CN103512965A - Detection method for impurities in amikacin sulfate injection - Google Patents
Detection method for impurities in amikacin sulfate injection Download PDFInfo
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Abstract
The invention provides a method for detecting the content of impurity D kanamycin in an amikacin sulfate injection. The method comprises the steps of: measuring an amikacin sulfate injection, adding water to conduct dilution to obtain a solution containing 1-5mg of amikacin sulfate per 1mL, performing a derivatization treatment on the solution and then taking it as a test solution; measuring a kanamycin reference sample, adding water to dilute it to a kanamycin solution with a concentration of 10-50 micrograms per 1mL, carrying out a derivatization treatment, and taking the solution as a kanamycin reference solution; measuring 5-100 microliters of the kanamycin reference solution, injecting it into a high performance liquid chromatograph, adjusting the detector sensitivity to make the peak height of a main component chromatographic peak account for 30-60% of a full range; measuring 10-100 microliters of the test solution and 10-100 microliters of the reference solution respectively, and injecting them into the high performance liquid chromatograph; and if the chromatogram of the test solution has a chromatographic peak consistent with the kanamycin retention time, acquiring the content of the impurity kanamycin in the test solution according to a kanamycin standard curve.
Description
Technical field
The present invention relates to the detection method of impurity in a kind of amikacin sulfate injection, be specifically related to the detection method to known impurities D kanamycins in amikacin sulfate injection with pre-column derivatization-HPLC method.
Background technology
The principal ingredient of amikacin sulfate injection is amikacin sulfate, this product as Escherichia coli, Klebsiella, Enterobacter etc. all have good action, can be used for treating various severe infections due to gram negative bacilli etc. to most enterobacteriaceae lactobacteriaceaes.
In European Pharmacopoeia, British Pharmacopoeia, all indicated and in amikacin sulfate injection, had four kinds of known impurities, be respectively impurity A, B, C, D, and in Chinese Pharmacopoeia, only controlled known impurities A, this impurity is the impurity producing in building-up process, in parenteral solution production and storage process, the amount of impurity A can not increase.And known impurities D kanamycins is degradation impurity, in preparation preparation and storage process, content can increase to some extent.And the renal toxicity of kanamycins and ototoxicity are all better than amikacin, after clinical practice kanamycins, also can produce hemopoietic system toxic reaction, cause that leucocyte reduces, strictly controlling its limit is the important step in chemical analysis work.
According to method under " Chinese Pharmacopoeia " version amikacin sulfate injection related substance item in 2010, known impurities D is carried out to methodological study, when main ingredient amikacin sulfate concentration is 10mg/mL, the average recovery rate of impurity D kanamycins is lower, is that 66.29%, RSD is 19.6%; In European Pharmacopoeia, British Pharmacopoeia, regulation derivatization sample should be stored under 10 ℃ of environment, and through investigating, at this temperature, sample stability is poor, and the content that records kanamycins in 24h has declined 76.5%.Visible, it is inaccurate adopting official method to carry out quantitative measurement to impurity D kanamycins.Therefore, provide the detection method ，Shi parties concerned of known impurities kanamycins in a kind of amikacin sulfate injection extremely to need.
Summary of the invention
Technical matters solved by the invention is the shortcoming of low, the poor stability of the recovery in existing detection method, provides a kind of accurate, stable detection method, for detection of the content of known impurities D kanamycins in amikacin sulfate injection.
A kind of method that detects the content of impurity D kanamycins in amikacin sulfate injection of one aspect of the present invention, said method comprising the steps of:
Measure amikacin sulfate injection, thin up is made the solution of sulfur acid amikacin 1-5mg in every 1mL, after derivatization treatment, as test solution;
Measure kanamycins reference substance, thin up becomes the solution that contains 10-50 μ g kanamycins in every 1mL, after derivatization treatment, as kanamycins reference substance solution;
Measure kanamycins reference substance solution 5-100 μ L and inject high performance liquid chromatograph, regulate detector sensitivity, the 30-60% that the peak height that makes major component chromatographic peak is full scale;
Measure each 10-100 μ L of test solution and reference substance solution, inject respectively high performance liquid chromatograph; In the chromatogram of test solution, if any the chromatographic peak consistent with kanamycins retention time, according to kanamycins typical curve, obtain the content of impurity kanamycins in test solution.
In a preferred embodiment of the present invention, described derivatization treatment comprises: measure the pending solution of 0.2mL to 10mL measuring bottle, add 1.0% 2,4,6-trinitro-benzene-sulfonic acid solution 2mL and pyridine 3mL; Then powerful jolting is 30 seconds; Measuring bottle is put to water bath heat preservation 2 hours, then in cold water cooling 2 minutes; Then in measuring bottle, add glacial acetic acid 2mL; Powerful jolting was diluted with water to scale after 30 seconds, and shook up.
In a preferred embodiment of the present invention, the temperature of described water-bath is 50-100 ℃, better 60-90 ℃, 70-80 ℃ more preferably, best 75 ℃.
