CN113640426A - Method for determining amine residue in acotiamide hydrochloride raw material medicine by gas chromatography - Google Patents
Method for determining amine residue in acotiamide hydrochloride raw material medicine by gas chromatography Download PDFInfo
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Abstract
The invention provides a method for determining amine residues in acotiamide hydrochloride bulk drug by gas chromatography, which comprises the following steps: A) dissolving acotiamide raw material medicine by adopting a solvent, and adding a strong base solution to obtain a solution to be detected; dissolving di-n-butylamine by using a solvent, and adding a strong base solution to obtain a di-n-butylamine reference substance solution; dissolving N, N-diisopropyl ethylenediamine by using a solvent, and adding a strong base solution to obtain an N, N-diisopropyl ethylenediamine reference solution; B) and (3) measuring the solution to be measured, the di-N-butylamine reference substance solution and the N, N-diisopropylethylenediamine reference substance solution by adopting a headspace-gas chromatography, and quantifying by adopting an external standard method. The detection method can quickly and accurately determine the contents of di-N-butylamine and N, N-diisopropylethylenediamine in acotiamide hydrochloride, and simultaneously detect two amine substances. Experiments show that the method has the advantages of good system applicability, strong specificity, high precision and accuracy and strong detection capability.
Description
Technical Field
The invention relates to the technical field of drug analysis and detection, in particular to a method for determining amine residues in acotiamide hydrochloride raw material drugs by using a gas chromatography.
Background
Acotiamide Hydrochloride (Acotiamide Hydrochloride Hydrate), the chemical name of which is N- [2- (bis-isopropylamino) ethyl ] -2- [ (2-hydroxy-4, 5-dimethoxybenzoyl) amino ] -4-thiazolecarboxamide Hydrochloride trihydrate, belongs to gastrointestinal motility drugs.
Acotiamide is the first FD (functional dyspepsia) therapeutic drug in the world, and clinical tests prove that the drug has good therapeutic effectiveness on patients diagnosed as FD according to the functional gastrointestinal tract disease diagnosis standard-ROMEIII, and has clinical application and market development values.
Acotiamide hydrochloride has the following structural formula:
the acotiamide hydrochloride can be synthesized by adopting N, N-diisopropyl ethylenediamine as a reaction material and di-N-butylamine as a reagent, but when the quality of a finished product is detected, the residue of the di-N-butylamine and the N, N-diisopropyl ethylenediamine in the acotiamide hydrochloride is not controlled. In the method for measuring 0861 residual solvent in the four parts of the Chinese pharmacopoeia of 2015 edition, the attached Table 1 does not control the limitation of di-N-butylamine and N, N-diisopropylethylenediamine, and has no corresponding measurement method guidance. Under different conditions, amino groups in di-N-butylamine and N, N-diisopropylethylenediamine react with hydrochloric acid in acotiamide hydrochloride to different degrees, so that sample detection is extremely difficult.
Because di-N-butylamine and N, N-diisopropylethylenediamine do not have ultraviolet absorption and are not suitable for detection by a liquid phase, the prior art takes development of a gas chromatography as a main detection means. For example, chinese patent application CN201610167810.5 discloses a method for determining the residual amount of N, N-diisopropylethylenediamine in acotiamide drug substance by using a gas chromatograph. The method adopts a DB-1 capillary chromatographic column for determination, the chromatographic column is a nonpolar capillary chromatographic column, and the separation effect is poor when two or more polar substances are separated. The research of technical personnel finds that the method provided by CN201610167810.5 can only be used for controlling the residual quantity of N, N-diisopropylethylenediamine, and cannot control the residual solution containing di-N-butylamine in the product, which directly influences the product quality of acotiamide hydrochloride and increases the risk of clinical medication.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a method for determining amine residues in acotiamide hydrochloride bulk drug by gas chromatography, wherein the method of the present invention is stable and reliable, and has a low detection limit.
The invention provides a method for determining amine residues in acotiamide hydrochloride bulk drug by gas chromatography, which comprises the following steps:
A) dissolving acotiamide raw material medicine by adopting a solvent, and adding a strong base solution to obtain a solution to be detected;
dissolving di-n-butylamine by using a solvent, and adding a strong base solution to obtain a di-n-butylamine reference substance solution;
dissolving N, N-diisopropyl ethylenediamine by using a solvent, and adding a strong base solution to obtain an N, N-diisopropyl ethylenediamine reference solution;
B) measuring the solution to be measured, a di-N-butylamine reference substance solution and an N, N-diisopropylethylenediamine reference substance solution by adopting a headspace-gas chromatography, and quantifying by adopting an external standard method;
preferably, the detection conditions of the method are as follows: a chromatographic column: a capillary chromatographic column with polyethylene glycol as a solid phase;
a detector: FID; and (3) sample introduction mode: carrying out headspace sample injection; the carrier gas is nitrogen;
temperature programming: the initial temperature is 40-60 ℃, the temperature is maintained for 4-10 min, then the temperature is increased to 200 ℃ at the speed of 15-30 ℃/min, and the temperature is maintained for 10-30 min.
Preferably, the solvent in step a) is dimethyl sulfoxide.
Preferably, the strong base of step a) is sodium hydroxide; the concentration of the strong alkali solution is 0.5-1.0 mol/L.
Preferably, the chromatographic column of step B) is CP-Wax or CP 7422; the specification of the chromatographic column is as follows: 25 m.times.0.320 mm.times.1.2. mu.m.
Preferably, the temperature programming in the step B) is specifically: the initial temperature is 60 deg.C, and maintained for 5min, and then the temperature is raised to 200 deg.C at a rate of 20 deg.C/min, and maintained for 15 min.
Preferably, said step B) is
The temperature of the detector is 280-320 ℃;
the temperature of a sample inlet is 230-260 ℃;
the equilibrium temperature of the headspace bottle is 90 ℃;
the equilibration time was 30 min.
Preferably, the flow rate of the carrier gas is 1.4-1.6 ml/min;
the temperature of a sample inlet is 250 ℃; the detector temperature was 300 ℃.
