CN113049687A - Method for detecting related substances of ambroxol hydrochloride raw material and injection - Google Patents
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Abstract
The invention provides an ambroxol bulk drug and a preparation with new impurity cis-4- [ [ (2-amino-3, 5-dibromophenyl) methylene ] amino ] cyclohexanol (impurity F); and provides a method for preparing the impurity; the invention also provides a detection method capable of simultaneously detecting six impurities such as impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and the like in the ambroxol bulk drug and the ambroxol injection, and the method is simple and convenient, high in sensitivity and stable; the separation degree of each impurity can reach more than 2.0, six impurities generated in the preparation, production and storage processes of the ambroxol bulk drug and the injection can be effectively detected, and the quality of the ambroxol bulk drug and the injection can be effectively reflected.
Description
Technical Field
The invention belongs to the technical field of drug analysis, and particularly relates to a method for detecting related substances of ambroxol hydrochloride raw materials and injection.
Background
Ambroxol hydrochloride is a new generation of respiratory mucus regulator and has excellent phlegm eliminating effect. Compared with the first and second generation expectorants, the ambroxol hydrochloride has strong mucolytic effect, and is characterized in that the ambroxol hydrochloride can stimulate alveolus II cells and promote the synthesis and secretion of surface active substances of alveolus, thereby effectively enhancing mucus transport and promoting sputum excretion, is the expectorant with the strongest clinical effect at present, can increase the concentration ratio of antibiotic lung tissues/blood plasma, shortens the time of antibiotic treatment and has slight adverse reaction when combined with antibiotics.
The european pharmacopoeia shows that 5 kinds of known impurities of ambroxol hydrochloride are respectively impurity a, impurity B, impurity C, impurity D and impurity E.
Chinese patent CN102262135A discloses a method for detecting impurities in ambroxol hydrochloride injection and a method for measuring the content of the impurities. The method is mainly used for detecting the impurity B and the impurity E in the ambroxol hydrochloride injection, and can not simultaneously detect five impurities (the impurity A, the impurity B, the impurity C, the impurity D and the impurity E).
Chinese patent CN106950291A discloses a method for detecting related substances in an ambroxol hydrochloride injection, which can detect five impurities in the ambroxol hydrochloride injection, and adopts gradient elution, and simultaneously succinic acid needs to be added into a mobile phase (PH 4.0), so that the gradient elution procedure is complicated, the requirements on instrument configuration are high, and the method setting is complex, therefore, the technique is not suitable for being applied to industrial production.
The quality control is needed in the preparation for related substances generated by the raw material process and degradation, so that the separation and analysis of the ambroxol hydrochloride and impurities thereof are realized by continuously analyzing the ambroxol raw material and the impurities in the preparation, and the method has important significance for the quality control of the amino acid ambroxol raw material and the preparation. In the existing detection method for related substances of ambroxol hydrochloride raw materials and preparations, most of the related substances only detect one or two impurities, and gradient elution is adopted, so that a plurality of impurities cannot be detected simultaneously. Therefore, research and improvement on impurities in ambroxol raw material medicines and preparations and detection methods thereof are still needed, so that the quality of the ambroxol raw materials and preparations is effectively controlled, and the clinical curative effect and the medication safety are ensured.
Disclosure of Invention
In order to overcome the defects in the prior art, the inventor conducts analysis, a large amount of experimental research and exploration through the preparation process of the ambroxol bulk drug, and finds that: in ambroxol bulk drug and preparation, impurity F may also exist, which is not mentioned in pharmacopoeia standards such as British Pharmacopoeia (BP), United States Pharmacopoeia (USP), European Pharmacopoeia (EP), Chinese pharmacopoeia and the like.
In view of the fact that the structure of the impurity F found in the ambroxol bulk drug preparation process is lack of clear characterization, and the synthesis preparation method and the analysis detection method are also lack, the invention researches the impurity F, designs the synthesis method to prepare the impurity F, and identifies the structure of the impurity F. The impurity F is a cis-form process impurity generated in the ambroxol hydrochloride raw material synthesis process, the research on the impurity is increased, and the medicine impurity can be more comprehensively controlled from a source system, so that the quality of the medicine is improved and ensured.
In view of six possible impurities, namely impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and the like, in the ambroxol bulk drug, effective, rapid and comprehensive detection of the contents of the impurities has significant significance for quality control of the ambroxol bulk drug and the ambroxol hydrochloride injection. The inventor finally develops a new method which is simple, easy to operate, comprehensive and efficient, is suitable for industrial production and can simultaneously detect six impurities of ambroxol hydrochloride raw materials or injection through carrying out experimental research on the flowing phase ratio, the pH value, the flow rate, the Chinese pharmacopoeia, other detection methods and the like and failing for many times, and the separation degree of adjacent peaks reaches more than 2.0.
The invention provides an impurity in ambroxol raw material or injection, named as impurity F, with chemical name of cis-4- [ [ (2-amino-3, 5-dibromophenyl) methylene]Amino group]Cyclohexanol of formula C13H16Br2N2O has the structure shown in the following formula I.
In another aspect, the invention provides a process for the preparation of cis-4- [ [ (2-amino-3, 5-dibromophenyl) methylene ] amino ] cyclohexanol.
A method for preparing cis-4- [ [ (2-amino-3, 5-dibromophenyl) methylene ] amino ] cyclohexanol comprises the following steps:
the preparation method comprises the following steps: adding 2-amino-3, 5-dibromobenzaldehyde, cis-4-aminocyclohexanol hydrochloride and methanol into a reaction kettle; stirring and reacting for 8-10h at 50-55 ℃, drying under reduced pressure to obtain a crude product, and recrystallizing the crude product by using acetone to obtain a final product cis-4- [ [ (2-amino-3, 5-dibromophenyl) methylene ] amino ] cyclohexanol.
Further:
the amount of 2-amino-3, 5-dibromobenzaldehyde to be used may be 1.1 to 2.0 molar equivalents, preferably 1.2 to 1.4 molar equivalents, relative to cis-4-aminocyclohexanol hydrochloride.
The weight ratio of the 2-amino-3, 5-dibromobenzaldehyde to the methanol is 1: 5-50, and the preferable weight ratio is 1: 10-20.
The purity of the final product cis-4- [ [ (2-amino-3, 5-dibromophenyl) methylene ] amino ] cyclohexanol is not less than 99%, preferably not less than 99.5%; more preferably not less than 99.6%.
Further, the invention provides application of cis-4- [ [ (2-amino-3, 5-dibromophenyl) methylene ] amino ] cyclohexanol (impurity F, the same below) in quality control of ambroxol bulk drug and ambroxol injection.
In some embodiments, the impurity F provided by the present invention is used as a quality control standard in ambroxol bulk drug and injection analysis assays.
The invention provides a detection method for comprehensively and accurately detecting six impurities in ambroxol, which is convenient to operate, and has the following structures. The method can more effectively control the quality of the ambroxol hydrochloride raw material and the preparation.