In a preferred embodiment of the present invention, the general thin up of described hydrochloric acid amikacin parenteral solution is made the solution of sulfur acid amikacin 1-4mg in every 1mL, better thin up is made the solution of sulfur acid amikacin 1-3mg in every 1mL, and preferably thin up is made the solution of sulfur acid amikacin 2mg in every 1mL.
In a preferred embodiment of the present invention, kanamycins reference substance thin up becomes the solution that contains 15-40 μ g kanamycins in every 1mL, better thin up becomes the solution that contains 15-30 μ g kanamycins in every 1mL, and preferably thin up becomes the solution that contains 20 μ g kanamycins in every 1mL
In a preferred embodiment of the present invention, measure kanamycins reference substance solution 10-90 μ L and inject high performance liquid chromatograph, better measure kanamycins reference substance solution 20-80 μ L and inject high performance liquid chromatograph, preferably measure kanamycins reference substance solution 40-60 μ L and inject high performance liquid chromatograph.
In a preferred embodiment of the present invention, the 30-60% that the peak height of major component chromatographic peak is full scale, the 40-60% of full scale preferably, more preferably the 45-55% of full scale, is preferably 55% of full scale.
In a preferred embodiment of the present invention, measure each 10-100 μ L of test solution and reference substance solution and inject respectively high performance liquid chromatograph, better measure each 10-90 μ L of test solution and reference substance solution and inject respectively high performance liquid chromatograph, measure each 20-80 μ L of test solution and reference substance solution and inject respectively high performance liquid chromatograph, measure each 40-60 μ L of test solution and reference substance solution and inject respectively high performance liquid chromatograph.
In a preferred embodiment of the present invention, above-mentioned test solution and reference substance solution leave in 5-40 ℃ of environment, more fortunately, in 10-30 ℃ of environment, better, in 20-25 ℃ of environment, are preferably room temperature.
Detection method of the present invention is compared the content of known impurities D kanamycins in the detection amikacin sulfate injection that existing detection method can be accurate, stable.
Accompanying drawing explanation
Fig. 1 is impurity D kanamycins contrast collection of illustrative plates (Specification Curve of Increasing: kanamycins contrast concentration 20 μ g/ml).
Fig. 2 is kanamycins content measuring standard curve (drafting of kanamycins content measuring standard curve).
Fig. 3 is that amikacin sulfate injection related substance kanamycins application of sample reclaims collection of illustrative plates (accuracy test: amikacin sulfate injection concentration 2mg/ml, the application of sample concentration 20 μ g/ml of kanamycins contrast).
Fig. 4 is that (embodiment 5, batch number: 110525) for amikacin sulfate injection related substance collection of illustrative plates.
Fig. 5 is that acid destroys collection of illustrative plates (specificity test: acid destroys sample).
Fig. 6 is that alkali destroys collection of illustrative plates (specificity test: alkali destroys sample).
Fig. 7 is (the specificity test: Oxidative demage sample) of Oxidative demage collection of illustrative plates.
Fig. 8 is (the specificity test: photo damage sample) of photo damage collection of illustrative plates.
Fig. 9 is (the specificity test: heat damage sample) of heat damage collection of illustrative plates.
Embodiment
" scope " disclosed herein is with the form of lower limit and the upper limit.Can be respectively one or more lower limits, and one or more upper limit.Given range limits by a selected lower limit and a upper limit.Selected lower limit and the upper limit define the border of special scope.All scopes that can limit by this way comprise with capable of being combined, and any lower limit can be combined to form a scope with any upper limit.For example, for special parameter, listed the scope of 60-120 and 80-110, be interpreted as that the scope of 60-110 and 80-120 also expects.In addition, if the minimum zone value 1 and 2 of listing, and if listed maximum magnitude value 3,4 and 5, scope below can all expect: 1-3,1-4,1-5,2-3,2-4 and 2-5.
In the present invention, unless have other explanation, the content range of each component of composition with and preferable range between can mutually be combined to form new technical scheme.
In the present invention, unless there are other explanations, " its combination " represents the multicomponent mixture of described each element, for example two kinds, three kinds, four kinds and until the multicomponent mixture of maximum possible.
In the present invention, unless there are other explanations, all " part " and percentage (%) all refer to percent by weight.
In the present invention, unless there are other explanations, in all compositions, the percentage sum of each component is 100%.
In the present invention, unless there are other explanations, numerical range " a-b " represents that the breviary that a closes to the arbitrary real array between b represents, wherein a and b are real numbers.For example numerical range " 0-5 " represents all to have listed the whole real numbers between " 0-5 " herein, and " 0-5 " just the breviary of these combinations of values represents.
In the present invention, unless there are other explanations, integer numerical range " a-b " represents that a represents to the breviary of the arbitrary integer combination between b, and wherein a and b are integers.For example integer numerical range " 1-N " represents 1,2 ... N, wherein N is integer.