Preferably, the linear relation between the concentration of the di-n-butylamine in the range of 0.0967 mu g/mL-201.48 mu g/mL and the peak area is good, the linear equation is 0.4569x +0.152, and the correlation coefficient is r 1; the linear relation between the peak area and the concentration of N, N-diisopropylethylenediamine is good in the range of 2.504 mu g/mL-200.32 mu g/mL, the linear equation is that y is 0.0325x-0.1532, and the correlation coefficient is that r is 0.9994.
Preferably, the quantitative limit concentration of the di-n-butylamine is 0.096 mu g/mL, and the quantitative limit is 0.00048%; the quantitative limit concentration of diisopropylethylenediamine is 2.508 μ g/mL, and the quantitative limit is 0.013%.
Preferably, the headspace sample injection comprises: and (3) split-flow sample injection, wherein the split-flow ratio is 10-20: 1.
Compared with the prior art, the invention provides a method for determining amine residues in acotiamide hydrochloride bulk drug by using a gas chromatography, which comprises the following steps: A) dissolving acotiamide raw material medicine by adopting a solvent, and adding a strong base solution to obtain a solution to be detected; dissolving di-n-butylamine by using a solvent, and adding a strong base solution to obtain a di-n-butylamine reference substance solution; dissolving N, N-diisopropyl ethylenediamine by using a solvent, and adding a strong base solution to obtain an N, N-diisopropyl ethylenediamine reference solution; B) and (3) measuring the solution to be measured, the di-N-butylamine reference substance solution and the N, N-diisopropylethylenediamine reference substance solution by adopting a headspace-gas chromatography, and quantifying by adopting an external standard method. The detection method can quickly and accurately detect the contents of di-N-butylamine and N, N-diisopropylethylenediamine in the acotiamide hydrochloride, simultaneously detect two amine substances, control the amine residue in the acotiamide hydrochloride raw material medicine, and provide a quality standard detection method for ensuring the quality of the acotiamide hydrochloride finished product. Experiments show that the method for determining the amine residue in the acotiamide hydrochloride bulk drug has the advantages of good system applicability, strong specificity, high precision and accuracy and strong detection capability, and is an efficient, sensitive and accurate determination method.
Drawings
FIG. 1 is a sample test result map with product lot number 201101-1 in example 9 of the present invention;
FIG. 2 is a spectrum of a test result of a sample with a product lot number of 201101-2 in example 9 of the present invention;
FIG. 3 is a spectrum of the test results of a sample with product lot number 201101-3 in example 9 of the present invention;
figure 4 is a chromatogram of a control tested using the method of patent application CN 201610167810.5.
Detailed Description
The invention provides a method for measuring amine residue in acotiamide hydrochloride bulk drug by gas chromatography, and the method can be realized by appropriately improving process parameters by referring to the content in the text. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention provides a method for determining amine residues in acotiamide hydrochloride bulk drug by gas chromatography, which comprises the following steps:
A) dissolving acotiamide raw material medicine by adopting a solvent, and adding a strong base solution to obtain a solution to be detected;
dissolving di-n-butylamine by using a solvent, and adding a strong base solution to obtain a di-n-butylamine reference substance solution;
dissolving N, N-diisopropyl ethylenediamine by using a solvent, and adding a strong base solution to obtain an N, N-diisopropyl ethylenediamine reference solution;
B) measuring the solution to be measured, a di-N-butylamine reference substance solution and an N, N-diisopropylethylenediamine reference substance solution by adopting a headspace-gas chromatography, and quantifying by adopting an external standard method;
the chromatographic conditions are as follows: a chromatographic column: a capillary chromatographic column with polyethylene glycol as a solid phase;
a detector: FID; and (3) sample introduction mode: carrying out headspace sample injection; the carrier gas is nitrogen;
temperature programming: the initial temperature is 40-60 ℃, the temperature is maintained for 4-10 min, then the temperature is increased to 200 ℃ at the speed of 15-30 ℃/min, and the temperature is maintained for 10-30 min.
The method for determining the amine residue in the acotiamide hydrochloride raw material medicine by using the gas chromatography provided by the invention is characterized in that the acotiamide raw material medicine is dissolved by using a solvent.
The solvent is dimethyl sulfoxide; the mass-volume ratio of the acotiamide raw material medicine to the solvent is 0.1 g: 3 mL;
the invention preferably dissolves in the headspace bottle.
After dissolution, adding a strong base solution to obtain a solution to be detected.
In one of the preferred embodiments of the present invention, the strong base is preferably sodium hydroxide; the concentration of the strong alkali solution is preferably 0.5-1.0 mol/L.
The inventor prefers to adopt strong alkali for treatment, and combines specific concentration, so that the detection result is more accurate and reliable.
Dissolving di-n-butylamine by using a solvent, and adding a strong base solution to obtain a di-n-butylamine reference substance solution;
the invention is preferably prepared by the following method:
taking di-n-butylamine, placing into a measuring flask, adding dimethyl sulfoxide to dissolve and dilute to a scale, and shaking uniformly to obtain a di-n-butylamine stock solution;
dissolving N, N-diisopropyl ethylenediamine by using a solvent, and adding a strong base solution to obtain an N, N-diisopropyl ethylenediamine reference solution;
the invention is preferably prepared by the following method:
taking N, N-diisopropyl ethylenediamine, putting into a measuring flask, adding dimethyl sulfoxide to dissolve and dilute to a scale, and shaking up to obtain N, N-diisopropyl ethylenediamine stock solution.
Precisely measuring a di-N-butylamine stock solution and an N, N-diisopropylethylenediamine stock solution, placing the di-N-butylamine stock solution and the N, N-diisopropylethylenediamine stock solution into a measuring flask, adding dimethyl sulfoxide to dilute the di-N-butylamine stock solution to a scale, and shaking the di-N-diisopropylethylenediamine stock solution uniformly to obtain a diluent; taking the solution, placing in a headspace bottle, adding dimethyl sulfoxide and sodium hydroxide solution, sealing, and shaking.
The dilution factor is not limited in the present invention, and may be in accordance with the detection concentration range of the present invention.
