CN104237223B - 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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- CN104237223B CN104237223B CN201410526312.6A CN201410526312A CN104237223B CN 104237223 B CN104237223 B CN 104237223B CN 201410526312 A CN201410526312 A CN 201410526312A CN 104237223 B CN104237223 B CN 104237223B
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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 analysis method of ferrum supplementing preparation, and in particular to a kind of to determine in iron-carbohydrate complex
The method of unstable ferrum (III) content.
Background technology
Ferrum element is one of requisite trace element of human body, be constitute hemoglobin, Myoglobin, cytochrome and
Various oxidasic important components, as the carrier of oxygen, it is ensured that the normal conveying of oxygen in body tissue.
Ferrum many histiocytes absorbed or absorb all be by classical transferrinss (transferrin, Tf) and
The approach of TfR (transferrin receptor, TfR), i.e. ferric iron are combined with Tf first, both combinations
Thing is combined with the TfR of cell surface again, and afterwards through steps such as endocytosis, acidifying, release and displacements, ferrum enters endochylema, finally quilt
Cell utilization, synthetic hemoglobin and other materials.
Enough hemoglobin can not be synthesized during iron deficiency, iron deficiency anemia is caused.Clinically, diagnosis of iron deficiency anemia into
Iron supplement therapy is given after founding and finding out the cause of disease immediately should.Generally, Oral Iron Preparations are first-selection, but Oral Iron Preparations often cause bright
Aobvious gastrointestinal upset, it has been reported that therefore nearly 1/5 patient interrupts treatment.Oral Iron Preparations are also disturbed by composition of food
And the impact of internal Blood lipids, especially many chronic diseases, kidney disease, heart disease and tumor patient, the absorption of ferrum and profit
With can also be affected.For this some patients, clinician is considered as giving intravenous iron.Additionally, iron deficiency anemia is with stomach
When intestinal tract disease patient needs ferrum reserve in fast quick-recovery body and is orally not enough to reach this target, also intravenous iron can be made
For first-selection.
Have been developed that various ferrum containing iron-carbohydrate complex supplement intravenous formulations, bag at present both at home and abroad
Include ferrous gluconate sodium injection, Iron Sucrose Injection, injection iron sucrose, Iron Dextran Injection, Iron Sorbitex injection
Liquid etc..By taking ferrous gluconate sodium injection as an example, it is colloidal suspension prepared by a kind of Ferrlecit and makees
For prodrug use, wherein Ferrlecit comprising with the colloidal ferric hydroxide granule of gluconic acid complexation (ferrum core).
By iron chloride alkali neutralisation number pH is obtained for 2, under the pH, saturation hydroxide ion forms colloid hydroxide to these ferrum cores
Ferrum, its after its formation with gluconic acid complexes ira situ.The complex of gained by the cellular uptake of reticuloendothelial system, tap a blast furnace by release
Ion.Iron ion is combined with transferrinss again, and transferrinss are further transported to bone marrow for erythropoiesis or transhipment
Iron promoter position into ferritin and bone marrow, spleen and liver.In general, the structure of ferrum core follows Coordinative Chemistry.Carbon aquation
The hydroxyl of compound replaces the hydrone combined by ferrum core appearance, so that carbohydrate is combined with ferrum core.
However, the ferrum is also not associated with or weak binding comprising some cores with complex in supplementing intravenous formulations
Ferrum element, form unstable ferrum.These unstable ferrum are easy to be lived by the such as albumen of the chelating agent in chemical reaction or blood plasma
Change, this can cause the quick release of ferrum supplementing preparation ferrum element after intravenously administrable, that is, dash forward and release effect.The release of unstable ferrum can
So that the rapid saturation of transferrinss and cause the appearance of non-Tf-Fe, rather than Tf-Fe can be catalyzed
Harmful response to oxidative stress, clinically causes possibly even fatal acute untoward reaction.
