CN103149170A - Solution concentration of nadroparin calcium determined by phenanthroline-zinc sulfate ultraviolet spectroscopy - Google Patents
Solution concentration of nadroparin calcium determined by phenanthroline-zinc sulfate ultraviolet spectroscopy Download PDFInfo
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Abstract
The invention discloses solution concentration of nadroparin calcium determined by phenanthroline-zinc sulfate ultraviolet spectroscopy. Under the environment of buffered solution, Ultraviolet absorbance degrees of mixed solution which includes Zn2+ions, phenanthroline and the nadroparin calcium are determined, and then the concentration of the nadroparin calcium is determined by quantity. According to the solution concentration of the nadroparin calcium determined by the phenanthroline-zinc sulfate ultraviolet spectroscopy, a phenanthroline-zinc sulfate ultraviolet spectroscopy test system is applied, quick quantitative determination of the solution concentration of the nadroparin calcium can be achieved. The linear range of an analytical method of the solution concentration of the nadroparin calcium determined by the phenanthroline-zinc sulfate ultraviolet spectroscopy is 1.184%-16.33%, meanwhile, the analytical method of the solution concentration of the nadroparin calcium determined by the phenanthroline-zinc sulfate ultraviolet spectroscopy has good anti-jamming capability and can be applied in the practical production process of nadroparin calcium medicines and capable of carrying out concentration determination to intermediate product solution or finished product solution.
Description
Technical field
The invention belongs to the production field of nadroparin calcium medicine, particularly relate to a kind of method of using Phen-zinc sulfate ultraviolet spectroscopy quantitative determination nadroparin calcium solution concentration.
Background technology
The mucopolysaccharide sulfuric ester that heparin (Heparin) alternately is comprised of GLUCOSAMINE, L-iduronic acid, N-acetyl-glucosamine and glucuronic acid, molecular weight is from 5 to 30kDa, and wherein sulfate radical accounts for 40%.Usually, heparin class medicine derives from pig, Roll mucous membrane and lungs at first.Now medically, heparin formally is applied to clinical treatment mainly as anticoagulant in nineteen fifty-three, is considered to the most effective and the anticoagulation medicine quantity maximum.What use at first is unfractionated heparin, i.e. Unfractionated Heparin.To last century Mo, by methods such as chemical cracking or biological enzymolysis, unfractionated heparin is prepared into mean molecular weight has been distributed as 3500 ~ 6500 heparin derivatives, be LMWHs (Low Molecular Weight Heparin), its drug effect has obviously obtained enhancing, becomes the novel heparin class medicine that is widely used.According to statistics, at present the whole world approximately has 1/3rd heparin bulk drug to be used for making the standard heparin preparation of sodium, all the other approximately 2/3rds as raw material for the production of Low-molecular-weight Heparins Calcium (low molecular sodium heparin) bulk drug.Further data show, in 2008, global heparin bulk drug sales volume was about 800,000,000 dollars, and global heparin class sales amount of medicine is near 6,000,000,000 dollars simultaneously; Estimate 2012, the sales volume of global heparin class medicine will be over 9,000,000,000 dollars.The general flow sheet of LMWHs class medicine as shown in Figure 1.
Wherein, nadroparin calcium (Nadroparin Calcium) is the LMWHs medicine that adopts the nitrous acid cracking process to prepare, and its principal character comprises: average molecular weight range is 3600-5000Da; The reduction end of molecular structure is 6-O-sulfuric acid-2, and 5-dehydration-PEARLITOL 25C, non-reduced end are 2-O-sulfuric acid-α-L-iduronic acid; It is 95～130IU/mg that anti-Xa tires, and AXa/A II a numerical value is 2.5～4.0.The nadroparin calcium medicine that our company produces reaches corresponding quality index, and its simple technological process of production as shown in Figure 2.In actual production process, need to carry out the techniques such as ultrafiltration, alcohol precipitation and drying, we have found the nadroparin calcium solution that obtains later at ultrafiltration technology, need to carrying out rapidly accurately, concentration detects, and according to the gained test result, it is carried out suitable concentration regulation and control, so extremely be conducive to the control of subsequent technique, especially alcohol precipitation process.But in the production run of reality, the nadroparin calcium solution that ultrafiltration technology obtains later on is difficult to by analytical approachs such as solution weight method or high performance liquid chromatographies, completes rapidly this Detection task.
