CN103245745A - Method for checking erythromycin A and imino ether in azithromycin for injection - Google Patents
Method for checking erythromycin A and imino ether in azithromycin for injection Download PDFInfo
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- CN103245745A CN103245745A CN2013101740193A CN201310174019A CN103245745A CN 103245745 A CN103245745 A CN 103245745A CN 2013101740193 A CN2013101740193 A CN 2013101740193A CN 201310174019 A CN201310174019 A CN 201310174019A CN 103245745 A CN103245745 A CN 103245745A
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Abstract
The invention relates to the field of medicines and in particular relates to a method for checking erythromycin A and imino ether in azithromycin for injection. The method comprises the following steps: taking an erythromycin A and imino ether reference substance, diluting the reference substance into a solution by using sodium chloride, sequentially carrying out the undue toxicity check method test according to the sequence of progressively increasing the injection of the solutions until a mouse death result occurs in the retest of testing the injection amount of a certain erythromycin A and imino ether, and setting the injection amount used in the previous test of the test to be the injection limit; quantitatively analyzing the content C1 of the erythromycin A and imino ether in the azithromycin for injection and content C of the azithromycin; calculating to obtain the impurity limit W of the erythromycin A and imino ether according to the maximum amount m of the azithromycin for injection each time and the following formula; if so, the content of the related substance reaches the safety level, otherwise the content of the related substance cannot reach the safety level. According to the method, the quantitative evaluation aim is realized, and the result can be used for other further researches.
Description
Technical field
The present invention relates to drug world, the inspection method of Erythromycin A iminoether in the azithromycin injection.
Background technology
The inspection of related substance is an important indicator estimating its security in the azithromycin injection.Related substance mainly refers to azithromycin B, azithromycin G in the azithromycin injection
x, Erythromycin A iminoether etc.
In the correlation technique, the inspection of Erythromycin A iminoether is to adopt thin-layered chromatography (TLC) in the azithromycin injection: with test sample (azithromycin injection) and contrast liquid point sample on silica gel g thin-layer plate respectively, judge by comparison both amount of speckle and shade whether its related substances reaches security level.
As seen, the Erythromycin A iminoether inspection method in the correlation technique can only realize the purpose of semi-quantitative assessment its related substances, can't realize quantitative evaluation, thereby can not more scientifically study, for example analysis of causes of its injection security etc.
Summary of the invention
The object of the present invention is to provide the inspection method of Erythromycin A iminoether in the azithromycin injection, to solve the above problems.
The inspection method of Erythromycin A iminoether in the azithromycin injection is provided in an embodiment of the present invention, has comprised the following steps:
Steps A: get Erythromycin A iminoether reference substance, and with sodium chloride injection with the solution of its dilution for same concentration or variable concentrations, the order that increases progressively according to Erythromycin A iminoether injection rate IR with these solution is carried out the test of undue toxicity inspection technique successively again, dead mouse occurs as a result the time in the retrial of the test of a certain Erythromycin A iminoether injection rate IR, used Erythromycin A iminoether injection rate IR is decided to be the injection rate IR limit of Erythromycin A iminoether in the previous test with this test;
Step B: the content C of the content C1 of Erythromycin A iminoether, azithromycin in the quantitative test azithromycin injection to be measured;
Step C: according to the each maximum consumption m of azithromycin injection to be measured, and following formula calculates, and obtains the l.o.i W of Erythromycin A iminoether:
Step D: will
Compare with described W; If
Then its related substances reaches security level in the described azithromycin injection to be measured, otherwise does not reach security level.
The inspection method of Erythromycin A iminoether in the azithromycin injection of the above embodiment of the present invention, can realize being specially the purpose of three kinds of related substances of Erythromycin A iminoether in the quantitative check azithromycin injection:
Described steps A has been measured the injection rate IR limit of Erythromycin A iminoether, and then obtains the l.o.i W of this material by described step C;
Obtain the content C of Erythromycin A iminoether again by the quantitative test of described step B
1, and the content C of the effective constituent azithromycin of this medicine;
Calculate the content ratio of related substance in the azithromycin injection to be measured-Erythromycin A iminoether and effective constituent azithromycin at last, namely
With this value and described W, compare again, if
Then Erythromycin A iminoether content reaches security level in the described azithromycin injection to be measured, otherwise does not reach security level;
Hence one can see that, and method of the present invention is by the l.o.i of quantitative measurement Erythromycin A iminoether, and the content of Erythromycin A iminoether in the azithromycin injection to be measured, realized the purpose of quantitative evaluation, and this result can be used for other further research.
