CN103604779B - A kind of protamine sulfate titration method - Google Patents
A kind of protamine sulfate titration method Download PDFInfo
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- CN103604779B CN103604779B CN201310338351.9A CN201310338351A CN103604779B CN 103604779 B CN103604779 B CN 103604779B CN 201310338351 A CN201310338351 A CN 201310338351A CN 103604779 B CN103604779 B CN 103604779B
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Abstract
" Chinese Pharmacopoeia " version regulation protamine sulfate in 2010 is tired employing bioassay method, relatively Heparin Standard product (S) and test sample (T) extend the degree of fresh rabbit blood or pig, rabbit plasma setting time, to measure tiring of test sample.The market price of rabbit plasma is expensive, 10 yuan/, often props up 0.5ml, measures a protamine sulfate sample, at least need 60.Patent of the present invention adopts ultraviolet spectrophotometer, measures solution transmittance under 550nm wavelength, measures tiring of protamine sulfate, and operation does not need rabbit plasma, and cost reduces, and faster and more convenient operation.
Description
Technical field
The present invention relates to biomedicine field, relate in particular to the mensuration that protamine sulfate is tired.
Background technology
" Chinese Pharmacopoeia " version annex VIIJ protamine sulfate bioassay method in 2010, details the assay method that protamine sulfate is tired, and judges that it is tired by rabbit plasma condensation situation.Concrete operation method is as follows:
(1) preparation of Heparin Standard product solution takes Heparin Standard product in right amount, adds 0.9% sodium chloride solution dissolve the solution making into several variable concentrations by tiring.
(2) preparation of protamine sulfate solution takes protamine sulfate, adds 0.9% sodium chloride solution, makes the solution of 1mg/ml.
(3) checking method gets small test tube 8,1st pipe and the 8th pipe are blank pipe, add 0.9% sodium chloride solution 0.2ml, 2nd ~ 7 pipes are test sample pipe, often pipe adds protamine sulfate solution 0.1ml, often pipe adds the Heparin Standard product dilution 0.1ml of above-mentioned a kind of concentration respectively again, mixes immediately.Then respectively at the blood plasma adding 0.7ml in above-mentioned each pipe, to put in 37 ± 0.5 DEG C of waters bath with thermostatic control preheating 5 ~ 10 minutes, often pipe adds 1% calcium chloride solution 0.1ml respectively, mixes immediately, avoids producing bubble, and starts the clock.Observe and record each pipe setting time.
Result judges that the setting time of two control tube must not differ more than 1.35 times.Be no more than in each pipe of two control tube average setting times 150% in the setting time of test sample pipe, using the highest pipe of heparin concentration as terminal pipe.Same repetition 5 times, 5 tests record the heparin concentration of terminal pipe, and difference must not be greater than 10 units.The mean value of 5 results, is the units with heparin in protamine sulfate 1mg.
The rabbit plasma used in inspection or in person prepare, or spend expensive price to buy, on market, the price of blood plasma is about 10 yuan/, often prop up 0.5ml, measure a protamine sulfate sample, repeat 5 times, at least 60 rabbit plasmas, more than 600 yuan of money, inspection cost is very high.In addition, complicated operation, time-consuming is long.
Summary of the invention
Order of the present invention there is provided a kind of protamine sulfate titration method.
Particularly, the protamine sulfate titration method that the object of this invention is to provide, inapplicable rabbit plasma, checks economical, easy to operate, quick.
The present invention adopts following assay method scheme to be achieved above-mentioned purpose:
(1) preparation of Heparin Standard product solution takes Heparin Standard product, adds 0.9% sodium chloride solution and dissolves and make into difference and to tire the Heparin Standard product dilution of concentration.
(2) preparation of protamine sulfate solution takes protamine sulfate, adds 0.9% sodium chloride solution, is diluted to the solution of 1mg/ml.
(3) checking method gets test tube, and often pipe adds protamine sulfate solution, more often pipe adds the Heparin Standard product dilution of isopyknic a kind of concentration, leaves standstill, gets supernatant, under 550nm wavelength, measure the transmittance of often pipe mixed liquor.
The concentration numbers of tiring of the Heparin Standard product of transmittance maximum tube, is tiring of protamine sulfate.
Compared with prior art, advantage of the present invention and good effect:
Through many experiments, the tiring of protamine sulfate that the present invention measures, tiring measured by the protamine sulfate bioassay method specified with " Chinese Pharmacopoeia " version in 2010 is completely the same.But carry out contrast to find, advantage of the present invention is that the experiment reagent 1. used is few, and do not need rabbit plasma, lime chloride, experimental cost is low; 2. swift to operate, the time is short; 3. test required instrument few, do not need water-bath, stopwatch etc.