On the other hand, the initial source of heparin class medicine is all mainly the animal materials such as pig intestinal mucosa both at home and abroad at present, has geographic difference, has larger variation range at aspects such as nitrogen content, sulfur content and glycosyl sequences.Simultaneously, the nadroparin calcium medicine in fact also is one and has stipulated molecular weight ranges and the macromolecule mixture with other main character, therefore the detection method for nadroparin calcium must possess stronger antijamming capability, otherwise in the testing process of actual sample, can produce larger analytical error, the gained test result can not be applied to control and instruct in technique subsequent technique.
Summary of the invention
The object of the invention is to provides a kind of Phen-zinc sulfate ultraviolet spectroscopy to measure the solution concentration of nadroparin calcium for the above-mentioned deficiency in existing nadroparin calcium actual production.
In order to achieve the above object, the present invention has adopted following technical scheme:
Phen-zinc sulfate ultraviolet spectroscopy is measured the solution concentration of nadroparin calcium, under the environment of buffer solution, by measuring Zn
2+The ultraviolet absorptivity of ion, Phen and nadroparin calcium three mixed solution comes the concentration of quantitative measurement nadroparin calcium.
Particularly, under the environment of buffer solution, with Zn
2+Solion, Phen solution and nadroparin calcium solution mix, Zn in mixed solution
2+The concentration range of ion is at 0.05 ~ 0.3mM, and the amount of substance of Phen is Zn
2+More than 2 times of the amount of substance of ion, the ultraviolet absorptivity of mensuration mixed solution;
During the Criterion working curve, what add mixed solution is the nadroparin calcium solution of concentration known, and the quality of nadroparin calcium is with respect to Zn
2+The amount of substance of ion is 11.84g/mmol～163.3g/mmol;
After the Criterion working curve, what add mixed solution is the nadroparin calcium solution of unknown concentration, calculates the concentration of nadroparin calcium solution according to the standard working curve of the absorbance of measuring and foundation, when the quality of the nadroparin calcium that adds with respect to Zn
2+The amount of substance of ion is not when the scope of 11.84g/mmol～163.3g/mmol, in the situation that do not change add the method for volume by concentrated or dilution to change to add mixed solution in the concentration of nadroparin calcium solution, measure its absorbance and utilize standard working curve to calculate the concentration of nadroparin calcium solution, make the quality of nadroparin calcium with respect to Zn
2+The amount of substance of ion is in the scope of 11.84g/mmol ~ 163.3g/mmol;
The concentration that it is pointed out that nadroparin calcium refers to the quality percent by volume of nadroparin calcium.
Particularly, the solvent of mixed solution is at least a in water, ethanol, methyl alcohol, isopropyl alcohol, n-propanol and normal butyl alcohol.
Particularly, buffer solution comprises the aqueous solution of at least a material in sodium acetate, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium carbonate, sodium bicarbonate and niter cake.
Preferably, buffer solution is sodium acetate solution, and the concentration of sodium acetate in mixed solution is 0.05 ~ 0.6M.
Preferably, the concentration of sodium acetate in mixed solution is 0.3M.
Particularly, Zn
2+Ion is from zinc sulfate, Zn
2+The concentration of ion is 0.2mM.
Preferably, the amount of substance of Phen is Zn
2+4 times of the amount of substance of ion.
When preferably, measuring the absorbance of mixed solution, ultraviolet wavelength is 302 ± 1nm.
