CN114609070A - Free Fe in hemoglobin oxygen carrier3+Content detection method - Google Patents
Free Fe in hemoglobin oxygen carrier3+Content detection method Download PDFInfo
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- 102000001554 Hemoglobins Human genes 0.000 title claims abstract description 71
- 108010054147 Hemoglobins Proteins 0.000 title claims abstract description 71
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 title claims abstract description 69
- 229910052760 oxygen Inorganic materials 0.000 title claims abstract description 69
- 239000001301 oxygen Substances 0.000 title claims abstract description 69
- 238000001514 detection method Methods 0.000 title claims abstract description 47
- 239000012488 sample solution Substances 0.000 claims abstract description 49
- 238000000825 ultraviolet detection Methods 0.000 claims abstract description 23
- 239000000243 solution Substances 0.000 claims description 87
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 46
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 claims description 41
- ZNNZYHKDIALBAK-UHFFFAOYSA-M potassium thiocyanate Chemical compound [K+].[S-]C#N ZNNZYHKDIALBAK-UHFFFAOYSA-M 0.000 claims description 41
- 229940116357 potassium thiocyanate Drugs 0.000 claims description 41
- 239000012086 standard solution Substances 0.000 claims description 39
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 35
- 238000002835 absorbance Methods 0.000 claims description 28
- 239000011550 stock solution Substances 0.000 claims description 26
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 claims description 25
- 229910001447 ferric ion Inorganic materials 0.000 claims description 25
- 239000000126 substance Substances 0.000 claims description 8
- XGGLLRJQCZROSE-UHFFFAOYSA-K ammonium iron(iii) sulfate Chemical compound [NH4+].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O XGGLLRJQCZROSE-UHFFFAOYSA-K 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims 1
- 239000000969 carrier Substances 0.000 abstract description 7
- 238000004458 analytical method Methods 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 101
- 239000012153 distilled water Substances 0.000 description 15
- 238000000034 method Methods 0.000 description 15
- 229910052742 iron Inorganic materials 0.000 description 12
- 238000011084 recovery Methods 0.000 description 9
- 239000012490 blank solution Substances 0.000 description 5
- 239000003633 blood substitute Substances 0.000 description 4
- 230000003252 repetitive effect Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- FPFSGDXIBUDDKZ-UHFFFAOYSA-N 3-decyl-2-hydroxycyclopent-2-en-1-one Chemical compound CCCCCCCCCCC1=C(O)C(=O)CC1 FPFSGDXIBUDDKZ-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 206010018901 Haemoglobinaemia Diseases 0.000 description 1
- 208000033866 Rare inborn errors of metabolism Diseases 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000012421 spiking Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
The present application provides a method for producing free Fe in hemoglobin-based oxygen carriers3+The content detection method can accurately detect free Fe in the hemoglobin oxygen carrier by specially processing the hemoglobin oxygen carrier to be detected and adopting the ultraviolet detection condition to detect the sample solution to be detected3+The content of (A); the detection method has the advantages of simple and convenient operation, high analysis speed, good specificity, high accuracy and the like, and can be used for controlling the quality of the hemoglobin oxygen carrier reliably and accurately.
Description
Technical Field
The application relates to the technical field of element content detection, in particular to free Fe in hemoglobin oxygen carrier3+And (4) a content detection method.
Background
Hemoglobin-Based Oxygen Carriers (HBOCs) are a novel blood substitute with an Oxygen carrying function, and have the advantages of long-term storage, no need of matching, convenience in transportation, low infection risk and the like. The blood substitute hemoglobin oxygen carrier prepared by taking bovine blood as a raw material comprises bound iron and free iron. The combined iron is an important constituent of hemoglobin, and the hemoglobin can completely carry and release oxygen after entering blood cells; and free ferric ion (free Fe)3+) It has no such function and cannot be absorbed by human body, if free Fe3+Excessive content of free Fe in HBOCs can cause hemoglobinemia, a relatively rare metabolic disease, which seriously affects the health of the body3+The content of (A) needs to be strictly controlled, and no method for detecting free Fe in HBOCs exists at present3 +The content detection method, therefore, the establishment of free Fe in hemoglobin oxygen carrier3+The content detection method can control the quality of hemoglobin oxygen carrier reliably and accurately.
