CN114324715A - Method for detecting related substances in 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone - Google Patents
Method for detecting related substances in 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone Download PDFInfo
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Abstract
The invention provides a method for detecting related substances in 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone. According to the test method provided by the invention, a 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone sample is mixed with a mobile phase A to form a test solution; mixing the impurity A and the impurity B to be detected with the mobile phase A to obtain a reference substance solution; mixing the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone sample, the reference stock solution and the mobile phase A to obtain a mixed solution; the 3 solutions are injected into a high performance chromatograph for HPLC test, and chromatographic conditions are controlled, so that the impurity A and the impurity B in the key material of the ivabradine hydrochloride can be effectively separated and detected, and the sensitivity is high and the reproducibility is good.
Description
Technical Field
The invention relates to the technical field of chemical analysis, in particular to a method for detecting related substances in 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone.
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 P4503a4, 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 hydrogenated to prepare the ivabradine hydrochloride.
As a starting material for synthesizing ivabradine hydrochloride, the production preparation and quality control of 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone have important significance.
The synthesis route of 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone reported in the prior art is as follows: the intermediate N- (2, 2-dimethoxyethyl) -3, 4-dimethoxyphenylacetamide (impurity B) is formed by reacting the reactant 3, 4-dimethoxyphenylacetic acid (impurity A) with aminoacetaldehyde dimethyl acetal, and then the material is formed by ring closure.
The chemical structural formulas of the impurity A and the impurity B are respectively as follows:
the impurity A and the impurity B are derived from a synthesis process of 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepine-2-ketone, and are key materials and intermediates in the synthesis of 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepine-2-ketone. In order to monitor the impurity transfer and removal conditions from the source and ensure the safety and effectiveness of clinical medication, the effective quality control of the impurity A and the impurity B in the ivabradine hydrochloride key material 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one is necessary.
In the prior art, only a synthetic route of 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepine-2-ketone is introduced, for example, patents US4584293A, CN101607939A and CN102276530A, and no method for analyzing and detecting related substances of 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepine-2-ketone is provided at present, so that a quantitative detection method of related substances of ivabradine hydrochloride key materials, which is simple and convenient to operate, high in sensitivity and good in reproducibility, is urgently needed to be established for continuously improving the safety and effectiveness of medicines.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for detecting related substances in 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one. The detection method provided by the invention can effectively separate impurities in 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepine-2-ketone and quantitatively detect the impurity A, and has high sensitivity and good reproducibility.
The invention provides a method for detecting related substances in 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone, which comprises the following steps:
a) mixing a 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample with a mobile phase A to obtain a test sample solution;
b) mixing the impurity A, the impurity B and the mobile phase A to obtain a reference substance solution;
c) mixing a 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample, a reference stock solution and a mobile phase A to obtain a mixed solution;
the reference product stock solution is a mixed solution of impurities A, B and a mobile phase A;
d) respectively injecting the test solution, the reference solution and the mixed solution into a high performance chromatograph for HPLC detection to respectively obtain HPLC chromatograms of the 3 solutions, and then obtaining the impurity content in the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample according to an external standard method;
the chromatographic conditions for HPLC detection are as follows: adopting a C18 chromatographic column, wherein the column temperature is 25-35 ℃, and the flow rate of a mobile phase is 0.9-1.1 mL/min; taking a mixed solution of potassium dihydrogen phosphate, triethylamine and acetonitrile as a mobile phase A, and taking the acetonitrile as a mobile phase B;
the impurity A is 3, 4-dimethoxy phenylacetic acid, and the impurity B is N- (2, 2-dimethoxy ethyl) -3, 4-dimethoxy phenyl acetamide;
the steps a), b) and c) are not limited in order.
Preferably, in the HPLC assay, the gradient elution procedure is: reducing the content of the mobile phase A from 100% to 75% in 0-40 min; the time is 40-50 min, and the mobile phase A is 75%; the mobile phase A rises from 75% to 100% in 50-52 min; 52-60 min, and 100% of mobile phase A.