A detection method for determining impurities A, B, C, D, E and F in ambroxol hydrochloride raw materials and injection by using high performance liquid chromatography comprises the following steps:
1) a chromatographic column: octane silane bonded silica gel is used as a filling agent, the specification is 4.6 multiplied by 250mm, and the grain diameter of the filling agent is 5 mu m;
2) a detector: an ultraviolet detector;
3) mobile phase: ph6.8-7.2 phosphate buffer-acetonitrile volume ratio: 48-52: 48-52;
4) detection wavelength: 248 nm; column temperature: 28-32 ℃; flow rate: 0.8 ml/min; and (3) elution: isocratic elution; sample introduction volume: 20 μ L.
5) The preparation and determination method of the reference substance solution comprises the following steps: accurately weighing a proper amount of reference substances of ambroxol hydrochloride, impurity A, impurity B, impurity C, impurity D, impurity E and impurity F into the same volumetric flask, adding methanol to dissolve and dilute the reference substances into reference substance solution of ambroxol, impurity A, impurity B, impurity C, impurity F, impurity D and impurity E; injecting the reference solution into a liquid chromatograph according to the chromatographic conditions, wherein the peak appearance sequence comprises impurity B, impurity A, ambroxol, impurity D, impurity E, impurity C and impurity F; the degree of separation of adjacent impurity peaks was not less than 2.0.
6) Preparation of a test solution: precisely taking out a proper amount of ambroxol hydrochloride raw material medicine or ambroxol hydrochloride injection, and adding methanol for dilution to serve as a test solution; injecting the sample solution into a liquid chromatograph according to the chromatographic conditions; calculating the contents of impurity A, impurity B, impurity C, impurity D, impurity E and impurity F by peak area according to an external standard method
Further:
the phosphate buffer solution is diammonium hydrogen phosphate solution with the concentration of 1.0-1.5 per mill; adjusting the pH value to 6.8-7.2 by using phosphoric acid; preferably 1.3-1.4 per mill of diammonium hydrogen phosphate solution; more preferably 1.32% diammonium hydrogen phosphate solution.
The mobile phase preferably has a pH value of 6.8-7.2 phosphate buffer solution-acetonitrile volume ratio: 50: 50; more preferably ph7.0 phosphate buffer to acetonitrile volume ratio: 50:50.
The preparation of the test solution comprises the following steps:
preparing an ambroxol hydrochloride raw material test solution: precisely weighing ambroxol hydrochloride raw material medicine, adding methanol to dilute into 1.0mg/ml solution as a test solution; sucking 20 μ L of sample solution, and injecting into liquid chromatograph; and calculating the contents of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E and the impurity F by peak areas according to an external standard method.
Preparing a test solution of ambroxol hydrochloride injection: precisely measuring 1ml of ambroxol hydrochloride injection, placing the ambroxol hydrochloride injection in a 100ml measuring flask, adding methanol to dissolve the ambroxol hydrochloride injection and diluting the ambroxol hydrochloride injection to a scale mark to obtain a test solution; sucking 20 μ L of sample solution, and injecting into liquid chromatograph; and calculating the contents of the impurity A, the impurity B, the impurity C, the impurity F, the impurity D and the impurity E by peak areas according to an external standard method.
The invention has the beneficial effects that:
1) the inventor carries out a great deal of experimental research and grope through the preparation process of ambroxol bulk drug, and finds that: a new impurity cis-4- [ [ (2-amino-3, 5-dibromophenyl) methylene ] amino ] cyclohexanol (impurity F) exists in an ambroxol bulk drug and a preparation; the preparation method of the impurity is simple to operate, the product purity is high, and the impurity is applied to the analysis and determination of ambroxol bulk drugs and injection as a quality control standard.
2) The invention provides a detection method capable of detecting six impurities in ambroxol bulk drug and ambroxol injection, and the detection method provided by the invention adopts equal elution without adding other reagents through the verification of a systematic analysis method, so that the method is simple and convenient, the sensitivity is high, and the detection method is stable; the separation degree of each impurity can reach more than 2.0, six impurities generated in the preparation, production and storage processes of the ambroxol bulk drug and the injection can be effectively detected, and the quality of the ambroxol bulk drug and the injection can be effectively reflected.
Drawings
FIG. 1: inventive example 1 impurity F nuclear magnetic spectrum was prepared.
FIG. 2: mass spectrum of impurity F prepared in example 1 of the present invention
FIG. 3: chromatogram of impurity F prepared in inventive example 1
FIG. 4: comparative example 4 chromatogram of impurity B detection method
FIG. 5: comparative example 4 chromatogram of detection method of impurity C
FIG. 6: comparative example 4 chromatogram of impurity E detection method
FIG. 7: comparative example 4 chromatogram of impurity F detection method
FIG. 8: comparative example 5 detection method chromatogram
FIG. 9: comparative example 6 detection method chromatogram
FIG. 10: comparative example 7 detection method chromatogram
FIG. 11: comparative example 8 detection method chromatogram
FIG. 12: comparative example 9 detection method chromatogram
FIG. 13: comparative example 10 detection method chromatogram
FIG. 14: example 1 detection method chromatogram
FIG. 15: example 12 detection method chromatogram
FIG. 16 chromatogram of detection method in example 13
In the map:
VWD, Single A, ultraviolet detector signal; 248nm, 250 nm: detecting the wavelength; retention Time: a retention time; area: peak area; area%: peak area percentage; the Theoretical plants: the number of theoretical plates; resolution: degree of separation.
Detailed Description
Comparative example 1 Synthesis of impurity F
In a reaction kettle, 23.9g (0.086eq) of 2-amino-3, 5-dibromobenzaldehyde, 10g (0.066eq) of cis-4-aminocyclohexanol hydrochloride and 350ml of ethyl acetate are added; stirring and reacting for 9h at 52 ℃ to obtain a crude product, and recrystallizing the crude product by using acetone to obtain 13.9g of a final product cis-4- [ [ (2-amino-3, 5-dibromophenyl) methylene ] amino ] cyclohexanol with the molar yield of 55.8%. The purity was 95.31%.
Comparative example 2 Synthesis of impurity F
In a reaction kettle, 23.9g (0.086eq) of 2-amino-3, 5-dibromobenzaldehyde, 10g (0.066eq) of cis-4-aminocyclohexanol hydrochloride and 350ml of methanol are added; stirring and reacting for 9h at 45 ℃ to obtain a crude product, and recrystallizing the crude product by using acetone to obtain 18.3g of a final product cis-4- [ [ (2-amino-3, 5-dibromophenyl) methylene ] amino ] cyclohexanol with the molar yield of 73.8%. The purity was 98.14%.
Comparative example 3 Synthesis of impurity F
In a reaction kettle, 23.9g (0.086eq) of 2-amino-3, 5-dibromobenzaldehyde, 10g (0.066eq) of cis-4-aminocyclohexanol hydrochloride and 350ml of methanol are added; stirring and reacting for 9h at the temperature of 60 ℃ to obtain a crude product, and recrystallizing the crude product by using acetone to obtain 20.7g of a final product cis-4- [ [ (2-amino-3, 5-dibromophenyl) methylene ] amino ] cyclohexanol with the molar yield of 83.2%. The purity is 98.87%.