Prior art discloses four kinds of measuring principles of ferrum：1) ferrous piperazine colorimetry principle：Serum levels of iron and transferrin knot
Synthesising complex, in acid medium, ferrum from complex dissociates out, then is reduced agent and is reduced into ferrous irons, and with ferrous piperazine
Aubergine compound is generated, has an absworption peak at wavelength 562nm, compared with the same titer for processing, you can try to achieve serum
The content of ferrum.2) duplex pyridine colorimetric method principle：Make in acid condition ferrum from separate out in protein bonding state.With
Oxammonium hydrochloride. makes ferric iron back in serum as reducing agent, and, into ferrous irons, the latter generates redness with the reaction of duplex pyridine developer and stings
Compound, the colorimetric assay at the 520nm.This law is easy to be quick, but poor sensitivity, and interference factor is more, is polluted the most with irony especially
Seriously, haemolysis can also affect result.3) luxuriant and rich with fragrance Lip river piperazine colorimetry principle：Trichloroacetic acid-hydrochloric acid mixed solution makes the ferrum in serum from fortune
Albumen precipitation is discharged and is made in ferritin, and the ferric iron for discharging can be reduced into ferrous irons, the latter and Fei with thioglycolic acid
Lip river piperazine is combined into aubergine compound, and absworption peak is 562nm.4) total iron binding capacity (TIBC)：Fill up in blood serum sample
The ferrum titer of amount makes fortune ferrum ferritin by ferrum saturation, excessive ferrum MgCO3Remove out, centrifuging and taking supernatant, by survey serum levels of iron
Method obtain the content of ferrum, as TIBC. (《Physiology》, People's Health Publisher, 2012).However, said method is only related to
And the ultimate principle that iron content is determined, can still there is relatively large deviation when being actually measured to concrete material at different conditions.
There is presently no the report with regard to determining unstable iron content method in ferrum intravenous formulations.Therefore, it is necessary to develop it is a kind of stable,
The reliable method for determining unstable iron content in vein iron preparation, and in this way based on the quality of ferrum supplementing preparation is entered
Row assessment.
The content of the invention
It is an object of the invention to provide a kind of method for determining unstable iron content in iron-carbohydrate complex, special
Not for the preparation containing ferrum-carbon aquo complex, such as vein ferrum supplementing preparation carries out the evaluation of external quality, ensure clinical
The safety of medication.
The present invention is achieved through the following technical solutions above-mentioned purpose：
A kind of method of unstable ferric iron content in measure iron-carbohydrate complex, including step：
Ferrum-carbon aquo complex is contacted with human serum, be ferrous irons by the unstable ferric iron back combined with human serum；
The ferrous irons that reduction is obtained generate colored compound with developer effect, measure the absorbance of colored compound；With ferric iron
Titer processes the concentration of iron-absorbance standard curve for obtaining through same reduction and chromogenic reaction and is compared, and is calculated
Unstable ferric content in ferrum-carbon aquo complex.
In the iron-carbohydrate complex, unstable ferrum is uncombined with the core of the complex or weak binding
Ferric ion.The weak binding ferrum be complex in be easy to participate in chemical reaction or anti-with other iron complex constituent components
The ferrum element answered.
In the present invention, described iron-carbohydrate complex can in the form of a solution or dosage form is present.Described
Unstable ferric iron is may reside in iron-carbohydrate complex, it is also possible to be present in comprising iron-carbohydrate complexation
In the solution or preparation of thing.
In one embodiment, the method for determining unstable ferric iron content in iron-carbohydrate complex,
Including step：
Make ferrum-carbon aquo complex mix with human serum, mixture is obtained respectively in more than three different time points of mixing,
Protein denaturant stopped reaction is added, then is ferrous irons by the ferric iron back combined with human serum；Then reduction is obtained
Ferrous irons generate colored compound with developer effect, measure its absorbance, and with incorporation time as abscissa, absorbance is vertical seat
Mark, obtains curvilinear equation, the absorption values when time of trying to achieve is 0, and the absorption values are substituted into by ferric iron titer Jing
Cross same reduction and chromogenic reaction processes the concentration of iron-absorbance standard curve equation for obtaining, be calculated unstable in sample
Ferric content.
In one embodiment, the method for determining unstable ferric iron content in iron-carbohydrate complex,
Including step：
1) foundation of standard curve：
The ferric iron standard substance serial solution of more than three kinds variable concentrations is prepared, a certain amount of ferrum standard strain is drawn respectively
Row solution, is reduced to ferrous irons under reducing agent effect in acid medium, and then developer is generated with ferrous irons effect
Colored compound, measures absorbance, and with ferric concentration as abscissa, absorbance is vertical coordinate, obtains calibration curve equation；
2) process of ferrum-carbon aquo complex sample：
Human serum and ferrum-carbon aquo complex sample mix are carried out into common incubation, wherein the total content of ferrum is changed in the sample
Calculate and 50～180 μ g/mL are preferably for people's serum iron, obtain a certain amount of respectively in more than three different time points of incubation
Sample, adds protein denaturant stopped reaction, under in acid medium, reducing agent is acted on by the ferric iron combined with human serum also
Originally it was ferrous irons, developer generates colored compound with ferrous irons effect, measures absorbance, with incubation time as abscissa, inhaled
Luminosity is vertical coordinate, obtains curvilinear equation, the absorption values when time of trying to achieve is 0, the absorption values is substituted into standard bent
Line equation, is calculated unstable ferric content in ferrum-carbon aquo complex sample.