Preferably, during the preparation mixed solution, zinc sulfate solution is 1.0mL, 2.0mM, the ethanolic solution of Phen is 1.0mL, 8.0mM, aqueous sodium acetate solution is 3.0mL, 1.0M, after fully mixing, adds 2.0mL nadroparin calcium solution and 3.0mL water for injection, fully mix and standing after, measure mixed solution in the absorbance of 302 ± 1nm.
The invention discloses Phen-zinc sulfate ultraviolet spectroscopy and measure the solution concentration of nadroparin calcium, this assay method is used Phen-zinc sulfate ultraviolet spectrum test system, realized the Quantitative detection to the nadroparin calcium solution concentration, the range of linearity of its analytical approach is 1.184%～16.33%; Simultaneously, the method has good antijamming capability to calcium ion, sodion and sulfate ion, can be applied in the actual production process of nadroparin calcium medicine, and middle product solution or final mean annual increment solution are carried out the concentration detection.
Description of drawings
Fig. 1 is LMWHs class medicament universal flow sheet;
Fig. 2 is the production technological process of nadroparin calcium medicine;
Fig. 3 is the ultraviolet spectrum detection figure of different batches nadroparin calcium sample in Phen-solution of zinc sulfate;
Fig. 4 is the variable concentrations calcium ion affects detection figure to what detect solution;
Fig. 5 is different sodium acetate solution consumptions affects detection figure to what detect solution;
Fig. 6 is different Phen solution usage affects detection figure to what detect solution;
Fig. 7 is different sodium nitrite solution consumptions affects detection figure to what detect solution;
Fig. 8 is the detection figure of variable concentrations nadroparin calcium sample in Phen-solution of zinc sulfate.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is done further explanation clearly and completely.
At first need to prove, in the present invention, production technology source liquaemin sample used is from the biochemical medicine company of Hebei Changshan incorporated company; The nadroparin calcium sample is from Shenzhen Sciprogen Biology Medicine Co., Ltd.Simultaneously, the main chemical reagent that uses in the present invention comprises: anhydrous sodium acetate (CH
3COONa, analyze pure, CAS127-09-3); Phen (C
12H
8N
2H
2O analyzes purely, when CAS5144-89-8 uses, is mixed with ethanolic solution); White vitriol (ZnSO
47H
2O, analyze pure, CAS7446-20-0); Lime chloride (CaCl
2, pharmaceutical grade, CAS10043-52-4).In embodiment, the concentration of nadroparin calcium solution refers to the quality percent by volume of nadroparin calcium.
Embodiment
Embodiment discloses the solution concentration of Phen-zinc sulfate ultraviolet spectroscopy mensuration nadroparin calcium, at first by experiment one check successively the applicability of Phen-zinc sulfate system, the anti-interference on calcium ion, the impact of sodium acetate solution consumption, the impact of Phen consumption and the impact of sodium nitrite solution consumption to testing five, but applicability variation range and detection principle so that this detection system to be described; Six standard working curves of setting up absorbance and nadroparin calcium solution concentration by experiment then, the reliability of the method and this standard working curve is verified in experiment seven by experiment.
The applicability of experiment one, Phen-zinc sulfate system
Get 6 15mL centrifuge tubes, wherein 5 are the sample test system, and 1 is blank system (not adding the nadroparin calcium sample).At first, in all centrifuge tubes, add respectively 1.0mL2.0mM solution of zinc sulfate, 1.0mL8.0mM Phen solution and 3.0mL1.0M sodium acetate solution, fully mix.Then, in the sample test system, add respectively the nadroparin calcium sample solution of the different lot numbers of 2.0mL, and 3.0mL water for injection, shake up; In the blank system, add 5.0mL water for injection, fully mix.After standing 15 minutes, as blank solution, in 200 ~ 400nm scope, scan respectively the ultraviolet spectrum of 5 sample test systems with the blank system.The gained test result as shown in Figure 3.
Can find from Fig. 3, in Phen-zinc sulfate system, the nadroparin calcium sample solution of 5 different batches all has obvious maximal ultraviolet absorption peak at 302 ± 1nm place.This shows, Phen-zinc sulfate system detects for the ultraviolet spectrum of different batches nadroparin calcium sample solution, and good system suitability is arranged.
