic peak to be 20-25% of the full scale, precisely measuring 20 mu L of test solution, control solution and mixed solution, respectively injecting into the phase chromatograph, and recording the chromatogram. FIG. 1 is a high performance liquid chromatogram of a test sample of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one in this example. Wherein, the measuring conditions of the high performance liquid chromatography comprise:
a chromatographic column: CHIRALPAK IC (type: 150mm long, 4.6mm inner diameter, cellulose surface covalently bonded silica gel filler, filler particle size 5 μm);
a detector: a UV detector;
detection wavelength: 287 nm;
column temperature: 35 ℃;
flow rate: 1 mL/min;
mobile phase: calculated by volume ratio, n-hexane: dichloromethane: diethylamine 78: 22: 0.5.
through detection, the content of 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one in the ivabradine hydrochloride intermediate sample is 0.031%. From the graph, it can be seen that the separation degree of the intermediate and the enantiomer of the ivabradine hydrochloride is more than 1.5.
Example 4
A sample of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one from the same lot as in example 3 was taken and tested for its purity of 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one, which differed from example 3 only in that: the detector used in the chromatographic conditions was a DAD detector and the other detection parameters and conditions were in accordance with example 3. In this example, the measurement conditions of the high performance liquid chromatography were the same as in example 3;
the peak purity of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one was 0.999995 and the peak purity of 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one was 0.999997.
Example 5
Selecting a 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one reference substance solution for detection limit and quantitative limit detection, wherein the signal-to-noise ratio is 10: 1 is the limit of quantitation, the signal-to-noise ratio is 3:1 is the limit of detection, and other detection parameters and conditions are consistent with those of example 3. In this example, the measurement conditions of the high performance liquid chromatography were the same as in example 3;
the detection result shows that the detection limit of 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one is 0.0337 mu g/ml, and the quantification limit is 0.1012 mu g/ml.
Example 6
3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one reference stock solution is selected to carry out linear relation detection, the quantitative limit concentration of the reference solution and the limit concentrations of 140%, 120%, 100%, 80% and 60% are prepared, and other detection parameters and conditions are consistent with the detection conditions of the example 3. In this example, the measurement conditions of the high performance liquid chromatography were the same as in example 3;
through detection, 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one has a linear regression equation of 30771x +926.89 and a linear correlation coefficient of 0.99949.
Example 7
Preparing a 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one reference substance solution with a quantitative limit concentration and a 100% concentration, continuously injecting samples for 6 times, and keeping other detection parameters and conditions consistent with the detection conditions of the example 3. In this example, the measurement conditions of the high performance liquid chromatography were the same as in example 3;
the detection shows that the precision of the 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one is 1.32 percent and 0.87 percent respectively.
Example 8
3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one reference solution and test solution were left for 0, 4, 8, 12, 16, 24 hours, stability was examined, and other parameters and conditions of detection were in accordance with the conditions of detection in example 3. In this example, the measurement conditions of the high performance liquid chromatography were the same as in example 3;
through detection, the peak area RSD of the 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one reference substance is 0.38%, the peak area RSD of the 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one in the test solution is 0.79%, the peak area RSD of the 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] octa-1, the peak area RSD of the main peak of 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one is 0.33%.
Example 9
6 parts of a sample of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one, obtained in the same batch as in example 3, were selected and subjected to reproducibility examination, and other detection parameters and conditions were in accordance with the detection conditions in example 3. In this example, the measurement conditions of the high performance liquid chromatography were the same as in example 3;
it was found that the 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one sample had a 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one content RSD of 2.6%.
Example 10
6 parts of a sample of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one, which was prepared in the same batch as in example 3, was selected to prepare 6 parts of a mixed solution, and other detection parameters and conditions were in accordance with the detection conditions in example 3. In this example, the measurement conditions of the high performance liquid chromatography were the same as in example 3;
as a result of detection, the recovery rate of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one in a sample of 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one was 95%, and the RSD was 2.3%.
