CN102841205A - Method for detecting fleroxacin bacterial endotoxin used for injection - Google Patents
Method for detecting fleroxacin bacterial endotoxin used for injection Download PDFInfo
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- CN102841205A CN102841205A CN2011102020241A CN201110202024A CN102841205A CN 102841205 A CN102841205 A CN 102841205A CN 2011102020241 A CN2011102020241 A CN 2011102020241A CN 201110202024 A CN201110202024 A CN 201110202024A CN 102841205 A CN102841205 A CN 102841205A
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- injection
- bacterial endotoxin
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- fleraxacin
- fleroxacin
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Abstract
The invention relates to a method for detecting fleroxacin bacterial endotoxin used for injection, comprising the steps of detecting fleroxacin used for injection by adopting a gel method or a luminous intensity determination method by utilizing tachypleus amebocyte lysate, diluting the fleroxacin used for injection with a bivalent cation solution and then detecting. The method disclosed by the invention is simple to operate, is sensitive and accurate, can eliminate interference and can avoid defects of a pyrogen inspection method, thus the method disclosed by the invention has positive significance to improvement of quality standard, delivery of finished product and safety of clinical use.
Description
Technical field
The present invention relates to the endotoxic method of a kind of bacterial detection, particularly relate to a kind of method that detects injection fleraxacin bacterial endotoxin.
Background technology
The injection fleraxacin is an antimicrobial, and clinical usage is drip-feed, and national Specification carries out pyrogen test with the rabbit method and controls its limit, and state's Extra Pharmacopoeia Martindale does not all record this kind.Whether rabbit method inspection pyrogen is about to the test sample of doses, and vein injects in the rabbit body, at the appointed time, observes the situation of rabbit fervescence, up to specification with the limit of judging contained pyrogen in the test sample.Because of the pyrogen reaction of rabbit is identical with the people, so method is the heat-original determinating method of comparatively generally acknowledging.But this method is time-consuming longer, and complex operation is unfavorable for animal protection, and the consistance of experimental result depends on animal situation, house condition and operation.
Pyrogen is the lipopolysaccharides of bacterial endotoxin.Whether the vitro pyrogen test method(s) of development in recent years is a limulus test, is the bacterial endotoxin that utilizes TAL to detect or quantize to be produced by gram-negative bacteria, up to specification to judge limiting the quantity of of bacterial endotoxin in the test sample.Its principle is to utilize amebocyte lysate of king crab and the agglutinating reaction between the endotoxin.TAL is a LAL, the commercially available detection of bacterial endotoxin that is specifically designed to.Limulus test can detect the endotoxin of 0.0001 μ g; Big ten times of its remolding sensitivity rabbit methods; And experimental implementation is simple, and experimental expenses is low, and the result is reliable rapidly; Thereby being specially adapted to pyrogen control and the non-detectable some drugs preparation of rabbit method in the production run, the inspection method of bacterial endotoxin also is a kind of universal method that various countries' pharmacopeia is admitted.The amount of bacterial endotoxin is with endotoxin unit (EU) expression, and its inspection method comprises two kinds, i.e. gel method and photometric means.
At present existing scholar's research Fleroxacin injection detection for bacterial endotoxin method all is to check that with Fleroxacin injection stoste or bacterial endotoxin water directly dilutes the method that Fleroxacin injection detects but they adopt.Disturb and exist when this method is used for the detection of bacterial endotoxin of injection fleraxacin, sample can not detect exactly, even change diluted sample concentration, the sensitivity of TAL and the pH value of sample, can not get rid of interference.Therefore, the method that needs a kind of bacterial endotoxin of new detection injection fleraxacin.
Summary of the invention
The purpose of this invention is to provide a kind of method that detects injection fleraxacin bacterial endotoxin, to solve the problems referred to above that prior art exists.
The present invention provides a kind of method that detects injection fleraxacin bacterial endotoxin; Comprise that utilizing TAL to pass through gel method or photometric means detects the injection fleraxacin, injection fleraxacin to be measured is detected in 37 ± 1 ℃ after with the bivalent cation solution dilution.Because bivalent cation solution, like calcium ion, magnesium ion etc. can be regulated the pH value of injection fleraxacin liquid to be measured, replenish metallic ion, improve the sensitivity of TAL reaction, guarantee that the reactivity of TAL is interference-free.
