CN104237223A - Method for determining unstable iron content in iron and carbohydrate complex - Google Patents
Method for determining unstable iron content in iron and carbohydrate complex Download PDFInfo
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- CN104237223A CN104237223A CN201410526312.6A CN201410526312A CN104237223A CN 104237223 A CN104237223 A CN 104237223A CN 201410526312 A CN201410526312 A CN 201410526312A CN 104237223 A CN104237223 A CN 104237223A
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Abstract
The invention provides a method for determining the unstable iron content in an iron and carbohydrate complex. The method comprises the steps of enabling the iron and carbohydrate complex to be in contact with human serum and reducing unstable iron which is combined with the human serum into ferrous iron; performing action on the ferrous iron obtained through reduction and a chromogenic agent to generate a colored compound and measuring the absorbance of the colored compound; performing comparison with the iron standard liquid through the same reduction and chromogenic reaction treatment and obtaining the unstable iron content in the iron and carbohydrate complex through calculation. According to the method for determining the unstable iron content in the iron and carbohydrate complex, the detection sensitivity is high, the precision is good, the accuracy is high, the unstable iron content in the iron and carbohydrate complex can be rapidly, accurately and quantitatively determined, the quality of iron supplements, particularly intravenous iron supplements, can be scientifically and effectively controlled, and the safety of clinical medication is ensured.
Description
Technical field
The present invention relates to the analytical approach of iron supplementing preparation, be specifically related to a kind of method measuring unstable iron (III) content in iron-carbohydrate complex.
Background technology
Ferro element is one of requisite trace element of human body, is to form haemoglobin, myoglobins, cytochrome and multiple oxidasic important component, as the carrier of oxygen, ensures the normal conveying of oxygen in body tissue.
Iron is absorbed at many histocytes or absorbs is all transferrins (transferrin by classics, and TfR (transferrin receptor Tf), TfR) approach, namely first ferric iron is combined with Tf, both bonds are combined with the TfR of cell surface again, and afterwards through steps such as endocytosis, acidifying, release and displacements, iron enters endochylema, finally utilized by cell, synthetic hemoglobin and other materials.
Enough haemoglobins can not be synthesized during asiderosis, cause hypoferric anemia.Clinically, diagnosis of iron deficiency anemia is set up and should be given iron supplement therapy immediately after finding out the cause of disease.Generally, Oral Iron Preparations is first-selected, but Oral Iron Preparations often causes obvious gastrointestinal discomfort, have report nearly 1/5 patient's therefore TD.Oral Iron Preparations is also subject to the impact of Blood lipids in the interference of composition of food and body, especially many chronic diseases, kidney trouble, heart disease and tumor patient, and the absorption of iron and utilizing also can be affected.For this part patient, clinician should consider to give intravenous iron.In addition, in hypoferric anemia companion enterogastric diseases needs of patients fast quick-recovery body, iron is stocked and oral when being not enough to reach this target, also can using intravenous iron as first-selection.
Develop the multiple iron containing iron-carbohydrate complex at present both at home and abroad and supplemented intravenous formulations, comprise iron gluconate sodium injection, iron sucrose injection, injection iron sucrose, iron dextran inj, iron sorbitex inj etc.For iron gluconate sodium injection, it is the colloidal suspension prepared of a kind of Ferrlecit and uses as prodrug, and wherein Ferrlecit comprises and the colloidal ferric hydroxide particle of gluconic acid complexing (iron core).These iron cores are by being 2 acquisitions by iron chloride alkali neutralisation number pH, and under this pH, saturated hydroxide ion forms colloidal ferric hydroxide, its after its formation with gluconic acid complexes ira situ.The compound of gained, by the cellular uptake of reticuloendothelial system, discharges ferric ion.Ferric ion is combined with transferrins again, transferrins so that be transported to marrow be used for RBC acceptor garland rate or be transported in ferritin and marrow, spleen and liver iron promoter position.In general, the structure of iron core follows Coordinative Chemistry.The hydroxyl of carbohydrates replaces the hydrone that iron core appearance combines, thus carbohydrates is combined with iron core.