If do not particularly not pointed out, this instructions term " a kind of " used refers to " at least one ".
If do not particularly not pointed out, the benchmark of percentage of the present invention (comprising percent by weight) is all the general assembly (TW) of described composition.
In this article, except as otherwise noted, the ratio of each component or weight all refer to dry weight.
In the present invention, if not special explanation, all embodiments mentioned in this article and preferred implementation can be combined to form new technical scheme mutually.
In the present invention, if not special explanation, all technical characterictics mentioned in this article and preferred feature can be combined to form new technical scheme mutually.
In the present invention, if not special explanation, " amikacin " mentioned in this article, " amikacin sulfate " etc. all represent amikacin sulfate, and it comprises the impurity conventionally comprising, for example impurity A, B, C and/or D.
The invention provides a kind of method that detects the content of impurity D kanamycins in amikacin sulfate injection, said method comprising the steps of:
Measure amikacin sulfate injection, thin up is made the solution of sulfur acid amikacin 1-5mg in every 1mL, after derivatization treatment, as test solution;
Measure kanamycins reference substance, thin up becomes the solution that contains 10-50 μ g kanamycins in every 1mL, after derivatization treatment, as kanamycins reference substance solution;
Measure kanamycins reference substance solution 5-100 μ L and inject high performance liquid chromatograph, regulate detector sensitivity, the 30-60% that the peak height that makes major component chromatographic peak is full scale;
Measure each 10-100 μ L of test solution and reference substance solution, inject respectively high performance liquid chromatograph; In the chromatogram of test solution, if any the chromatographic peak consistent with kanamycins retention time, according to kanamycins typical curve, obtain the content of impurity kanamycins in test solution.
In the present invention, described hydrochloric acid amikacin parenteral solution is conventional.For example, hydrochloric acid amikacin parenteral solution can be purchased from Zhu Yi Jin pharmaceutcal corporation, Ltd of upper Hisense etc.
In the present invention, described derivatization treatment is known, and those of ordinary skill in the art can directly carry out derivatization treatment according to prior art.For example, derivatization treatment can be referring to the operation under Chinese Pharmacopoeia version amikacin assay item in 2010.In a preferred embodiment of the present invention, described derivatization treatment comprises: measure the pending solution of 0.2mL to 10mL measuring bottle, add 1.0% 2,4,6-trinitro-benzene-sulfonic acid solution 2mL and pyridine 3mL; Then powerful jolting is 30 seconds; Measuring bottle is put to water bath heat preservation 2 hours, then in cold water (2-10 ℃) cooling 2 minutes; Then in measuring bottle, add glacial acetic acid 2mL; Powerful jolting was diluted with water to scale after 30 seconds, and shook up.In another preferred embodiment of the present invention, the temperature of described water-bath is 50-100 ℃, better 60-90 ℃, 70-80 ℃ more preferably, best 75 ℃.
In the present invention, the general thin up of described hydrochloric acid amikacin parenteral solution is made the solution of sulfur acid amikacin 1-5mg in every 1mL, better thin up is made the solution of sulfur acid amikacin 1-4mg in every 1mL, better thin up is made the solution of sulfur acid amikacin 1-3mg in every 1mL, and preferably thin up is made the solution of sulfur acid amikacin 2mg in every 1mL.
In the present invention, the general thin up of kanamycins reference substance becomes the solution that contains 10-50 μ g kanamycins in every 1mL, better thin up becomes the solution that contains 15-40 μ g kanamycins in every 1mL, better thin up becomes the solution that contains 15-30 μ g kanamycins in every 1mL, and preferably thin up becomes the solution that contains 20 μ g kanamycins in every 1mL
In the present invention, general quantity is got kanamycins reference substance solution 5-100 μ L and is injected high performance liquid chromatograph, better measure kanamycins reference substance solution 10-90 μ L and inject high performance liquid chromatograph, better measure kanamycins reference substance solution 20-80 μ L and inject high performance liquid chromatograph, preferably measure kanamycins reference substance solution 40-60 μ L and inject high performance liquid chromatograph.
In the present invention, the peak height of major component chromatographic peak is generally the 30-60% of full scale, the 40-60% of full scale preferably, and more preferably the 45-55% of full scale, is preferably as 55% of full scale.
In the present invention, generally measure each 10-100 μ L of test solution and reference substance solution and inject respectively high performance liquid chromatograph, better measure each 10-90 μ L of test solution and reference substance solution and inject respectively high performance liquid chromatograph, measure each 20-80 μ L of test solution and reference substance solution and inject respectively high performance liquid chromatograph, measure each 40-60 μ L of test solution and reference substance solution and inject respectively high performance liquid chromatograph.In a preferred embodiment of the present invention, the consumption of described test solution and reference substance solution is identical or different.In a preferred embodiment of the present invention, the consumption of described test solution and reference substance solution is identical.