Comparative example 4: detection method
The detection was carried out under the following chromatographic conditions.
1) A chromatographic column: bonding octyl silane as filler; specification 4.6X 250mm, filler particle size 5 μm;
2) a detector: an ultraviolet detector;
2) mobile phase: 2mg/ml diammonium phosphate (pH adjusted to 4.0 with phosphoric acid) -methanol (48: 52);
3) the detection wavelength is 250 nm; the column temperature is 30 ℃; the flow rate is 1.0 ml/min; isocratic elution;
5) preparation of control solutions:
preparing an ambroxol hydrochloride impurity B reference substance solution: precisely weighing about 11.25mg of the reference substance of the ambroxol hydrochloride impurity B into a 100ml measuring flask, dissolving and diluting the reference substance into a scale by using a mobile phase, and shaking up to a constant volume to obtain a reference substance solution; 20. mu.L of the control solution was aspirated and injected into a liquid chromatograph. The liquid chromatogram is shown in FIG. 4.
Preparing an ambroxol hydrochloride impurity C reference substance solution: precisely weighing about 5mg of the reference substance of the ambroxol hydrochloride impurity C into a 25ml measuring flask, dissolving and diluting the reference substance to a scale by using a mobile phase, and shaking up to a constant volume to obtain a reference substance solution; 20. mu.L of the control solution was aspirated and injected into a liquid chromatograph. The liquid chromatogram is shown in FIG. 5.
Preparing an ambroxol hydrochloride impurity E reference substance solution: precisely weighing about 15mg of the reference substance of the ambroxol hydrochloride impurity E into a 100ml measuring flask, dissolving and diluting the reference substance to a scale by using a mobile phase, and shaking up to a constant volume to obtain a reference substance solution; 20. mu.L of the control solution was aspirated and injected into a liquid chromatograph. The liquid chromatogram is shown in FIG. 6.
Preparing an ambroxol hydrochloride impurity F reference substance solution: precisely weighing about 10mg of an ambroxol hydrochloride impurity F reference substance (prepared in example 1) in a measuring flask of 100ml, dissolving and diluting the reference substance to a scale by using a mobile phase, and shaking up to a constant volume to obtain a reference substance solution; 20. mu.L of the control solution was aspirated and injected into a liquid chromatograph. The liquid chromatogram is shown in FIG. 7
FIG. 4: impurity B peaks were 2, with retention times of 11.150 minutes and 23.917 minutes respectively. The mobile phase is adopted to dissolve the impurity reference substance, so that the stability of the impurity B is obviously influenced, the impurity B is degraded, and 2 peaks appear.
FIG. 5: under the chromatographic conditions, no impurity C peak exists. Impurity C is unstable under these conditions and cannot be detected.
FIG. 6: under the chromatographic check, no impurity E peak exists. Impurity E is unstable under these conditions and cannot be detected.
FIG. 7: under this chromatographic condition, the impurity F peak was abnormal. The impurity E is unstable under such conditions and cannot be accurately detected
From comparative example 4 it can be seen that: under the chromatographic condition, the content of each impurity cannot be accurately quantified, and the method is not suitable for detecting related substances in ambroxol raw materials and injection.
Comparative example 5: detection method
A method for detecting related substances of ambroxol hydrochloride bulk drugs in 'Chinese pharmacopoeia' 2015 edition is adopted;
1) a chromatographic column: octadecylsilane chemically bonded silica is used as a filler, the specification is 4.6mm multiplied by 250mm, and the particle size of the filler is 5 mu m;
2) a detector: an ultraviolet detector;
3) mobile phase: 0.01mol/L diammonium hydrogen phosphate solution (pH adjusted to 7.0 with phosphoric acid) -acetonitrile (50: 50) as mobile phase;
4) the detection wavelength is 248 nm; the flow rate is 1.0 ml/min; isocratic elution;
5) preparation of control solutions: accurately weighing appropriate amounts of an ambroxol hydrochloride reference substance, an impurity A, an impurity B, an impurity D and an impurity E reference substance into the same 100ml measuring flask, dissolving and diluting the reference substance to a scale by adding methanol, and shaking up the solution with constant volume to obtain a reference substance solution of ambroxol, the impurity A, the impurity B, the impurity D and the impurity E; sucking 20 μ L of reference solution, injecting into a liquid chromatograph, and collecting the reference solution containing impurity B, impurity A, ambroxol, impurity D and impurity E; the liquid chromatogram is shown in FIG. 8.
The peak separation degrees of impurity B and impurity A were 0.91, which was unsatisfactory.
Comparative example 6: detection method
The detection was carried out under the following chromatographic conditions.
1) A chromatographic column: octadecylsilane chemically bonded silica is used as a filler, the specification is 4.6mm multiplied by 250mm, and the particle size of the filler is 5 mu m;
2) a detector: an ultraviolet detector;
3) mobile phase: 0.01mol/L diammonium hydrogen phosphate solution (pH adjusted to 7.0 with phosphoric acid) -acetonitrile (50: 50) as mobile phase;
4) the detection wavelength is 248 nm; the flow rate is 0.8 ml/min; isocratic elution;
5) preparation of control solutions: accurately weighing appropriate amounts of an ambroxol hydrochloride reference substance, an impurity A, an impurity B, an impurity D and an impurity E reference substance into the same 100ml measuring flask, dissolving and diluting the reference substance to a scale by adding methanol, and shaking up the solution with constant volume to obtain a reference substance solution of ambroxol, the impurity A, the impurity B, the impurity D and the impurity E; sucking 20 μ L of reference solution, injecting into a liquid chromatograph, and collecting the reference solution containing impurity B, impurity A, ambroxol, impurity D and impurity E; the liquid chromatogram is shown in FIG. 9.
The peak separation of impurity B and impurity a was 1.14; the degree of separation is not satisfactory.
As can be seen from comparative examples 4 and 5: the chromatographic column has great influence on the detection of impurities, and the separation degree of the impurity B and the impurity A cannot meet the requirement by using the chromatographic column with octadecylsilane chemically bonded silica as a filler, so that the contents of the impurity B and the impurity A in the ambroxol raw material and the injection cannot be accurately detected; is not suitable for detecting related substances in ambroxol raw materials and injection.
Comparative example 7: detection method
The detection was carried out under the following chromatographic conditions.
1) A chromatographic column: the octyl silane is bonded into a filler with the specification of 4.6 multiplied by 250mm and the particle size of 5 mu m;
2) a detector: an ultraviolet detector;
3) mobile phase: 0.01mol/L diammonium phosphate solution (pH adjusted to 6.5 with phosphoric acid) -acetonitrile (50: 50);
4) detection wavelength: 248 nm; column temperature: 30 ℃; flow rate: 1.0 ml/min; and (3) elution: isocratic elution.