The invention also comprises a blank solution preparation, which comprises the following steps: taking dimethyl sulfoxide and sodium hydroxide solution, placing in a headspace bottle, sealing, and shaking up to obtain the final product.
And (3) measuring the solution to be measured, the di-N-butylamine reference substance solution and the N, N-diisopropylethylenediamine reference substance solution by adopting headspace-gas chromatography.
The invention adopts the blank solution, the reference substance and the liquid to be detected to carry out the gas chromatography determination, so that the detection result is more accurate.
The detection instrument is not limited in the present invention, and may be Agilent 7890A.
The chromatographic conditions of the invention are specifically as follows:
a chromatographic column: polyethylene glycol is a capillary chromatographic column of solid phase.
In some of the preferred embodiments of the invention, the chromatographic column is CP-Wax or CP 7422; the specification of the chromatographic column is as follows: 25 m.times.0.320 mm.times.1.2. mu.m.
The present inventors have found that only the above-mentioned chromatographic column can solve the technical problem of detecting two amine residues according to the present invention.
A detector: FID; and (3) sample introduction mode: carrying out headspace sample injection; the headspace sample injection of the invention comprises the following steps: and (3) split-flow sample injection, wherein the split-flow ratio is 10-20: 1.
The carrier gas is nitrogen; the flow rate of the carrier gas is preferably 1.4-1.6 ml/min.
The temperature of the detector is preferably 280-320 ℃; more preferably 290-310 ℃; most preferably 300 deg.c.
The preferred injection port temperature is 230-260 ℃; more preferably 240 to 250 ℃.
The headspace bottle equilibrium temperature was 90 ℃.
The equilibration time was 30 min.
In some of the preferred embodiments of the present invention,
temperature programming: the initial temperature is 40-60 ℃, the temperature is maintained for 4-10 min, then the temperature is increased to 200 ℃ at the speed of 15-30 ℃/min, and the temperature is maintained for 10-30 min.
In some of the preferred embodiments of the present invention,
the temperature programming specifically comprises the following steps: the initial temperature is 60 deg.C, and maintained for 5min, and then the temperature is raised to 200 deg.C at a rate of 20 deg.C/min, and maintained for 15 min.
According to the invention, through the control of the temperature programming, the separation degrees are all larger than 1.5, and the separation degree is high.
In the embodiment of the invention, the contents of N, N-diisopropylethylenediamine and di-N-butylamine in qualified products are respectively not more than 0.5 percent by peak area calculation according to an external standard method.
According to the invention, the linear relation between the concentration of di-n-butylamine in the range of 0.0967 mu g/mL-201.48 mu g/mL and the peak area is good, the linear equation is 0.4569x +0.152, and the correlation coefficient is r 1; the linear relation between the peak area and the concentration of N, N-diisopropylethylenediamine is good in the range of 2.504 mu g/mL-200.32 mu g/mL, the linear equation is that y is 0.0325x-0.1532, and the correlation coefficient is that r is 0.9994.
The method has the advantages of large linear range and good linear result.
The quantitative limit concentration of the di-n-butylamine is 0.096 mu g/mL, and the quantitative limit is 0.00048%; the quantitative limit concentration of diisopropylethylenediamine is 2.508 μ g/mL, and the quantitative limit is 0.013%.
The detection limit concentration of the di-n-butylamine is 0.032 mu g/mL, and the detection limit is 0.00016%. The detection limit concentration of the N, N-diisopropyl ethylenediamine is 0.5005 mu g/mL, and the quantification limit is 0.0025%.
The method has low detection limit and quantification limit, can detect amine residue with lower content, and is beneficial to the improvement of product quality.
The invention can detect two amine substances simultaneously, successfully controls the amine residue in the acotiamide hydrochloride raw material medicine, and provides an effective detection method for ensuring the quality of the finished product of the acotiamide hydrochloride.
The method can be used for simultaneously detecting the separation of N, N-dimethylformamide, triethylamine, NN dimethyl acetamide, isopropanol, ethanol, dichloromethane, toluene, dichloromethane, di-N-butylamine, NN-diisopropylethylenediamine and DMSO, and has a wider detection range compared with the prior art.
The invention provides a method for determining amine residues in acotiamide hydrochloride bulk drug by gas chromatography, which comprises the following steps: A) dissolving acotiamide raw material medicine by adopting a solvent, and adding a strong base solution to obtain a solution to be detected; dissolving di-n-butylamine by using a solvent, and adding a strong base solution to obtain a di-n-butylamine reference substance solution; dissolving N, N-diisopropyl ethylenediamine by using a solvent, and adding a strong base solution to obtain an N, N-diisopropyl ethylenediamine reference solution; B) measuring the solution to be measured, a di-N-butylamine reference substance solution and an N, N-diisopropylethylenediamine reference substance solution by adopting a headspace-gas chromatography, and quantifying by adopting an external standard method; the chromatographic conditions are as follows: a chromatographic column: a capillary chromatographic column with polyethylene glycol as a solid phase; a detector: FID; and (3) sample introduction mode: carrying out headspace sample injection; the carrier gas is nitrogen; temperature programming: the initial temperature is 40-60 ℃, the temperature is maintained for 4-10 min, then the temperature is increased to 200 ℃ at the speed of 15-30 ℃/min, and the temperature is maintained for 10-30 min. The detection method can quickly and accurately detect the contents of di-N-butylamine and N, N-diisopropylethylenediamine in the acotiamide hydrochloride, simultaneously detect two amine substances, control the amine residue in the acotiamide hydrochloride raw material medicine, and provide a quality standard detection method for ensuring the quality of the acotiamide hydrochloride finished product. Experiments show that the method for determining the amine residue in the acotiamide hydrochloride bulk drug has the advantages of good system applicability, strong specificity, high precision and accuracy and strong detection capability, and is an efficient, sensitive and accurate determination method.
In order to further illustrate the present invention, the following describes the method for determining the amine residue in the acotiamide hydrochloride bulk drug by gas chromatography provided by the present invention in detail with reference to the examples.