Commercially available obtainable aseptic pyrogen-free bivalent cation solution has the anticoagulant for storage of whole blood special diluting agent that Zhanjiang Andusi Biology Co., Ltd. produces at present.
The present invention detects the method for injection fleraxacin bacterial endotoxin; Can avoid the deficiency of above pyrogen test method, and easy and simple to handle, sensitive and accurate; Can get rid of interference, the raising of quality standard, the clearance of finished product and the security of clinical use are all had positive meaning.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
Embodiment 1
1, experiment material:
1.1 test sample: injection fleraxacin
Production unit: Singapore and Malaysia, Shenyang pharmaceutcal corporation, Ltd
Specification: 0.1g lot number: 20100703,20100704,20100705;
Specification: 0.2g lot number: 20101221,20101222,20101223;
1.2 TAL
1.3 bacterial endotoxin working standard (CSE)
Production unit: Nat'l Pharmaceutical & Biological Products Control Institute, lot number 150601-201065,180 EU/ that tire prop up.
1.4 bacterial endotoxin inspection water (BET water)
Production unit: Zhanjiang Andusi Biology Co., Ltd., specification: 5ml, lot number: 1002050.
1.5 anticoagulant for storage of whole blood special diluting agent
Production unit: Zhanjiang Andusi Biology Co., Ltd., specification: 5ml, lot number 1008310.
2, limit value is confirmed
The clinical usage and dosage of these article a: 0.2-0.4g (in fleraxacin) is diluted in that lucifuge slowly instils in 5% glucose injection of 250-500ml.Calculate limit value L=K/M=0.75Eu/mg (K is 5Eu/kgh, and M is 400mg/60Kgh), draft limit value and be: the amount that contains bacterial endotoxin among every 1mg should be less than 0.75 EU.
3, experimental technique and result
3.1 sensitivity of the limulus reagent inspection test
Get the bacterial endotoxin working standard, add the bacterial endotoxin inspection water dissolving of ormal weight, be diluted to the bacterial endotoxin standard solution of 4 concentration then.Wait that with what fully dissolve checking TAL adds in the endotoxin standard solution, the line space of going forward side by side contrasts in vain, carries out the sign sensitivity of TAL and checks, with the sensitivity of proof TAL and the correctness of this method of operating personnel.The result sees table 1.
Conclusion: the sensitivity inspection test result of used 2 producer's each batch TALs, all up to specification.
3.2 disturb the primary dcreening operation test
3.2.1 the calculating of minimum effective dilute concentration (MVC)
Minimum effective dilute concentration MVC=λ/L (L is bacterial endotoxin limit value 0.75 EU/mg of test sample, and λ is that TAL indicates sensitivity).When λ was 0.5,0.25,0.125,0.06 or 0.03 EU/mL, minimum effective dilute concentration MVC that test sample is corresponding was respectively MVC
0.5=0.667 mg/mL, MVC
0.25=0.333 mg/mL, MVC
0.125=0.167 mg/mL, MVC
0.06=0.08 mg/mL, MVC
0.03=0.04 mg/mL.
3.2.2 disturb the primary dcreening operation test
With BET water with test sample be diluted to 0.667mg/mL, 0.333 mg/mL, 0.167 mg/mL,
0.08mg/mL with 0.04mg/mL concentration, this serial solution is designated as NPC.Prepare the standard endotoxin solution that all contains 0.5 EU/mL concentration in this NPC series solution simultaneously, this serial solution is designated as PPC.Get the TAL that sensitivity is 0.25 EU/mL, react in 37 ℃ ± 1 ℃ with above-mentioned NPC, PPC respectively, each concentration is parallel does two pipes, and establishes positive control (PC) and negative control (NC), and the result sees table 2.
Conclusion: can judge tentatively that by above interference primary dcreening operation test findings test sample possibly tested noiseless effect to the BET of two kinds of TALs when 0.08mg/ mL concentration.3.3. interference test
3.3.1 interference test 1
For the final situation of confirming interference, carry out following interference test, with BET water test sample is diluted to concentration 0.08,0.04mg/mL and carries out interference test respectively.