Disclosure of Invention
The purpose of the application is to provide free Fe in hemoglobin oxygen carrier3+Content detection method to realize free Fe in hemoglobin oxygen carrier3+And (4) accurately detecting the content. The specific technical scheme is as follows:
the present application provides a method for producing free Fe in hemoglobin-based oxygen carriers3+The content detection method comprises the following steps:
(1) establishment of Fe3+Standard curve of (2):
taking an ammonium ferric sulfate standard product, adding water and concentrated sulfuric acid to prepare Fe3+Standard stock solution with the concentration of 80-120 mug/mL; wherein, water and concentrateThe volume ratio of the sulfuric acid is 100: (0.15-0.25);
taking standard stock solutions with different volumes, adding water, concentrated sulfuric acid, potassium persulfate solution and potassium thiocyanate solution, and preparing 5-10 Fe solutions with different concentrations3+The standard solution of (4); wherein, the standard substance solution contains Fe3+The concentration of (A) is 0.12-5 mug/mL; the volume ratio of the concentrated sulfuric acid to the potassium persulfate solution to the potassium thiocyanate solution is 1: (1.5-2.5): (15-25), the volume ratio of the total volume of the concentrated sulfuric acid, the potassium persulfate solution and the potassium thiocyanate solution to the volume of the standard solution is (0.15-0.2): 1;
under the ultraviolet detection condition, taking each standard solution for detection to obtain Fe in each standard solution3+Absorbance of (a);
using Fe in each standard solution3+The absorbance of (2) is the ordinate, and Fe in each standard solution is used3+The concentration of (A) is the abscissa, and Fe is established3+A standard curve of (a);
(2) obtaining free Fe in hemoglobin oxygen carrier to be detected3+Absorbance of (a):
taking a hemoglobin oxygen carrier to be detected, adding water, concentrated sulfuric acid, a potassium persulfate solution and a potassium thiocyanate solution, and preparing a sample solution to be detected; wherein the volume ratio of the concentrated sulfuric acid to the potassium persulfate solution to the potassium thiocyanate solution is 1: (1.5-2.5): (15-25), the volume ratio of the total volume of the concentrated sulfuric acid, the potassium persulfate solution and the potassium thiocyanate solution to the volume of the hemoglobin oxygen carrier to be detected and the volume of the sample solution to be detected is (0.15-0.2): (0.005-0.015): 1;
under the same ultraviolet detection condition as the step (1), taking the sample solution to be detected for detection to obtain free Fe in the sample solution to be detected3+Absorbance of (a);
(3) determination of free Fe in hemoglobin oxygen carrier to be measured3+The content of (A):
according to the established Fe3+From Fe in the sample solution to be measured3+Obtaining Fe in the sample solution to be detected3+The concentration C1 is calculated according to the formula C-C1 XN to obtain the free Fe in the hemoglobin oxygen carrier to be measured3+The content of (A); it is composed ofWherein N is V1/V2, V1 is the volume of the sample solution to be measured, and V2 is the volume of the hemoglobin oxygen carrier to be measured.
Free Fe in hemoglobin oxygen carrier3+The content detection method is characterized in that the hemoglobin oxygen carrier to be detected is specially treated, the treatment method is simple, and the ultraviolet detection condition is adopted to detect the sample solution to be detected, so that the free Fe in the hemoglobin oxygen carrier as the blood substitute can be accurately detected3+The content of (2) can not damage substances existing in a form of bound iron, and can eliminate the interference of the bound iron in the hemoglobin oxygen carrier on free iron; the detection method has the advantages of simplicity and convenience in operation, cost saving, high analysis speed, good specificity, high accuracy and the like, so that the method can be used for controlling the quality of the hemoglobin oxygen carrier reliably and accurately.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present application, and it is also obvious for a person skilled in the art to obtain other embodiments according to the drawings.