The examples described below, unless otherwise indicated, are all temperatures set forth in degrees Celsius. The gas chromatograph used Agilent 7890A and the electronic balance used XPE204 or ML 204. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagent is commercially available from Aldrich, Arco, Alfa, MERK, Scharlau, Allantin, West Longsu science, etc.
Example 1 System applicability of the method of the invention
(1) Chromatographic conditions
The type of the chromatograph: agilent GC or equivalent;
a detector: FID;
a chromatographic column: capillary chromatographic column (CP-Wax, 25m × 0.320mm × 1.2 μm) with polyethylene glycol as solid phase;
temperature rising procedure: the initial temperature is 60 ℃, the temperature is maintained for 5 minutes, then the temperature is increased to 200 ℃ at the speed of 20 ℃/min, and the temperature is maintained for 15 minutes;
detector temperature: 300 ℃;
sample inlet temperature: 250 ℃;
headspace bottle equilibrium temperature: 90 ℃;
and (3) sample introduction mode: and (4) headspace.
(2) System applicability solution formulation
Preparing a di-n-butylamine stock solution: 503.7mg of di-n-butylamine is taken and placed in a 50ml measuring flask, dimethyl sulfoxide is added for dissolution and dilution to the scale, and the mixture is shaken up to be used as a di-n-butylamine stock solution;
preparing a di-n-butylamine reference substance stock solution: precisely measuring 1ml of di-n-butylamine stock solution, placing the di-n-butylamine stock solution into a 20ml measuring flask, adding dimethyl sulfoxide to dilute to a scale, and shaking up to be used as a reference substance stock solution;
preparing a N, N-diisopropyl ethylenediamine stock solution: taking 505.2mg of N, N-diisopropyl ethylenediamine, putting the N, N-diisopropyl ethylenediamine into a 50ml measuring flask, adding dimethyl sulfoxide to dissolve and dilute the N, N-diisopropyl ethylenediamine to a scale, and shaking up the solution to be used as a stock solution of the N, N-diisopropyl ethylenediamine;
preparing a reference stock solution of N, N-diisopropylethylenediamine: precisely measuring 1ml of N, N-diisopropylethylenediamine stock solution, placing the stock solution in a 20ml measuring flask, adding dimethyl sulfoxide to dilute to a scale, and shaking up to obtain a reference stock solution.
(3) Experimental procedures and results
Taking 2ml of dimethyl sulfoxide, placing the dimethyl sulfoxide in a headspace bottle, adding 2ml of 0.5mol/L sodium hydroxide solution, mixing uniformly, cooling to room temperature, taking 1ml of each reference substance stock solution, sealing, and shaking uniformly to obtain the final product; 6 parts are prepared in parallel, 6 needles are continuously injected, and the measurement results are shown in the following table.
Table 1: system applicability test results
As can be seen from Table 1, the separation degrees between di-N-butylamine and the adjacent components are all greater than 1.5, the peak area RSD is 0.5%, the separation degrees between N, N-diisopropylethylenediamine and the adjacent components are all greater than 1.5, and the peak area RSD is 6.7% and less than 15.0%, which indicates that the system applicability of the method of the invention is good.
Example 2 specificity verification of the method of the invention
(1) Chromatographic conditions
The same as in example 1.
(2) Experimental procedures and results
Preparation of a blank solution: taking 3ml of dimethyl sulfoxide, adding 2ml of 0.5mol/L sodium hydroxide solution, placing in a headspace bottle, sealing, and shaking up to obtain the product.
Di-n-butylamine peak positioning solution: taking about 501.7mg of di-n-butylamine, placing the di-n-butylamine in a 50ml measuring flask, adding dimethyl sulfoxide to dissolve and dilute the di-n-butylamine to a scale, and shaking up the solution to be used as a di-n-butylamine stock solution; taking 1ml, placing in a 20ml measuring flask, adding dimethyl sulfoxide for diluting to scale, shaking up, taking 2ml of dimethyl sulfoxide, placing in a headspace flask, adding 2ml of 0.5mol/L sodium hydroxide solution, mixing well, cooling to room temperature, taking 1ml of the above solution, sealing, and shaking up to obtain the final product.
N, N-diisopropylethylenediamine peak positioning solution: taking about 500.8mg of N, N-diisopropyl ethylenediamine, putting the N, N-diisopropyl ethylenediamine into a 50ml measuring flask, adding dimethyl sulfoxide to dissolve and dilute the N, N-diisopropyl ethylenediamine to a scale, and shaking up the solution to be used as a stock solution of the N, N-diisopropyl ethylenediamine; taking 1ml, placing in a 20ml measuring flask, adding dimethyl sulfoxide for diluting to scale, shaking up, taking 2ml of dimethyl sulfoxide, placing in a headspace flask, adding 2ml of 0.5mol/L sodium hydroxide solution, mixing well, cooling to room temperature, taking 1ml of the above solution, sealing, and shaking up to obtain the final product.
Ethanol peak localization solution: taking about 500.9mg of ethanol, placing the ethanol into a 50ml measuring flask, adding dimethyl sulfoxide to dissolve and dilute the ethanol to a scale, and shaking up to obtain ethanol stock solution; placing 1ml in a 20ml measuring flask, adding dimethyl sulfoxide to dilute to scale, shaking up, taking 2ml of dimethyl sulfoxide, placing in a headspace flask, adding 2ml of 0.5mol/L sodium hydroxide solution, mixing well, cooling to room temperature, taking 1ml of the above solution, sealing, and shaking up to obtain the final product.
Isopropanol peak localization solution: taking about 502.9mg of isopropanol, placing the isopropanol in a 50ml measuring flask, adding dimethyl sulfoxide to dissolve and dilute the isopropanol to a scale, and shaking up the solution to be used as isopropanol stock solution; placing 1ml in a 20ml measuring flask, adding dimethyl sulfoxide to dilute to scale, shaking up, taking 2ml of dimethyl sulfoxide, placing in a headspace flask, adding 2ml of 0.5mol/L sodium hydroxide solution, mixing well, cooling to room temperature, taking 1ml of the above solution, sealing, and shaking up to obtain the final product.