Example 11
A sample of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one from the same lot as in example 3 was taken and tested for its 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one content, which differed from example 3 only in that: the flow rate in the measurement conditions of the high performance liquid chromatography was 0.8mL/min, and other measurement parameters and conditions were the same as those in example 3. Wherein, the measuring conditions of the high performance liquid chromatography comprise:
the detection shows that the content of the 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one is 0.029%.
Example 12
A sample of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one from the same lot as in example 3 was taken and tested for its 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one content, which differed from example 3 only in that: the flow rate in the measurement conditions of the high performance liquid chromatography was 1.2mL/min, and other measurement parameters and conditions were the same as those in example 3.
The detection shows that the content of the 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one is 0.033%.
Example 13
A sample of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one from the same lot as in example 3 was taken and tested for its 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one content, which differed from example 3 only in that: the column temperature in the measurement conditions of the high performance liquid chromatography was 30 ℃ and other measurement parameters and conditions were the same as those in example 3.
The detection shows that the content of the 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one is 0.029%.
Example 14
A sample of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one from the same lot as in example 3 was taken and tested for its 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one content, which differed from example 3 only in that: the column temperature in the measurement conditions of the high performance liquid chromatography was 40 ℃ and other measurement parameters and conditions were the same as those in example 3.
The detection shows that the content of the 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one is 0.034%.
Example 15
A sample of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one from the same lot as in example 3 was taken and tested for its 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one content, which differed from example 3 only in that: the mobile phase proportion in the determination conditions of the high performance liquid chromatography is as follows: calculated by volume ratio, n-hexane: dichloromethane: diethylamine 76: 24: 0.5, the other detection parameters and conditions were consistent with those of example 3.
The detection shows that the content of the 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one is 0.033%.
Example 16
A sample of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one from the same lot as in example 3 was taken and tested for its 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one content, which differed from example 3 only in that: the mobile phase proportion in the determination conditions of the high performance liquid chromatography is as follows: calculated by volume ratio, n-hexane: dichloromethane: diethylamine 80: 20: 0.5, the other detection parameters and conditions were consistent with those of example 3.
The detection shows that the content of the 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one is 0.031%.
From the results of examples 3 to 16, it can be seen that in the detection of 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one in the key intermediate of ivabradine hydrochloride, parameters for chromatographic conditions, such as mobile phase ratio, flow rate, column temperature, etc., can be adjusted within various parameter ranges to compensate for the enantiomer 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one is effectively separated, and the detection result is effective and accurate.
Comparative example 1
A sample of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one from the same lot as in example 3 was taken and tested for its 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one content, which differed from example 3 only in that:
the mobile phase in the determination conditions of the high performance liquid chromatography is n-hexane: anhydrous ethanol: triethylamine 85:15:0.5, and other detection parameters and conditions were consistent with those of example 3.
As a result of detection, the content of 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one cannot be detected.
Comparative example 2
A sample of 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one from the same lot as in example 3 was taken and tested for its 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] oct-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one content, which differed from example 3 only in that: the chromatographic column used in the determination conditions of the high performance liquid chromatography was an OD-RH (chiralcel, 150 mm. times.4.6 mm, 5 μm) chiral column, and other detection parameters and conditions were consistent with those of example 3.
As a result of detection, the content of 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one cannot be detected.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A method for detecting enantiomer in ivabradine hydrochloride intermediate, wherein the ivabradine hydrochloride intermediate is 3- [3- [ [ [ (7S) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one, and the enantiomer thereof is 3- [3- [ [ [ (7R) -3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl ] methyl ] methylamino ] propyl ] -1, 3-dihydro-2H-benzazepin-2-one, and is characterized in that:
respectively preparing a sample solution to be detected: injecting a reference substance solution of enantiomer, a test substance solution of ivabradine hydrochloride intermediate and a mixed solution of the ivabradine hydrochloride intermediate and enantiomer into a high performance chromatograph for measurement, wherein the measurement conditions comprise that: the chromatographic column is CHIRALPAK IC chromatographic column, the column temperature is 25-40 ℃, and the detection wavelength is 280-290 nm; the mobile phase is a mixture of n-hexane, dichloromethane and diethylamine, the n-hexane: dichloromethane: the volume ratio of the diethylamine is (76-80): 20-24): 0.5, and the flow rate of the mobile phase is 0.8-1.2 mL/min.
