CN102692494B - Endotoxin detection method for nano-particle size analyzer - Google Patents
Endotoxin detection method for nano-particle size analyzer Download PDFInfo
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- CN102692494B CN102692494B CN 201210196656 CN201210196656A CN102692494B CN 102692494 B CN102692494 B CN 102692494B CN 201210196656 CN201210196656 CN 201210196656 CN 201210196656 A CN201210196656 A CN 201210196656A CN 102692494 B CN102692494 B CN 102692494B
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- endotoxin
- particle size
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- standard solution
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- 239000002158 endotoxin Substances 0.000 title claims abstract description 60
- 238000001514 detection method Methods 0.000 title claims abstract description 27
- 239000002105 nanoparticle Substances 0.000 title abstract 3
- 239000002245 particle Substances 0.000 claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 23
- 239000012086 standard solution Substances 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000012360 testing method Methods 0.000 claims description 10
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 239000000523 sample Substances 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 5
- 231100000284 endotoxic Toxicity 0.000 claims description 5
- 230000002346 endotoxic effect Effects 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 4
- 238000011156 evaluation Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 3
- 239000012488 sample solution Substances 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 4
- 239000000243 solution Substances 0.000 abstract description 2
- 239000012895 dilution Substances 0.000 abstract 1
- 238000010790 dilution Methods 0.000 abstract 1
- 238000000611 regression analysis Methods 0.000 abstract 1
- 241000239222 Tachypleus Species 0.000 description 9
- 239000006166 lysate Substances 0.000 description 9
- 239000000463 material Substances 0.000 description 5
- 240000007164 Salvia officinalis Species 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 235000005412 red sage Nutrition 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 230000001754 anti-pyretic effect Effects 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 238000004848 nephelometry Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000239218 Limulus Species 0.000 description 1
- 230000004523 agglutinating effect Effects 0.000 description 1
- 230000002605 anti-dotal effect Effects 0.000 description 1
- 239000002221 antipyretic Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 238000013499 data model Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 108010048121 pro-clotting enzyme Proteins 0.000 description 1
- 239000002510 pyrogen Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an endotoxin detection method for a nano-particle size analyzer, and belongs to the field of bacterial endotoxin detection. According to the characteristic that the endotoxin can be agglomerated into nano-colloidal particles in a water solution, the method comprises the followings steps: preparing a series of endotoxin standard solutions with at least three dilution concentrations; detecting the particle sizes of the standard solutions by adopting the nano-particle size analyzer; performing regression analysis on the detected particle size data and the concentration of a corresponding standard solution, and establishing a standard curve; and finally calculating the content of the endotoxin of a sample according to the standard curve and the endotoxin particle size in the sample to be detected. The method has the advantages of quick detection speed, no consumption of detection reagent, low detection cost and high reliability.
Description
Technical field
The present invention relates to a kind of endotoxin detection method, relate in particular to a kind of method that nanometer particle size analyser detects for endotoxin, belong to the detection of bacterial endotoxin field.
Background technology
Bacterial endotoxin is lipopolysaccharides, is also referred to as liposome, and it is the composition of Gram-negative bacteria mantle, extensively is present in occurring in nature, and this material enters blood of human body can cause heating, is commonly called as pyrogen reaction.Produce serious bad reaction because this type of material may cause the people, therefore in drug injection, need strict control.
Detection of bacterial endotoxin can be divided into qualitative detection and quantitatively detect two classes.Conventional detection method is the rabbit method, and the test sample vein is injected in the rabbit body, observes the situation of change of body temperature in official hour.The method disturbing factor is many, and poor sensitivity particularly may also there will be false negative to the medicine of antipyretic effect or the injection of antipyretic and antidotal type.
Endotoxin in pharmacopeia detects and all adopts limulus reagent test, and wherein: the tachypleus amebocyte lysate gel method is under suitable condition (temperature, pH value and noiseless material), and bacterial endotoxin activates the proclotting enzyme in tachypleus amebocyte lysate, makes tachypleus amebocyte lysate produce agglutinating reaction and forms gel; The tachypleus amebocyte lysate nephelometry is linear with endotoxin concns according to the variation of the turbidity in tachypleus amebocyte lysate and endotoxin course of reaction, thereby measures endotoxin content.These method preparatory stages are long, detect consuming timely, and cost is high, can not realize rapidly, continuously, the online detection.