Triethylamine peak positioning solution: taking 104.2mg of triethylamine, placing the triethylamine in a 50ml measuring flask, adding dimethyl sulfoxide to dissolve and dilute the triethylamine to a scale, and shaking up the solution to be used as triethylamine stock solution; placing 1ml in a 20ml measuring flask, adding dimethyl sulfoxide to dilute to scale, shaking up, taking 2ml of dimethyl sulfoxide, placing in a headspace flask, adding 2ml of 0.5mol/L sodium hydroxide solution, mixing well, cooling to room temperature, taking 1ml of the above solution, sealing, and shaking up to obtain the final product.
Acetic acid peak localization solution: taking about 501.9mg of acetic acid, placing the acetic acid into a 50ml measuring flask, adding dimethyl sulfoxide to dissolve and dilute the acetic acid to a scale, and shaking up the solution to be used as an acetic acid stock solution; placing 1ml in a 20ml measuring flask, adding dimethyl sulfoxide to dilute to scale, shaking up, taking 2ml of dimethyl sulfoxide, placing in a headspace flask, adding 2ml of 0.5mol/L sodium hydroxide solution, mixing well, cooling to room temperature, taking 1ml of the above solution, sealing, and shaking up to obtain the final product.
Dichloromethane peak localization: taking about 63.0mg of dichloromethane, putting the dichloromethane into a 50ml measuring flask, adding dimethyl sulfoxide to dissolve and dilute the dichloromethane to a scale, and shaking up the dichloromethane to be used as dichloromethane stock solution; placing 1ml in a 20ml measuring flask, adding dimethyl sulfoxide to dilute to scale, shaking up, taking 2ml of dimethyl sulfoxide, placing in a headspace flask, adding 2ml of 0.5mol/L sodium hydroxide solution, mixing well, cooling to room temperature, taking 1ml of the above solution, sealing, and shaking up to obtain the final product.
Toluene peak localization solution: taking about 94.1mg of toluene, placing the toluene in a 50ml measuring flask, adding dimethyl sulfoxide to dissolve and dilute the toluene to a scale, and shaking up the solution to be used as a toluene stock solution; placing 1ml in a 20ml measuring flask, adding dimethyl sulfoxide to dilute to scale, shaking up, taking 2ml of dimethyl sulfoxide, placing in a headspace flask, adding 2ml of 0.5mol/L sodium hydroxide solution, mixing well, cooling to room temperature, taking 1ml of the above solution, sealing, and shaking up to obtain the final product.
N, N-dimethylformamide peak localization solution: taking about 88.4mg of N, N-dimethylformamide, putting the N, N-dimethylformamide into a 50ml measuring flask, adding dimethyl sulfoxide to dissolve and dilute the N, N-dimethylformamide to a scale, and shaking up the solution to be used as a stock solution of the N, N-dimethylformamide; taking 2ml of dimethyl sulfoxide, placing the dimethyl sulfoxide into a headspace bottle, adding 2ml of 0.5mol/L sodium hydroxide solution, mixing uniformly, cooling to room temperature, taking 1ml of the solution, sealing, and shaking uniformly to obtain the compound.
N, N-dimethylacetamide peak localization solution: taking about 92.9mg of N, N-dimethylacetamide, putting the N, N-dimethylacetamide in a 50ml measuring flask, adding dimethyl sulfoxide to dissolve and dilute the N, N-dimethylacetamide to a scale, and shaking up to obtain N, N-dimethylacetamide stock solution; taking 2ml of dimethyl sulfoxide, placing the dimethyl sulfoxide into a headspace bottle, adding 2ml of 0.5mol/L sodium hydroxide solution, mixing uniformly, cooling to room temperature, taking 1ml of the solution, sealing, and shaking uniformly to obtain the compound.
Impurity mixed solution: precisely measuring 1ml of a di-N-butylamine stock solution, an N, N-diisopropylethylenediamine stock solution, triethylamine, N-dimethylformamide, N-dimethylacetamide, toluene, ethanol, isopropanol, dichloromethane and acetic acid stock solution respectively, putting the two solutions into a 20ml measuring flask, adding dimethyl sulfoxide to dilute the solutions to a scale, and shaking the solutions uniformly to obtain an impurity mixed stock solution. Taking 2ml of dimethyl sulfoxide, placing the dimethyl sulfoxide into a headspace bottle, adding 2ml of 0.5mol/L sodium hydroxide solution, mixing uniformly, cooling to room temperature, taking 1ml of the solution, sealing, and shaking uniformly to obtain the compound.
Preparation of a special solution: placing 0.1010g of the product in a headspace bottle, adding 2ml of dimethyl sulfoxide and 2ml of 0.5mol/L sodium hydroxide solution, mixing, cooling to room temperature, adding 1ml of impurity mixed stock solution, sealing, and shaking.
Preparation of a test solution: taking a proper amount of acotiamide hydrochloride (self-made), placing the acotiamide hydrochloride in a headspace bottle, adding 2mL of dimethyl sulfoxide, adding 2mL of 0.5mol/L sodium hydroxide solution with a certain concentration, and sealing to obtain the acotiamide hydrochloride.
Sampling blank solution, special solution, sample solution and impurity positioning solution respectively for 1 needle, injecting into gas chromatograph, recording chromatogram, and detecting results are shown in Table 2.
Table 2: result of specialization
As can be seen from Table 2, the blank solvent peak had no interference with the di-N-butylamine peak and the N, N-diisopropylethylenediamine peak, the degree of separation between the di-N-butylamine peak and the adjacent solvent peak was 36.49, and the degree of separation between the N, N-diisopropylethylenediamine peak and the adjacent solvent peak was 36.30 (as shown in Table 2), both of which were greater than 1.5; in the special solution, chromatographic peaks of N, N-dimethylformamide and N, N-dimethylacetamide coincide, acetic acid does not show a peak under the chromatographic condition, the separation degrees between other solvent peaks are all larger than 1.5, and no interference is caused on detection of a di-N-butylamine peak and N, N-diisopropylethylenediamine, so that the method for detecting di-N-butylamine and N, N-diisopropylethylenediamine in acotiamide hydrochloride has good specificity.