In the present invention, although be not specifically limited, above-mentioned test solution and reference substance solution can leave in 5-40 ℃ of environment, more fortunately, in 10-30 ℃ of environment, better, in 20-25 ℃ of environment, are preferably room temperature.
Process of the test involved in the present invention (with reference to two of " Chinese Pharmacopoeia " versions in 2010 and " British Pharmacopoeia " version in 2009) is as follows:
1, the foundation of its related substances assay method
Specificity test (destructive test)
Get 10 of test samples, mix, precision measures this product 1mL in 50mL measuring bottle, by following each condition preparation test sample.
Do not destroy: thin up is also settled to scale, mix the rear derivatization method that shines and process, shake up, standby;
Acid destroys: the hydrochloric acid solution that adds 0.1mol/L is diluted to scale, mixes, and room temperature lucifuge is placed 10 days, according to derivatization method, processes, and shakes up, standby;
Alkali destroys: the sodium hydroxide solution that adds 0.1mol/L is diluted to scale, mixes, and room temperature lucifuge is placed 10 days, according to derivatization method, processes, and shakes up, standby;
Oxidative demage: add 3% superoxol and be diluted to scale, mix, room temperature lucifuge is placed 10 days, processes according to derivatization method, shakes up, standby;
Illumination destroys: thin up is also settled to scale, mixes, and puts (4500 ± 500Lx) under high light and irradiates 10 days, according to derivatization method, processes, and shakes up, standby;
High temperature destroys: thin up is also settled to scale, mixes, and is placed in 80 ℃ of baking ovens and heats 10 days, lets cool, and according to derivatization method, processes, and shakes up, standby.
Get respectively each 50 μ L of above-mentioned solution, injection liquid chromatography, records chromatogram, and does not destroy sample and compares.Result shows, amikacin is comparatively stable under oxidation and ultraviolet lighting condition, under strong acid, highly basic and hot conditions, destroy, all can produce degradation impurity kanamycins, other impurity peaks have no significant change, kanamycins peak is separated with amikacin main peak and other each impurity peaks good, illustrate that the method specificity is strong, and separation preferably also detects the content of impurity kanamycins in amikacin sulfate injection.
The mensuration of quantitative limit
Get kanamycins reference substance appropriate, after stepwise dilution, according to derivatization method, process, sample introduction is measured, and presses 10:1 signal to noise ratio (S/N ratio) as quantitative limit, and result is 2ng.
(3) serviceability test
According to derivatization method, respectively need testing solution and reference substance solution are processed, sample introduction is measured, and investigates its durability when certain change occurs for chromatographic condition and mobile phase.
Serviceability test result
From result, when wavelength, chromatographic column, mobile phase pH value, mobile phase ratio change within the specific limits, kanamycins percentage composition does not have significant change, illustrates that the method tolerance is good.
(4) drafting of typical curve
Get kanamycins reference substance appropriate (about 37.5mg), accurately weighed, put in 50mL measuring bottle, be dissolved in water, be diluted to scale, accurate absorption 0.1,0.25,0.5,1,2,3,4mL put in 25mL measuring bottle respectively, be diluted with water to scale, precision measures each 0.2mL of above-mentioned solution, after processing, gets respectively 50 μ L injection liquid chromatographies according to derivatization method, record chromatogram, the kanamycins solution concentration (C) of take is horizontal ordinate, and the peak area (A) of take is ordinate, carries out linear regression.
Result shows, in 2-80 μ g/ml concentration range, the peak area of kanamycins and concentration linear relationship are good, in this concentration range, can carry out Accurate Determining to kanamycins content.
Kanamycins content measuring standard curve
(5) system suitability test
Get kanamycins reference substance appropriate, accurately weighed, be dissolved in water and quantitatively dilute and make the solution that contains 40 μ g in every 1mL, according to derivatization method, process, parallel six parts, sample introduction, records retention time and peak area respectively, calculates RSD value.The relative standard deviation of retention time and peak area is all less than 2.0%, and illustrative system precision is good.
Kanamycins (40 μ g/ml) system flexibility
(6) replica test
It is appropriate that precision measures this product, dilute with water is made the solution that contains 2mg in every 1mL, separately get kanamycins reference substance appropriate, accurately weighed, water dissolves and quantitatively dilutes and make the solution that contains 2 μ g in every 1mL, precision measures each 0.2mL of above-mentioned solution, according to derivatization method, process, parallel six parts, difference sample introduction, calculate RSD value, investigate repeatability.
Kanamycins repeatability
Result shows, the RSD value of kanamycins chromatographic peak area is 2.4%, is less than 15%, and repeatability is up to specification.
(7) precision test in the middle of
According to the method under replica test item, in same date not, different tests personnel, use identical a collection of amikacin sulfate injection and kanamycins reference substance, measure its content on different instruments, calculate RSD value, investigate its centre precision.
Precision in the middle of kanamycins
Result shows, the RSD value of middle precision test is 6.9%, is less than 20%, and middle precision is good.