Ferrum in the total content of above-mentioned ferrum is the total content of stable and unstable ferrum in sample；More specifically, for stable and
Unstable ferric total content.
The standard substance for preparing ferric iron titer can be crystalline sulfuric acid ferrum ammonium (molecular formula FeNH4(SO4)2〃
12H2O).The concentration of the ferric iron standard substance serial solution can be 0.1～100 μ g/ml, preferably 5～80 μ g/ml, for example
5th, other numerical value in 10,20,30,40,50,60,70,80 μ g/ml and above range.Ferrum standard substance series titer is preferred
For more than 3 kinds variable concentrations in above-mentioned concentration range, for example, prepare the titer of 3 kinds, 4 kinds, 5 kinds, 6 kinds variable concentrations.
Carbohydrate in the ferrum-carbon aquo complex is selected from gluconate, sucrose, dextran, dextrin and mountain
One or more in pears alcohol.
The ratio of the sample of the ferrum-carbon aquo complex and human serum mixing is that the total content of ferrum is with human serum in sample
Concentration of iron is calculated as 50～180 μ g/ml, preferably 65～170 μ g/ml, such as 70,80,90,100,110,120,130,140,150,
Any number in the above ranges such as 160 μ g/ml.
The sample of the ferrum-carbon aquo complex is 10～60min, preferably 30～60min with the common incubation time of human serum.
The time point of the measurement is such as 10,20 or 60 minutes in three different time points being incubated；Can also be and incubate
Four different time points educated, such as 10,20,30,45min.Can also be more than 4 time points.Different time points measurement can be with
Accuracy is improved, and is easy to monitoring.
The protein denaturant is selected from carbamide, thiourea, 3- [3- (gallbladder amido propyl) dimethylamino] propane sulfonic acid inner salt
(CHAPS), one or more in dithiothreitol, DTT (DTT), IPG buffer.It is preferred that thiourea, is playing albuminous degeneration effect
Copper ion interference is shielded simultaneously.
The addition of the acid medium is so that system pH is controlled to 1～4 is advisable, it is ensured that reduction reaction is complete.It is acid to be situated between
It is of fine quality to elect citric acid as.
The reducing agent is selected from ascorbic acid, reproducibility flavin mononucleotide (FMN), dithionite, hydroxyl acetate, hydrogen
Quinone, lactic acid or lactate, citric acid or citrate, bicarbonate, pyruvate, succinic acid or succinate, Fructose, half Guang
One or more in propylhomoserin and Sorbitol.Preferably faintly acid reducing agent ascorbic acid.Reducing agent exist amount be relative to
At least about 10 times excess of iron-carbohydrate complex, preferably 30 times excess, more preferably 50 times excess.
The developer is ferrous piperazine.
In a preferred embodiment, acid medium and protein denaturant are added simultaneously, and by reducing agent and is shown
Toner is added simultaneously, does not so interfere with testing result, can also simplify step, convenient to operate.Additionally, the developer with
Reducing agent is simultaneously added, and the ferrous irons that reduction is produced can be made to react with developer immediately generation colored compound, while
Promote the carrying out of reduction reaction.The time of the reduction reaction and chromogenic reaction is about 30～60min, preferred 60min.
The colored compound be with spectrophotometer at wavelength 562nm mensuration absorbance.