But described iron supplements in intravenous formulations and also comprises some and be not combined with the core of compound or the ferro element of weak binding, forms unstable iron.These unstable iron be easy to by the complexing agent in chemical reaction or blood plasma as albumen activate, this can cause the quick release of iron supplementing preparation ferro element after intravenously administrable, namely prominently releases effect.The release of unstable iron can make transferrins saturated rapidly and cause the appearance of non-Tf-Fe, but not Tf-Fe can the harmful response to oxidative stress of catalysis, cause clinically even may be fatal acute bad reaction.
Prior art discloses four kinds of measuring principles of iron: 1) ferrous piperazine colourimetry principle: serum levels of iron and transferrin are combined into compound, in acid medium, iron dissociates out from compound, be reduced agent again and be reduced into ferrous iron, and generate aubergine compound with ferrous piperazine, an absorption peak is had at wavelength 562nm place, compare with the same titer processed, the content of serum levels of iron can be tried to achieve.2) duplex pyridine colorimetric method principle: make in acid condition iron from protein bound state free out.Make ferric iron back in serum become ferrous iron with oxammonium hydrochloride as reductive agent, the latter and duplex pyridine developer react and generate red chelate, at 520nm place colorimetric assay.This law is fast easy, but poor sensitivity, disturbing factor is more, and especially pollute the most serious with irony, haemolysis also can affect result.3) luxuriant and rich with fragrance Lip river piperazine colourimetry principle: trichloroacetic acid-hydrochloric acid mixed solution makes the iron in serum discharge from transferrin and make albumen precipitation, the ferric iron discharged can be reduced into ferrous iron with thioglycolic acid, the latter and Fei Luo piperazine are combined into aubergine compound, and absorption peak is 562nm.4) total iron binding capacity (TIBC): add enough iron titers and make fortune iron ferritin saturated by iron in blood serum sample, excessive iron MgCO
3remove out, centrifuging and taking supernatant, obtain the content of iron by the method surveying serum levels of iron, be TIBC. (" physiology ", People's Health Publisher, 2012).But said method only relates to the ultimate principle that iron content measures, and still can there is relatively large deviation when reality measures concrete material at different conditions.At present also not about the report measuring unstable iron content method in iron intravenous formulations.Therefore, be necessary to develop a kind of stable, method of measuring unstable iron content in vein iron preparation reliably, and in this way based on the quality of iron supplementing preparation is assessed.
Summary of the invention
The object of this invention is to provide a kind of method measuring unstable iron content in iron-carbohydrate complex, be used in particular for the preparation containing iron-carbon aquo complex, such as vein iron supplementing preparation carries out the evaluation of external quality, ensures the safety of clinical application.
The present invention is achieved through the following technical solutions above-mentioned purpose:
Measure a method for unstable ferric iron content in iron-carbohydrate complex, comprise step:
Iron-carbon aquo complex is contacted with human serum, is ferrous iron by the unstable ferric iron back be combined with human serum; The ferrous iron obtain reduction and developer effect generate colored compound, measure the absorbance of colored compound; To reduce and concentration of iron-absorbance standard curve that chromogenic reaction process obtains compares through same with ferric iron titer, calculate unstable ferric content in iron-carbon aquo complex.
In described iron-carbohydrate complex, unstable iron is be not combined with the core of described complex compound or the ferric ion of weak binding.Described weak binding iron is be easy in complex compound participate in chemical reaction or form the ferro element of component reaction with other iron complexs.
In the present invention, described iron-carbohydrate complex can in the form of a solution or dosage form exist.Described unstable ferric iron may reside in iron-carbohydrate complex, also may reside in the solution or preparation comprising iron-carbohydrate complex.