Description of drawings
Fig. 1 is the high-efficient liquid phase chromatogram of first sample described in the test example of the present invention;
Fig. 2 is the high-efficient liquid phase chromatogram of second sample described in the test example of the present invention.
Embodiment
Also by reference to the accompanying drawings the present invention is described in further detail below by specific embodiment.
Embodiment
Embodiments of the invention provide the inspection method of Erythromycin A iminoether in the azithromycin injection, comprise the following steps:
Step 101: get Erythromycin A iminoether reference substance, and with sodium chloride injection with the solution of its dilution for same concentration or variable concentrations, the order that increases progressively according to Erythromycin A iminoether injection rate IR with these solution is carried out the test of undue toxicity inspection technique successively again, dead mouse occurs as a result the time in the retrial of the test of a certain Erythromycin A iminoether injection rate IR, used Erythromycin A iminoether injection rate IR is decided to be the injection rate IR limit of Erythromycin A iminoether in the previous test with this test;
Step 102: the content C of Erythromycin A iminoether in the quantitative test azithromycin injection to be measured
1, azithromycin content C;
Step 103: according to the each maximum consumption m of azithromycin injection to be measured, and following formula calculates, and obtains the l.o.i W of Erythromycin A iminoether:
Step 104: will
Compare with described W; If
Then Erythromycin A iminoether content reaches security level in the described azithromycin injection to be measured, otherwise does not reach security level.
The inspection method of Erythromycin A iminoether in the azithromycin injection of above-described embodiment, can realize being specially the purpose of three kinds of related substances of Erythromycin A iminoether in the quantitative check azithromycin injection:
Described steps A has been measured the injection rate IR limit of Erythromycin A iminoether, and then obtains the l.o.i W of this material by described step C;
Obtain the content C of Erythromycin A iminoether again by the quantitative test of described step B
1, and the content C of the effective constituent azithromycin of this medicine;
Calculate the content ratio of related substance in the azithromycin injection to be measured-Erythromycin A iminoether and effective constituent azithromycin at last, namely
With this value and described W, compare again, if
Then Erythromycin A iminoether content reaches security level in the described azithromycin injection to be measured, otherwise does not reach security level;
Hence one can see that, and method of the present invention is by the l.o.i of quantitative measurement Erythromycin A iminoether, and the content of Erythromycin A iminoether in the azithromycin injection to be measured, realized the purpose of quantitative evaluation, and this result can be used for other further research.As seen, the present invention provides a new science approach for the safety evaluatio of Erythromycin A iminoether in the medicine.
Wherein, described undue toxicity inspection technique is referring to Pharmacopoeia of the People's Republic of China version appendix in 2010 XIC: 5 of healthy mices getting body weight and be 17-20g, reference substance solution is injected mouse tail vein, should 4-5 in second at the uniform velocity injection finish dead mouse situation in 48 hours after the record administration; During if any death, other gets 10 retrials of healthy mice of body weight 18-19g, dead mouse situation in 48 hours after the record administration.
Described m can be different at different treatment targets, and common people are 0.5g with the m of this medicine.(can referring to the instructions of specific product)
Described quantitative analysis method can be adopted high efficiency liquid phase chromatographic analysis method, and perhaps other is suitable for the method for quantitative measurment Erythromycin A iminoether and effective constituent.
The described step 101 of said method is the solution of variable concentrations with sodium chloride injection with reference substance dilution, gets the order that these solution of same dose increase progressively according to concentration again and carries out the test of undue toxicity inspection technique successively.Adopt the mode of this " dosage is identical, concentration is different " to change injection rate IR, can reduce the influence of the other factors between each test group, the accuracy of raising method.
When described step 102 used high efficiency liquid phase chromatographic analysis method to carry out quantitative test, for further accuracy, the science of this inspection method, this method can also be improved in the following areas.
(1) preferably, described high performance liquid chromatography detects C
1, the used filling agent of C is octadecylsilane chemically bonded silica, used flowing is the mixture of acetonitrile-phosphate buffer that volume ratio is 45:55 mutually; The detection wavelength is 210nm.Through evidence, adopt this analysis condition, analysis result accuracy and degree of accuracy are higher, and detection time is short.