5) Preparation of control solutions: accurately weighing appropriate reference substances of ambroxol hydrochloride, impurity A, impurity B, impurity C, impurity D, impurity E and impurity F into the same 100ml measuring flask, dissolving with methanol, diluting to scale, and shaking to constant volume to obtain reference substance solution of ambroxol, impurity A, impurity B, impurity C, impurity D, impurity E and impurity F; sucking 20 μ L of reference solution, injecting into a liquid chromatograph, and collecting the reference solution containing impurity B, impurity A, ambroxol, impurity D, impurity E, impurity C and impurity F; the liquid chromatogram is shown in FIG. 10.
As can be seen in fig. 10: six impurities can be detected simultaneously, and the peak separation degree of the impurity B and the impurity A is 0.60.
Comparative example 8: the detection of the relevant substances was carried out under the following chromatographic conditions
1) A chromatographic column; 2) a detector; 3) comparative example 7 with the same flow;
4) detection wavelength: 248 nm; column temperature: 30 ℃; flow rate: 0.8 ml/min; and (3) elution: isocratic elution.
5) Preparation of control solutions: accurately weighing appropriate reference substances of ambroxol hydrochloride, impurity A, impurity B, impurity C, impurity D, impurity E and impurity F into the same 100ml measuring flask, dissolving with methanol, diluting to scale, and shaking to constant volume to obtain reference substance solution of ambroxol, impurity A, impurity B, impurity C, impurity D, impurity E and impurity F; sucking 20 μ L of reference solution, injecting into a liquid chromatograph, and collecting the reference solution containing impurity B, impurity A, ambroxol, impurity D, impurity E, impurity C and impurity F; the liquid chromatogram is shown in FIG. 11.
As can be seen in fig. 11: six impurities can be detected simultaneously, and the peak separation degree of the impurity B and the impurity A is 0.69.
Comparative example 9: the detection of the relevant substances was carried out under the following chromatographic conditions
1) A chromatographic column: the octyl silane is bonded into a filler with the specification of 4.6 multiplied by 250mm and the particle size of 5 mu m;
2) a detector: an ultraviolet detector;
3) mobile phase: 0.01mol/L diammonium phosphate solution (pH adjusted to 7.0 with phosphoric acid) -acetonitrile (45: 55);
4) the detection wavelength is 248 nm; the column temperature is 30 ℃; the flow rate is 0.8 ml/min; and (3) elution: isocratic elution.
5) Preparation of control solutions: accurately weighing appropriate reference substances of ambroxol hydrochloride, impurity A, impurity B, impurity C, impurity D, impurity E and impurity F into the same 100ml measuring flask, dissolving with methanol, diluting to scale, and shaking to constant volume to obtain reference substance solution of ambroxol, impurity A, impurity B, impurity C, impurity D, impurity E and impurity F; sucking 20 μ L of reference solution, injecting into a liquid chromatograph, and collecting the reference solution containing impurity B, impurity A, ambroxol, impurity D, impurity E, impurity C and impurity F; the liquid chromatogram is shown in FIG. 12.
As can be seen from fig. 12: the separation degrees of the impurity B and the impurity A are 1.34, and the separation degrees of the impurity B and the ambroxol are 1.74, which is not satisfactory.
Comparative example 10: the detection of the relevant substances was carried out under the following chromatographic conditions
1) A chromatographic column; 2) detector same as and comparative example 9
3) Mobile phase: 0.01mol/L diammonium phosphate solution (pH adjusted to 7.0 with phosphoric acid) -acetonitrile (55: 45);
4) the detection wavelength is 248 nm; the column temperature is 30 ℃; the flow rate is 0.8 ml/min; and (3) elution: isocratic elution.
5) Preparation of control solutions: accurately weighing appropriate reference substances of ambroxol hydrochloride, impurity A, impurity B, impurity C, impurity D, impurity E and impurity F into the same 100ml measuring flask, dissolving with methanol, diluting to scale, and shaking to constant volume to obtain reference substance solution of ambroxol, impurity A, impurity B, impurity C, impurity D, impurity E and impurity F; sucking 20 μ L of reference solution, injecting into a liquid chromatograph, and collecting the reference solution containing impurity B, impurity A, ambroxol, impurity D, impurity E, impurity C and impurity F; the liquid chromatogram is shown in FIG. 13.
As can be seen from fig. 13: the peak separation of impurity B and impurity a was 1.97, the separation was unsatisfactory.
Example 1: synthesis and Structure confirmation of impurity F
In a reaction kettle, 23.9g (0.086eq) of 2-amino-3, 5-dibromobenzaldehyde, 10g (0.066eq) of cis-4-aminocyclohexanol hydrochloride and 350ml of methanol are added; stirring and reacting for 9h at 52 ℃ to obtain a crude product, and recrystallizing the crude product by using acetone to obtain 18.5g of a final product cis-4- [ [ (2-amino-3, 5-dibromophenyl) methylene ] amino ] cyclohexanol with the molar yield of 74.6%. The purity is 99.73%. The nuclear magnetic spectrum, mass spectrum and chromatogram are shown in FIG. 1, FIG. 2 and FIG. 3.
Example 2: synthesis of impurity F
22.1g (0.079eq) of 2-amino-3, 5-dibromobenzaldehyde, 10g (0.066eq) of cis-4-aminocyclohexanol hydrochloride and 220ml of methanol are added into a reaction kettle; stirring and reacting for 8h at 55 ℃ to obtain a crude product, and recrystallizing the crude product by using acetone to obtain 18.1g of a final product cis-4- [ [ (2-amino-3, 5-dibromophenyl) methylene ] amino ] cyclohexanol with a molar yield of 72.8%. The purity is 99.68%.
Example 3: synthesis of impurity F
In a reaction kettle, 25.8g (0.092eq) of 2-amino-3, 5-dibromobenzaldehyde, 10g (0.066eq) of cis-4-aminocyclohexanol hydrochloride and 510ml of methanol are added; stirring and reacting for 10h at 50 ℃ to obtain a crude product, and recrystallizing the crude product by using acetone to obtain 16.7g of a final product cis-4- [ [ (2-amino-3, 5-dibromophenyl) methylene ] amino ] cyclohexanol with the molar yield of 67.4%. The purity is 99.71%.
Example 4: the detection method of the invention is a system applicability test
1) A chromatographic column: the octyl silane is bonded into a filler with the specification of 4.6 multiplied by 250mm and the particle size of 5 mu m;
2) a detector: an ultraviolet detector;
3) mobile phase: pH7.0 phosphate buffer (1.32% diammonium phosphate solution, pH adjusted to 7.0 with phosphoric acid) -acetonitrile volume ratio: 50: 50;
4) detection wavelength: 248 nm; column temperature: 30 ℃; flow rate: 0.8 ml/min; and (3) elution: isocratic elution.