The bacterial endotoxin working standard is diluted with water to 0.125,0.06,0.03,0.015 and 0.0075EU/ml with bacterial endotoxin inspection; Use respectively that concentration is 0.08, the 0.04mg/mL need testing solution replaces bacterial endotoxin inspection water that the bacterial endotoxin working standard is diluted to 0.125,0.06,0.03,0.015 and 0.0075 EU/ml.Carry out interference test, the result sees table 3.
Interference test is 1 result show: when test sample is diluted to 0.08 and 0.04 mg/mL with BET water, and its E
tBe 2E
sPossibly also there is inhibiting effect to a certain degree in inspection to the test sample of these 2 concentration to bacterial endotoxin.
3.3.2 the photometry disturbed condition is judged test
For the further conclusive evidence test sample disturbed condition that test exists to BET, observe the influence that test sample is tested BET with photometry, and adopt proper method to get rid of and disturb, test as follows.Respectively test sample is diluted to 0.08mg/ml with BET water and anticoagulant for storage of whole blood special diluting agent; Use sensitivity is that the TAL of 0.06 EU/mL carries out photometry interference judgement test, and the result shows with the sample of anticoagulant for storage of whole blood special diluting agent dilution noiseless basically; There is inhibiting effect to a certain degree in test to BET and with the water-reducible test sample of BET.
3.3.3 formal interference test
Be final and confirm to use the anticoagulant for storage of whole blood special diluting agent that the TAL that to use 2 producer's sensitivity be 0.25 EU/mL carries out following formal interference test to the disturbed condition that these article BET tests.With BET water CSE is diluted to 0.5,0.25,0.125,0.06 EU/mL; With the anticoagulant for storage of whole blood special diluting agent test sample is diluted to 0.333 mg/mL, replaces bacterial endotoxin inspection water that the bacterial endotoxin working standard is diluted to 0.5,0.25,0.125 and 0.06 EU/mL with it.Carry out interference test, the result sees table 4.
Formal interference test result shows: E
SAll at 0.5 λ～2 λ, and E
tAll at 0.5E
S～2E
S; When affirmation is diluted to 0.333 EU/mL with the anticoagulant for storage of whole blood special diluting agent with test sample, noiseless to the bacterial endotoxin inspection.
3.4 the bacterial endotoxin of test sample inspection
The bacterial endotoxin working standard is diluted with water to 0.5EU/ml with the bacterial endotoxin inspection; Test sample is processed the solution of 40mg/ml with bacterial endotoxin inspection water, be diluted to 0.333mg/ml with the anticoagulant for storage of whole blood special diluting agent again and process NPC, use 0.667mg/ml test liquid and 1.0 EU/ml endotoxin solution equivalent to dilute and process PPC.The result sees table 5.
The result: 2 each 3 lot sample article of specification carry out the bacterial endotoxin inspection by drafting limit value, and the result is all up to specification.
Embodiment 2
The bacterial endotoxin working standard is diluted with water to 0.25EU/ml with the bacterial endotoxin inspection; The injection fleraxacin is diluted to 0.667mg/ml with the anticoagulant for storage of whole blood special diluting agent processes NPC, use the dilution of 1.333mg/ml test liquid and 0.50 EU/ml endotoxin solution equivalent to process PPC.The negative reaction of result: NPC and NC, the positive reaction of PPC and PC, the bacterial endotoxin check result is up to specification.
Embodiment 3
The bacterial endotoxin working standard is diluted with water to 0.5EU/ml with the bacterial endotoxin inspection; The injection fleraxacin is diluted to the solution of 20mg/ml with the anticoagulant for storage of whole blood special diluting agent; Process NPC with bacterial endotoxin inspection water prepared and diluted to 0.08mg/ml again, PPC is processed in use 0.16mg/ml test liquid and the dilution of 1.0 EU/ml endotoxin solution equivalent.The negative reaction of result: NPC and NC, the positive reaction of PPC and PC, the bacterial endotoxin check result is up to specification.
Embodiment 4
Add the solution that the injection water is processed 5mmol/L with calcium hydroxide, filter, sterilization detects aseptic and pyrogen is qualified.
The bacterial endotoxin working standard is diluted with water to 0.125EU/ml with the bacterial endotoxin inspection; The injection fleraxacin is diluted to 0.333mg/ml with above-mentioned aqua calcis processes NPC, use the dilution of 0.667mg/ml test liquid and 0.25 EU/ml endotoxin solution equivalent to process PPC.The negative reaction of result: NPC and NC, the positive reaction of PPC and PC, the bacterial endotoxin check result is up to specification.