FIG. 1 is Fe3+Scanning spectrogram under the ultraviolet detection condition with the scanning wavelength of 250-650 nm.
FIG. 2 shows the formation of Fe3+Standard graph of (2).
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the description herein are intended to be within the scope of the present disclosure.
The present application provides a method for producing free Fe in hemoglobin-based oxygen carriers3+The content detection method comprises the following steps:
(1) establishment of Fe3+Standard curve of (2):
taking an ammonium ferric sulfate standard product, adding water and concentrated sulfuric acid to prepare Fe3+Standard stock solution with the concentration of 80-120 mug/mL; wherein, the volume ratio of water to concentrated sulfuric acid is 100: (0.15-0.25);
taking standard stock solutions with different volumes, adding water, concentrated sulfuric acid, potassium persulfate solution and potassium thiocyanate solution, and preparing 5-10 Fe solutions with different concentrations3+The standard solution of (4); wherein, the standard substance solution contains Fe3+The concentration of (A) is 0.12-5 mug/mL; the volume ratio of the concentrated sulfuric acid to the potassium persulfate solution to the potassium thiocyanate solution is 1: (1.5-2.5): (15-25), the volume ratio of the total volume of the concentrated sulfuric acid, the potassium persulfate solution and the potassium thiocyanate solution to the volume of the standard solution is (0.15-0.2): 1;
under the ultraviolet detection condition, taking each standard solution for detection to obtain Fe in each standard solution3+Absorbance of (a);
using Fe in each standard solution3+The absorbance of (A) is plotted as the ordinate, and Fe in each standard solution is used3+The concentration of (A) is the abscissa, and Fe is established3+A standard curve of (a);
(2) obtaining free Fe in hemoglobin oxygen carrier to be detected3+Absorbance of (a):
taking a hemoglobin oxygen carrier to be detected, adding water, concentrated sulfuric acid, a potassium persulfate solution and a potassium thiocyanate solution, and preparing a sample solution to be detected; wherein the volume ratio of the concentrated sulfuric acid to the potassium persulfate solution to the potassium thiocyanate solution is 1: (1.5-2.5): (15-25), the volume ratio of the total volume of the concentrated sulfuric acid, the potassium persulfate solution and the potassium thiocyanate solution to the volume of the hemoglobin oxygen carrier to be detected and the volume of the sample solution to be detected is (0.15-0.2): (0.005-0.015): 1;
under the same ultraviolet detection condition as the step (1), taking the sample solution to be detected for detection to obtain free Fe in the sample solution to be detected3+Absorbance of (a);
(3) determination of free Fe in hemoglobin oxygen carrier to be measured3+The content of (A):
according to establishedFe3+From Fe in the sample solution to be measured3+Obtaining Fe in the sample solution to be detected3+The concentration C1 is calculated according to the formula C-C1 XN to obtain the free Fe in the hemoglobin oxygen carrier to be measured3+The content of (A); wherein N is V1/V2, V1 is the volume of the sample solution to be measured, and V2 is the volume of the hemoglobin oxygen carrier to be measured.
In the application, in the standard solution in the step (1), except for adding a standard stock solution and concentrated sulfuric acid, a potassium persulfate solution and a potassium thiocyanate solution with a certain volume ratio, the rest is added with water to make up the volume of the standard solution; and (3) adding a hemoglobin oxygen carrier to be detected and concentrated sulfuric acid, a potassium persulfate solution and a potassium thiocyanate solution in a certain volume ratio into the sample solution to be detected in the step (2), and supplementing the rest of the volume of the sample solution to be detected with water.
In the present application, the water may be distilled water or deionized water, and the present application is not particularly limited thereto as long as the object of the present application can be achieved.