(2) preferably, the pH value of described phosphate buffer is 8.2-8.4, wherein more preferably is 8.2.Through evidence, adopt the damping fluid of this pH value, detection time is shorter, has improved detection efficiency.Wherein, preferably, the compound method of described phosphate buffer is: get the 0.05mol/L dipotassium hydrogen phosphate solution, regulate pH value to 8.2 with the phosphoric acid solution of 0.2g/mL, the used two kinds of reagent costs of this method are low, and easily acquisition, reduced experimentation cost.
(3) preferably, be certain density solution with acetonitrile with azithromycin injection dilution to be measured, as first sample; Again first test sample is diluted n doubly as second sample; Inject described first sample to high performance liquid chromatograph, detect C
1In high performance liquid chromatograph, inject described second sample, detect C; Described n is 50-150.Azithromycin and the Erythromycin A iminoether solubleness height in acetonitrile is more conducive to detect.And the solution that adopts variable concentrations simultaneously detects Erythromycin A iminoether and effective constituent respectively, can improve the accuracy of method: the impurity in the common azithromycin injection to be measured (Erythromycin A iminoether etc.) content is lower, be that the concentration of Erythromycin A iminoether in the same solution and the concentration of azithromycin have big difference, if adopt the solution of same concentration to detect this two kinds of materials simultaneously, can exceed the detectability of instrument or too near the detectability of instrument, the accuracy of reduction method.
(4) preferably, the concentration of azithromycin injection is 8-10mg/mL in described first sample, and described n=100.Through evidence, accuracy and the precision of this detection method are higher.In like manner, preferably, the injection rate IR of described first sample and second sample is 50 μ L.
In order to illustrate in greater detail the performance of method of the present invention, below also provide concrete test example.
The test example
Test method:
Undue toxicity inspection technique test: get Erythromycin A iminoether reference substance, make the solution of variable concentrations with sodium chloride injection, get 5 of healthy mices (body weight 17-20g), need testing solution is injected mouse tail vein, should at the uniform velocity inject second at 4-5 and finish, every mouse gives need testing solution 0.5ml respectively, dead mouse situation in 48 hours after the record administration; During if any death, other gets 10 retrials of healthy mice of body weight 18-19g, dead mouse situation in 48 hours after the record administration.
The high performance liquid chromatography test: precision takes by weighing azithromycin injection 98.21mg, adds acetonitrile dissolving and dilution and makes the solution that concentration is 9.821mg/ml, as first sample; Precision is measured 1ml, puts in the 100ml volumetric flask,, shake up to scale with dilution in acetonitrile, and as second sample, be filling agent with octadecylsilane chemically bonded silica; (volume ratio is 45:55) for flowing phase with phosphate buffer (get the 0.05mol/L dipotassium hydrogen phosphate solution, the phosphoric acid solution with 20% is regulated pH value to 8.2)-acetonitrile; The detection wavelength is 210nm, and precision is measured each 50 μ L of first sample and second sample, injects liquid chromatograph respectively, 2 times of record chromatogram (peak of azithromycin) retention time to the major component peak.
Because the absorptivity of two kinds of materials of Erythromycin A iminoether and azithromycin under same liquid phase testing conditions is different, so for the ease of the comparison of later stage two kinds of material concentrations, this test has also detected the correction factor f of Erythromycin A iminoether with respect to azithromycin.
The measuring method of f: getting the reference substance of azithromycin and two materials of impurity Erythromycin A iminoether, each is an amount of, put and add mobile phase in the same volumetric flask { phosphate buffer (is got the 0.05mol/L dipotassium hydrogen phosphate solution, phosphoric acid solution with 20% is regulated pH value to 8.2)-acetonitrile (45:55) } dissolving, and be diluted to the solution that two kinds of materials respectively contain about 10mg/ml, as mixed solution.Measure above-mentioned mixed solution 50 μ l and inject liquid chromatograph, the record chromatogram, read the correction factor that each material peak-to-peak area is calculated as follows each impurity:
M
Impurity: the title sample quality of impurity
M
Archie: the title sample quality of azithromycin
A
Impurity: the peak area of impurity
A
Archie: the peak area of azithromycin
Test findings:
The test of undue toxicity inspection technique: as shown in Table 1, Erythromycin A iminoether injection rate IR limit is 1.75mg.
This test is with artificial medicine use object in the future, and the each maximum consumption m of its azithromycin injection is 0.5g.The l.o.i that obtains the Erythromycin A iminoether thus is 0.35%(1.75/500).
The undue toxicity experiment of table one Erythromycin A iminoether reference substance
The high performance liquid chromatography test:
As depicted in figs. 1 and 2, the retention time of Erythromycin A iminoether, azithromycin is respectively: 4.216,15.630.Fig. 1 is the chromatogram of described first sample; Fig. 2 is the chromatogram of described second sample.