(8) accuracy test
It is appropriate that precision measures amikacin sulfate injection, and dilute with water is made the solution that contains 2mg in every 1mL, parallel two parts, according to derivatization method, processes; Separately get kanamycins reference substance appropriate, accurately weighed, be dissolved in water and quantitatively dilute and make the solution that contains respectively 2,20,80 μ g in every 1mL, parallel two parts, according to derivatization method, process; Precision measures sample solution and each 0.2mL of reference substance solution respectively, and 3 parts of the parallel preparations of each concentration are processed according to derivatization method.Measure respectively each 50 μ L injection liquid chromatographies of above-mentioned solution, record chromatogram, calculate recovery rate.
Kanamycins accuracy
Result shows, impurity kanamycins is within the scope of 0.1-4.0%, and average average recovery is 97.34%, and relative standard deviation is 6.36%, prove that the accuracy of the method mensuration kanamycins content is high.
Below in conjunction with embodiment, specifically describe the present invention, but scope of the present invention is not limited to this.
Sample and reagent: amikacin sulfate injection (lot number: 080703,090601,100525,110524,110525,110526, manufacturing enterprise: Zhu Yi Jin pharmaceutcal corporation, Ltd of upper Hisense);
Kanamycins reference substance (lot number: 130556-200501, Chinese pharmaceutical biological product is identified institute);
2,4,6-trinitro-benzene-sulfonic acid (lot number: 031M5021/080M5000, Sigma-Aldrich);
Pyridine (lot number: T20110418 ，AR， Chemical Reagent Co., Ltd., Sinopharm Group);
Glacial acetic acid (lot number: T20110114 ，AR， Chemical Reagent Co., Ltd., Sinopharm Group);
Potassium dihydrogen phosphate (lot number: 201006010 ，AR， Chemical Reagent Co., Ltd., Sinopharm Group);
Potassium hydroxide (lot number: F201101110 ，AR， Chemical Reagent Co., Ltd., Sinopharm Group);
Methyl alcohol (lot number: 150808110, HPLC, Merk);
Instrument and equipment: Agilent 1200 high performance liquid chromatographs; Chromatographic column (Agilent ZORBAXSB-C185 μ m 150 * 4.6mm Column).
Embodiment 1
It is appropriate that precision measures amikacin sulfate injection, and dilute with water is made the solution that contains 2mg in every 1mL, as test sample stoste.Precision measures in above-mentioned solution 0.2mL to 10mL measuring bottle, add 1.0% 2,4,6-trinitro-benzene-sulfonic acid solution 2mL, pyridine 3mL, close plug.Powerful jolting 30 seconds, puts 75 ℃ of water bath heat preservations 2 hours, in cold water cooling 2 minutes, adds glacial acetic acid 2mL, close plug.Powerful jolting 30 seconds, is diluted with water to scale, shakes up, and parallel six parts, as need testing solution.
Get kanamycins reference substance appropriate, accurately weighed, be dissolved in water and quantitatively dilute and make the solution that contains respectively 2,20,80 μ g in every 1mL, in contrast product stoste.Precision measures in above-mentioned each solution 0.2mL to 10mL measuring bottle respectively, add 1.0% 2,4,6-trinitro-benzene-sulfonic acid solution 2mL, pyridine 3mL, close plug, powerful jolting 30 seconds, put 75 ℃ of water bath heat preservations 2 hours, in cold water cooling 2 minutes, add glacial acetic acid 2mL, close plug, powerful jolting 30 seconds, is diluted with water to scale, shakes up, parallel two parts of each concentration, in contrast product solution.
Precision measures in test sample stoste, each 0.2mL to 10mL measuring bottle of reference substance stoste respectively, parallel three parts of each concentration, add 1.0% 2,4,6-trinitro-benzene-sulfonic acid solution 2mL, pyridine 3mL, close plug, powerful jolting 30 seconds, put 75 ℃ of water bath heat preservations 2 hours, in cold water cooling 2 minutes, add glacial acetic acid 2mL, close plug, powerful jolting 30 seconds, is diluted with water to scale, shakes up.
According to high performance liquid chromatography (the Chinese Pharmacopoeia appendix V D of version II portion in 2010), with octadecylsilane chemically bonded silica, be filling agent; 0.27% potassium dihydrogen phosphate (potassium hydroxide solution with 2.2% regulates pH to the 6.5)-methyl alcohol (30:70) of take is mobile phase; Detection wavelength is 340nm.Theoretical cam curve is calculated and is not less than 3500 by amikacin.The degree of separation of kanamycins and amikacin main peak and other each impurity peaks all should meet the requirements.
Measure respectively each 50 μ L injection liquid chromatographies of above-mentioned need testing solution, contrast solution and each application of sample solution, record chromatogram, calculate the recovery that adds kanamycins.
Kanamycins average recovery test (sample concentration 2mg/mL)
The average average recovery of kanamycins is that 97.33%, RSD value is 6.4%.