Experiment two, Phen-zinc sulfate system are to the anti-interference of calcium ion
Get 7 15mL centrifuge tubes, wherein 6 are the sample test system, and 1 is blank system (not adding the nadroparin calcium sample).At first, in all centrifuge tubes, add respectively 1.0mL2.0mM solution of zinc sulfate, 1.0mL8.0mM Phen solution and 3.0mL1.0M sodium acetate solution, fully mix. then, in 6 sample test systems, all add 2.00mL10% nadroparin calcium sample solution, shake up.Then, in 6 sample test systems, add respectively 0.00,0.05,0.10,0.15,0.20, the 5.0M calcium chloride solution of 0.25mL, and 3.00,2.95,2.90,2.85,2.80, the water for injection of 2.75mL; In the blank system, add 5.0mL water for injection, fully mix.After standing 15 minutes, as blank solution, in 200 ~ 400nm scope, scan respectively the ultraviolet spectrum of 6 sample test systems with the blank system.The gained test result as shown in Figure 4.
Can find from Fig. 4, concentration range is the calcium ion that adds of 2.5%-12.5%, on the not significantly impact of test result of Phen-zinc sulfate ultraviolet spectrum system.This shows, Phen-zinc sulfate ultraviolet spectrum system has very strong Anti-Jamming to the calcium ion concentration in nadroparin calcium sample test solution.
Experiment three, sodium acetate solution consumption are on the impact of Phen-zinc sulfate ultraviolet spectrum test system
Get 7 15mL centrifuge tubes, wherein 6 are the sample test system, and 1 is blank system (not adding the nadroparin calcium sample).At first, in all centrifuge tubes, add respectively 1.0mL2.0mM solution of zinc sulfate and 1.0mL8.0mM Phen solution, fully mix.Then, in 6 sample test systems, all add 2.00mL10% nadroparin calcium sample solution, shake up.Then, in 6 sample test systems, add respectively 1.0,1.5,2.0,3.0,3.5, the 1.0M sodium acetate solution of 4.0mL, and 5.0,4.5,4.0,3.0,2.5, the water for injection of 2.0mL; In the blank system, add 3.0mL sodium acetate solution and 5.0mL water for injection, fully mix.After standing 15 minutes, as blank solution, in 200 ~ 400nm scope, scan respectively the ultraviolet spectrum of 6 sample test systems with the blank system.The gained test result as shown in Figure 5.
Can find that from Fig. 5 different sodium acetate solution consumptions to the test result of Phen-zinc sulfate ultraviolet spectrum system, only can produce smaller change.This shows, the sodium acetate solution consumption is very little on the impact of Phen-zinc sulfate ultraviolet spectrum test system.But, in order to strengthen Phen-zinc sulfate ultraviolet spectrum system, to the anti-interference of specimen solution acidity and salt content, in testing program, keeping the addition of sodium acetate solution is 3.0mL.
Experiment four, Phen solution usage are on the impact of Phen-zinc sulfate ultraviolet spectrum test system
Get 7 15mL centrifuge tubes, wherein 6 are the sample test system, and 1 is blank system (not adding the nadroparin calcium sample).At first, in all centrifuge tubes, add respectively 1.0mL2.0mM solution of zinc sulfate and 3.0mL sodium acetate solution, fully mix.Then, in 6 sample test systems, all add 2.00mL10% nadroparin calcium sample solution, shake up.Then, in 1-6 sample test system, add respectively 0,0.5,0.8,1.0,1.2, the 8.0mM Phen solution of 1.5mL, and 4.0,3.5,3.2,3.0,2.8, the water for injection of 2.5mL; In the blank system, add 1.0mL8.0mM Phen solution and 5.0mL water for injection, fully mix.After standing 15 minutes, as blank solution, in 200 ~ 400nm scope, scan respectively the ultraviolet spectrum of 6 sample test systems with the blank system.Acquired results as shown in Figure 6.