In the present application, through the special treatment to the hemoglobin oxygen carrier that awaits measuring, obtain the sample solution that awaits measuring makes free Fe in the hemoglobin oxygen carrier that awaits measuring3+Can be connected with SCN-Formation of the blood Red Complex Fe (SCN)3Measuring the absorbance of the hemoglobin by ultraviolet detection, and calculating the free Fe in the hemoglobin oxygen carrier to be measured by a standard curve method3+The content of (A); in the present application, free Fe in the hemoglobin oxygen carrier to be measured3+The content of (b) is expressed as a concentration.
Free Fe in hemoglobin oxygen carrier provided by the application3+The content detection method is simple by special treatment of the hemoglobin oxygen carrier to be detected, and the ultraviolet detection condition is adopted to detect the sample solution to be detected, so that the free Fe in the hemoglobin oxygen carrier can be accurately detected3+The content of (a).
Preferably, the standard stock solution contains Fe3+The concentration is 90-110. mu.g/mL.
In some embodiments of the present application, the mass fraction of the concentrated sulfuric acid is equal to or greater than 70%, the mass fraction of the potassium persulfate solution is 0.8-1.2%, and the mass fraction of the potassium thiocyanate solution is 8-12%.
In the application, potassium persulfate is weighed and dissolved by adding water to obtain a potassium persulfate solution with the mass fraction of 0.8-1.2%; for example, 10g of potassium persulfate is weighed and dissolved in 990mL of water to obtain a 1% potassium persulfate solution. In the application, potassium thiocyanate is weighed and dissolved in water to obtain a potassium thiocyanate solution with the mass fraction of 8-12%; for example, 10g of potassium thiocyanate is weighed and dissolved in 90mL of water to obtain a 10% by mass potassium thiocyanate solution.
The inventor finds that when concentrated sulfuric acid, potassium persulfate solution and potassium thiocyanate solution with the mass fraction within the range of the application are adopted, the obtained hemoglobin oxygen carrier to be tested has free Fe3+The accuracy of the content detection result is good.
In some embodiments of the present application, in the step (1), the volume ratio of the concentrated sulfuric acid to the potassium persulfate solution to the potassium thiocyanate solution is 1: (1.8-2.2): (18-22); in the step (2), the volume ratio of the concentrated sulfuric acid to the potassium persulfate solution to the potassium thiocyanate solution is 1: (1.8-2.2): (18-22).
In some embodiments of the present application, in step (1), the volume ratio of the total volume of the concentrated sulfuric acid, the potassium persulfate solution, the potassium thiocyanate solution and the standard solution is (0.17-0.19): 1; in the step (2), the volume ratio of the total volume of the concentrated sulfuric acid, the potassium persulfate solution and the potassium thiocyanate solution to the volume of the hemoglobin oxygen carrier to be detected and the volume of the sample solution to be detected is (0.17-0.19): (0.008-0.012): 1.
in some embodiments of the present application, the standard solution is formulated, allowed to stand for 8-12min, and centrifuged for detection.
In some embodiments of the present application, the sample solution to be tested is prepared, left standing for 8-12min, and centrifuged for detection.
The centrifugation is not particularly limited in the present application as long as the clarification of the solution for detection after centrifugation can be ensured without affecting the measurement of absorbance, and for example, the centrifugation can be performed at 12000r/min of 8000- "for 3-5 min.
In some embodiments of the present application, the ultraviolet detection conditions include: the detection wavelength is 460-500 nm; preferably, the detection wavelength is 470-490 nm.
The inventor finds in research that by adopting the detection wavelength of the method, the free Fe in the hemoglobin oxygen carrier to be detected can be accurately obtained3+And (5) detecting the content.
The instruments and reagents required for the examples of the present application are described below.
The instrument comprises the following steps: ultraviolet spectrophotometer, electronic balance (sensitive quantity 0.01 mg); reagent: ammonium ferric sulfate standard, concentrated sulfuric acid, potassium persulfate and potassium thiocyanate. The reagents referred to in the following examples are commercially available or obtained according to a method known in the art unless otherwise specified.