It is appropriate that precision measures amikacin sulfate injection, and dilute with water is made the solution that contains 5mg in every 1mL, as test sample stoste.Precision measures in solution 0.2mL to 10mL measuring bottle, add 1.0% 2,4,6-trinitro-benzene-sulfonic acid solution 2mL, pyridine 3mL, close plug.Powerful jolting 30 seconds, puts 75 ℃ of water bath heat preservations 2 hours, in cold water cooling 2 minutes, adds glacial acetic acid 2mL, close plug.Powerful jolting 30 seconds, is diluted with water to scale, shakes up, and parallel six parts, as need testing solution.
Get kanamycins reference substance appropriate, accurately weighed, be dissolved in water and quantitatively dilution make in every 1mL respectively the solution containing 5,80,200 μ g, product stoste in contrast, precision measures in above-mentioned each solution 0.2mL to 10mL measuring bottle respectively, add 1.0% 2,4,6-trinitro-benzene-sulfonic acid solution 2mL, pyridine 3mL, close plug, powerful jolting 30 seconds, put 75 ℃ of water bath heat preservations 2 hours, in cold water cooling 2 minutes, add glacial acetic acid 2mL, close plug, powerful jolting 30 seconds, is diluted with water to scale, shakes up, parallel two parts of each concentration, in contrast product solution.
Precision measures in test sample stoste, each 0.2mL to 10mL measuring bottle of reference substance stoste respectively, parallel three parts of each concentration, add 1.0% 2,4,6-trinitro-benzene-sulfonic acid solution 2mL, pyridine 3mL, close plug, powerful jolting 30 seconds, put 75 ℃ of water bath heat preservations 2 hours, in cold water cooling 2 minutes, add glacial acetic acid 2mL, close plug, powerful jolting 30 seconds, is diluted with water to scale, shakes up.
Measure respectively each 20 μ L injection liquid chromatographies (chromatographic condition is with embodiment 1) of above-mentioned need testing solution, contrast solution and each application of sample solution, record chromatogram, calculate the recovery that adds kanamycins.
Kanamycins average recovery test (sample concentration 5mg/mL)
The average average recovery of kanamycins is that 91.12%, RSD value is 9.6%.
It is appropriate that precision measures amikacin sulfate injection, and dilute with water is made the solution that contains 10mg in every 1mL, as test sample stoste, precision measures in solution 0.2mL to 10mL measuring bottle, add 1.0% 2,4,6-trinitro-benzene-sulfonic acid solution 2mL, pyridine 3mL, close plug, powerful jolting 30 seconds, put 75 ℃ of water bath heat preservations 2 hours, in cold water cooling 2 minutes, add glacial acetic acid 2mL, close plug, powerful jolting 30 seconds, is diluted with water to scale, shake up, parallel six parts, as need testing solution.
Get kanamycins reference substance appropriate, accurately weighed, be dissolved in water and quantitatively dilution make in every 1mL respectively the solution containing 5,200,400 μ g, product stoste in contrast, precision measures in above-mentioned each solution 0.2mL to 10mL measuring bottle respectively, add 1.0% 2,4,6-trinitro-benzene-sulfonic acid solution 2mL, pyridine 3mL, close plug, powerful jolting 30 seconds, put 75 ℃ of water bath heat preservations 2 hours, in cold water cooling 2 minutes, add glacial acetic acid 2mL, close plug, powerful jolting 30 seconds, is diluted with water to scale, shakes up, parallel two parts of each concentration, in contrast product solution.
Precision measures in test sample stoste, each 0.2mL to 10mL measuring bottle of reference substance stoste respectively, parallel three parts of each concentration, add 1.0% 2,4,6-trinitro-benzene-sulfonic acid solution 2mL, pyridine 3mL, close plug, powerful jolting 30 seconds, put 75 ℃ of water bath heat preservations 2 hours, in cold water cooling 2 minutes, add glacial acetic acid 2mL, close plug, powerful jolting 30 seconds, is diluted with water to scale, shakes up.
Measure respectively each 20 μ L injection liquid chromatographies (chromatographic condition is with embodiment 1) of above-mentioned need testing solution, contrast solution and each application of sample solution, record chromatogram, calculate the recovery that adds kanamycins.
Kanamycins average recovery test (sample concentration 10mg/mL)
The average average recovery of kanamycins is that 66.29%, RSD value is 19.6%, and accuracy is starkly lower than the inventive method.
Sample stability test
It is appropriate that precision measures amikacin sulfate injection, and dilute with water is made the solution that contains 2mg in every 1mL; Get kanamycins reference substance appropriate, accurately weighed, be dissolved in water and quantitatively dilute and make the solution that contains 80 μ g in every 1mL; Precision measures in each 0.2mL to 10mL measuring bottle of above-mentioned solution respectively, add 1.0% 2,4,6-trinitro-benzene-sulfonic acid solution 2mL, pyridine 3mL, close plug, powerful jolting 30 seconds, puts 75 ℃ of water bath heat preservations 2 hours, in cold water cooling 2 minutes, add glacial acetic acid 2mL, close plug, powerful jolting 30 seconds, be diluted with water to scale, shake up.