Preferably, the preparation process of the mobile phase A comprises the following steps:
s1, mixing the potassium dihydrogen phosphate solution with triethylamine to obtain a phosphate solution;
s2, mixing the phosphate solution with acetonitrile to obtain a mobile phase A;
the potassium dihydrogen phosphate solution is an aqueous solution of potassium dihydrogen phosphate; the concentration of the potassium dihydrogen phosphate solution is 0.02 mol/L;
the triethylamine accounts for 0.1 percent of the phosphate solution by mass;
the volume ratio of the phosphate solution to the acetonitrile is (80-90) to (20-10).
Preferably, in step S2, the phosphate solution is adjusted to pH 4.5 with phosphoric acid and then mixed with acetonitrile.
Preferably, in the step a), the dosage ratio of the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample to the mobile phase A is (0.02-1) g: 1L;
in the step B), the dosage ratio of the total mass of the impurities A and B to the mobile phase A is (0.0001-0.01) g: 1L; wherein the mass ratio of the impurity A to the impurity B is 1: 1;
in the step c), the ratio of the total mass of the impurities A and B to the amount of the mobile phase in the reference product stock solution is (0.001-0.1) g: 1L; wherein the mass ratio of the impurity A to the impurity B is 1: 1.
Preferably, the C18 chromatographic column is a YMC-Triart C18 chromatographic column.
Preferably, the size of the C18 chromatographic column is: the length is 250mm, the inner diameter is 4.6mm, and the grain size of the filler is 5 μm;
the detection wavelength of the HPLC detection is 230 nm.
Preferably, in the step a), the dosage ratio of the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample to the mobile phase A is 0.2 g: 1L;
in the step B), the ratio of the total mass of the impurities A and B to the dosage of the mobile phase A is 0.001 g: 1L;
in the step c), the ratio of the total mass of the impurities A and B to the amount of the mobile phase in the reference substance stock solution is 0.01g to 1L.
Preferably, the column temperature is 30 ℃;
the flow rate of the mobile phase was 1 mL/min.
Preferably, the volume ratio of the phosphate solution to the acetonitrile is 85: 15.
According to the test method provided by the invention, a 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone sample is mixed with a mobile phase A to form a test solution; mixing the impurity A and the impurity B to be detected with the mobile phase A to obtain a reference substance solution; mixing the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone sample, the reference stock solution and the mobile phase A to obtain a mixed solution; the 3 solutions are injected into a high performance chromatograph for HPLC test, the chromatographic conditions are controlled, the result can effectively separate the impurity A and the impurity B in the key material of the ivabradine hydrochloride, the impurity A is quantitatively detected, and the sensitivity is high and the reproducibility is good.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a high performance liquid chromatogram of a control solution of impurity A and impurity B in example 1;
FIG. 2 is a high performance liquid chromatogram of a test solution of 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one of example 1;
FIG. 3 is a high performance liquid chromatogram of the mixed solution of example 1.
Detailed Description
The invention provides a method for detecting related substances in 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone, which comprises the following steps:
a) mixing a 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample with a mobile phase A to obtain a test sample solution;
b) mixing the impurity A, the impurity B and the mobile phase A to obtain a reference substance solution;
c) mixing a 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample, a reference stock solution and a mobile phase A to obtain a mixed solution;
the reference product stock solution is a mixed solution of impurities A, B and a mobile phase A;
d) respectively injecting the test solution, the reference solution and the mixed solution into a high performance chromatograph for HPLC detection to respectively obtain HPLC chromatograms of the 3 solutions, and then obtaining the impurity content in the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample according to an external standard method;
the chromatographic conditions for HPLC detection are as follows: adopting a C18 chromatographic column, wherein the column temperature is 25-35 ℃, and the flow rate of a mobile phase is 0.9-1.1 mL/min; taking a mixed solution of potassium dihydrogen phosphate, triethylamine and acetonitrile as a mobile phase A, and taking the acetonitrile as a mobile phase B;
the impurity A is 3, 4-dimethoxy phenylacetic acid, and the impurity B is N- (2, 2-dimethoxy ethyl) -3, 4-dimethoxy phenyl acetamide;
the steps a), b) and c) are not limited in order.