Example 3 accuracy verification of the method of the invention
(1) Chromatographic conditions
The same as in example 1.
(2) Experimental procedures and results
80% recovery solution: precisely measuring 0.8ml of each of a di-N-butylamine stock solution and an N, N-diisopropylethylenediamine stock solution under the item of 'system applicability', putting the di-N-butylamine stock solution and the N, N-diisopropylethylenediamine stock solution into a 20ml measuring flask, adding dimethyl sulfoxide to dilute to a scale, and shaking up to be used as 80% reference substance stock solutions; taking about 0.1g of acotiamide hydrochloride, placing the acotiamide hydrochloride in a headspace bottle, adding 2ml of dimethyl sulfoxide for dissolving, adding 2ml of 0.5mol/L sodium hydroxide solution, uniformly mixing, cooling to room temperature, taking 1ml of 80% reference substance stock solution, sealing, and shaking uniformly to obtain the acotiamide hydrochloride. 3 parts are prepared in parallel.
100% recovery solution: precisely measuring 1ml of each of a di-N-butylamine stock solution and an N, N-diisopropylethylenediamine stock solution, putting the di-N-butylamine stock solution and the N, N-diisopropylethylenediamine stock solution into a 20ml measuring flask, adding dimethyl sulfoxide to dilute the di-N-butylamine stock solution to a scale, and shaking the solution uniformly to serve as 100% reference substance stock solutions; taking about 0.1g of acotiamide hydrochloride, placing the acotiamide hydrochloride in a headspace bottle, adding 2ml of dimethyl sulfoxide for dissolving, adding 2ml of 0.5mol/L sodium hydroxide solution, uniformly mixing, cooling to room temperature, taking 1ml of 100% reference substance stock solution, sealing, and shaking uniformly to obtain the acotiamide hydrochloride. 3 parts are prepared in parallel.
120% recovery solution: precisely measuring 1.2ml of each of a di-N-butylamine stock solution and an N, N-diisopropylethylenediamine stock solution, putting the di-N-butylamine stock solution and the N, N-diisopropylethylenediamine stock solution into a 20ml measuring flask, adding dimethyl sulfoxide to dilute the solutions to scales, and shaking the solutions uniformly to obtain 120% reference substance stock solutions; taking about 0.1g of acotiamide hydrochloride, placing the acotiamide hydrochloride in a headspace bottle, adding 2ml of dimethyl sulfoxide for dissolving, adding 2ml of 0.5mol/L sodium hydroxide solution, uniformly mixing, cooling to room temperature, taking 1ml of 120% reference substance stock solution, sealing, and shaking uniformly to obtain the acotiamide hydrochloride. 3 parts are prepared in parallel.
Preparation of control solutions: taking about 500mg of di-n-butylamine, putting the di-n-butylamine in a 50ml measuring flask, adding dimethyl sulfoxide to dissolve and dilute the di-n-butylamine to a scale, and shaking the mixture uniformly to obtain a di-n-butylamine stock solution; taking about 500mg of N, N-diisopropyl ethylenediamine, putting the N, N-diisopropyl ethylenediamine into a 50ml measuring flask, adding dimethyl sulfoxide to dissolve and dilute the N, N-diisopropyl ethylenediamine to a scale, and shaking up to obtain a stock solution of the N, N-diisopropyl ethylenediamine; precisely measuring 1ml of each of a di-N-butylamine stock solution and an N, N-diisopropylethylenediamine stock solution, placing the two solutions in a 20ml measuring flask, adding dimethyl sulfoxide to dilute the solutions to a scale, shaking up the solutions, taking 1ml of the solution, placing the solution in a headspace flask, adding 2ml of dimethyl sulfoxide, adding 2ml of 0.5mol/L sodium hydroxide solution, sealing and shaking up the solution to obtain the product.
Preparation of a test solution: the same as in example 2.
Taking the reference solution, the test solution and the accuracy solution, injecting into a gas chromatograph with 1 needle respectively, recording the chromatogram, and the detection results are shown in tables 3 and 4.
Table 3: results of recovery of di-n-butylamine
Table 4: results for recovery of N, N-diisopropylethylenediamine
As can be seen from tables 3 and 4, the recovery rates of di-N-butylamine were between 92% and 95%, the average recovery rate was 93%, the RSD was 1.1% and less than 15.0% (N ═ 9), the recovery rates of N, N-diisopropylethylenediamine were between 87% and 104%, the average recovery rate was 98%, the RSD was 5.3% and less than 15.0% (N ═ 9). The method is proved to have good recovery rate for detecting di-N-butylamine and N, N-diisopropylethylenediamine in acotiamide hydrochloride.
Example 4 validation of the precision of the method of the invention
(1) Chromatographic conditions
The same as in example 1.
(2) Repetitive experimental procedures and results
Control solution: taking 2ml of dimethyl sulfoxide, placing the dimethyl sulfoxide into a headspace bottle, adding 2ml of 0.5mol/L sodium hydroxide solution, mixing uniformly, cooling to room temperature, taking 1ml of a 'reference substance stock solution' under the system applicability, sealing, and shaking uniformly to obtain the product.
Test solution: taking about 0.1g of the product, placing the product in a headspace bottle, adding 2ml of dimethyl sulfoxide, adding 2ml of 0.5mol/L sodium hydroxide solution, mixing uniformly, cooling to room temperature, adding 1ml of a 'control stock solution' under the system applicability, sealing, and shaking uniformly to obtain the product. 6 parts are prepared in parallel. The results are shown in the following table.
Table 5: results of repeated measurements
As can be seen from Table 5, the average of the di-n-butylamine content in 6 test sample solutions was 0.46%, and the RSD was 2.0%; the average content of N, N diisopropyl ethylenediamine is 0.53%, RSD is 2.9%, and both are less than 15.0%. The method is proved to have good repeatability for detecting di-N-butylamine and N, N-diisopropylethylenediamine in acotiamide hydrochloride.
