CN106222237A - Gatifloxacin eye drop Sterility Test Methods - Google Patents
Gatifloxacin eye drop Sterility Test Methods Download PDFInfo
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- CN106222237A CN106222237A CN201610635722.3A CN201610635722A CN106222237A CN 106222237 A CN106222237 A CN 106222237A CN 201610635722 A CN201610635722 A CN 201610635722A CN 106222237 A CN106222237 A CN 106222237A
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- flushing liquor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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Abstract
The invention discloses a kind of gatifloxacin eye drop Sterility Test Methods, relate to enzyme or the mensuration of microorganism or the method for inspection.Gatifloxacin eye drop Sterility Test Methods of the present invention comprises the following steps: filter gatifloxacin eye drop after extracting the thin film of a small amount of aseptic filter cylinder of flushing liquor moistening；After gatifloxacin eye drop all filters, by flushing liquor suction by several times, the 1/10 of the total flushing dose of each suction, filtration and washing filter membrane；Before suction finally a flushing liquor, aseptic filter cylinder base apertures is covered tightly；Then the last a flushing liquor of extraction, then the nertralizer of suction every part flushing liquor 1/10 volume, after the two mixing, is rinsed filter cylinder inwall, filters；Add culture medium to cultivate at different conditions, and testing result.The present invention change nertralizer coal addition position by science, thus save flushing liquor and washing time, reduce test fussy degree, substantially increase test efficiency.Test method science of the present invention, simple to operate, result of the test is accurate, has broad application prospects.
Description
Technical field
The present invention relates to enzyme or the mensuration of microorganism or the method for inspection, specifically a kind of gatifloxacin eye drop sterility test
Method.
Background technology
Gatifloxacin eye drop is applicable to sensitive microbial acute bacterial conjunctivitis, is mainly composed of Gatifloxacin.
Gatifloxacin is the 8-BAY 128039 class medicine that Xing Lin company of Japan is pioneering, the MIC to major part gram positive bacteria90For
0.1-0.78 mg/L, its activity is 2-16 times of ciprofloxacin and ofloxacin；MIC to major part gram negative bacteria90For
0.025-0.39 mg/L, its activity is suitable with ciprofloxacin and ofloxacin；Excellent to anaerobe, mycoplasma, chlamydial activity
In ciprofloxacin and ofloxacin.
The antibacterial activity widely in view of its strength, must make its antibacterial activity disappear, to ensure sample during sterility test
In microorganism injury-free, make assay accurately and reliably.Quinolinones can be with metal ion (Fe2+ ,Mg2+ ,Mn2+) etc.
Complexation, forms stable hexatomic ring complex, covers its active group, make antibacterial activity disappear.Based on above character, existing
Gatifloxacin Sterility Test Methods be to use flushing liquor to rinse antipathogenic composition, and with in manganese sulfate solution and Gatifloxacin is active
The mode of group is to eliminate its biocidal property.But, the flushing fluid that the method uses is big, to filter membrane be likely to be present in medicinal liquid
Microorganism cause damage, affect the accuracy of testing result；Meanwhile, the test period of the method is long, and test efficiency is low, no
Meet the requirement of modern production.
Summary of the invention
It is big that the present invention is contemplated to solve existing Gatifloxacin Sterility Test Methods flushing liquor usage amount, and test period is long
Etc. problem, a kind of gatifloxacin eye drop Sterility Test Methods proposed.
The present invention realizes according to techniques below scheme.
A kind of gatifloxacin eye drop Sterility Test Methods, comprises the following steps:
I. filter gatifloxacin eye drop after extracting the thin film of a small amount of aseptic filter cylinder of flushing liquor moistening；
II. after gatifloxacin eye drop all filters, by flushing liquor suction by several times, the 1/10 of the total flushing dose of each suction, fall
Low wriggling pump speed, filtration and washing filter membrane；
III. before suction finally a flushing liquor, aseptic filter cylinder base apertures is covered tightly；Then the last a flushing liquor of extraction,
The nertralizer of suction every part flushing liquor 1/10 volume again, after the two mixing, is rinsed filter cylinder inwall, filters；
IV. add culture medium and cultivate at different conditions, and testing result.
The composition of described flushing liquor is peptone dry powder and tween 80, and the mass fraction of the two is than for 1:1.
The preparation method of described flushing liquor is: claims 1 part of peptone dry powder, tween 80 1 part, is dissolved in 1000 parts of water, adjusts
PH makes the pH value after sterilizing be 7.1 ± 0.2,115-121 DEG C of autoclaving 15-30 minute.
Described total flushing dose is 500ml.
Described nertralizer is 0.1mol/L manganese sulfate solution.
Described culture medium is THIOGLYCOLLIC ACID salt broth or pancreas cheese soya peptone fluid medium；THIOGLYCOLLIC ACID salt fluid
Cultivating and cultivate 14 days based on 30-35 DEG C, pancreas cheese soya peptone liquid culture is cultivated 14 days based on 20-25 DEG C.