In one embodiment, the method for unstable ferric iron content in described mensuration iron-carbohydrate complex, comprises step:
Iron-carbon aquo complex is mixed with human serum, obtains potpourri respectively in more than three different time points of mixing, add protein denaturant stopped reaction, then be ferrous iron by the ferric iron back be combined with human serum; Then ferrous iron reduction obtained and developer effect generate colored compound, record its absorbance, take incorporation time as horizontal ordinate, absorbance is ordinate, obtain curvilinear equation, the absorption values when time of trying to achieve is 0, substitutes into the concentration of iron-absorbance standard curve equation obtained through same reduction and chromogenic reaction process by ferric iron titer, calculates unstable ferric content in sample by this absorption values.
In one embodiment, the method for unstable ferric iron content in described mensuration iron-carbohydrate complex, comprises step:
1) foundation of typical curve:
The ferric iron standard items serial solution of preparation more than three kinds variable concentrations, draw a certain amount of iron standard items serial solution respectively, ferrous iron is reduced under reductive agent effect in acid medium, then developer and ferrous iron effect are generated colored compound, record absorbance, with ferric concentration for horizontal ordinate, absorbance is ordinate, obtains typical curve equation;
2) process of iron-carbon aquo complex sample:
Human serum and iron-carbon aquo complex sample mix are hatched altogether, in wherein said sample, the total content of iron is scaled human serum concentration of iron and is preferably 50 ~ 180 μ g/mL, a certain amount of sample is obtained respectively in more than three different time points of hatching, add protein denaturant stopped reaction, be ferrous iron by the ferric iron back be combined with human serum under reductive agent effect in acid medium, developer and ferrous iron effect generate colored compound, record absorbance, take incubation time as horizontal ordinate, absorbance is ordinate, obtain curvilinear equation, the absorption values when time of trying to achieve is 0, this absorption values is substituted into typical curve equation, calculate unstable ferric content in iron-carbon aquo complex sample.
Iron in the total content of above-mentioned iron is total content that is stable in sample and unstable iron; More specifically, be stable and unstable ferric total content.
The standard items of described preparation ferric iron titer can be crystalline sulfuric acid iron ammonium (molecular formula FeNH
4(SO
4)
2〃 12H
2o).The concentration of described ferric iron standard items serial solution can be 0.1 ~ 100 μ g/ml, preferably 5 ~ 80 μ g/ml, such as, other numerical value in 5,10,20,30,40,50,60,70,80 μ g/ml and above-mentioned scope.Iron standard items series titer is preferably more than 3 kinds variable concentrations in above-mentioned concentration range, such as, prepare the titer of 3 kinds, 4 kinds, 5 kinds, 6 kinds variable concentrations.
Carbohydrates in described iron-carbon aquo complex be selected from gluconate, sucrose, dextran, dextrin and sorbierite one or more.
The ratio that the sample of described iron-carbon aquo complex mixes with human serum is, in sample, the total content of iron counts 50 ~ 180 μ g/ml with human serum concentration of iron, preferably 65 ~ 170 μ g/ml, such as, any number in the above-mentioned scopes such as 70,80,90,100,110,120,130,140,150,160 μ g/ml.
The sample of described iron-carbon aquo complex and the common incubation time of human serum are 10 ~ 60min, preferably 30 ~ 60min.
The time point of described measurement is three different time points of hatching, such as 10,20 or 60 minutes; Can also be four different time points of hatching, as 10,20,30,45min.Can also be more than 4 time points.Different time points is measured can improve accuracy, and is easy to monitoring.
Described protein denaturant be selected from urea, thiocarbamide, 3-[3-(courage amido propyl) dimethylamino] propane sulfonic acid inner salt (CHAPS), dithiothreitol (DTT) (DTT), IPG damping fluid one or more.Preferred thiocarbamide, shields copper ion interference while playing albuminous degeneration effect.
The addition of described acid medium is advisable to make system pH control to be 1 ~ 4, ensures that reduction reaction is complete.Acid medium is preferably citric acid.
Described reductive agent be selected from ascorbic acid, reductibility flavin mononucleotide (FMN), hyposulfite, hydroxyl acetate, quinhydrones, lactic acid or lactate, citric acid or citrate, supercarbonate, acetonate, succinic acid or succinate, fructose, halfcystine and sorbierite one or more.Be preferably faintly acid reductive agent ascorbic acid.Reductive agent exist amount be excessive at least about 10 times relative to iron-carbohydrate complex, preferably 30 times excessive, more preferably 50 times excessive.