According to the test method provided by the invention, a 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone sample is mixed with a mobile phase A to form a test solution; mixing the impurity A and the impurity B to be detected with the mobile phase A to obtain a reference substance solution; mixing the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone sample, the reference stock solution and the mobile phase A to obtain a mixed solution; the 3 solutions are injected into a high performance chromatograph for HPLC test (namely high performance liquid chromatography detection), the chromatographic conditions are controlled, the result can effectively separate the impurity A and the impurity B in the key material of the ivabradine hydrochloride, and the impurity A is quantitatively detected, and the sensitivity is high and the reproducibility is good.
With respect to step a): and mixing the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample with the mobile phase A to obtain a test solution.
In the invention, the mobile phase A is the mobile phase A in the subsequent HPLC detection. In the invention, the dosage ratio of the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample to the mobile phase A is preferably (0.02-1) g: 1L, and more preferably 0.2 g: 1L. And mixing the two substances uniformly to obtain a test solution.
With respect to step b): mixing the impurity A, the impurity B and the mobile phase A to obtain a reference substance solution.
In the invention, the impurity A and the impurity B are two main impurities to be detected in a 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepine-2-ketone sample, wherein the impurity A is 3, 4-dimethoxy phenylacetic acid, and the impurity B is N- (2, 2-dimethoxy ethyl) -3, 4-dimethoxy phenylacetamide.
The mobile phase A is the mobile phase A in the subsequent HPLC detection.
In the invention, the dosage ratio of the total mass of the impurity A and the impurity B to the mobile phase A is preferably (0.0001-0.01) g: 1L, and more preferably 0.001 g: 1L. Wherein, the mass ratio of the impurity A to the impurity B is preferably 1: 1. Mixing the above three materials uniformly to obtain reference solution.
With respect to step c): and mixing the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample, the reference stock solution and the mobile phase A to obtain a mixed solution.
In the invention, the reference stock solution is also a mixed solution of impurity A, impurity B and mobile phase A; the dosage ratio of the total mass of the impurities A and B to the mobile phase A is preferably (0.001-0.1) g: 1L, and more preferably 0.01 g: 1L. Wherein, the mass ratio of the impurity A to the impurity B is preferably 1: 1.
In the invention, the dosage ratio of the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample to the mobile phase A is preferably (0.02-1) g: 1L, and more preferably 0.2 g: 1L. The dosage ratio of the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample to the reference substance storage solution is preferably (0.02-1) g: 1L, and more preferably 2 g: 1L. And uniformly mixing the three substances to obtain a mixed solution.
The present invention does not limit the order of step a), step b) and step c).
With respect to step d): and respectively injecting the test solution, the reference solution and the mixed solution into a high performance chromatograph for HPLC detection to respectively obtain HPLC chromatograms of the 3 solutions, and then obtaining the impurity content in the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample according to an external standard method.
In the present invention, preferably, the reference solution is injected into the HPLC, the detection sensitivity is adjusted such that the height of the main component chromatographic peak is 20% to 25% of the full scale range, and then the sample solution, the reference solution and the mixed solution are measured and injected into the HPLC for HPLC detection. The amount of the control solution injected first is preferably 20 μ L, and after adjustment, the sample injection amount is preferably 20 μ L when the sample solution, the control solution and the mixed solution are injected.
In the invention, the chromatographic conditions of HPLC detection are as follows: adopting a C18 chromatographic column, wherein the column temperature is 25-35 ℃, and the flow rate of a mobile phase is 0.9-1.1 mL/min; the method comprises the following steps of taking a mixed solution of potassium dihydrogen phosphate, triethylamine (namely TEA) and acetonitrile as a mobile phase A, and taking the acetonitrile as a mobile phase B.
Wherein:
the C18 chromatographic column is preferably a YMC-Triart C18 chromatographic column. The size of the C18 chromatography column is preferably: the length is 250mm, the inner diameter is 4.6mm, and the grain size of the filler is 5 μm.
The column temperature is 25-35 ℃, and preferably 30 ℃. In some embodiments of the invention, the column temperature is 25 ℃, 30 ℃ or 35 ℃.