Embodiment 5
Add the solution that the injection water is processed 13mmol/L with magnesium chloride, filter, sterilization detects aseptic and pyrogen is qualified.
The bacterial endotoxin working standard is diluted with water to 0.5EU/ml with the bacterial endotoxin inspection; The injection fleraxacin is diluted to 0.167mg/ml with above-mentioned magnesium chloride solution processes NPC, use the dilution of 0.333mg/ml test liquid and 1.0 EU/ml endotoxin solution equivalent to process PPC.The negative reaction of result: NPC and NC, the positive reaction of PPC and PC, the bacterial endotoxin check result is up to specification.
Embodiment 6
Use lime chloride and magnesium sulphate to add the injection water and process to process respectively and contain lime chloride and the magnesium sulphate solution as 6mmol/L and 3mmol/L, filtration is sterilized, and detects aseptic and pyrogen is qualified.
The bacterial endotoxin working standard is diluted with water to 0.25EU/ml with the bacterial endotoxin inspection; The injection fleraxacin is diluted to 0.333mg/ml with above-mentioned mixed solution processes NPC, use the dilution of 0.667mg/ml test liquid and 0.5 EU/ml endotoxin solution equivalent to process PPC.The negative reaction of result: NPC and NC, the positive reaction of PPC and PC, the bacterial endotoxin check result is up to specification.
Embodiment 7 test samples are with the result of limulus reagent test inspection and pyrogen test result's contrast
Get the injection fleraxacin sample of using in the foregoing description, detect with the pyrogen method, testing result is qualified.This shows; What Bacterial endotoxins inspection that the present invention is used and pyrogen method detected comes to the same thing; Explained that this method can substitute original pyrogen test; And this law is easy and simple to handle, and is sensitive and accurate, and the raising of quality standard, the clearance of finished product and the security of clinical use are all had positive meaning.
Claims (3)
1. method that detects injection fleraxacin bacterial endotoxin comprises and utilizes TAL to pass through gel method or photometric means detects the injection fleraxacin, it is characterized in that injection fleraxacin to be measured is detected after with the bivalent cation solution dilution.
2. method according to claim 1 is characterized in that, described bivalent cation solution is that calcium ion, magnesium ion and other have one or more the formed solution in the compound of identical valent ion.
3. method according to claim 1 is characterized in that, described bivalent cation solution is the anticoagulant for storage of whole blood special diluting agent.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105445467A (en) * | 2015-11-27 | 2016-03-30 | 中国大冢制药有限公司 | Method for detecting bacterial endotoxin of sodium metabisulfite |
| CN108027374A (en) * | 2015-07-28 | 2018-05-11 | 博斯特尔莱布尼茨林根岑特鲁姆研究中心 | For measuring endotoxic improved bacterial endotoxin test |
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| CN1536363A (en) * | 2003-04-09 | 2004-10-13 | 中国科学院大连化学物理研究所 | Method for determing sensitivity of limulus reagent |
| US20050069972A1 (en) * | 2003-04-18 | 2005-03-31 | Associates Of Cape Cod, Inc. | Kit for detecting endotoxin |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108027374A (en) * | 2015-07-28 | 2018-05-11 | 博斯特尔莱布尼茨林根岑特鲁姆研究中心 | For measuring endotoxic improved bacterial endotoxin test |
| US10585097B2 (en) | 2015-07-28 | 2020-03-10 | Forschungszentrum Borstel Leibniz Lungerzentrum | Bacterial endotoxin test for the determination of endotoxins |
| CN108027374B (en) * | 2015-07-28 | 2020-07-07 | 博斯特尔莱布尼茨林根岑特鲁姆研究中心 | Improved bacterial endotoxin test for assaying endotoxin |
| CN105445467A (en) * | 2015-11-27 | 2016-03-30 | 中国大冢制药有限公司 | Method for detecting bacterial endotoxin of sodium metabisulfite |
| CN105445467B (en) * | 2015-11-27 | 2017-07-28 | 中国大冢制药有限公司 | The detection method of sodium pyrosulfite bacterial endotoxin |
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Application publication date: 20121226 |