Described developer is ferrous piperazine.
In a preferred embodiment, acid medium and protein denaturant are added simultaneously, and reductive agent and developer are added simultaneously, so namely can not affect testing result, can also step be simplified, handled easily.In addition, described developer and reductive agent add simultaneously, and the ferrous iron that reduction can be made to produce reacts with developer generation colored compound immediately, promote the carrying out of reduction reaction simultaneously.The time of described reduction reaction and chromogenic reaction is about 30 ~ 60min, preferred 60min.
Described colored compound is measuring absorbance with spectrophotometer at wavelength 562nm place.
In a kind of concrete embodiment, the method for unstable ferric iron content in described mensuration iron-carbohydrate complex, comprises step:
1) foundation of typical curve:
At room temperature, the iron standard items serial solution of preparation 0.1 ~ 100 μ g/mL scope more than 3 kinds variable concentrations, the aqueous solution (as 700 μ l) containing citric acid (as 200mM) is added respectively in described iron standard items serial solution (as 100 μ l), add the aqueous solution (as 350 μ L) containing ascorbic acid (as 150mM) and ferrous piperazine (as 6mM) again, after reaction, (as 60min) measures absorbance with spectrophotometer in 562nm place, take concentration of iron as horizontal ordinate X, absorbance is ordinate Y, obtains typical curve equation Y=aX+b;
2) mensuration of the unstable iron content of iron-carbon aquo complex sample:
At room temperature, human serum and iron-carbon aquo complex sample mix are hatched, wherein in sample, the total content of iron is scaled serum iron is 50 ~ 180 μ g/mL, the different time points sample thief (as 100 μ l) of three respectively in the process of hatching or more adds the aqueous solution (as 700 μ l) containing thiocarbamide (as 115mM) and citric acid (as 200mM), then the aqueous solution (as 350 μ L) containing ascorbic acid (as 150mM) and ferrous piperazine (as 6mM) is added, after reaction, (as 60min) measures absorbance with spectrophotometer in 562nm place, take incubation time as horizontal ordinate X
1, absorbance is ordinate Y
1carry out linear regression, obtain equation Y
1=a
1x
1+ b
1, the absorption values when time that obtains thus is 0 is b
1, by sample absorbance numerical value b
1substitute into typical curve equation, calculate the content obtaining unstable iron in sample.
Same as above, preferably, described standard items are crystalline sulfuric acid iron ammonium; The concentration of described iron standard items serial solution is preferably 5 ~ 80 μ g/ml; The ratio that described iron-carbon aquo complex sample mixes with human serum is, in sample, the total content of iron counts 65 ~ 170 μ g/ml with human serum concentration of iron.
In the present invention, in iron-carbon aquo complex, unstable ferric reduction meets first order kinetics, above-mentioned detection method of the present invention, the preparation arbitrarily containing iron-carbon aquo complex can be detected, it can be oral or the formulation such as injection, as iron gluconate sodium injection, iron sucrose injection, iron sorbitex inj, iron dextran inj, dextriferron parenteral solution, injection iron cacodylate etc., detection sensitivity is high, precision is good, accuracy is high, can be quick, accurately, measure unstable iron content in iron-carbohydrate quantitatively, science and effective control can be carried out to the quality of (especially vein) iron supplementing preparation, ensure the safety of clinical application.
Embodiment
Following examples may be used for understanding the features and advantages of the invention further, but should not be construed as and limit the scope of the invention.Any improvement of making on basis of the present invention and change, all within protection scope of the present invention.
Except indicating especially, the instrument that embodiment is used and reagent are common commercially available prod.
Embodiment 1:
The preparation (100 μ g/ml) of iron stock solution: accurately take crystalline sulfuric acid iron ammonium [FeNH
4(SO
4)
2〃 12H
2o] (Aladdin Industrial Corporation) 0.860g, with adding the concentrated sulphuric acid (AR) 2.0ml after deionized water dissolving, adding deionized water in immigration 1L volumetric flask and being diluted to scale as stock solution.