The pilot study of another some method is more, also fails to form application, or can not overcome and need to add reagent or use the shortcoming such as expensive instrument, can't realize the direct-detection to sample.
Summary of the invention
The present invention is directed to the defect of prior art, and propose a kind of method that nanometer particle size analyser detects for endotoxin, to reduce costs and to improve detection speed.
The method comprises the steps:
Step 1: the endotoxin standard solution of preparation series concentration adopts the nanometer particle size analyser to carry out the particle diameter detection to these endotoxin standard solution;
Step 2: the particle size data of detection gained and the concentration of corresponding endotoxin standard solution are done to regretional analysis, Criterion curve ln (d)=kC+b, in formula: d is particle diameter, and C is endotoxin concns, and k is correction coefficient, and b is correction factor;
Step 3: according to typical curve, calculate the endotoxin content in testing sample.
Series concentration in described step 1 comprises at least three dilute concentrations; The curve evaluation coefficient of the typical curve in described step 2 is greater than 0.98.
Technique effect:
1, the sense cycle of each sample, at 2～5 minutes, has shortened the time that endotoxin detects greatly, and detection speed is fast.
2, detect reagent (tachypleus amebocyte lysate) without consuming, can significantly reduce endotoxic testing cost.
3, detection method is easy, and testing result is comparatively accurate, and reliability is high, is applicable to the endotoxin assay in water for injection, injection semi-manufacture and finished product.
Embodiment
Below the invention will be further described.
Because endotoxic structure one end is hydrophobic fat chain, an end is hydrophilic sugar chain, the hydrophobic side of surfactant-like and water-wet side.Bacterial endotoxin exists with the form of reuniting in aqueous solution, and the reunion molecular weight is in several thousand to 1,000,000 scope, and according to the feature of the nanometre glue particle of solution, its particle size increases with the increase of concentration.The nanometer particle size analyser is the instrument of the detection particle diameter commonly used, and the particle diameter of bacterial endotoxin molecular group can be detected by particle size analyzer be that we are serendipitous.
The inventive method specifically comprises the steps:
Step 1: adopt normative reference, international standard, national standard or working stamndard endotoxin as the standard endotoxin, determine required standard endotoxin series concentration, this series concentration comprises at least three dilute concentrations, the endotoxin standard solution of preparation series concentration, adopt the nanometer particle size analyser to carry out the particle diameter detection to these endotoxin standard solution.
Step 2: the particle size data of detection gained and the concentration of corresponding endotoxin standard solution are done to regretional analysis, Criterion curve ln (d)=kC+b, the curve evaluation coefficient of this typical curve need be greater than 0.98, in formula: d is particle diameter (unit: nm), C is endotoxin concns (unit: EU/ml), k is correction coefficient, and b is correction factor.
Step 3: detect under the same conditions the endotoxin particle diameter in testing sample solution, according to typical curve, calculate the endotoxin content in testing sample.
One embodiment of the present of invention below are provided:
The foundation of I, bacterial endotoxin typical curve
Equipment and materials: a, Ma Erwen Nano ZS ZEW3600 type laser diffraction analyzer; B, bacterial endotoxin working standard (lot number: 150601-201070, specification: 140EUAmp
-1, Nat'l Pharmaceutical & Biological Products Control Institute); C, bacterial endotoxin check water (lot number: 100130, Zhanjiang Bo Kang sea life company limited).
Method step: get the working stamndard endotoxin, use and check that water is mixed with the endotoxin standard solution that concentration is 100EU/ml, re-use and check that water progressively is diluted to the endotoxin standard solution of series concentration, series concentration is respectively 50EU/ml, 10EU/ml, 1EU/ml, 0.25EU/ml.