Embodiment
Embodiment 1:
(1) preparation of Heparin Standard product solution
Take Heparin Standard product 150.05mg, unit titer 180iu/mg, add 0.9% sodium chloride solution and dissolve and make it to become difference and to tire each 25ml of Heparin Standard product dilution of concentration, concentration of tiring is respectively 145iu/ml, 150iu/ml, 155iu/ml, 160iu/ml, 165iu/ml, 170iu/ml.
(2) preparation of protamine sulfate solution
Take protamine sulfate 122.52mg, lot number 110901, add 0.9% sodium chloride solution, be diluted to the solution of 1mg/ml.
(3) checking method
Get 6, test tube, in each test tube, add protamine sulfate solution 5ml, then the corresponding concentration adding 5ml in each test tube is respectively 145iu/ml, 150iu/ml, 155iu/ml, 160iu/ml, 165iu/ml, 170iu/ml Heparin Standard product dilution, leave standstill, get supernatant, under 500nm wavelength, measure the transmittance of often pipe mixed liquor, repeat 3 times.
The results are shown in following table:
Detected by transmittance, the transmittance of the concentration 155iu/ml pipe of tiring of Heparin Standard product is the highest, is tiring as 155iu/mg of protamine sulfate.
(4) existing checking method
Get 8, test tube, the 1st pipe and the 8th pipe are blank pipe, add 0.9% sodium chloride solution 0.2ml, 2nd ~ 7 pipes are test sample pipe, often pipe adds protamine sulfate solution 0.1ml, more often pipe adds the Heparin Standard product dilution 0.1ml of above-mentioned a kind of concentration respectively, mixes immediately.Then respectively at the blood plasma adding 0.7ml in above-mentioned each pipe, to put in 37 ± 0.5 DEG C of waters bath with thermostatic control preheating 5 ~ 10 minutes, often pipe adds 1% calcium chloride solution 0.1ml respectively, mixes immediately, avoids producing bubble, and starts the clock.Observe and record each pipe setting time.
Embodiment 2:
(1) preparation of Heparin Standard product solution
Take Heparin Standard product 152.35mg, unit titer 180iu/mg, add 0.9% sodium chloride solution and dissolve and make it to become difference and to tire each 25ml of Heparin Standard product dilution of concentration, concentration is respectively 145iu/ml, 150iu/ml, 155iu/ml, 160iu/ml, 165iu/ml, 170iu/ml.
(2) preparation of protamine sulfate solution
Take protamine sulfate 125.30mg, lot number 110902, add 0.9% sodium chloride solution, be diluted to the solution of 1mg/ml.
(3) checking method
Get 6, test tube, in each test tube, add protamine sulfate solution 5ml, then the corresponding concentration adding 5ml in each test tube is respectively 145iu/ml, 150iu/ml, 155iu/ml, 160iu/ml, 165iu/ml, 170iu/ml Heparin Standard product dilution, leave standstill, get supernatant, under 450nm wavelength, measure the transmittance of mixed liquor in each test tube, repeat 3 times.
The results are shown in following table:
Detected by transmittance, the transmittance of the concentration 150iu/ml pipe of tiring of Heparin Standard product is the highest, is tiring as 150iu/mg of protamine sulfate.
(4) existing checking method
Get 8, test tube, the 1st pipe and the 8th pipe are blank pipe, add 0.9% sodium chloride solution 0.2ml, 2nd ~ 7 pipes are test sample pipe, often pipe adds protamine sulfate solution 0.1ml, more often pipe adds the Heparin Standard product dilution 0.1ml of above-mentioned a kind of concentration respectively, mixes immediately.Then respectively at the blood plasma adding 0.7ml in above-mentioned each pipe, to put in 37 ± 0.5 DEG C of waters bath with thermostatic control preheating 5 ~ 10 minutes, often pipe adds 1% calcium chloride solution 0.1ml respectively, mixes immediately, avoids producing bubble, and starts the clock.Observe and record each pipe setting time.
As can be seen from embodiment 1,2, adopt protamine sulfate of the present invention to tire the method for inspection, simple and easy to do, required testing reagent is few, and cost is low, and valence value measures accurately same.