Iron standard serial solution is prepared: pipette iron stock solution 5,10,20,30,40ml respectively, join respectively in 100ml volumetric flask, add deionized water and be settled to scale, shake up as iron standard serial solution, concentration is respectively 5,10,20,30,40 μ g/ml.
Iron typical curve is set up: at room temperature, the each 100 μ l of iron standard serial solution are added respectively in 1.5mlEP pipe, then add containing 200mM lemon aqueous acid (reagent I) 700 μ l, finally add aqueous solution (reagent II) the 350 μ L containing 150mM ascorbic acid and the ferrous piperazine of 6mM, after reaction 60min, measure absorbance with spectrophotometer (Shimadzu UV2401) in 562nm place.Take concentration as horizontal ordinate x, absorbance is that ordinate y carries out linear regression, draws and show that typical curve equation is: y=0.0158x+0.0023, r=0.9985.
The detection of unstable iron content in iron gluconate preparation of sodium: at room temperature, in 1.5mL EP pipe, human serum (Biosharp) and iron gluconate sodium sucrose compound parenteral solution (SANOFI AVENTIS US LLC) are hatched, it is 166.70 μ g/mL that chalybeate addition is scaled serum iron, respectively at incubation time 10, 20, 30, 45min, getting 100 μ l samples adds containing 115mM thiocarbamide and 200mM lemon aqueous acid (reagent I) 700ul, then aqueous solution (reagent II) the 350 μ L containing 150mM ascorbic acid and the ferrous piperazine of 6mM is added, absorbance is measured with spectrophotometer in 562nm place after reaction 60min.
Result calculates: take incubation time as horizontal ordinate x
1, absorbance is ordinate y
1carry out linear regression, draw and show that curvilinear equation is y
1=0.0118x
1+ 0.0869, r=0.9978.When the time that obtains thus is 0, absorbance is 0.0869, and bringing iron typical curve into, to calculate unstable concentration of iron be 5.354 μ g/ml.
Get same batch glucose acid ferrisodium formulation samples and repeat above-mentioned experimentation 5 times altogether, carry out survey fixed sum data and calculate, result is as shown in table 1.
Table 1
Embodiment 2:
The foundation of iron typical curve: with embodiment 1.
The detection method of unstable iron content in iron gluconate preparation of sodium: similar embodiment 1, when hatching unlike human serum and iron gluconate sodium sucrose compound parenteral solution, it is 100.0 μ g/mL that chalybeate addition is scaled serum iron.Get same batch glucose acid ferrisodium formulation samples and repeat experimentation altogether 5 times, carry out survey fixed sum data and calculate, result is as shown in table 2.
Table 2
Embodiment 3:
The foundation of iron typical curve: similar embodiment 1, replaces ascorbic acid unlike in reagent II with sorbierite.
The detection method of unstable iron content in iron gluconate preparation of sodium: similar embodiment 1, when hatching unlike human serum and iron gluconate sodium sucrose compound parenteral solution, it is 40.0 μ g/mL that chalybeate addition is scaled serum iron, replace thiocarbamide with CHAPS (Hao Yang bio tech ltd, Shanghai) in reagent I, in reagent II, replace ascorbic acid with sorbierite.Get same batch glucose acid ferrisodium formulation samples and repeat experimentation altogether 5 times, carry out survey fixed sum data and calculate, result is as shown in table 3.
Table 3
Embodiment 4:
The foundation of iron typical curve: with embodiment 1.
The detection method of unstable iron content in iron gluconate preparation of sodium: similar embodiment 1, when hatching unlike human serum and iron gluconate sodium sucrose compound parenteral solution, it is 100.0 μ g/mL that chalybeate addition is scaled serum iron, replaces thiocarbamide in reagent I with dithiothreitol (DTT) (Lian Shuo bio tech ltd, Shanghai).