Determination of detection wavelength
0.08631g of ferric ammonium sulfate standard substance are precisely weighed and placed in a 100mL volumetric flask, distilled water is added for dissolution, 4 drops of concentrated sulfuric acid (about 0.2mL) with the mass fraction of 98 percent are added, the volume is determined by distilled water, and Fe is prepared3+A standard stock solution with a concentration of 100 μ g/mL;
sucking 1mL of standard stock solution, placing in a 25mL volumetric flask, adding 5mL of distilled water, 0.2mL of concentrated sulfuric acid with the mass fraction of 98%, 0.4mL of potassium persulfate solution with the mass fraction of 1%, and 4.0mL of potassium thiocyanate solution with the mass fraction of 10%, diluting to constant volume with distilled water, standing for 10min, centrifuging, and preparing Fe3+A standard solution to be tested;
taking Fe under the ultraviolet detection condition of setting the scanning wavelength to be 250-650nm3+Pouring the standard solution to be detected into a cuvette, and detecting by an ultraviolet spectrophotometer to obtain Fe3+The scanning spectrum of (1) is shown in FIG. 1, and Fe can be seen from FIG. 13+The standard solution to be detected has maximum absorption at 480nm, the detection wavelength of the standard solution is 480nm, and the Fe in the application is confirmed according to the determination of the maximum absorption wavelength in the figure 13+The content detection method has good specificity.
Example 1
(1) EstablishingFe3+Standard curve of (2):
0.08631g of ferric ammonium sulfate standard substance are precisely weighed and placed in a 100mL volumetric flask, distilled water is added for dissolution, 4 drops of concentrated sulfuric acid (about 0.2mL) with the mass fraction of 98 percent are added, the volume is determined by distilled water, and Fe is prepared3+A standard stock solution with a concentration of 100 μ g/mL;
precisely sucking 0, 0.2, 0.4, 0.6, 0.8 and 1.0mL of standard stock solution, respectively placing in a 25mL volumetric flask, adding 5mL of distilled water, 0.2mL of concentrated sulfuric acid with the mass fraction of 98%, 0.4mL of potassium persulfate solution with the mass fraction of 1% and 4.0mL of potassium thiocyanate solution with the mass fraction of 10%, diluting to constant volume with distilled water, standing for 10min, centrifuging, preparing blank solution and 5 solutions containing different Fe3+Standard solutions of concentrations as shown in table 1;
under the ultraviolet detection condition that the detection wavelength is 480nm, pouring each standard solution into a cuvette, and detecting by an ultraviolet spectrophotometer to obtain Fe in each standard solution3+Absorbance of (a); wherein, a blank solution with 0mL of standard stock solution is added as a blank for zero adjustment;
using Fe in each standard solution3+The absorbance of (a) is given as the ordinate (y), and Fe in each standard solution is used3+Is the abscissa (x), Fe is established3+As shown in FIG. 2, Fe was obtained3+The results of the linear equation and the correlation coefficient are shown in Table 1, and it can be seen that the correlation coefficient R is2Not less than 0.99, and has good linear relation.
TABLE 1 Fe3+Linear equation and correlation coefficient of
(2) Obtaining free Fe in the hemoglobin oxygen carrier to be detected3+Absorbance of (a):
precisely absorbing 0.25mL of hemoglobin oxygen carrier to be detected, placing the hemoglobin oxygen carrier in a 25mL volumetric flask, adding 5mL of distilled water, 0.2mL of concentrated sulfuric acid with the mass fraction of 98%, 0.4mL of potassium persulfate solution with the mass fraction of 1%, and 4.0mL of potassium thiocyanate solution with the mass fraction of 10%, fixing the volume by using distilled water, standing for 10min, centrifuging, and preparing a sample solution to be detected;
under the same ultraviolet detection condition as the step (1), pouring the sample solution to be detected into a cuvette, and detecting by an ultraviolet spectrophotometer to obtain free Fe in the sample solution to be detected3+Absorbance of (a); wherein, a blank solution with 0mL of standard stock solution is added as a blank for zero adjustment;
(3) determination of free Fe in hemoglobin oxygen carrier to be measured3+The content of (A):
according to the established Fe3+From Fe in the sample solution to be measured3+Obtaining Fe in the sample solution to be detected3+The concentration of C1 is calculated according to the formula C1 XN C1 XV 1/V2 0.1666 μ g/mL × 25mL/0.25mL 16.66 μ g/mL to obtain the free Fe in the hemoglobin oxygen carrier to be measured3+The content of (A); wherein V1 is the volume of the sample solution to be detected of 25mL, and V2 is the volume of the hemoglobin oxygen carrier to be detected of 0.25 mL.