Present invention obtains following beneficial effect.
It is big that the present invention effectively solves existing Gatifloxacin Sterility Test Methods flushing liquor usage amount, and test period is long
Etc. problem.Using the method for the invention to carry out Gatifloxacin sterility test, flushing liquor consumption is by original 800ml(Chen Ying, Xing
Be lost China etc. the foundation of gatifloxacin eye drop Sterility Test Methods. Tianjin pharmacy, 2009,21 (4): 7-9) be reduced to present
500ml, reduces 37.5%, and experimentation cost reduces accordingly；After the nertralizer joining day moves forward to flushing liquor, the joining day is more
Science；Test period is reduced to 25min by original 60min, decreases 58.3%, and test efficiency is improved.So, use
Inspection method of the present invention, 500ml flushing liquor and 5ml nertralizer can also play the effect reducing antipathogenic composition, and save
Save flushing liquor and washing time, reduce test fussy degree, substantially increase test efficiency；Flushing liquor consumption reduces also simultaneously
Reduce damage filter membrane and be originally present in the probability of microorganism in medicinal liquid, improve the accuracy of testing result.The present invention
Test method science, simple to operate, result of the test is accurate, is more suitable for the sterility test of gatifloxacin eye drop, has wide
Wealthy application prospect.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described further.
One, test prepares
1. test sample: gatifloxacin eye drop
2. strain: staphylococcus aureus (CMCC(B) 26003)
Escherichia coli (CMCC(B) 44102)
Clostridium sporogenes (CMCC(B) 64941)
Bacillus subtilis (CMCC(B) 63501)
Candida albicans (CMCC(F) 98001)
Aspergillus niger (CMCC(F) 98003)
Above reference culture is purchased from Nat'l Pharmaceutical & Biological Products Control Institute or Tianjin City Medicine Inspection Inst..
3. culture medium
4. culture medium, flushing liquor, nertralizer compound method
5. test apparatus
6. the preparation of bacteria suspension:
A. bacterium is increased: inoculate escherichia coli, staphylococcus aureus, bacillus subtilis respectively in the cultivation of pancreas cheese soya peptone agar
On base flat board, clostridium sporogenes is in THIOGLYCOLLIC ACID salt broth, and 30-35 DEG C of calorstat is cultivated 18-24 hour；Inoculation white
Candidiasis, Aspergillus niger are in Sabouraud glucose agar, and 20-25 DEG C of calorstat, it is little that Candida albicans cultivates 24-48
Time, Aspergillus niger 5-7 days, by the 3-5ml 0.9% aseptic NaCl solution containing 0.05% Tween 80 by Aspergillus niger spores eluting, with pipe
Mouth is collected spore suspension with the capillary tube that sterile absorbent is cotton and is preserved in sterile test tube, standby.
B. bacterium solution dilution: escherichia coli, staphylococcus aureus, bacillus subtilis, clostridium sporogenes, Candida albicans
Bacterium, the fresh cultured thing of Aspergillus niger press 10 times of dilutions by 0.9% aseptic NaCl solution, make test bacterium solution；The test every milli of bacterium solution
Rise bacteria containing amount less than 100cfu.
The most freshly prepd bacteria suspension should use at ambient temperature in 2 hours, and 2 ~ 8 DEG C of cold preservations can preserve 24 hours.
Two, test method
1. film preparation
Extract a small amount of flushing liquor (making thin film moistening in aseptic filter cylinder), by every filtration yield 50ml(10 bottle/cylinder) filter confession
Test product.After test sample all filters, by flushing liquor (500ml/ cylinder) suction by several times, each 50ml/ cylinder, shake gently, filter
Rinse filter membrane (reduce pump speed be 30 revolutions per seconds with abundant rinse).Flushing liquor suction by several times, when exhausting 400ml, by aseptic filter
Cylinder base apertures covers tightly, and extracts 50ml flushing liquor, then suction 5ml 0.1mol/L manganese sulfate nertralizer, closes pump, by 50ml flushing liquor
Mix with 5ml nertralizer, gently shaking (filter cylinder inwall being rinsed, it is to avoid dead angle), filter, then extract last 50ml flushing
Liquid also adds less than 100cfu test organisms, and full dose filters, in addition culture medium to filter cylinder, as test group.
Separately take one and add equivalent test organisms as a control group equipped with same volume culture medium filter cylinder.3 ~ 5 are cultivated by set point of temperature
My god.Each strain operates with method.
2. cultivate
Addition escherichia coli, staphylococcus aureus and the THIOGLYCOLLIC ACID salt broth filter cylinder of clostridium sporogenes, 30-35 DEG C
Calorstat is cultivated 3-5 days；Add the trypticase soybean broth filter of bacillus subtilis, Candida albicans and Aspergillus niger
Cylinder, 20-25 DEG C of calorstat is cultivated 3-5 days.