Get same batch glucose acid ferrisodium formulation samples and repeat experimentation altogether 5 times, carry out survey fixed sum data and calculate, result is as shown in table 4.
Table 4
Embodiment 5:
The foundation of iron typical curve: with embodiment 1.
The detection of unstable iron content in Astrafer: at room temperature, in 1.5mL EP pipe, human serum and iron dextran inj (Pharmacosmos A/S) are hatched, it is 66.70 μ g/ml that chalybeate addition is scaled serum iron, respectively at incubation time 10, 20, 30, 45min, getting 100 μ l samples adds containing 115mM thiocarbamide and 200mM lemon aqueous acid (reagent I) 700 μ l, then aqueous solution (reagent II) 350uL containing 150mM ascorbic acid and the ferrous piperazine of 6mM is added, absorbance is measured with spectrophotometer in 562nm place after reaction 60min.
Data calculate: take incubation time as horizontal ordinate x
2, absorbance is ordinate y
2carry out linear regression, draw and show that curvilinear equation is y
2=0.000986x
2+ 0.0180, r=0.9973.When the time that obtains thus is 0, absorbance is 0.0180, and bringing iron typical curve into, to calculate unstable concentration of iron be 0.994 μ g/ml.
Get and repeat above-mentioned experimentation 5 times with a batch Astrafer sample, carry out survey fixed sum data and calculate, result is as shown in table 5.
Table 5
Embodiment 6:
The foundation of iron typical curve: with embodiment 1.
The detection of unstable iron content in Astrafer: with embodiment 5, when hatching unlike human serum and iron dextran inj, it is 100.00 μ g/mL that chalybeate addition is scaled serum iron.Get and repeat experimentation altogether 5 times with a batch Astrafer sample, carry out survey fixed sum data and calculate, result is as shown in table 6.
Table 6
Embodiment 7:
The foundation of iron typical curve: similar embodiment 1, replaces ascorbic acid unlike in reagent II with sorbierite.
The detection of unstable iron content in Astrafer: similar embodiment 5, when hatching unlike human serum and iron dextran inj, it is 200.0 μ g/mL that chalybeate addition is scaled serum iron, replace thiocarbamide with CHAPS in reagent I, in reagent II, replace ascorbic acid with sorbierite.Get and repeat experimentation altogether 5 times with a batch Astrafer sample, carry out survey fixed sum data and calculate, result is as shown in table 7.
Table 7
Embodiment 8:
The foundation of iron typical curve: with embodiment 4.
The detection method of unstable iron content in Astrafer: similar embodiment 5, when hatching unlike human serum and iron dextran inj, it is 100.0 μ g/mL that chalybeate addition is scaled serum iron, replaces thiocarbamide in reagent I with dithiothreitol (DTT).Get and repeat experimentation altogether 5 times with a batch Astrafer sample, carry out survey fixed sum data and calculate, result is as shown in table 8.
Table 8
The experimental result display of above-described embodiment, when human serum and iron-carbohydrate complex are hatched, it is 50 ~ 180 μ g/mL that chalybeate addition is scaled serum iron, and protein denaturant is thiocarbamide, and when reductive agent is ascorbic acid, accuracy in detection is high, and error is little.
More than provide only minority embodiment of the present invention, not specified operating conditions can be carried out according to this area conventional method.Some operating process, the such as preparation of iron standard solution, the preparation of reagent I and II, the acquisition of curvilinear equation, data calculating etc. can be carried out according to conventional method known in the art, need not be confined to the concrete restriction in embodiment.Unless stated otherwise, described operating process is all at room temperature carried out, preferably 10 ~ 35 DEG C.Only otherwise the improvement made under departing from essence of the present invention and spiritual prerequisite or equivalence change all belong to the scope of protection of the invention.
Claims (10)
1. measure a method for unstable ferric iron content in iron-carbohydrate complex, it is characterized in that, described method comprises the steps:
Iron-carbon aquo complex is contacted with human serum, is ferrous iron by the unstable ferric iron back be combined with human serum; The ferrous iron obtain reduction and developer effect generate colored compound, measure the absorbance of colored compound; To reduce and concentration of iron-absorbance standard curve that chromogenic reaction process obtains compares through same with ferric iron titer, calculate unstable ferric content in iron-carbon aquo complex.