(3) Reproducibility test procedures and results
The same batch of samples (AK9170702) was weighed and prepared into 12 test solutions, respectively, which were prepared as in example 2.
The experimental procedure was repeated by different experimenters at different times.
Table 6: results of repeated measurements
As can be seen from Table 6, the average of the di-n-butylamine content in 12 test sample solutions was 0.47% and the RSD was 3.4%; the average content of N, N diisopropyl ethylenediamine is 0.54%, the RSD is 8.1%, and the content is less than 15.0%. The method is proved to have good reproducibility for detecting di-N-butylamine and N, N-diisopropylethylenediamine in acotiamide hydrochloride.
The method for detecting the di-N-butylamine and the N, N-diisopropylethylenediamine in the acotiamide hydrochloride has good repeatability and intermediate precision.
Example 5 Linear verification of the method of the invention
(1) Chromatographic conditions
The same as in example 1.
(2) Experimental procedures and results
Blank solvent: taking 3ml of dimethyl sulfoxide, adding 2ml of 0.5mol/L sodium hydroxide solution, placing in a headspace bottle, sealing, and shaking up to obtain the product.
Di-n-butylamine stock solution: 503.7mg of di-n-butylamine is taken and placed in a 50ml measuring flask, dissolved and diluted to the scale by adding dimethyl sulfoxide, and shaken up to be used as a di-n-butylamine stock solution.
N, N-diisopropylethylenediamine stock solution: 500.8mg of N, N-diisopropyl ethylenediamine is taken and placed in a 50ml measuring flask, and dimethyl sulfoxide is added to dissolve and dilute the solution to the scale mark, and the solution is shaken up to be used as a stock solution of the N, N-diisopropyl ethylenediamine.
Taking the stock solution to prepare linear solutions according to the following table, respectively injecting the linear solutions into a gas chromatograph, and performing linear regression by taking the concentration as an X axis and the peak area as a Y axis. The results are shown in tables 8 and 10, respectively.
Table 7: preparation of di-n-butylamine linear solution
Table 8: linear results of di-n-butylamine
Table 9: preparation of N, N-diisopropyl ethylenediamine linear solution
Table 10: linear results for N, N-diisopropylethylenediamine
As can be seen from tables 8 and 10, di-N-butylamine has a good linear relationship with the peak area in the concentration range of 0.0967 μ g/mL to 201.48 μ g/mL, the linear equation is y 0.4569x +0.152, the correlation coefficient is r 1, N-diisopropylethylenediamine has a good linear relationship with the peak area in the concentration range of 2.504 μ g/mL to 200.32 μ g/mL, the linear equation is y 0.0325x-0.1532, and the correlation coefficient is r 0.9994.
EXAMPLE 6 method of the present methodQuantitative limit verification
(1) Chromatographic conditions
The same as in example 1.
(2) Experimental procedures and results
And (3) referring to the signal-to-noise ratio in the linear solution, and gradually diluting until the concentration of the solution is the quantitative limit concentration when the S/N of the signal-to-noise ratio is about 10, wherein the solution is the quantitative limit solution.
Samples were continuously fed through 6 needles, and the results are shown in Table 11.
Table 11: quantitative limit result
As can be seen from Table 11, the 6-point SNR mean value of the quantitation limit of di-n-butylamine is 11.8, the peak area RSD is 5.1% and is less than 15.0%, the concentration of the quantitation limit is 0.096. mu.g/mL, and the quantitation limit is 0.00048%. The N, N-diisopropylethylenediamine quantification limit of 6 needles of the signal-to-noise ratio mean value is 11.0, the peak area RSD is 13.1 percent and is less than 15.0 percent, the quantification limit concentration is 2.508 mu g/mL, and the quantification limit is 0.013 percent.
Example 7 detection Limit verification of the method of the invention
(1) Chromatographic conditions
The same as in example 1.
(2) Experimental procedures and results
And (3) diluting the quantitative limiting solution step by step until the S/N ratio is about 3, wherein the concentration of the solution is the detection limiting concentration, and the solution is the detection limiting solution. Samples were continuously fed through 3 needles, and the results are shown in Table 10.
Table 12: limit of detection result
As can be seen from Table 12, the average of the SNR values of the 3-pin detection limits of di-n-butylamine is 3.7, the concentration of the detection limit is 0.032 μ g/mL, and the detection limit is 0.00016%. The average value of the signal-to-noise ratio of 3 pins of the detection limit of N, N-diisopropyl ethylenediamine is 3.5, the concentration of the detection limit is 0.5005 mu g/mL, and the limit of quantitation is 0.0025%.
Example 8 durability verification of the inventive method
(1) Chromatographic conditions
The same as in example 1.
(2) Experimental procedures and results
The test solution and the control solution in example 3 were taken, the initial column temperature, the flow rate, and the like were finely adjusted, and the durability of the detection method was examined, and the results are shown in Table 13.
Table 13: durability results
As can be seen from Table 13, after the chromatographic conditions were adjusted, the degrees of separation of the di-N-butylamine and the adjacent solvent peaks in the test solution were all greater than 1.5, and the degrees of separation of the N, N-diisopropylethylenediamine and the adjacent solvent peaks were all greater than 1.5, as compared with the results obtained without changing the conditions. The method for detecting the di-N-butylamine and the N, N-diisopropylethylenediamine in the acotiamide hydrochloride has good durability.
In summary, the experimental results of the methodology verification of the analysis method for the amine residue in the acotiamide hydrochloride bulk drug show that the system applicability, specificity, precision, accuracy, linearity, detection limit, quantification limit and durability of the method all meet the acceptable standards, which indicates that the method can obtain accurate and reliable experimental results for the determination of di-N-butylamine and N, N-diisopropylethylenediamine in acotiamide hydrochloride, and the specific summary is shown in table 14.
Table 14: summary of the verification
EXAMPLE 9 three batches of samples
1. Chromatographic conditions
The instrument model is as follows: Agilent-GC2
A chromatographic column: CP-Wax 25 m.times.0.32 mm,1.2 μm, 20190806-GC-01
Temperature rising procedure: the starting temperature was 60 ℃ for 5 minutes, then the temperature was raised to 200 ℃ at a rate of 20 ℃/min for 15 minutes.