3. method applicability result of the test
4. conclusion
Above employment and suitability test (E & ST) result shows: test group compared with matched group, test organisms well-grown, this test method and used
Test material is on test organisms growth nothing impact, therefore this test method may be used for the sterility test of this product.
The most daily Sterility Test Methods is established
Extract a small amount of flushing liquor (making thin film moistening in aseptic filter cylinder), filter test sample by every filtration yield 50ml.Wait to supply
After test product all filters, by flushing liquor (500ml/ cylinder) suction by several times, each 50ml/ cylinder, shake gently, filtration and washing filter membrane
(reduce pump speed be 30 revolutions per seconds with abundant rinse).Flushing liquor suction by several times, when exhausting 450ml, by aseptic filter cylinder base apertures
Cover tightly, extract 50ml flushing liquor, then suction 5ml 0.1mol/L manganese sulfate nertralizer, close pump, by 50ml flushing liquor and 5ml
Mediating recipe mixes, and shakes gently, is rinsed filter cylinder inwall, it is to avoid dead angle, filters.It is separately added into THIOGLYCOLLIC ACID salt fluid to cultivate
Base and pancreas cheese soya peptone fluid medium, THIOGLYCOLLIC ACID salt broth is in 30-35 DEG C of cultivation 14 days, pancreas cheese soya peptone liquid
Cultivate and cultivate 14 days based on 20-25 DEG C.Observed result, result see table (table 5).
Claims (6)
1. a gatifloxacin eye drop Sterility Test Methods, it is characterised in that: comprise the following steps:
I. filter gatifloxacin eye drop after extracting the thin film of a small amount of aseptic filter cylinder of flushing liquor moistening；
II. after gatifloxacin eye drop all filters, by flushing liquor suction by several times, the 1/10 of the total flushing dose of each suction, fall
Low wriggling pump speed, filtration and washing filter membrane；
III. before suction finally a flushing liquor, aseptic filter cylinder base apertures is covered tightly；Then the last a flushing liquor of extraction,
The nertralizer of suction every part flushing liquor 1/10 volume again, after the two mixing, is rinsed filter cylinder inwall, filters；
IV. add culture medium and cultivate at different conditions, and testing result.
A kind of gatifloxacin eye drop Sterility Test Methods the most according to claim 1, it is characterised in that: described flushing liquor
Composition be peptone dry powder and tween 80, the mass fraction of the two is than for 1:1.
A kind of gatifloxacin eye drop Sterility Test Methods the most according to claim 2, it is characterised in that: described flushing liquor
Preparation method be: claim 1 part of peptone dry powder, tween 80 1 part, be dissolved in 1000 parts of water, adjust pH make sterilizing after pH value be
7.1 ± 0.2,115-121 DEG C of autoclaving 15-30 minute.
A kind of gatifloxacin eye drop Sterility Test Methods the most according to claim 1, it is characterised in that: described total flushing
Amount is 500ml.
A kind of gatifloxacin eye drop Sterility Test Methods the most according to claim 1, it is characterised in that: described nertralizer
For 0.1mol/L manganese sulfate solution.
A kind of gatifloxacin eye drop Sterility Test Methods the most according to claim 1, it is characterised in that: described culture medium
For THIOGLYCOLLIC ACID salt broth or pancreas cheese soya peptone fluid medium；THIOGLYCOLLIC ACID salt broth is in 30-35 DEG C of training
Supporting 14 days, pancreas cheese soya peptone liquid culture is cultivated 14 days based on 20-25 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610635722.3A CN106222237A (en) | 2016-08-05 | 2016-08-05 | Gatifloxacin eye drop Sterility Test Methods |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610635722.3A CN106222237A (en) | 2016-08-05 | 2016-08-05 | Gatifloxacin eye drop Sterility Test Methods |
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| Publication Number | Publication Date |
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| CN106222237A true CN106222237A (en) | 2016-12-14 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201610635722.3A Pending CN106222237A (en) | 2016-08-05 | 2016-08-05 | Gatifloxacin eye drop Sterility Test Methods |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107164454A (en) * | 2017-07-03 | 2017-09-15 | 中国大冢制药有限公司 | Benza microbial limit tests |
| CN107287276A (en) * | 2017-07-03 | 2017-10-24 | 中国大冢制药有限公司 | Compound sodium acetate electrolyte injection Sterility Test Methods |
-
2016
- 2016-08-05 CN CN201610635722.3A patent/CN106222237A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 陈莹: "加替沙星滴眼液无菌检查方法的建立", 《天津药学》 * |
| 马仕洪: "加替沙星无菌检查方法的建立与标准操作探讨", 《药物分析杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107164454A (en) * | 2017-07-03 | 2017-09-15 | 中国大冢制药有限公司 | Benza microbial limit tests |
| CN107287276A (en) * | 2017-07-03 | 2017-10-24 | 中国大冢制药有限公司 | Compound sodium acetate electrolyte injection Sterility Test Methods |
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Application publication date: 20161214 |