2. method according to claim 1, is characterized in that, described method comprises the steps:
Iron-carbon aquo complex is mixed with human serum, obtains potpourri respectively in more than three different time points of mixing, add protein denaturant stopped reaction, then be ferrous iron by the ferric iron back be combined with human serum; Then ferrous iron reduction obtained and developer effect generate colored compound, record its absorbance, take incorporation time as horizontal ordinate, absorbance is ordinate, obtain curvilinear equation, the absorption values when time of trying to achieve is 0, substitutes into the concentration of iron-absorbance standard curve equation obtained through same reduction and chromogenic reaction process by ferric iron titer, calculates the content of unstable iron in sample by this absorption values.
Preferred further, comprise the steps:
1) foundation of typical curve:
The ferric iron standard items serial solution of preparation more than three kinds variable concentrations, ferrous iron is reduced under reductive agent effect in acid medium, then developer and ferrous iron effect are generated colored compound, record absorbance, with ferric concentration for horizontal ordinate, absorbance is ordinate, obtains typical curve equation;
2) process of iron-carbon aquo complex sample:
Human serum and iron-carbon aquo complex sample mix are hatched altogether, in wherein said sample, the total content of iron is scaled human serum concentration of iron and is preferably 50 ~ 180 μ g/mL, a certain amount of sample is obtained respectively in more than three different time points of hatching, add protein denaturant stopped reaction, be ferrous iron by the Fe3+ reduction be combined with human serum under reductive agent effect in acid medium, developer and ferrous iron effect generate colored compound, record absorbance, take incubation time as horizontal ordinate, absorbance is ordinate, obtain curvilinear equation, the absorption values when time of trying to achieve is 0, this absorption values is substituted into typical curve equation, calculate the content of unstable iron in sample.
3. method as claimed in claim 1 or 2, wherein said protein denaturant be selected from urea, thiocarbamide, 3-[3-(courage amido propyl) dimethylamino] propane sulfonic acid inner salt, dithiothreitol (DTT), IPG damping fluid one or more; Preferred thiocarbamide.
Described acid medium is citric acid.
Described reductive agent be selected from reductibility flavin mononucleotide (FMN), hyposulfite, hydroxyl acetate, quinhydrones, lactic acid or lactate, citric acid or citrate, supercarbonate, acetonate, succinic acid or succinate, fructose, halfcystine, sorbierite and ascorbic acid one or more.Be preferably faintly acid reductive agent ascorbic acid.
Described developer is ferrous piperazine.
4. the method as described in any one of claim 1-3, wherein said acid medium and protein denaturant add simultaneously, and then developer and reductive agent add simultaneously, and the ferrous iron that reduction is produced reacts with developer generation colored compound immediately; The time of described reduction reaction and chromogenic reaction is about 60min.
The time of described reduction reaction and chromogenic reaction is about 30 ~ 60min respectively, preferred 60min.
Preferably, the addition of described acid medium makes system pH control is 1 ~ 4.
Described reductive agent consumption is excessive at least about 10 times relative to iron-carbohydrate complex, preferably 30 times excessive, more preferably 50 times excessive.
5. the method as described in any one of claim 1-4, wherein said colored compound is measuring absorbance with spectrophotometer at wavelength 562nm place.