5) Preparation of control solutions: accurately weighing appropriate reference substances of ambroxol hydrochloride, impurity A, impurity B, impurity C, impurity D, impurity E and impurity F into the same 100ml measuring flask, dissolving with methanol, diluting to scale, and shaking to constant volume to obtain reference substance solution of ambroxol, impurity A, impurity B, impurity C, impurity D, impurity E and impurity F; sucking 20 μ L of reference solution, injecting into a liquid chromatograph, and collecting the reference solution containing impurity B, impurity A, ambroxol, impurity D, impurity E, impurity C and impurity F; the degrees of separation of adjacent impurity peaks are not less than 2.0. The liquid chromatogram is shown in FIG. 14.
Table 1: retention time of each impurity peak
| Name (R) | Retention time (minutes) |
| Ambroxol hydrochloride impurity B | 7.518 |
| Ambroxol hydrochloride impurity A | 8.445 |
| Ambroxol hydrochloride | 9.304 |
| Ambroxol hydrochloride impurity D | 10.930 |
| Ambroxol hydrochloride impurity E | 17.278 |
| Ambroxol hydrochloride impurity C | 25.461 |
| Ambroxol hydrochloride impurity F | 28.479 |
Example 5: the detection method of the invention is a specificity test
The chromatographic conditions (column, detector, mobile phase, detection wavelength, column temperature, flow rate, elution, sample volume) were the same as in example 4.
Blank solvent: methanol and water
Ambroxol hydrochloride impurity A, impurity B, impurity C, impurity D, impurity E and impurity F reference stock solution: precisely weighing about 10mg of each impurity reference substance of ambroxol hydrochloride, respectively placing the reference substances into a 100ml measuring flask, adding methanol to dissolve and dilute the reference substances to a scale, and shaking up to obtain the ambroxol hydrochloride.
Ambroxol hydrochloride reference solution: precisely weighing 25mg of ambroxol hydrochloride reference substance, placing the ambroxol hydrochloride reference substance into a 25ml measuring flask, adding methanol to dissolve and dilute the reference substance to a scale, and shaking up the solution to obtain the ambroxol hydrochloride reference substance.
Ambroxol hydrochloride impurity A, impurity B, impurity C, impurity D, impurity E and impurity F reference solution: precisely measuring 1ml of reference substance stock solution of each impurity of ambroxol hydrochloride, respectively placing into 100ml measuring flasks, adding methanol for dissolving, diluting to scale, and shaking uniformly to obtain the ambroxol hydrochloride.
Adding a standard test solution: taking 100mg of ambroxol hydrochloride raw material medicine, precisely weighing, placing in a 100ml measuring flask, precisely weighing 1ml of ambroxol hydrochloride impurity A, impurity B, impurity C, impurity D, impurity E and impurity F stock solution respectively, placing in the same 100ml measuring flask, diluting to scale with methanol, and uniformly shaking to a constant volume.
Table 2: specificity test data
And (4) conclusion: the blank solvent has no interference to the impurity determination; the separation degrees of the ambroxol hydrochloride in the sample solution to be tested, the adjacent impurity peaks and the impurity peaks are all more than 2.0. The retention time of the ambroxol hydrochloride and each impurity in the reference solution is consistent with that of the added standard test solution. The method provided by the invention has specificity in detecting ambroxol bulk drug.
Example 6: the detection method of the invention is a specificity test
The chromatographic conditions (column, detector, mobile phase, detection wavelength, column temperature, flow rate, elution, sample volume) were the same as in example 4.
Blank solution of auxiliary materials: according to the prescription of the ambroxol hydrochloride injection, a proper amount of sodium chloride, citric acid and disodium hydrogen phosphate are taken to prepare an auxiliary material solution for testing.
Ambroxol hydrochloride impurity A, impurity B, impurity C, impurity D, impurity E and impurity F reference stock solution: precisely weighing about 10mg of each impurity reference substance of ambroxol hydrochloride, respectively placing the reference substances into a 100ml measuring flask, adding methanol to dissolve and dilute the reference substances to a scale, and shaking up to obtain the ambroxol hydrochloride.
Ambroxol hydrochloride reference solution: precisely weighing 25mg of ambroxol hydrochloride reference substance, placing the ambroxol hydrochloride reference substance into a 25ml measuring flask, adding methanol to dissolve and dilute the reference substance to a scale, and shaking up the solution to obtain the ambroxol hydrochloride reference substance.
Ambroxol hydrochloride impurity A, impurity B, impurity C, impurity F, impurity D and impurity E reference substance solution: precisely measuring 1ml of reference substance stock solution of each impurity of ambroxol hydrochloride, respectively placing into 100ml measuring flasks, adding methanol for dissolving, diluting to scale, and shaking uniformly to obtain the ambroxol hydrochloride.
Adding a standard test solution: precisely measuring 5ml of ambroxol hydrochloride injection, placing the ambroxol hydrochloride injection into a 100ml measuring flask, precisely measuring 1ml of each stock solution of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E and the impurity F of the ambroxol hydrochloride, placing the ambroxol hydrochloride injection, the impurity D, the impurity E and the impurity F into the same 100ml measuring flask, diluting the ambroxol hydrochloride injection to a scale with methanol, and shaking up to a constant volume.
Table 3: specificity test data
And (4) conclusion: the blank auxiliary material solution has no interference to the impurity determination; the separation degrees of the ambroxol hydrochloride in the sample solution to be tested, the adjacent impurity peaks and the impurity peaks are all more than 2.0. The retention time of the ambroxol hydrochloride and each impurity in the reference solution is consistent with that of the added standard test solution. . The method provided by the invention has specificity in detecting the ambroxol injection.
Example 7: forced degradation test
The chromatographic conditions (column, detector, mobile phase, detection wavelength, column temperature, flow rate, elution, sample volume) were the same as in example 4.
Carrying out forced degradation experiments under the conditions of high temperature, acid, alkali, oxidation and illumination on the ambroxol hydrochloride bulk drug, detecting a sample solution which is not degraded and degraded, and carrying out peak purity inspection on the ambroxol hydrochloride peak so as to confirm that chromatographic peaks of main components of all samples do not contain other component peaks.
After the ambroxol hydrochloride raw material is treated by the following table, the solution containing about 1mg of ambroxol hydrochloride in 1ml is prepared by diluting with methanol.
TABLE 4 forced degradation test conditions and solution preparation
TABLE 5 forced degradation test data
And (4) conclusion: the peak purity of the main peak of the forced degradation sample is 100 percent; the sum of the content of the main component and the content of the total impurities is compared with the sum of the content of the undegraded sample and the total impurities, and the ratio is 100 percent. After the ambroxol bulk drug is forcedly degraded, the detection method provided by the invention can detect forcedly degraded impurities.
Example 8: linear and range test
The chromatographic conditions (column, detector, mobile phase, detection wavelength, column temperature, flow rate, elution, sample volume) were the same as in example 4.