The correction factor that test records the Erythromycin A iminoether is 0.42.
At last, the peak area of Erythromycin A iminoether in described first sample be multiply by its correction factor respectively, because the concentration of described first sample is 100 times of described second sample, peak area with azithromycin in described second sample multiply by 100 again, peak area with above-mentioned corrected impurity is divided by again, obtain the content ratio of Erythromycin A iminoether and azithromycin: 0.179%, the l.o.i of this value and abnormal toxicity test gained is compared: 0.179%＜0.35%.
The content of the azithromycin injection product Erythromycin A iminoether that as seen, checks in this test example reaches security level.
The above is the preferred embodiments of the present invention only, is not limited to the present invention, and for a person skilled in the art, the present invention can have various changes and variation.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. the inspection method of Erythromycin A iminoether in the azithromycin injection is characterized in that, comprises the following steps:
Steps A: get Erythromycin A iminoether reference substance, and with sodium chloride injection with the solution of its dilution for same concentration or variable concentrations, the order that increases progressively according to Erythromycin A iminoether injection rate IR with these solution is carried out the test of undue toxicity inspection technique successively again, dead mouse occurs as a result the time in the retrial of the test of a certain Erythromycin A iminoether injection rate IR, used Erythromycin A iminoether injection rate IR is decided to be the injection rate IR limit of Erythromycin A iminoether in the previous test with this test;
Step B: the content C of Erythromycin A iminoether in the quantitative test azithromycin injection to be measured
1, azithromycin content C;
Step C: according to the each maximum consumption m of azithromycin injection to be measured, and following formula calculates, and obtains the l.o.i W of Erythromycin A iminoether:
Step D: will
Compare with described W; If
Then its related substances reaches security level in the described azithromycin injection to be measured, otherwise does not reach security level.
2. method according to claim 1, it is characterized in that, in the described steps A, be the solution of variable concentrations with sodium chloride injection with the dilution of Erythromycin A iminoether reference substance, get these solution of same dose again and carry out the test of undue toxicity inspection technique successively according to the order that concentration increases progressively.
3. according to each described method of claim 1-2, it is characterized in that the method for the quantitative test among the described step B is:
High efficiency liquid phase chromatographic analysis method.
4. method according to claim 3 is characterized in that, described high performance liquid chromatography detects C
1, the used filling agent of C is octadecylsilane chemically bonded silica, used flowing is the mixture of acetonitrile-phosphate buffer that volume ratio is 45:55 mutually; The detection wavelength is 210nm.
5. method according to claim 4 is characterized in that, the pH value of described phosphate buffer is 8.0-8.4.
6. method according to claim 5 is characterized in that, the pH value of described phosphate buffer is 8.2.
7. method according to claim 6 is characterized in that, the compound method of described phosphate buffer is: get the 0.05mol/L dipotassium hydrogen phosphate solution, regulate pH value to 8.2 with the phosphoric acid solution of 0.2g/mL.
8. method according to claim 7 is characterized in that, described high efficiency liquid phase chromatographic analysis method is:
With acetonitrile azithromycin injection to be measured being diluted is certain density solution, as first sample; Again first test sample is diluted n doubly as second sample; Inject described first sample to high performance liquid chromatograph, detect C
1In high performance liquid chromatograph, inject described second sample, detect C;
Described n is 50-150.
9. method according to claim 8 is characterized in that, the concentration of azithromycin injection is 8-10mg/mL in described first sample, and described n=100.
10. method according to claim 9 is characterized in that, the injection rate IR of described first sample and second sample is 50 μ L.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111474279A (en) * | 2020-05-20 | 2020-07-31 | 中国检验检疫科学研究院 | Method and kit for detecting macrolide antibiotic compounds |
| CN111474278A (en) * | 2020-05-20 | 2020-07-31 | 中国检验检疫科学研究院 | Method and kit for detecting metabolites of macrolide compounds |
-
2013
- 2013-05-10 CN CN2013101740193A patent/CN103245745A/en active Pending
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111474279A (en) * | 2020-05-20 | 2020-07-31 | 中国检验检疫科学研究院 | Method and kit for detecting macrolide antibiotic compounds |
| CN111474278A (en) * | 2020-05-20 | 2020-07-31 | 中国检验检疫科学研究院 | Method and kit for detecting metabolites of macrolide compounds |
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Application publication date: 20130814 |