The detection wavelength of the HPLC detection is preferably 230 nm.
The detector for the HPLC detection is preferably: and a UV detector.
The speed of the mobile phase is 0.9-1.1 mL/min, preferably 1 mL/min. In some embodiments of the invention, the velocity of the mobile phase is 0.9mL/min, 1mL/min, or 1.1 mL/min.
The mobile phase A is a mixed solution of potassium dihydrogen phosphate, triethylamine and acetonitrile. The preparation process preferably comprises the following steps: s1, mixing the potassium dihydrogen phosphate solution with triethylamine to obtain a phosphate solution; and S2, mixing the phosphate solution with acetonitrile to obtain a mobile phase A. Wherein the potassium dihydrogen phosphate solution is an aqueous solution of potassium dihydrogen phosphate; the concentration of the potassium dihydrogen phosphate solution is preferably 0.02 mol/L. The content of the triethylamine in the phosphate solution is preferably 0.1% by mass. The volume ratio of the phosphate solution to the acetonitrile is preferably (80-90) to (20-10), and more preferably 85 to 15.
In step S2, the phosphate solution is preferably adjusted to pH 4.5 with phosphoric acid and then mixed with acetonitrile to form mobile phase a.
Mobile phase B was acetonitrile.
In the present invention, the amount of the sample to be subjected to HPLC detection is preferably 20. mu.L.
In the invention, in the HPLC detection, the gradient elution procedure is as follows: reducing the content of the mobile phase A from 100% to 75% in 0-40 min; the time is 40-50 min, and the mobile phase A is 75%; the mobile phase A rises from 75% to 100% in 50-52 min; 52-60 min, and 100% of mobile phase A.
And respectively obtaining HPLC (high performance liquid chromatography) maps of the test solution, the reference solution and the mixed solution through the HPLC detection, and obtaining the content of the impurity A in the test sample through an impurity reference external standard method. In the invention, the impurity content in the test sample is obtained by an external standard method, and is specifically obtained by the following calculation formula 1:
in formula 1:
Wto pairWeighing the reference substance in mg;
a is the purity of the reference substance;
Ato pairPeak area of the control solution;
Asample (A)Is the peak area of the test solution;
Dsample (A)Is the dilution multiple of the test sample;
Wsample (A)Weighing sample amount of the sample in mg;
Dto pairIs the dilution factor of the control.
The above-mentioned weighing means the weighing of the solid sample into the volumetric flask, for example, the solid of the impurity reference substance is weighed into the volumetric flask (mass W)To pair) Then diluting the solution to a scale by using a mobile phase to form a reference substance initial solution; then putting the reference substance primary solution into another volumetric flask, and diluting with mobile phaseAnd (4) obtaining a reference substance solution when the scale is reached. Wherein the weighing in the first step is the weighing amount of the reference substance; the reference solution entering the HPLC test of step d) is the second diluted liquid.
The dilution factor is a factor by which a solid sample is dissolved and diluted with a solution in a volumetric flask, and is 10 when 1g of the solid sample is diluted to a scale of 10mL in a 10mL volumetric flask. Further, as in example 1, 2mg of the control was weighed out into a 200mL volumetric flask, the mobile phase was added to the scale (at this time, the dilution was 100 times, and the volume of the control was small enough to be negligible compared with the volume of the solution), 5mL of the solution was weighed out from the volumetric flask, the solution was placed into a 50mL volumetric flask, the mobile phase was added to the scale and the solution was diluted to the scale (at this time, the solution was diluted by 10 times, and the total dilution ratio was 200 × 10 ═ 2000), and the final dilution ratio was 2000.
Compared with the prior art, the test method provided by the invention has the following beneficial effects:
(1) the invention provides a method for controlling specific impurities of an ivabradine hydrochloride key material, in particular to a method for separating and detecting 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepine-2-ketone, impurities A and impurities B.
(2) The detection method adopting the impurity reference substance has the advantages that 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, all verification results meet the requirements of relevant regulations and guiding principles, and the actual detection effect is good.