Preparation of solution
Ambroxol hydrochloride impurity a reference stock solution: accurately weighing about 10mg of the reference substance of the ambroxol hydrochloride impurity A, placing the reference substance into a 100ml measuring flask, adding methanol to dissolve and dilute the reference substance to a scale, and shaking up the reference substance to obtain the ambroxol hydrochloride impurity A. (0.1mg/ml)
Ambroxol hydrochloride impurity B reference stock solution: accurately weighing about 10mg of reference substance of ambroxol hydrochloride impurity B, placing the reference substance in a 100ml measuring flask, adding methanol to dissolve and dilute the reference substance to a scale, and shaking up the reference substance to obtain the ambroxol hydrochloride impurity B. (0.1mg/ml)
Ambroxol hydrochloride impurity C reference stock solution: accurately weighing about 10mg of reference substance of ambroxol hydrochloride impurity C, placing the reference substance into a 100ml measuring flask, adding methanol to dissolve and dilute the reference substance to a scale, and shaking up the reference substance to obtain the ambroxol hydrochloride impurity C. (0.1mg/ml)
Ambroxol hydrochloride impurity F control stock solution: precisely weighing about 10mg of the reference substance of the ambroxol hydrochloride impurity F, placing the reference substance into a 100ml measuring flask, adding methanol to dissolve and dilute the reference substance to a scale, and shaking up the reference substance to obtain the ambroxol hydrochloride impurity F. (0.1mg/ml)
Ambroxol hydrochloride impurity D reference solution: accurately weighing about 10mg of reference substance of ambroxol hydrochloride impurity D, placing the reference substance into a 100ml measuring flask, adding methanol to dissolve and dilute the reference substance to a scale, and shaking up the reference substance to obtain the ambroxol hydrochloride impurity D. (0.1mg/ml)
Ambroxol hydrochloride impurity E reference substance solution: accurately weighing about 10mg of reference substance of ambroxol hydrochloride impurity E, placing the reference substance into a 100ml measuring flask, adding methanol to dissolve and dilute the reference substance to a scale, and shaking up the reference substance to obtain the ambroxol hydrochloride impurity E. (0.1mg/ml)
Linear stock solution: precisely measuring 1ml of the ambroxol hydrochloride control solution, 1ml of the ambroxol hydrochloride impurity A, 1ml of the ambroxol hydrochloride impurity B, 1ml of the ambroxol hydrochloride impurity C, 1ml of the ambroxol hydrochloride impurity D, 1ml of the ambroxol hydrochloride impurity E and 1ml of the ambroxol hydrochloride impurity F into the same 50ml measuring flask, diluting the solution to a scale with methanol, and carrying out constant volume shaking to obtain a linear stock solution.
Linear 1 (20%): precisely measuring 2ml of linear 5 solution, placing the linear 5 solution into a 10ml measuring flask, adding methanol to dilute the linear 5 solution to a scale, and shaking up the linear 5 solution to obtain the water-soluble chitosan.
Linear 2 (40%): weighing 4ml of linear 5 solution, placing the linear 5 solution into a 10ml measuring flask, adding methanol to dilute the linear 5 solution to the scale, and shaking up the linear 5 solution to obtain the water-soluble chitosan.
Linear 3 (60%): weighing 6ml of linear 5 solution, placing the linear 5 solution into a 10ml measuring flask, adding methanol to dilute the linear 5 solution to the scale, and shaking up the linear 5 solution to obtain the water-soluble chitosan.
Linear 4 (80%): weighing 8ml of linear 5 solution, placing the linear 5 solution into a 10ml measuring flask, adding methanol to dilute the linear 5 solution to the scale, and shaking up the linear 5 solution to obtain the water-soluble chitosan.
Linear 5 (100%): and (3) placing 5ml of the precise linear stock solution into a 10ml measuring flask, adding methanol to dilute to the scale, and shaking up to obtain the product.
Linear 6 (200%): the linear stock solution was a linear 6 solution.
(II) Linear test data
Table 6: data of linear test
And (4) conclusion: the peak area of each impurity and the concentration thereof form a good linear relation, and the correlation coefficient r is more than 0.99.
Example 9: detection limit and quantification limit
The chromatographic conditions (column, detector, mobile phase, detection wavelength, column temperature, flow rate, elution, sample volume) were the same as in example 4.
Precisely transferring a proper amount of the ambroxol hydrochloride reference solution and each impurity reference linear stock solution in the embodiment 8, gradually diluting with methanol, measuring, recording a chromatogram, and measuring the S/N (signal-to-noise ratio, the same below) of each impurity peak; when the S/N is 3-10, the corresponding concentration is the detection limit concentration; when the S/N is more than or equal to 10, the corresponding concentration is the quantitative limit concentration. The data are as follows:
table 7: limit of detection experimental data
| Sample (I) | Percentage content (%) | Concentration (μ g/ml) | Signal-to-noise ratio (S/N) |
| Ambroxol hydrochloride | 0.0015 | 0.015 | 4.5 |
| Ambroxol hydrochloride impurity A | 0.0020 | 0.020 | 8.3 |
| Ambroxol hydrochloride impurity B | 0.0042 | 0.042 | 5.0 |
| Ambroxol hydrochloride impurity C | 0.0104 | 0.104 | 6.0 |
| Ambroxol hydrochloride impurity D | 0.0044 | 0.044 | 3.8 |
| Ambroxol hydrochloride impurity E | 0.0041 | 0.041 | 3.5 |
| Ambroxol hydrochloride impurity F | 0.0106 | 0.106 | 5.0 |
Table 8: quantitative limit experimental data
| Sample (I) | Percentage content (%) | Concentration (μ g/ml) | Signal-to-noise ratio (S/N) |
| Ambroxol hydrochloride | 0.0030 | 0.030 | 17 |
Table 9: quantitative limit precision experimental data
| |
1 |
2 nd needle | No. 3 needle | The 4 th needle | The 5 th needle | The 6 th needle | RSD(％) |
| Ambroxol hydrochloride | 23403 | 21912 | 22320 | 22944 | 22447 | 21784 | 2.8 |
Example 10: accuracy test
The chromatographic conditions (column, detector, mobile phase, detection wavelength, column temperature, flow rate, elution, sample volume) were the same as in example 4.
Preparation of solution
Preparation of a test solution: taking about 25mg of ambroxol hydrochloride, precisely weighing, placing in a 25ml measuring flask, adding methanol for dissolving, diluting to scale, and shaking up to obtain the ambroxol hydrochloride.
Accuracy stock solution: precisely measuring 2ml of the ambroxol hydrochloride impurity A, impurity B, impurity C, impurity D, impurity E and impurity F stock solutions prepared in the embodiment 8 respectively, putting the ambroxol hydrochloride impurity A, the impurity B, the impurity C, the impurity D, the impurity E and the impurity F stock solutions into a same 100ml measuring flask, diluting the solution to a scale with methanol, shaking up the solution in a constant volume manner, and preparing 3 parts in parallel, wherein the 3 parts are marked as S1, S2 and S3; obtaining the accuracy stock solution.