Open the particle size analyzer power supply, more than preheating 30min, serial endotoxin standard solution is joined in detection cell and detected successively to high concentration by low concentration,, peak area strong to the peak in measured particle diameter distribution plan and particle size distribution range carry out the data model computing, obtain the logarithm ln (d) of endotoxin particle diameter.Using ln (d) as ordinate Y, and the endotoxin concns of usining carries out regretional analysis as horizontal ordinate X, and linear equation is: ln (d)=0.061C+0.846, R
2=0.996, the curve evaluation coefficient is greater than 0.98, and typical curve is set up, and the typical curve result is as shown in table 1.
Table 1
II, measure respectively the endotoxin content of red sage roo drip liquid intermediate by this method and dynamic turbidimetric
Equipment and materials: a, Ma Erwen Nano ZS ZEW3600 type laser diffraction analyzer; B, BET-16M bacteria endotoxin detector; C, dynamic turbidimetric tachypleus amebocyte lysate (lot number: 0711010, λ=0.03EUmL
-1, specification: 0.6mLAmp
-1, Zhanjiang Bo Kang sea life company limited); D, bacterial endotoxin working standard (lot number: 2007-1, specification: 150EUAmp
-1, Nat'l Pharmaceutical & Biological Products Control Institute); E, bacterial endotoxin check water (lot number: 070130, specification: 5mLAmp
-1, Zhanjiang Bo Kang sea life company limited); F, bacterial endotoxin indicator (lot number: 071114, specification: 3000EUAmp
-1, Zhanjiang Bo Kang sea life company limited); G, red sage roo drip liquid intermediate (lot number: 05110209, Shanghai Worldbest & Anhui Jinhui Pharmaceutical Co., Ltd.).
Method: get the red sage roo drip liquid intermediate, adopt respectively this method (calculating by the typical curve in the part I) and tachypleus amebocyte lysate Nephelometric Determination endotoxin content, result is as shown in table 2.
Table 2
Result shows, the result that this method and tachypleus amebocyte lysate nephelometry record is comparatively approaching, and testing result is comparatively accurate, and this has also illustrated the reliability of this method.
Claims (3)
1. one kind by endotoxic method in nanometer particle size analyser detection of drugs injection, it is characterized in that:
The method comprises the steps:
Step 1: use the endotoxin standard solution that checks water preparation 100EU/ml, re-use and check the endotoxin standard solution that is diluted with water to series concentration, described series concentration comprises at least three dilute concentrations, adopts the nanometer particle size analyser to carry out the particle diameter detection to the endotoxin standard solution of series concentration;
Step 2: the particle size data of detection gained and the concentration of corresponding endotoxin standard solution are done to regretional analysis, Criterion curve ln (d)=kC+b, in formula: d is particle diameter, and C is endotoxin concns, and k is correction coefficient, and b is correction factor;
Step 3: detect under the same conditions the endotoxin particle diameter in testing sample solution, according to typical curve, calculate the endotoxin content in testing sample.
2. according to claim 1 by endotoxic method in nanometer particle size analyser detection of drugs injection, it is characterized in that: described series concentration is 50EU/ml, 10EU/ml, 1EU/ml, 0.25EU/ml.
3. according to claim 1 and 2 by endotoxic method in nanometer particle size analyser detection of drugs injection, it is characterized in that: the curve evaluation coefficient of the typical curve in described step 2 is greater than 0.98.
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| CN111077307B (en) * | 2018-10-21 | 2022-08-23 | 厦门鲎试剂生物科技股份有限公司 | Novel method for rapidly detecting sepsis by using gram-negative bacterial infection |
| CN113804593B (en) * | 2020-06-11 | 2024-05-24 | 北京科兴生物制品有限公司 | Detection method and application of split vaccine splitting effect |
| CN112505279B (en) * | 2020-12-04 | 2021-08-13 | 北京师范大学 | Method for detecting endotoxin concentration in biochemical tail water by using nanotube membrane pressure difference |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101903776A (en) * | 2007-12-19 | 2010-12-01 | 兴和株式会社 | Method for measuring endotoxin and reagent kit for measuring endotoxin |
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| US4221865A (en) * | 1978-10-06 | 1980-09-09 | Baxter Travenol Laboratories, Inc. | Method for determining endotoxin concentration |
| JP5401115B2 (en) * | 2009-02-13 | 2014-01-29 | 興和株式会社 | Measuring method and measuring apparatus for biologically active substances derived from living organisms |
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