(3) The invention has strong practicability, the detection limits of the impurity A and the impurity B are 0.0998ng/ml and 34.75ng/ml respectively in the detection process, the sensitivity requirement is met, and the practicability is strong. The detection process is simple and quick.
(4) In the high performance liquid chromatography according to the present invention, the effective values are within the range included in the measurement conditions, that is, the following values: after any value is taken within each parameter range, the impurities A and B can be effectively separated. In the actual detection process, the parameter adjustment of the detection personnel is facilitated, the influence of human errors on the detection result is avoided, and the method is suitable for popularization and application.
For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The following 14 examples were tested on samples of the same batch of 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one (batch 115B3-00-190801 sp).
Example 1
S1, preparing a test solution of 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one:
weighing 10mg of 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample, adding 50ml of mobile phase A, uniformly mixing, and preparing a solution containing 0.2mg of 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one in each 1ml of the solution as a test solution for later use.
In the above process, the sample weight W of the sampleSample (A)10mg, dilution factor D of the test sampleSample (A)Is 50.
S2, preparing reference stock solution of impurity A and impurity B:
weighing appropriate amount of impurity A and impurity B reference substances, precisely weighing, dissolving mobile phase A, and quantitatively diluting to obtain solution containing 10 μ g impurity A and 10 μ g impurity B per 1ml, and using as reference substance stock solution of impurity A and impurity B.
S3, preparing reference solutions of impurity A and impurity B:
weighing reference substances of impurities A and B, storing appropriate amount, dissolving mobile phase A, and quantitatively diluting to obtain solution containing about 1 μ g of impurities A and 1 μ g of impurities B in each 1ml as reference substance solution of impurities A and B.
In the above process, the reference substance is weighed to obtain the sample weight WTo pairAt 2mg, dilution factor D of controlTo pair2000 (2 mg of the solid sample is weighed firstly and added into a 200mL volumetric flask to be diluted to a scale mark, then 5mL of the solution is weighed into a 50mL volumetric flask to be diluted to a scale mark, the dilution multiple is 200 multiplied by 10 to 2000), and the purity of the impurity A reference substance a is 99.98%.
S4, preparation of a mixed solution:
weighing 10mg of 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one, adding into a 50mL volumetric flask, precisely weighing 5mL of the reference stock solution obtained in the step S2, adding into the volumetric flask, diluting with a mobile phase A to 50mL, mixing, and shaking up to obtain a mixed solution for later use.
S5, injecting 20 mul of the reference solution into a high performance liquid chromatograph, adjusting the detection sensitivity to enable the peak height of the main component chromatographic peak to be 20% -25% of the full scale, precisely measuring 20 mul of each of the test solution, the reference solution and the mixed solution, injecting into the phase chromatograph, and recording the chromatogram. FIG. 1, FIG. 2 and FIG. 3 are HPLC chromatograms of a reference solution of impurity A and impurity B, a test sample of 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one and a mixed solution in this example, respectively. Wherein the determination conditions of the high performance liquid chromatography comprise:
a chromatographic column: YMC-Triart C18 (length 250mm, inner diameter 4.6mm, filler particle diameter 5 μm);
a detector: a UV detector;
detection wavelength: 230 nm;
column temperature: 30 ℃;
flow rate: 1 ml/min;
mobile phase A: mixing a potassium dihydrogen phosphate solution (0.02mol/L) with triethylamine to obtain a phosphate solution (the mass percentage of the triethylamine in the phosphate solution is 0.1%); after adjusting the pH to 4.5 with phosphoric acid, the phosphate solution was mixed with acetonitrile in a volume ratio of 85: 15 to obtain mobile phase a.
Mobile phase B: and (3) acetonitrile.