It is appropriate that precision measures amikacin sulfate injection, and dilute with water is made the solution that contains 5mg in every 1mL; Get kanamycins reference substance appropriate, accurately weighed, be dissolved in water and quantitatively dilute and make the solution that contains 200 μ g in every 1mL; Precision measures in each 0.2mL to 10mL measuring bottle of above-mentioned solution respectively, add 1.0% 2,4,6-trinitro-benzene-sulfonic acid solution 2mL, pyridine 3mL, close plug, powerful jolting 30 seconds, puts 75 ℃ of water bath heat preservations 2 hours, in cold water cooling 2 minutes, add glacial acetic acid 2mL, close plug, powerful jolting 30 seconds, be diluted with water to scale, shake up.
It is appropriate that precision measures amikacin sulfate injection, and dilute with water is made the solution that contains 10mg in every 1mL; Get kanamycins reference substance appropriate, accurately weighed, be dissolved in water and quantitatively dilute and make the solution that contains 400 μ g in every 1mL; Precision measures in each 0.2mL to 10mL measuring bottle of above-mentioned solution respectively, add 1.0% 2,4,6-trinitro-benzene-sulfonic acid solution 2mL, pyridine 3mL, close plug, powerful jolting 30 seconds, puts 75 ℃ of water bath heat preservations 2 hours, in cold water cooling 2 minutes, add glacial acetic acid 2mL, close plug, powerful jolting 30 seconds, be diluted with water to scale, shake up.
Measure respectively each 50 μ L injection liquid chromatographies (chromatographic condition is with embodiment 1) of above-mentioned application of sample solution, regulate the temperature of injector to be respectively 10,25 and 35 ℃, investigate the stability of sample solution in 24h.
10 ℃ of stability
25 ℃ of stability
35 ℃ of stability
According to official method, main ingredient amikacin sulfate concentration is 10mg/mL, and storage environment is while being 10 ℃, the less stable of sample solution, and after 24h, the content of kanamycins is reduced to 23.5%; And adopt this inventive method to detect, and stability of solution is obviously improved, and after 24h, the content of kanamycins is without marked change.
Measure the content of impurity D kanamycins in amikacin sulfate injection
It is appropriate that precision measures this product, and dilute with water is made the solution that contains 2mg in every 1mL, and precision measures in this solution 0.2mL to 10mL measuring bottle, add 1.0% 2,4,6-trinitro-benzene-sulfonic acid solution 2mL, pyridine 3mL, close plug, powerful jolting 30 seconds, puts 75 ℃ of water bath heat preservations 2 hours, in cold water cooling 2 minutes, add glacial acetic acid 2mL, close plug, powerful jolting 30 seconds, be diluted with water to scale, shake up, as need testing solution.
Separately get kanamycins reference substance appropriate, accurately weighed, be dissolved in water and quantitatively dilute and make the solution that contains 20 μ g in every 1mL, according to derivatization method, process, in contrast product solution.
According to high performance liquid chromatography (two appendix V D of Chinese Pharmacopoeia version in 2010), with octadecylsilane chemically bonded silica, be filling agent; 0.27% potassium dihydrogen phosphate (potassium hydroxide solution with 2.2% regulates pH to the 6.5)-methyl alcohol (30:70) of take is mobile phase; Detection wavelength is 340nm.
Precision measures reference substance solution 50 μ L injection liquid chromatographies, regulates detector sensitivity, makes the peak height of major component chromatographic peak be about 50% of full scale.
Precision measures need testing solution and each 50 μ L of reference substance solution again, inject respectively high performance liquid chromatograph, record chromatogram, in the chromatogram of need testing solution, if any the chromatographic peak consistent with kanamycins retention time, its area must not be greater than 1.5 times (1.5%) of contrast solution main peak area in contrast solution.
Measurement result:
Theoretical cam curve is calculated and is greater than 3500 by amikacin.The degree of separation of kanamycins and amikacin main peak and other each impurity peaks all should meet the requirements.
Kanamycins content in six batches of amikacin sulfate injections
In six batch samples, the content of kanamycins is all up to specification.
Claims (9)
1. a method that detects the content of impurity D kanamycins in amikacin sulfate injection, said method comprising the steps of:
Measure amikacin sulfate injection, thin up is made the solution of sulfur acid amikacin 1-5mg in every 1mL, after derivatization treatment, as test solution;
Measure kanamycins reference substance, thin up becomes the solution that contains 10-50 μ g kanamycins in every 1mL, after derivatization treatment, as kanamycins reference substance solution;
Measure kanamycins reference substance solution 5-100 μ L and inject high performance liquid chromatograph, regulate detector sensitivity, the 30-60% that the peak height that makes major component chromatographic peak is full scale;
Measure each 10-100 μ L of test solution and reference substance solution, inject respectively high performance liquid chromatograph; In the chromatogram of test solution, if any the chromatographic peak consistent with kanamycins retention time, according to kanamycins typical curve, obtain the content of impurity kanamycins in test solution.