Methodology investigation
Detection limit and quantification limit
In the present application, Fe3+When the concentration is detected under ultraviolet detection conditions, the theoretical detection limit absorbance is 0.005, and the theoretical quantitative limit absorbance is 0.015.
Fe was prepared by precisely sucking 10. mu.L of the stock solution of the standard in example 1 by the same preparation method as that of the stock solution of the standard in example 13+The standard solution with the concentration of 0.04 mu g/mL is detected according to the ultraviolet detection condition of the example 1 to obtain Fe in the standard solution3+The absorbance of (a) was 0.0052, and the ratio thereof to the theoretical detection limit absorbance was in the range of 80 to 120%, and the concentration of the detection limit was determined to be 0.04. mu.g/mL.
Fe was prepared by precisely sucking 30. mu.L of the stock solution of the standard in example 1 by the same preparation method as that of the stock solution of the standard in example 13+The standard solution with the concentration of 0.12 mu g/mL is detected according to the ultraviolet detection condition of the example 1 to obtain Fe in the standard solution3+The absorbance of (A) is 0.0155, and the ratio of the absorbance of (A) to the theoretical limit absorbance of (B) is 8Ranging from 0 to 120%, the concentration defining the limit of quantitation being 0.12. mu.g/mL.
Precision test
Repeatability: person 1 prepares 6 parts of sample solutions to be tested respectively on different dates in the same manner as in example 1, and detects free Fe in the 6 parts of sample solutions to be tested according to the ultraviolet detection conditions in example 13+Obtaining free Fe in each sample solution to be measured3+The concentration C1 of the hemoglobin was calculated according to the calculation formula of example 1 to obtain 6 parts of free Fe in the hemoglobin oxygen carrier to be measured3+And the average value and the relative standard deviation (RSD,%) thereof were calculated, and the results are shown in table 2.
As can be seen from Table 2, 6 parts of hemoglobin oxygen carriers to be measured prepared by person 1 contained free Fe3+The RSD content is 1.8 percent, namely the repetitive RSD of the person 1 is 1.8 percent, and the RSD is less than or equal to 6 percent; 6 parts of hemoglobin oxygen Carriers prepared by person 2 for measurement of free Fe3+The RSD content is 0.9 percent, namely the repetitive RSD of the personnel 2 is 0.9 percent, and the RSD is less than or equal to 6 percent; the results show that the method of the application has good repeatability.
Intermediate precision: the intermediate precision RSD value is the sum of the repetitive RSD values of different personnel, and according to the table 2, the result of repetitive detection of different personnel operating at different time can be known, the calculated intermediate precision RSD is 2.7%, and the RSD is less than or equal to 6%, and the result shows that the intermediate precision of the method is good.
TABLE 2 repeatability and intermediate precision results
Durability test
Stability: placing the prepared standard stock solutions for 0h, 2h, 6h, 8h, 10h and 24h at room temperature respectively, precisely sucking 0.6mL of the standard stock solutions at each time point, placing the standard stock solutions in a 25mL volumetric flask, adding 5mL of distilled water, 0.2mL of concentrated sulfuric acid with the mass fraction of 98%, 0.4mL of potassium persulfate solution with the mass fraction of 1% and 4.0mL of potassium thiocyanate solution with the mass fraction of 10%, fixing the volume to 25mL by using the distilled water, placing for 10min, and centrifuging to obtain standard substance solutions at each time point; and the UV detection conditions of example 1 were respectively determined (blank solution with 0mL of standard stock solution added as blank zero adjustment), and the Fe content in the standard solution at each time point was recorded3+The absorbance of (c) was calculated, and the RSD (%) was calculated, and the results are shown in table 3. As can be seen from Table 3, Fe was contained in the standard solutions at the respective time points3+The absorbance RSD is 0.42 percent, and the RSD is less than or equal to 6 percent, which shows that the standard stock solution has good stability and can be stored for at least 24 hours at room temperature.