See table 1 for elution procedure:
table 1 elution procedure for HPLC detection in example 1
| T(min) | A | B％ | |
| 0 | 100 | 0 | |
| 40 | 75 | 25 | |
| 50 | 75 | 25 | |
| 52 | 100 | 0 | |
| 60 | 100 | 0 |
And according to the HPLC detection result, obtaining the content of the impurities in the test sample by an impurity reference product external standard method. The formula is as follows:
in formula 1:
Wto pairWeighing the reference substance in mg;
a is the purity of the reference substance;
Ato pairPeak area of the control solution;
Asample (A)Is the peak area of the test solution;
Dsample (A)Is the dilution multiple of the test sample;
Wsample (A)Weighing sample amount of the sample in mg;
Dto pairIs the dilution factor of the control.
The detection shows that the content of the impurity A in the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone sample is 0.024%, and the impurity B is not detected. The graph shows that the separation degree of the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepine-2-ketone and the impurity A and the impurity B is more than 1.5, and the method can effectively separate the impurity A and the impurity B from a sample.
Example 2 reliability of the method of the invention
The procedure of example 1 was followed except that the detector used in the HPLC assay was different and the other assay conditions were the same as in example 1. In this example, the measurement conditions of the high performance liquid chromatography include:
a chromatographic column: YMC-Triart C18 (length 250mm, inner diameter 4.6mm, filler particle diameter 5 μm);
a detector: a DAD detector;
detection wavelength: 230 nm;
column temperature: 30 ℃;
flow rate: 1 ml/min;
mobile phase: the same as in example 1.
The detection result shows that the peak purity of the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone is 0.999999, the peak purity of the impurity A is 0.999999, and the peak purity of the impurity B is 1.000000. The peak purity is determined by judging whether one peak contains other compounds or not based on different light absorption of different compounds, and if the peak is a single compound, the peak purity is more than 0.999; according to the detection results, the purity of the detected main component and each impurity peak reaches more than 0.999, and the peaks are proved to be single and contain no other compounds, so that the detection method provided by the invention is proved to have high reliability, and the method can effectively separate the impurity A from the impurity B.
Example 3 quantitation and detection limits of the methods of the invention
The control solution in example 1 is used for detection of detection limit and quantification limit, the signal-to-noise ratio is 10: 1 as the quantification limit, the signal-to-noise ratio is 3: 1 as the detection limit, other detection conditions are the same as those in example 1, and the determination conditions of the high performance liquid chromatography comprise:
a chromatographic column: YMC-Triart C18 (length 250mm, inner diameter 4.6mm, filler particle diameter 5 μm);
a detector: a UV detector;
detection wavelength: 230 nm;
column temperature: 30 ℃;
flow rate: 1 ml/min;
mobile phase: the same as in example 1.
The detection result shows that the limit of quantitation of the impurity A is 29.96ng/ml, the limit of detection is 9.98ng/ml, the limit of quantitation of the impurity B is 62.99ng/ml, and the limit of detection is 34.75 ng/ml. The detection result proves that the detection method provided by the invention has better sensitivity.
Example 4 linearity of the process of the invention
A reference stock solution containing the impurity A and the impurity B is selected to be subjected to linear relation detection, and the quantitative limit concentrations and the limit concentrations of 200%, 150%, 100%, 80% and 50% of the reference solution are prepared, wherein the detection conditions of the embodiment are consistent with those of the embodiment 1. In this example, the measurement conditions of the high performance liquid chromatography include:
a chromatographic column: YMC-Triart C18 (length 250mm, inner diameter 4.6mm, filler particle diameter 5 μm);
a detector: a UV detector;
detection wavelength: 230 nm;
column temperature: 30 ℃;
flow rate: 1 ml/min;
mobile phase: the same as in example 1.
The detection result shows that the linear regression equation y of the impurity A is 44533x-82.926, the linear correlation coefficient is 0.99994, the linear regression equation y of the impurity B is 33141x +372.48, and the linear correlation coefficient is 0.99987. The detection result proves that the detection method provided by the invention meets the requirement of a verification scheme (the regression coefficient should be not less than 0.999).
EXAMPLE 5 precision of the method of the invention
Preparing reference solutions of impurity A and impurity B with quantitative limit concentration and 100% concentration, and continuously injecting samples for 6 times, wherein the detection conditions of the embodiment are consistent with those of embodiment 1. The determination conditions of the high performance liquid chromatography include:
a chromatographic column: YMC-Triart C18 (length 250mm, inner diameter 4.6mm, filler particle diameter 5 μm);
a detector: a UV detector;
detection wavelength: 230 nm;
column temperature: 30 ℃;
flow rate: 1 ml/min;
mobile phase: the same as in example 1.