A detector: FID
Detector temperature: 300 deg.C
Sample inlet temperature: 250 deg.C
Flow rate: 1.5ml/min
The split ratio is as follows: 15:1
And (3) sample introduction mode: headspace sampling
Headspace equilibrium time: 30 minutes
Headspace equilibrium temperature: 90 deg.C
And (3) quantitative ring: 150 ℃ C
Transmission line: 160 deg.C
2. Solution preparation
0.5mol/L sodium hydroxide solution: 1.02635g of sodium hydroxide is weighed into a 50ml measuring flask, dissolved and diluted to the scale by adding water, and shaken up.
Blank solution: precisely measuring 3ml of dimethyl sulfoxide, adding 2ml of 0.5mol/L sodium hydroxide solution, placing in a top empty bottle, sealing, and shaking up.
Test solution: weighing about 99.7mg of each of the samples with the batch numbers of 201101-1, 201101-2 and 201101-3, placing the samples into a headspace bottle, adding 3ml of dimethyl sulfoxide to dissolve the samples, adding 2ml of 0.5mol/L sodium hydroxide solution, sealing and shaking up the samples.
Di-n-butylamine stock solution: 0.5029g of di-n-butylamine are weighed into a 50ml measuring flask, and dimethyl sulfoxide is added to dilute the solution to the scale and the solution is shaken up.
N, N-diisopropylethylenediamine stock solution: 0.5014g of N, N-diisopropylethylenediamine is weighed and placed in a 50ml measuring flask, dimethyl sulfoxide is added to dilute the mixture to the scale, and the mixture is shaken up.
Control solution: precisely measuring 1ml of the di-N-butylamine stock solution and the N, N-diisopropylethylenediamine stock solution respectively, placing the two solutions into a 20ml measuring flask, adding dimethyl sulfoxide to dilute the solutions to a scale, and shaking the solutions uniformly.
Control solution 1: precisely measuring 1ml of the reference solution, placing in a headspace bottle, adding 2ml of dimethyl sulfoxide, adding 2ml of 0.5mol/L sodium hydroxide solution, sealing, and shaking.
3. Testing
Injecting a sample into the blank solution, the reference solution 1 and the sample solution respectively.
4. Computing
Calculating content by external standard method
5. Results
Comparative example 1
The test results are shown in FIG. 4, in which the concentration of the control (mixed control) was measured at 100. mu.g/m using the test method and conditions disclosed in patent application CN 201610167810.5.
It can be seen from FIG. 4 that N, N-diisopropylethylenediamine and di-N-butylamine did not show peaks.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for determining amine residues in acotiamide hydrochloride bulk drug by gas chromatography comprises the following steps:
A) dissolving acotiamide raw material medicine by adopting a solvent, and adding a strong base solution to obtain a solution to be detected;
dissolving di-n-butylamine by using a solvent, and adding a strong base solution to obtain a di-n-butylamine reference substance solution;
dissolving N, N-diisopropyl ethylenediamine by using a solvent, and adding a strong base solution to obtain an N, N-diisopropyl ethylenediamine reference solution;
B) and (3) measuring the solution to be measured, the di-N-butylamine reference substance solution and the N, N-diisopropylethylenediamine reference substance solution by adopting a headspace-gas chromatography, and quantifying by adopting an external standard method.
2. The method according to claim 1, wherein the detection conditions of the method are that the chromatographic column: a capillary chromatographic column with polyethylene glycol as a solid phase;
a detector: FID; and (3) sample introduction mode: carrying out headspace sample injection; the carrier gas is nitrogen;
temperature programming: the initial temperature is 40-60 ℃, the temperature is maintained for 4-10 min, then the temperature is increased to 200 ℃ at the speed of 15-30 ℃/min, and the temperature is maintained for 10-30 min.
3. The method according to claim 1, wherein the solvent of step a) is dimethyl sulfoxide.
4. The method of claim 1, wherein the strong base of step a) is sodium hydroxide; the concentration of the strong alkali solution is 0.5-1.0 mol/L.
5. The method according to claim 1, wherein the chromatographic column of step B) is CP-Wax or CP 7422; the specification of the chromatographic column is as follows: 25 m.times.0.320 mm.times.1.2. mu.m.
6. The method according to claim 1, wherein the temperature programming of step B) is specifically: the initial temperature is 60 deg.C, and maintained for 5min, and then the temperature is raised to 200 deg.C at a rate of 20 deg.C/min, and maintained for 15 min.
7. The method of claim 1, wherein step B) is performed
The temperature of the detector is 280-320 ℃;
the temperature of a sample inlet is 230-260 ℃;
the equilibrium temperature of the headspace bottle is 90 ℃;
the equilibration time was 30 min.
8. The method of claim 6, wherein the carrier gas flow rate is 1.4 to 1.6 ml/min;
the temperature of a sample inlet is 250 ℃; the detector temperature was 300 ℃.
9. The method according to claim 1, wherein di-n-butylamine has a good linear relationship with peak area in the concentration range of 0.0967 μ g/mL to 201.48 μ g/mL; the linear relation between the peak area and the N, N-diisopropyl ethylenediamine in the concentration range of 2.504 mu g/mL-200.32 mu g/mL is good;
the quantitative limit concentration of the di-n-butylamine is 0.096 mu g/mL, and the quantitative limit is 0.00048%; the quantitative limit concentration of diisopropylethylenediamine is 2.508 μ g/mL, and the quantitative limit is 0.013%.
10. The method of claim 1, wherein the headspace injection is: and (3) split-flow sample injection, wherein the split-flow ratio is 10-20: 1.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116500172A (en) * | 2023-06-29 | 2023-07-28 | 成都普康唯新生物科技有限公司 | Detection method of amine solvent in acidic substrate |
| CN116500172B (en) * | 2023-06-29 | 2023-09-05 | 成都普康唯新生物科技有限公司 | Detection method of amine solvent in acidic substrate |
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