TABLE 3 stability results
| Time (h) | Absorbance of the |
| 0 | 0.2788 |
| 2 | 0.2786 |
| 6 | 0.2785 |
| 8 | 0.2796 |
| 10 | 0.2815 |
| 24 | 0.2803 |
| RSD(％) | 0.42 |
Recovery test
Precisely sucking 0.25mL of hemoglobin oxygen carrier to be measured, placing into 25mL volumetric flasks respectively, dividing into 3 groups, each group having 3 samples, precisely adding the above standard stock solution (Fe) into each group3+Concentration 100 mug/mL) of 0.1mL, 0.2mL, 0.4mL, respectively adding 5mL of distilled water, 0.2mL of concentrated sulfuric acid with mass fraction of 98%, 0.4mL of potassium persulfate solution with mass fraction of 1% and 4.0mL of potassium thiocyanate solution with mass fraction of 10%, fixing the volume to 25mL by using distilled water, standing for 10min, and centrifuging to obtain 9 standard sample solutions; and the ultraviolet detection conditions of the example 1 are respectively detected (blank solution with 0mL of standard stock solution is added as blank zero adjustment), so as to obtain free Fe in each standard sample solution3+Calculating to obtain free Fe in each standard sample solution3+The concentration of (c); from free Fe in each spiked sample solution3 +Concentration of-free Fe in each sample solution to be measured3+The measured spiked concentration is calculated and obtained according to the formula: the recovery ratio (%) was calculated as the recovery ratio of each spiked sample solution as measured/actual spiked concentration × 100%, and the results are shown in table 4. As can be seen from Table 4, the recovery rates of 3 different spiking concentrations are 102.6%, 93.4% and 96.1%, respectively, and the range of 80-115% meeting the recovery rate requirement indicates that the method of the present application has high accuracy.
TABLE 4 recovery results
Analysis of results based on recovery: if the method of the application can destroy the bound iron, the recovery rate result measured after the standard addition hardly meets the requirement of the range of 80-115%; the recovery rate of the method is 93.4-102.6%, and the method proves that the method does not damage substances existing in the form of bound iron, can eliminate the interference of the bound iron in the hemoglobin oxygen carrier on free iron, and can accurately measure the free Fe in the hemoglobin oxygen carrier3+The content of (A) is strong in pertinence and good in specificity.
Free Fe in hemoglobin oxygen carrier provided by the application3+The content detection method can accurately detect the free Fe in the hemoglobin oxygen carrier of the blood substitute3+The content of (a) can eliminate the interference of bound iron in hemoglobin oxygen carriers on free iron; the detection method has the advantages of simplicity and convenience in operation, high analysis speed, good specificity, high accuracy and the like.
The above description is only for the preferred embodiment of the present application and is not intended to limit the scope of the present application. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application are included in the protection scope of the present application.