The detection results show that the RSD values of the impurity A with the quantitative limit concentration and the 100% concentration precision are respectively 3.2% and 0.3%, and the RSD values of the impurity B with the quantitative limit concentration and the 100% concentration precision are respectively 3.2% and 0.2%. The detection result proves that the detection method provided by the invention has higher precision.
EXAMPLE 6 durability of the method of the invention
Impurity control solution and sample solution are prepared according to example 1, and are placed for 0h, 6h, 12h, 18h, 24h, 37h, 50h, 63h and 72h, stability is checked, and basis is provided for placing time of the impurity control solution and the sample solution during detection. In this example, the detection conditions were the same as those in example 1, and the measurement conditions of high performance liquid chromatography included:
a chromatographic column: YMC-Triart C18 (length 250mm, inner diameter 4.6mm, filler particle diameter 5 μm);
a detector: a UV detector;
detection wavelength: 230 nm;
column temperature: 30 ℃;
flow rate: 1 ml/min;
mobile phase: the same as in example 1.
The detection result shows that the peak areas RSD of the reference substances of the impurity A and the impurity B are respectively 2.0 percent and 0.6 percent, and the impurity A and the impurity B in the test solution do not increase. The detection results prove that the reference solution and the test solution have good stability and are stable within 72 hours.
Example 7 reproducibility of the method of the invention
6 parts of 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone sample with the same batch number as that of the example 1 is selected for repeatability investigation, the detection conditions are consistent with those of the example 1, and the determination conditions of the high performance liquid chromatography comprise:
a chromatographic column: YMC-Triart C18 (length 250mm, inner diameter 4.6mm, filler particle diameter 5 μm);
a detector: a UV detector;
detection wavelength: 230 nm;
column temperature: 30 ℃;
flow rate: 1 ml/min;
mobile phase: the same as in example 1.
The detection result shows that the average content of the impurity A in the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone sample is 0.024%, the RSD is 6.2%, and the impurity B is not detected. The detection result proves that the detection method provided by the invention has better precision.
Example 8 sample recovery according to the method of the invention
6 parts of a 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample which is prepared in the same batch as in example 1 is selected to prepare 6 parts of a mixed solution, the detection conditions are consistent with those in example 1, and the determination conditions of the high performance liquid chromatography comprise:
a chromatographic column: YMC-Triart C18 (length 250mm, inner diameter 4.6mm, filler particle diameter 5 μm);
a detector: a UV detector;
detection wavelength: 230 nm;
column temperature: 30 ℃;
flow rate: 1 ml/min;
mobile phase: the same as in example 1.
The detection result shows that the recovery rate of the impurity A in the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone sample is 100.8 percent, the RSD is 0.4 percent, the recovery rate of the impurity B is 100.6 percent, and the RSD is 0.6 percent. The detection result proves that the detection method provided by the invention has better accuracy.
Example 9
The procedure of example 1 was followed except that the flow rate parameters were varied in the chromatographic conditions and the other detection conditions were identical to those of example 1. The method comprises the following specific steps:
a chromatographic column: YMC-Triart C18 (length 250mm, inner diameter 4.6mm, filler particle diameter 5 μm);
a detector: a UV detector;
detection wavelength: 230 nm;
column temperature: 30 ℃;
flow rate: 0.9 ml/min;
mobile phase: the same as in example 1.
The detection result shows that the contents of the impurity A and the impurity B are not detected.
From the detection results of the embodiments 1 to 8, the detection method provided by the present invention can effectively separate the impurity a and the impurity B from the sample, and quantitatively detect the impurity a, and the detection method of the present invention has good reliability, sensitivity, linearity, precision, durability, repeatability and accuracy. From the test results of example 9, it is known that impurities a and B can be effectively separated, but the content of impurity a cannot be obtained, when the chromatographic condition parameters are changed, only under the preferred chromatographic condition parameters of the present invention, the content of impurity a can be obtained as well as impurities a and B.