Preparation of 50% limit level solution: precisely weighing 10mg of the product, placing the product in a 10ml measuring flask, preparing three parts in parallel, measuring 2.5ml of each of accurate stock solutions S1, S2 and S3, placing the accurate stock solutions in the measuring flask, adding methanol to dilute the accurate stock solutions to a scale, and shaking the accurate stock solutions uniformly to obtain a 50% horizontal limit solution.
Preparation of 100% limit level solution: precisely weighing 10mg of the product, placing the product in a 10ml measuring flask, preparing three parts in parallel, precisely weighing 5ml of each of accurate stock solutions S1, S2 and S3, placing the accurate stock solutions in the measuring flask, adding methanol to dilute the accurate stock solutions to a scale, and shaking the accurate stock solutions uniformly to obtain 100% horizontal limit solutions.
Preparation of 200% limit level solution: accurately weighing 10mg of the product, placing the product in a 10ml measuring flask, preparing three parts in parallel, adding accuracy stock solutions S1, S2 and S3 respectively, diluting to a scale, and shaking uniformly to obtain a 200% horizontal limit solution.
(II) accuracy test data
Table 10: accuracy test data
The standard recovery rate of the ambroxol hydrochloride impurity A, the ambroxol hydrochloride impurity B, the ambroxol hydrochloride impurity C, the ambroxol hydrochloride impurity D, the ambroxol hydrochloride impurity E and the ambroxol hydrochloride impurity F in 9 parts of test solution is 87.5-111.8%, and the RSD is 0.4-5.8%, which meets the regulation.
Example 11: durability test
The chromatographic conditions (column, detector, mobile phase, detection wavelength, column temperature, flow rate, elution, sample volume) were the same as in example 2.
Under otherwise identical conditions, the buffer pH (adjusted to 6.8 and 7.2, respectively), the mobile phase ratio (52: 48 and 48:52), the column temperature (adjusted to 28 ℃ and 32 ℃ respectively), the different brands of chromatography columns were varied and the system applicability was examined
6.8.2 validation requirements:
the separation degree between the ambroxol hydrochloride peak and the ambroxol hydrochloride impurity B in the system applicability solution chromatogram should be larger than 4.0, and the signal-to-noise ratio of the main component peak height in the sensitivity solution chromatogram should be larger than 10.
6.8.3 validated data:
table 11: durability test data
Changing the pH of the phosphate buffer solution to 6.8 and 7.2; column temperatures were varied to 28 ℃ and 32 ℃, respectively, and phosphate buffer: adjusting the proportion of acetonitrile to be 52:48 and 48:52, and replacing a Waters 100-5-C8 chromatographic column, wherein the separation degrees of ambroxol hydrochloride and ambroxol hydrochloride impurity B in a system applicability solution chromatogram are both greater than 4.0, and the peak signal-to-noise ratio of ambroxol hydrochloride is both greater than 10, which meets the specification.
Example 12: preparation and detection method of ambroxol hydrochloride bulk drug
The preparation of the amine bromxol hydrochloride raw material medicine comprises the following steps: the amine bromxol hydrochloride is prepared by the following synthetic route.
The specific process steps are as follows:
(1) 55.6kg of o-aminodibromobenzaldehyde, 23.6kg of (E) -aminocyclohexanol and 128kg of toluene are put into a condensation kettle; and (3) heating to 110 ℃, keeping the temperature for 4h, cooling to room temperature for crystallization, filtering, and drying in vacuum to obtain an intermediate I.
(2) Putting the intermediate 1 obtained in the step (1) and 200Kg of methanol into a hydrogenation kettle; 1.2Kg of platinum carbon, the valve was closed, then replaced with nitrogen 2 times, and the oxygen content was determined to be below 0.5%. Heating to 60 deg.C, introducing hydrogen gas, maintaining the pressure at 1.2MPa for 5 hr, cooling to room temperature, and discharging hydrogen gas; cooling to 20 ℃, preserving the heat for 4 hours, and centrifuging to obtain the ambroxol.
(3) Adding the ambroxol obtained in the step (2) into a reaction kettle, cooling to 10 ℃ by using 160Kg of ethanol solution, dropwise adding 189Kg of 20% hydrochloric acid, preserving the temperature for 2h after dropwise adding, and centrifuging; the ambroxol hydrochloride raw material medicine is obtained.
The impurity detection is carried out on the prepared ambroxol bulk drug, and the detection method comprises the following steps:
1) a chromatographic column: the octyl silane is bonded into a filler with the specification of 4.6 multiplied by 250mm and the particle size of 5 mu m;
2) a detector: an ultraviolet detector;
3) mobile phase: pH7.0 phosphate buffer (1.32% diammonium phosphate solution, pH adjusted to 7.0 with phosphoric acid) -acetonitrile volume ratio: 50: 50;
4) detection wavelength: 248 nm; column temperature: 30 ℃; flow rate: 0.8 ml/min; and (3) elution: isocratic elution.
5) The preparation and determination method of the reference substance solution comprises the following steps: accurately weighing 25mg of ambroxol hydrochloride reference substance, 10mg of impurity A reference substance, 10mg of impurity B reference substance, 10mg of impurity C reference substance, 10mg of impurity F reference substance, 10mg of impurity D reference substance and 10mg of impurity E reference substance into the same volumetric flask, dissolving and diluting the components to the scales by using methanol in the same 100ml measuring flask, and shaking up to the constant volume to obtain a reference substance solution; absorbing 20 μ L of the reference solution, injecting into a liquid chromatograph, wherein the peak emergence order is impurity B, impurity A, ambroxol, impurity D, impurity E, impurity C and impurity F, and the separation degree of adjacent impurity peaks is not lower than 2.0.
6) Preparation of test solution: precisely weighing ambroxol hydrochloride raw material medicine, adding methanol to dilute into 1.0mg/ml solution as a test solution; 20. mu.L of the sample solution was aspirated and injected into a liquid chromatograph. And calculating the contents of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E and the impurity F by peak areas according to an external standard method. The chromatogram is shown in FIG. 15.
Example 13: method for detecting related substances of ambroxol hydrochloride injection
Ambroxol raw material medicine: meets the quality standard of raw material medicaments in the ambroxol of the 2015 edition of Chinese pharmacopoeia.
Preparing an ambroxol injection: ambroxol injection was prepared according to example 1 in CN 110200905A.
The impurity detection is carried out on the prepared ambroxol injection, and the detection method comprises the following steps:
1) a chromatographic column: the octyl silane is bonded into a filler with the specification of 4.6 multiplied by 250mm and the particle size of 5 mu m;
2) a detector: an ultraviolet detector;
3) mobile phase: pH7.0 phosphate buffer (1.32% diammonium phosphate solution, pH adjusted to 7.0 with phosphoric acid) -acetonitrile volume ratio: 50: 50;
4) detection wavelength: 248 nm; column temperature: 30 ℃; flow rate: 0.8 ml/min; and (3) elution: isocratic elution.