Can find that from Fig. 6 different Phen solution usage has the impact of highly significant to Phen-zinc sulfate ultraviolet test system.During less than the Phen solution usage in the blank system, the gained ultraviolet spectrum drops on the minus zone of absorbance when the consumption of Phen solution; And when the consumption of Phen solution was equal to or greater than Phen solution usage in the blank system, the absorbance of gained ultraviolet spectrum was at Zheng Qu, and became multiple to increase.This has illustrated, the nadroparin calcium specimen derives from Phen and is combined in the time of nadroparin calcium molecule, zinc ion at the maximal ultraviolet absorption peak at 302nm place, and the not just combination of zinc ion and nadroparin calcium molecule.
Experiment five, sodium nitrite solution consumption are on the impact of Phen-zinc sulfate ultraviolet spectrum test system
Get 7 15mL centrifuge tubes, wherein 6 are the sample test system, and 1 is blank system (not adding the nadroparin calcium sample).At first, in all centrifuge tubes, add respectively 1.0mL2.0mM solution of zinc sulfate, 1.0mL8.0mM Phen solution and 3.0mL1.0M sodium acetate solution, fully mix.Then, in 6 sample test systems, all add 2.00mL10% nadroparin calcium sample solution, shake up.Then, in 1-6 sample test system, add respectively 0.00,0.05,0.10,0.15,0.20, the 2.0M sodium nitrite solution of 0.25mL, and 3.00,2.95,2.90,2.85,2.80, the water for injection of 2.75mL; In the blank system, add 5.0mL water for injection, fully mix. after standing 15 minutes, as blank solution, in 200 ~ 400nm scope, scan respectively the ultraviolet spectrum of 6 sample test systems with the blank system.The gained test result is as shown in Figure 7.
Can find from Fig. 7, along with the increase of sodium nitrite solution consumption, the gained ultraviolet spectrum fades away at the maximum absorption band at 302nm place, simultaneously at new maximum absorption band of 354nm place's appearance.This has illustrated, sodium nitrite solution is very large on the impact of Phen-zinc sulfate ultraviolet test system, can make its maximum absorption band produce significant Red Shift Phenomena.Chemical structure characteristic according to nadroparin calcium, its reason is likely in test system, the a large amount of nitrite ions that add have oxidation, change the hydroxyl in the nadroparin calcium molecule into aldehyde radical or carboxyl, thereby increased the conjugative effect of colour developing group, and then made its maximal ultraviolet absorption peak produce Red Shift Phenomena.Simultaneously, this has also illustrated, the hydroxyl in the nadroparin calcium molecule has participated in the formation of whole color development system.
To experiment five, can prove that Phen-zinc sulfate system can be used as measuring the concentration of nadroparin calcium solution by above-mentioned experiment one.Then two groups of following experiments confirm the reliability of this working curve by experiment with the standard working curves of model Phen-zinc sulfate system ultraviolet spectroscopy nadroparin calcium solution concentration.
Experiment six, Phen-zinc sulfate ultraviolet spectrum test system are to the standard working curve of nadroparin calcium
Get 7 15mL centrifuge tubes, wherein 6 are the sample test system, and 1 is blank system (not adding the nadroparin calcium sample).At first, in all centrifuge tubes, add respectively 1.0mL2.0mM solution of zinc sulfate, 1.0mL8.0mM Phen solution and 3.0mL1.0M sodium acetate solution, fully mix (Zn in other embodiments
2+The concentration range of ion in the whole mixed system after adding nadroparin calcium and water for injection, is 0.05 ~ 0.3mM; In other embodiments, the amount of substance of Phen is Zn
2+More than 2 times of the amount of substance of ion).Then, in the sample test system, add respectively the nadroparin calcium sample solution of 2.0mL variable concentrations, concentration successively 5.69%, 7.86%, 9.61%, 11.84%, 14.14%, 16.33%, and 3.0mL water for injection shake up; In the blank system, add 5.0mL water for injection, fully mix.After standing 15 minutes, as blank solution, in 200 ~ 400nm scope, scan respectively the ultraviolet spectrum of 6 sample test systems with the blank system.The gained test result as shown in Figure 8, in Fig. 8 from top to bottom shown in the concentration of the nadroparin calcium solution that adds of curve be followed successively by: 5.69%, 7.86%, 9.61%, 11.84%, 14.14% and 16.33%.