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
- The method for detecting related substances in 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-ketone is characterized by comprising the following steps of:a) mixing a 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample with a mobile phase A to obtain a test sample solution;b) mixing the impurity A, the impurity B and the mobile phase A to obtain a reference substance solution;c) mixing a 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample, a reference stock solution and a mobile phase A to obtain a mixed solution;the reference product stock solution is a mixed solution of impurities A, B and a mobile phase A;d) respectively injecting the test solution, the reference solution and the mixed solution into a high performance chromatograph for HPLC detection to respectively obtain HPLC chromatograms of the 3 solutions, and then obtaining the impurity content in the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample according to an external standard method;the chromatographic conditions for HPLC detection are as follows: adopting a C18 chromatographic column, wherein the column temperature is 25-35 ℃, and the flow rate of a mobile phase is 0.9-1.1 mL/min; taking a mixed solution of potassium dihydrogen phosphate, triethylamine and acetonitrile as a mobile phase A, and taking the acetonitrile as a mobile phase B;the impurity A is 3, 4-dimethoxy phenylacetic acid, and the impurity B is N- (2, 2-dimethoxy ethyl) -3, 4-dimethoxy phenyl acetamide;the steps a), b) and c) are not limited in order.
- 2. The detection method according to claim 1, wherein in the HPLC detection, the gradient elution procedure is as follows: reducing the content of the mobile phase A from 100% to 75% in 0-40 min; the time is 40-50 min, and the mobile phase A is 75%; the mobile phase A rises from 75% to 100% in 50-52 min; 52-60 min, and 100% of mobile phase A.
- 3. The detection method according to claim 1, wherein the preparation process of the mobile phase A comprises:s1, mixing the potassium dihydrogen phosphate solution with triethylamine to obtain a phosphate solution;s2, mixing the phosphate solution with acetonitrile to obtain a mobile phase A;the potassium dihydrogen phosphate solution is an aqueous solution of potassium dihydrogen phosphate; the concentration of the potassium dihydrogen phosphate solution is 0.02 mol/L;the triethylamine accounts for 0.1 percent of the phosphate solution by mass;the volume ratio of the phosphate solution to the acetonitrile is (80-90) to (20-10).
- 4. The method for detecting according to claim 3, wherein in step S2, the phosphate solution is adjusted to pH 4.5 with phosphoric acid and then mixed with acetonitrile.
- 5. The detection method according to claim 1, wherein in the step a), the dosage ratio of the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample to the mobile phase A is (0.02-1) g: 1L;in the step B), the dosage ratio of the total mass of the impurities A and B to the mobile phase A is (0.0001-0.01) g: 1L; wherein the mass ratio of the impurity A to the impurity B is 1: 1;in the step c), the ratio of the total mass of the impurities A and B to the amount of the mobile phase in the reference product stock solution is (0.001-0.1) g: 1L; wherein the mass ratio of the impurity A to the impurity B is 1: 1.
- 6. The method of claim 1, wherein the C18 column is a YMC-Triart C18 column.
- 7. The detection method according to claim 1 or 6, wherein the size of the C18 chromatographic column is: the length is 250mm, the inner diameter is 4.6mm, and the grain size of the filler is 5 μm;the detection wavelength of the HPLC detection is 230 nm.
- 8. The detection method according to claim 5, wherein in the step a), the dosage ratio of the 7, 8-dimethoxy-1, 3-dihydro-2H-3-benzazepin-2-one sample to the mobile phase A is 0.2 g: 1L;in the step B), the ratio of the total mass of the impurities A and B to the dosage of the mobile phase A is 0.001 g: 1L;in the step c), the ratio of the total mass of the impurities A and B to the amount of the mobile phase in the reference substance stock solution is 0.01g to 1L.
- 9. The detection method according to claim 1, wherein the column temperature is 30 ℃;the flow rate of the mobile phase was 1 mL/min.
- 10. The assay of claim 3, wherein the volume ratio of phosphate solution to acetonitrile is 85: 15.
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