2. the method for claim 1, is characterized in that, described derivatization treatment comprises: measure the pending solution of 0.2mL to 10mL measuring bottle, add 1.0% 2,4,6-trinitro-benzene-sulfonic acid solution 2mL and pyridine 3mL; Then powerful jolting is 30 seconds; Measuring bottle is put to water bath heat preservation 2 hours, then in cold water cooling 2 minutes; Then in measuring bottle, add glacial acetic acid 2mL; Powerful jolting was diluted with water to scale after 30 seconds, and shook up.
3. the method for claim 1, is characterized in that, the temperature of described water-bath is 50-100 ℃, better 60-90 ℃, 70-80 ℃ more preferably, best 75 ℃.
4. the method for claim 1, it is characterized in that, the general thin up of described hydrochloric acid amikacin parenteral solution is made the solution of sulfur acid amikacin 1-4mg in every 1mL, better thin up is made the solution of sulfur acid amikacin 1-3mg in every 1mL, and preferably thin up is made the solution of sulfur acid amikacin 2mg in every 1mL.
5. the method for claim 1, it is characterized in that, kanamycins reference substance thin up becomes the solution that contains 15-40 μ g kanamycins in every 1mL, better thin up becomes the solution that contains 15-30 μ g kanamycins in every 1mL, and preferably thin up becomes the solution that contains 20 μ g kanamycins in every 1mL.
6. the method for claim 1, it is characterized in that, measure kanamycins reference substance solution 10-90 μ L and inject high performance liquid chromatograph, better measure kanamycins reference substance solution 20-80 μ L and inject high performance liquid chromatograph, preferably measure kanamycins reference substance solution 40-60L and inject high performance liquid chromatograph.
7. the method for claim 1, is characterized in that, the 30-60% that the peak height of major component chromatographic peak is full scale, and the 40-60% of full scale preferably, more preferably the 45-55% of full scale, is preferably 55% of full scale.
8. the method for claim 1, it is characterized in that, measure each 10-100 μ L of test solution and reference substance solution and inject respectively high performance liquid chromatograph, better measure each 10-90 μ L of test solution and reference substance solution and inject respectively high performance liquid chromatograph, measure each 20-80 μ L of test solution and reference substance solution and inject respectively high performance liquid chromatograph, measure each 40-60 μ L of test solution and reference substance solution and inject respectively high performance liquid chromatograph.
9. the method for claim 1, is characterized in that, above-mentioned test solution and reference substance solution leave in 5-40 ℃ of environment, more fortunately, in 10-30 ℃ of environment, better, in 20-25 ℃ of environment, is preferably room temperature.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111337607A (en) * | 2019-08-02 | 2020-06-26 | 嘉圣生物医药(嘉兴)有限公司 | On-line solid phase extraction liquid chromatography for detecting kanamycin content in blood |
| CN115322234A (en) * | 2022-08-24 | 2022-11-11 | 江苏吴中医药集团有限公司 | Amikacin impurity, and preparation method and application thereof |
| CN117269389A (en) * | 2023-11-23 | 2023-12-22 | 成都市海通药业有限公司 | Quality detection method of amikacin sulfate injection |
-
2012
- 2012-06-20 CN CN201210206258.8A patent/CN103512965A/en active Pending
Non-Patent Citations (5)
| Title |
|---|
| HSIN-HUA YEH 等: "Rapid and selective micellar electrokinetic chromatography for simultaneous determination of amikacin, kanamycin A, and tobramycin with UV detection and application in drug formulations", 《ELECTROPHORESIS》 * |
| 吴宏伟 等: "HPLC-ELSD法测定硫酸阿米卡星的含量", 《中国药品标准》 * |
| 王明娟 等: "硫酸阿米卡星注射液及有关物质的HPLC法分析", 《中国抗生素杂志》 * |
| 袁耀佐等: "HPLC柱前衍生化法测定硫酸阿米卡星的含量", 《中国药学会学术年会论文集》 * |
| 陈冠华 等: "硫酸阿米卡星含量及有关物质的HPLC-ELSD测定", 《中国医药工业杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111337607A (en) * | 2019-08-02 | 2020-06-26 | 嘉圣生物医药(嘉兴)有限公司 | On-line solid phase extraction liquid chromatography for detecting kanamycin content in blood |
| CN115322234A (en) * | 2022-08-24 | 2022-11-11 | 江苏吴中医药集团有限公司 | Amikacin impurity, and preparation method and application thereof |
| CN117269389A (en) * | 2023-11-23 | 2023-12-22 | 成都市海通药业有限公司 | Quality detection method of amikacin sulfate injection |
| CN117269389B (en) * | 2023-11-23 | 2024-02-09 | 成都市海通药业有限公司 | Quality detection method of amikacin sulfate injection |
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