Claims (9)
1. Free Fe in hemoglobin oxygen carrier3+The content detection method comprises the following steps:
(1) establishment of Fe3+Standard curve of (2):
taking an ammonium ferric sulfate standard product, adding water and concentrated sulfuric acid to prepare Fe3+Standard stock solution with the concentration of 80-120 mug/mL; wherein, the volume ratio of water to concentrated sulfuric acid is 100: (0.15-0.25);
taking standard stock solutions with different volumes, adding water, concentrated sulfuric acid, potassium persulfate solution and potassium thiocyanate solution, and preparing 5-10 Fe solutions with different concentrations3+The standard solution of (4); wherein, the standard substance solution contains Fe3+The concentration of (A) is 0.12-5 mug/mL; the volume of the concentrated sulfuric acid, the potassium persulfate solution and the potassium thiocyanate solutionThe ratio is 1: (1.5-2.5): (15-25), the volume ratio of the total volume of the concentrated sulfuric acid, the potassium persulfate solution and the potassium thiocyanate solution to the volume of the standard solution is (0.15-0.2): 1;
under the ultraviolet detection condition, taking each standard solution for detection to obtain Fe in each standard solution3+Absorbance of (a);
using Fe in each standard solution3+The absorbance of (A) is plotted as the ordinate, and Fe in each standard solution is used3+The concentration of (A) is the abscissa, and Fe is established3+A standard curve of (a);
(2) obtaining free Fe in hemoglobin oxygen carrier to be detected3+Absorbance of (a):
taking a hemoglobin oxygen carrier to be detected, adding water, concentrated sulfuric acid, a potassium persulfate solution and a potassium thiocyanate solution, and preparing a sample solution to be detected; wherein the volume ratio of the concentrated sulfuric acid to the potassium persulfate solution to the potassium thiocyanate solution is 1: (1.5-2.5): (15-25), the volume ratio of the total volume of the concentrated sulfuric acid, the potassium persulfate solution and the potassium thiocyanate solution to the volume of the hemoglobin oxygen carrier to be detected and the volume of the sample solution to be detected is (0.15-0.2): (0.005-0.015): 1;
under the same ultraviolet detection condition as the step (1), taking the sample solution to be detected for detection to obtain free Fe in the sample solution to be detected3+Absorbance of (a);
(3) determination of free Fe in hemoglobin oxygen carrier to be measured3+The content of (A):
according to the established Fe3+From Fe in the sample solution to be measured3+Obtaining Fe in the sample solution to be detected3+The concentration C1 is calculated according to the formula C-C1 XN to obtain the free Fe in the hemoglobin oxygen carrier to be measured3+The content of (A); wherein, N is V1/V2, V1 is the volume of the sample solution to be measured, and V2 is the volume of the hemoglobin oxygen carrier to be measured.
2. The assay of claim 1, wherein Fe is present in the standard stock solution3+The concentration is 90-110. mu.g/mL.
3. The detection method according to claim 1, wherein the mass fraction of the concentrated sulfuric acid is not less than 70%, the mass fraction of the potassium persulfate solution is 0.8-1.2%, and the mass fraction of the potassium thiocyanate solution is 8-12%.
4. The detection method according to claim 1, wherein in the step (1), the volume ratio of the concentrated sulfuric acid to the potassium persulfate solution to the potassium thiocyanate solution is 1: (1.8-2.2): (18-22); in the step (2), the volume ratio of the concentrated sulfuric acid to the potassium persulfate solution to the potassium thiocyanate solution is 1: (1.8-2.2): (18-22).
5. The detection method according to claim 1, wherein in step (1), the volume ratio of the total volume of the concentrated sulfuric acid, the potassium persulfate solution, the potassium thiocyanate solution and the standard solution is (0.17-0.19): 1; in the step (2), the volume ratio of the total volume of the concentrated sulfuric acid, the potassium persulfate solution and the potassium thiocyanate solution to the volume of the hemoglobin oxygen carrier to be detected and the volume of the sample solution to be detected is (0.17-0.19): (0.008-0.012): 1.
6. the detection method according to claim 1, wherein the standard solution is prepared, left for 8-12min, centrifuged and detected.
7. The detection method according to claim 1, wherein the sample solution to be detected is prepared, then is kept still for 8-12min, and is detected after centrifugation.
8. The detection method according to any one of claims 1 to 7, wherein the ultraviolet detection condition includes: the detection wavelength is 460-500 nm.
9. The detection method according to claim 8, wherein the detection wavelength is 470-490 nm.
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| US6194218B1 (en) * | 1999-01-22 | 2001-02-27 | The Fredric Rieders Family Renaissance Foundation | Blood methemoglobin analysis |
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