5) The preparation and determination method of the reference substance solution comprises the following steps: accurately weighing 25mg of ambroxol hydrochloride reference substance, 10mg of impurity A reference substance, 10mg of impurity B reference substance, 10mg of impurity C reference substance, 10mg of impurity F reference substance, 10mg of impurity D reference substance and 10mg of impurity E reference substance into the same volumetric flask, dissolving and diluting the components to the scales by using methanol in the same 100ml measuring flask, and shaking up to the constant volume to obtain a reference substance solution; absorbing 20 μ L of the reference solution, injecting into a liquid chromatograph, wherein the peak emergence order is impurity B, impurity A, ambroxol, impurity D, impurity E, impurity C and impurity F, and the separation degree of adjacent impurity peaks is not lower than 2.0.
6) Preparation of test solution: precisely measuring 1ml of ambroxol hydrochloride injection, placing the ambroxol hydrochloride injection in a 100ml measuring flask, adding methanol to dissolve the ambroxol hydrochloride injection and diluting the ambroxol hydrochloride injection to a scale mark to obtain a test solution; sucking 20 μ L of sample solution, and injecting into liquid chromatograph; and calculating the contents of the impurity A, the impurity B, the impurity C, the impurity F, the impurity D and the impurity E by peak areas according to an external standard method. The chromatogram is shown in FIG. 16. The content of impurity B is 0.03%.
The above-described embodiments are intended to illustrate the technical idea and advantages of the invention, and the invention may also be subject to other variants, as known to the skilled person, which serve merely as illustrations of the scope of protection of the invention described above, and to the skilled person in the art who is within the scope of protection of the invention defined by the present invention there are many conventional variants and other embodiments, which are all within the scope of protection of the invention covered by the present invention.
Claims (10)
1. Ambroxol raw materialOr impurities in the injection, named impurity F, with the chemical name of cis-4- [ [ (2-amino-3, 5-dibromophenyl) methylene]Amino group]Cyclohexanol of formula C13H16Br2N2O has the structure shown in the following formula I.
2. A process for the preparation of cis-4- [ [ (2-amino-3, 5-dibromophenyl) methylene ] amino ] cyclohexanol as claimed in claim 1, comprising the steps of: adding 2-amino-3, 5-dibromobenzaldehyde, cis-4-aminocyclohexanol hydrochloride and methanol into a reaction kettle; stirring and reacting for 8-10h at 50-55 ℃, drying under reduced pressure to obtain a crude product, and recrystallizing the crude product by using acetone to obtain a final product cis-4- [ [ (2-amino-3, 5-dibromophenyl) methylene ] amino ] cyclohexanol.
3. The method according to claim 2, wherein the 2-amino-3, 5-dibromobenzaldehyde is used in an amount of 1.2 to 1.4 molar equivalents relative to cis-4-aminocyclohexanol hydrochloride.
4. The use of the impurity F as claimed in as a quality control standard in the analytical determination of ambroxol bulk drugs and injections.
5. A detection method for determining impurities A, B, C, D, E and F in ambroxol hydrochloride raw materials and injection by using high performance liquid chromatography comprises the following steps:
1) a chromatographic column: octane silane bonded silica gel is used as a filling agent, the specification is 4.6 multiplied by 250mm, and the grain diameter of the filling agent is 5 mu m;
2) a detector: an ultraviolet detector;
3) mobile phase: ph6.8-7.2 phosphate buffer-acetonitrile volume ratio: 48-52: 48-52;
4) detection wavelength: 248 nm; column temperature: 28-32 ℃; flow rate: 0.8 ml/min; and (3) elution: isocratic elution; sample introduction volume: 20 μ L.
6. The detection method of claim 5, wherein the detection method further comprises:
5) the preparation and determination method of the reference substance solution comprises the following steps: accurately weighing a proper amount of reference substances of ambroxol hydrochloride, impurity A, impurity B, impurity C, impurity D, impurity E and impurity F into the same volumetric flask, adding methanol to dissolve and dilute the reference substances into reference substance solution of ambroxol, impurity A, impurity B, impurity C, impurity F, impurity D and impurity E; injecting the reference solution into a liquid chromatograph according to the chromatographic conditions, wherein the peak appearance sequence comprises impurity B, impurity A, ambroxol, impurity D, impurity E, impurity C and impurity F; the degree of separation of adjacent impurity peaks was not less than 2.0.
6) Preparing an ambroxol hydrochloride raw material medicine and an injection test solution: precisely taking out a proper amount of ambroxol hydrochloride raw material medicine or ambroxol hydrochloride injection, and adding methanol for dilution to serve as a test solution; injecting the reference solution into a liquid chromatograph according to the chromatographic conditions; and calculating the contents of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E and the impurity F by peak areas according to an external standard method.
7. The assay of claim 6, wherein the phosphate buffer is a diammonium phosphate solution at a concentration of 1.0% o to 1.5% o; adjusting the pH value to 6.8-7.2 by using phosphoric acid; preferably 1.3-1.4 per mill of diammonium hydrogen phosphate solution; more preferably 1.32% diammonium hydrogen phosphate solution.
8. The detection method according to claim 6, wherein the mobile phase is phosphate buffer solution-acetonitrile at pH6.8-7.2 in volume ratio: 50: 50; more preferably ph7.0 phosphate buffer to acetonitrile volume ratio: 50:50.
9. The detection method according to claim 6, wherein the preparation of the ambroxol hydrochloride raw material test solution: precisely weighing ambroxol hydrochloride raw material medicine, adding methanol to dilute into 1.0mg/ml solution as a test solution; sucking 20 μ L of sample solution, and injecting into liquid chromatograph; and calculating the contents of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E and the impurity F by peak areas according to an external standard method.
10. The preparation method according to claim 6, wherein the preparation of the test solution of ambroxol hydrochloride injection is as follows: precisely measuring 1ml of ambroxol hydrochloride injection, placing the ambroxol hydrochloride injection in a 100ml measuring flask, adding methanol to dissolve the ambroxol hydrochloride injection and diluting the ambroxol hydrochloride injection to a scale mark to obtain a test solution; sucking 20 μ L of sample solution, and injecting into liquid chromatograph; and calculating the contents of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E and the impurity F by peak areas according to an external standard method.
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| CN114354780A (en) * | 2021-12-15 | 2022-04-15 | 南京工业大学 | Method for detecting impurity content in oral solution of ambroxol |
| CN117630202A (en) * | 2023-10-25 | 2024-03-01 | 山东泰合医药科技有限公司 | Method for detecting buspirone hydrochloride intermediate impurities |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114354780A (en) * | 2021-12-15 | 2022-04-15 | 南京工业大学 | Method for detecting impurity content in oral solution of ambroxol |
| CN114354780B (en) * | 2021-12-15 | 2022-07-26 | 南京工业大学 | Method for detecting impurity content in ammonia bromine terro oral solution |
| CN117630202A (en) * | 2023-10-25 | 2024-03-01 | 山东泰合医药科技有限公司 | Method for detecting buspirone hydrochloride intermediate impurities |
| CN117630202B (en) * | 2023-10-25 | 2024-06-11 | 山东泰合医药科技有限公司 | Method for detecting buspirone hydrochloride intermediate impurities |
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