6. the method described in any one of claim 1-5, comprises step:
1) foundation of typical curve:
At room temperature, the iron standard items serial solution of preparation 0.1 ~ 100 μ g/mL scope more than 3 kinds variable concentrations, the aqueous solution (as 700 μ l) containing citric acid (as 200mM) is added respectively in described iron standard items serial solution (as 100 μ l), add the aqueous solution (as 350 μ L) containing ascorbic acid (as 150mM) and ferrous piperazine (as 6mM) again, after reaction, (as 60min) measures absorbance with spectrophotometer in 562nm place, take concentration of iron as horizontal ordinate X, absorbance is ordinate Y, obtains typical curve equation Y=aX+b;
2) mensuration of the unstable iron content of iron-carbon aquo complex sample:
At room temperature, human serum and iron-carbon aquo complex sample mix are hatched, wherein in sample, the total content of iron is scaled serum iron is 50 ~ 180 μ g/mL, the different time points sample thief (as 100 μ l) of three respectively in the process of hatching or more, add the aqueous solution (as 700 μ l) containing thiocarbamide (as 115mM) and citric acid (as 200mM), then the aqueous solution (as 350 μ L) containing ascorbic acid (as 150mM) and ferrous piperazine (as 6mM) is added, after reaction, (as 60min) measures absorbance with spectrophotometer in 562nm place, take incubation time as horizontal ordinate X
1, absorbance is ordinate Y
1carry out linear regression, obtain equation Y
1=a
1x
1+ b
1, the absorption values when time that obtains thus is 0 is b
1, by sample absorbance numerical value b
1substitute into typical curve equation, calculate the content obtaining unstable iron in sample.
Same as above, preferably, described standard items are crystalline sulfuric acid iron ammonium; The concentration of described iron standard items serial solution is preferably 5 ~ 80 μ g/ml; The ratio that described iron-carbon aquo complex sample mixes with human serum is, in sample, the total content of iron counts 65 ~ 170 μ g/ml with human serum concentration of iron.
7. the method as described in any one of claim 1-6, the carbohydrates in wherein said iron-carbohydrate complex be selected from gluconate, sucrose, dextran, dextrin and sorbierite one or more.
The standard items of described preparation ferric iron titer can be crystalline sulfuric acid iron ammonium (molecular formula FeNH
4(SO
4)
212H
2o).
The concentration of described ferric iron standard items serial solution can be 0.1 ~ 100 μ g/ml, preferably 5 ~ 80 μ g/ml, such as, other numerical value in 5,10,20,30,40,50,60,70,80 μ g/ml and above-mentioned scope.Iron standard items series titer is preferably more than 3 kinds variable concentrations in above-mentioned concentration range, such as, prepare the titer of 3 kinds, 4 kinds, 5 kinds, 6 kinds variable concentrations.
The ratio that the sample of described iron-carbon aquo complex mixes with human serum is, in sample, the total content of iron counts 50 ~ 180 μ g/ml with human serum concentration of iron, preferably 65 ~ 170 μ g/ml, such as, any number in the above-mentioned scopes such as 70,80,90,100,110,120,130,140,150,160 μ g/ml.
8. the method as described in any one of claim 1-7, the common incubation time of wherein said iron-carbohydrate complex sample and human serum is 10 ~ 60min, preferably 30 ~ 60min.
9. the method as described in any one of claim 1-8, wherein said standard items are crystalline sulfuric acid iron ammonium; The concentration of described iron standard items serial solution is 5 ~ 80 μ g/ml.
10. the method as described in any one of claim 1-9, the ratio that wherein said iron-carbon aquo complex sample mixes with human serum is, in sample, the total content of iron counts 65 ~ 170 μ g/ml with human serum concentration of iron.
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| CN110118739A (en) * | 2019-06-05 | 2019-08-13 | 四川旭虹光电科技有限公司 | The measuring method of ferrous iron and ferric iron content in a kind of glass |
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| US3607097A (en) * | 1967-08-09 | 1971-09-21 | Philips Corp | Analyzer for liquid samples |
| CN102037015A (en) * | 2007-09-21 | 2011-04-27 | 健泰科生物技术公司 | Inhibition of angiogenesis |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110118739A (en) * | 2019-06-05 | 2019-08-13 | 四川旭虹光电科技有限公司 | The measuring method of ferrous iron and ferric iron content in a kind of glass |
| CN110118739B (en) * | 2019-06-05 | 2021-09-10 | 四川虹科创新科技有限公司 | Method for measuring content of ferrous iron and ferric iron in glass |
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