2. The method for detecting enantiomers of an ivabradine hydrochloride intermediate according to claim 1, characterized in that: the n-hexane: dichloromethane: the volume ratio of diethylamine is 78: 22: 0.5.
3. the method for detecting enantiomers of an ivabradine hydrochloride intermediate according to claim 1, characterized in that: the temperature of the chromatographic column is 35 ℃, and the detection wavelength is 287 nm.
4. The method for detecting enantiomers of an ivabradine hydrochloride intermediate according to claim 1, characterized in that: the flow rate of the mobile phase was 1 mL/min.
5. The method for detecting enantiomers of an ivabradine hydrochloride intermediate according to claim 1, characterized in that: the method for preparing the sample solution to be detected comprises the following steps:
(1) test solution for preparing ivabradine hydrochloride intermediate
Weighing an ivabradine hydrochloride intermediate sample, mixing the ivabradine hydrochloride intermediate sample with a mobile phase, and preparing a test solution of the ivabradine hydrochloride intermediate for later use;
(2) enantiomeric control stock solutions
Weighing an enantiomer sample, mixing the enantiomer sample with a mobile phase, and preparing a reference substance stock solution of the enantiomer for later use;
(3) control solutions for enantiomeric preparations
Weighing the prepared enantiomer reference substance stock solution, mixing with a mobile phase, and preparing into enantiomer reference substance solution for later use;
(4) preparing a mixed solution of ivabradine hydrochloride intermediate and enantiomer
Weighing an ivabradine hydrochloride intermediate sample, weighing a reference substance stock solution of an enantiomer, adding into a volumetric flask, diluting by adopting a mobile phase, uniformly mixing, and preparing into a mixed solution for later use;
(5) respectively sucking equivalent enantiomer reference substance solution, ivabradine hydrochloride intermediate sample solution and ivabradine hydrochloride intermediate and enantiomer mixed solution, and injecting into high performance liquid chromatograph for determination.
6. The method for detecting enantiomers of an ivabradine hydrochloride intermediate according to claim 5, wherein the method comprises the following steps: in the step (1), g/L is calculated according to the mass-volume ratio, and the ratio of the ivabradine hydrochloride intermediate to the mobile phase is (0.1-5): 1.
7. the method for detecting enantiomers of an ivabradine hydrochloride intermediate according to claim 5, wherein the method comprises the following steps: in the step (2), g/L is calculated according to the mass-to-volume ratio, and the ratio of the enantiomer to the mobile phase is (0.001-0.1): 1; in the step (3), the mass-to-volume ratio of g/L is (0.0001-0.01): 1.
8. the method for detecting enantiomers of an ivabradine hydrochloride intermediate according to claim 5, wherein the method comprises the following steps: in the step (4), g/L is calculated according to the mass-volume ratio, and the ratio of the ivabradine hydrochloride intermediate to the mobile phase is (0.1-5): 1; the ratio of the enantiomer to the mobile phase is (0.0001-0.01): 1.
9. the method for detecting enantiomers of an ivabradine hydrochloride intermediate according to claim 5, wherein the method comprises the following steps: in the step (5), in the measuring conditions of the high performance liquid chromatography, the specification of the CHIRALPAK IC chromatographic column is as follows: the length is 150mm, the inner diameter is 4.6mm, the cellulose surface is covalently bonded with a silica gel filler, and the particle size of the filler is 5 μm.
10. Use of the method for detecting enantiomers of an ivabradine hydrochloride intermediate according to any one of claims 1 to 9 in drug detection analysis.
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