The above is only preferred embodiment of the present invention, and be not restriction the present invention being made to other form, any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the Equivalent embodiments of equivalent variations.But everyly do not depart from technical solution of the present invention content, any simple modification, equivalent variations and the remodeling done above embodiment according to technical spirit of the present invention, still belong to the protection domain of technical solution of the present invention.
Claims (8)
1. a protamine sulfate assay method of tiring, is characterized in that comprising the following steps:
(1) preparation of Heparin Standard product solution
Take Heparin Standard product, add sodium chloride solution and dissolve and make it to become difference and to tire the Heparin Standard product dilution of concentration, unit is in iu/mg;
(2) preparation of protamine sulfate solution
Take protamine sulfate testing sample, add sodium chloride solution, be diluted to protamine sulfate solution;
(3) checking method
Get test tube, the protamine sulfate solution obtained in step (2) is added respectively in every test tube, the difference adding preparation in above-mentioned steps (1) again in every test tube is respectively tired the Heparin Standard product dilution of concentration, leave standstill, get supernatant, measure the transmittance of mixed liquor in each test tube, the protamine sulfate solution wherein in each test tube is equal with the volume of Heparin Standard product dilution;
According to the valence value of tiring corresponding to concentration numbers of the Heparin Standard product dilution in the test tube that transmittance is the highest, calculate the unit titer of protamine sulfate sample, in iu/mg.
2. assay method according to claim 1, is characterized in that: in described step (1) and (2), the concentration of sodium chloride solution used is 0.8 quality %-1.2 quality %.
3. assay method according to claim 1, is characterized in that: the concentration of the protamine sulfate solution in described step (2) is 0.6-1.2mg/ml.
4. assay method according to claim 3, is characterized in that: the concentration of the protamine sulfate solution in described step (2) is 1mg/ml.
5. assay method according to claim 1, is characterized in that: the difference in described step (1) tire concentration Heparin Standard product dilution between concentration difference of tiring can be identical or different.
6. assay method according to claim 1, is characterized in that: the difference in described step (1) tire concentration Heparin Standard product dilution between concentration difference of tiring be 3-8iu/ml.
7. assay method according to claim 1, is characterized in that: the wavelength measuring the light of transmittance in described step (3) is 400nm-600nm.
8. assay method according to claim 1, is characterized in that: the tire concentration range of tiring of Heparin Standard product dilution of concentration of difference is 100iu/ml-200iu/ml in described step (1).
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3915640A (en) * | 1974-08-06 | 1975-10-28 | Warner Lambert Co | Method and composition for detecting fibrin monomers and fibrin degradation products |
| US4175182A (en) * | 1978-07-03 | 1979-11-20 | Research Corporation | Separation of high-activity heparin by affinity chromatography on supported protamine |
| US4551308A (en) * | 1981-07-09 | 1985-11-05 | International Technidyne Corp. | Apparatus for analyzing the influence of additive reagents upon the coagulation of blood and related methods |
| WO1998038263A1 (en) * | 1997-02-27 | 1998-09-03 | St. John's University | Optical membrane films for polycation detection |
| CN101357935A (en) * | 2008-09-03 | 2009-02-04 | 河北科技大学 | Method for separating purified protamine using reverse micelle method |
| CN102445483A (en) * | 2011-09-30 | 2012-05-09 | 中国科学院烟台海岸带研究所 | Method for detecting heparins |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ATE552494T1 (en) * | 2003-07-09 | 2012-04-15 | Univ Auburn | REVERSIBLE ELECTOCHEMICAL SENSORS FOR POLYIONS |
-
2013
- 2013-07-30 CN CN201310338351.9A patent/CN103604779B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3915640A (en) * | 1974-08-06 | 1975-10-28 | Warner Lambert Co | Method and composition for detecting fibrin monomers and fibrin degradation products |
| US4175182A (en) * | 1978-07-03 | 1979-11-20 | Research Corporation | Separation of high-activity heparin by affinity chromatography on supported protamine |
| US4551308A (en) * | 1981-07-09 | 1985-11-05 | International Technidyne Corp. | Apparatus for analyzing the influence of additive reagents upon the coagulation of blood and related methods |
| WO1998038263A1 (en) * | 1997-02-27 | 1998-09-03 | St. John's University | Optical membrane films for polycation detection |
| CN101357935A (en) * | 2008-09-03 | 2009-02-04 | 河北科技大学 | Method for separating purified protamine using reverse micelle method |
| CN102445483A (en) * | 2011-09-30 | 2012-05-09 | 中国科学院烟台海岸带研究所 | Method for detecting heparins |
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