Can find from Fig. 8, Phen-zinc sulfate ultraviolet test system within concentration range 5.69% ~ 16.33%, presents good linear relationship to the nadroparin calcium sample, and namely the quality of nadroparin calcium is with respect to Zn
2+The amount of substance of ion can quantitatively detect when the scope of 56.9g/mmol ~ 163.3g/mmol exactly, and the standard working curve equation that calculates is: y=2.6362x+0.0644, R
2=0.9987, wherein y represents the absorbance at 302nm place, the concentration of the nadroparin calcium that the x representative adds.Show according to the further result of study of the method, Phen-zinc sulfate ultraviolet test system to the total range of linearity of nadroparin calcium sample concentration is: 1.184% ~ 16.33%, namely work as the quality of nadroparin calcium with respect to Zn
2+The amount of substance of ion is applicable to the detection of nadroparin calcium actual sample in production run when the scope of 11.84g/mmol ~ 163.3g/mmol.
Experiment seven, Phen-zinc sulfate ultraviolet spectrum test system are to the detection of nadroparin calcium actual sample
On the basis of experiment six, separately get 3 15mL centrifuge tubes, as the actual sample test system, i.e. then repeated test three times averages.At first, in all centrifuge tubes, add respectively 1.0mL2.0mM solution of zinc sulfate, 1.0mL8.0mM Phen solution and 3.0mL1.0M sodium acetate solution, fully mix.Then, in the actual sample test system, add respectively the nadroparin calcium solution through 2 times of dilutions that 2.0mL produces (as shown in Figure 2, the actual product here is the intermediate product solution between ultrafiltration technology and alcohol precipitation process) in actual production process, and 3.0mL water for injection, shake up.After standing 15 minutes, as blank solution, in 200 ~ 400nm scope, scan its ultraviolet spectrum with the blank system, record the absorbance at 302nm place.With the standard working curve equation of gained absorbance substitution in experiment six, calculate its concentration, test result is listed in table 1.
The detection of table 1 Phen-zinc sulfate ultraviolet spectrum test system to the nadroparin calcium actual sample
It is pointed out that in above-mentioned all experiments, can replace with at least a in sodium acetate, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium carbonate, sodium bicarbonate and niter cake as the sodium acetate of buffer solution solute.At least a in comprising water, ethanol, methyl alcohol, isopropyl alcohol, n-propanol and normal butyl alcohol of available solute when the solution of preparation Phen.
By above-described embodiment as can be known, Phen disclosed by the invention-zinc sulfate ultraviolet spectroscopy is measured the solution concentration of nadroparin calcium and is used Phen-zinc sulfate ultraviolet spectrum test system, realized the Quantitative detection to the nadroparin calcium solution concentration, the range of linearity of its analytical approach is 1.184%～16.33%; Simultaneously, the method has good antijamming capability to calcium ion, sodion and sulfate ion, can be applied in the actual production process of nadroparin calcium medicine, and middle product solution or final mean annual increment solution are carried out the concentration detection.
It is to be noted, the method not only is applicable to the concentration determination of nadroparin calcium medicine, and the solution concentration that can be applied to other several heparin class medicines quantitatively detects, Dalteparin Sodium, Enoxaparin Sodium, Parnaparin Sodium, spit of fland liquaemin such as on present market even comprise the liquaemin raw material of upstream etc.Simultaneously, based on the test philosophy of ultraviolet spectrum, the Phen in the present invention-zinc sulfate system can be deformed into simply: Phen-copper sulphate system, Phen-ammonium ferric sulfate system, Phen-silver nitrate system etc.
Above-described embodiment is only to the illustrating of technical scheme of the present invention, and is not intended to limit the present invention, and everyly the present invention is done being equal to of same design replaces or conversion, all belongs to protection scope of the present invention.
Claims (10)
1. Phen-zinc sulfate ultraviolet spectroscopy is measured the solution concentration of nadroparin calcium, it is characterized in that: under the environment of buffer solution, by measuring Zn
2+The ultraviolet absorptivity of ion, Phen and nadroparin calcium three mixed solution comes the concentration of quantitative measurement nadroparin calcium.
2. assay method according to claim 1, is characterized in that: under the environment of buffer solution, with Zn
2+Solion, Phen solution and nadroparin calcium solution mix, Zn described in mixed solution
2+The concentration range of ion is at 0.05 ~ 0.3mM, and the amount of substance of described Phen is Zn
2+More than 2 times of the amount of substance of ion, the ultraviolet absorptivity of the described mixed solution of mensuration;
During the Criterion working curve, what add mixed solution is the nadroparin calcium solution of concentration known, and the quality of described nadroparin calcium is with respect to described Zn
2+The amount of substance of ion is 11.84g/mmol ~ 163.3g/mmol;
After the Criterion working curve, add mixed solution for the nadroparin calcium solution of unknown concentration, calculate the concentration of described nadroparin calcium solution according to the standard working curve of the absorbance of measuring and foundation, when the quality of the nadroparin calcium that adds with respect to described Zn
2+The amount of substance of ion is not when the scope of 11.84g/mmol ~ 163.3g/mmol, in the situation that do not change add the method for volume by concentrated or dilution to change to add mixed solution in the concentration of nadroparin calcium solution, measure its absorbance and utilize standard working curve to calculate the concentration of described nadroparin calcium solution, make the quality of nadroparin calcium with respect to described Zn
2+The amount of substance of ion is in the scope of 11.84g/mmol ~ 163.3g/mmol;
The concentration of described nadroparin calcium refers to the quality percent by volume of nadroparin calcium.
3. assay method according to claim 1 and 2 is characterized in that: the solvent of described mixed solution is at least a in water, ethanol, methyl alcohol, isopropyl alcohol, n-propanol and normal butyl alcohol.
4. assay method according to claim 1 and 2, it is characterized in that: described buffer solution comprises the aqueous solution of at least a material in sodium acetate, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium carbonate, sodium bicarbonate and niter cake.
5. assay method according to claim 4, it is characterized in that: described buffer solution is sodium acetate solution, the concentration of sodium acetate in described mixed solution is 0.05 ~ 0.6M.
6. assay method according to claim 4, it is characterized in that: the concentration of described sodium acetate in described mixed solution is 0.3M.
7. assay method according to claim 1 and 2, is characterized in that: described Zn
2+Ion is from zinc sulfate, described Zn
2+The concentration of ion is 0.2mM.
8. assay method according to claim 1 and 2, it is characterized in that: the amount of substance of described Phen is described Zn
2+4 times of the amount of substance of ion.
9. assay method according to claim 1 and 2 is characterized in that: when measuring the absorbance of described mixed solution, ultraviolet wavelength is 302 ± 1nm.
10. assay method according to claim 5, it is characterized in that: during the preparation mixed solution, described zinc sulfate solution is 1.0mL, 2.0mM, the ethanolic solution of described Phen is 1.0mL, 8.0mM, described aqueous sodium acetate solution is 3.0mL, 1.0M, after fully mixing, adds 2.0mL nadroparin calcium solution and 3.0mL water for injection, fully mix and standing after, measure described mixed solution in the absorbance of 302 ± 1nm.
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| CN103698426A (en) * | 2013-12-12 | 2014-04-02 | 中国海洋大学 | Method for degrading chondroitin sulfate and hyaluronic acid to obtain chondroitin sulfate disaccharide and hyaluronic acid disaccharide and detecting chondroitin sulfate disaccharide and hyaluronic acid disaccharide |
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