In a kind of specific embodiment, unstable ferric iron content in the measure iron-carbohydrate complex
Method, including step：
1) foundation of standard curve：
At room temperature, the ferrum standard substance serial solution of 0.1～100 μ g/mL more than 3 kinds variable concentrations of scope is prepared, respectively
The aqueous solution (such as 700 μ l) containing citric acid (such as 200mM) is added in the ferrum standard substance serial solution (such as 100 μ l), then
The aqueous solution (such as 350 μ L) containing ascorbic acid (such as 150mM) and ferrous piperazine (such as 6mM) is added, (such as 60min) uses light splitting after reaction
Photometer mensuration absorbance at the 562nm, with concentration of iron as abscissa X, absorbance is vertical coordinate Y, obtains calibration curve equation Y
=aX+b；
2) measure of the unstable iron content of ferrum-carbon aquo complex sample：
At room temperature, human serum is incubated with ferrum-carbon aquo complex sample mix, the total content of ferrum wherein in sample
It is 50～180 μ g/mL to be scaled serum iron, and the different time points of three during incubation or more take sample respectively
(such as 100 μ l) add the aqueous solution (such as 700 μ l) containing thiourea (such as 115mM) and citric acid (such as 200mM), are subsequently adding containing anti-bad
The aqueous solution (such as 350 μ L) of hematic acid (such as 150mM) and ferrous piperazine (such as 6mM), after reaction (such as 60min) with spectrophotometer in
Mensuration absorbance at 562nm, with incubation time as abscissa X1, absorbance is vertical coordinate Y1Linear regression is carried out, equation Y is obtained1
=a1X1+b1, it is b thus to obtain absorption values of time when being 01；By sample absorbance numerical value b1Substitute into calibration curve equation,
The content of unstable ferrum in sample is obtained in calculating.
It is same as above, it is preferable that the standard substance are crystalline sulfuric acid ferrum ammonium；The concentration of the ferrum standard substance serial solution is excellent
Elect 5～80 μ g/ml as；The ratio of the ferrum-carbon aquo complex sample and human serum mixing is that the total content of ferrum is with people in sample
Serum iron is calculated as 65～170 μ g/ml.
In the present invention, unstable ferric reduction in ferrum-carbon aquo complex meets first order kineticss, and the present invention's is above-mentioned
Detection method, can detect any preparation containing ferrum-carbon aquo complex, can be oral or the dosage form, such as glucose such as injection
Sour ferrum sodium injection, Iron Sucrose Injection, iron sorbitex injection, Iron Dextran Injection, dextriferron injection, compound recipe card
Rail injection liquid etc., detection sensitivity is high, precision is good, and accuracy is high, can quickly, accurately, quantitatively determine ferrum-carbon hydrate
Unstable iron content in thing, can carry out science and effectively control, ensure to the quality of (especially vein) ferrum supplementing preparation
The safety of clinical application.
Specific embodiment
Following examples can be used for further appreciating that the features and advantages of the invention, but should not be construed as limiting the invention
Protection domain.Any improvement made on the basis of the present invention and change, all within protection scope of the present invention.
In addition to especially indicating, the instrument and reagent used by embodiment is ordinary commercial products.
Embodiment 1：
The preparation (100 μ g/ml) of ferrum stock solution：Crystalline sulfuric acid ferrum ammonium [FeNH is weighed accurately4(SO4)2〃12H2O]
(Aladdin Industrial Corporation) 0.860g, enriching sulphuric acid (AR) 2.0ml after deionized water dissolving, moves into
Deionized water is added to be diluted to scale as stock solution in 1L volumetric flasks.
Ferrum standard serial solution is prepared：Ferrum stock solution 5,10,20,30,40ml is pipetted respectively, is added separately to 100ml appearances
In measuring bottle, plus deionized water is settled to scale, shakes up as ferrum standard serial solution, and concentration is respectively 5,10,20,30,40 μ g/
ml。
Ferrum standard curve is set up：At room temperature, each 100 μ l of ferrum standard serial solution are separately added in 1.5mlEP pipes, so
The aqueous acid of Fructus Citri Limoniae containing 200mM (reagent I) 700 μ l are added afterwards, are eventually adding the ferrous piperazine of ascorbic acid containing 150mM and 6mM
350 μ L of aqueous solution (reagent II), after reaction 60min with spectrophotometer (Shimadzu UV2401) at the 562nm mensuration absorbance.With
Concentration is abscissa x, and absorbance is that vertical coordinate y carries out linear regression, draws and show that calibration curve equation is：Y=0.0158x+
0.0023, r=0.9985.
The detection of unstable iron content in ferrous gluconate preparation of sodium：At room temperature, in 1.5mL EP pipes, human serum
(Biosharp) with compound composition injection (the SANOFI AVENTIS US LLC) incubation of ferrous gluconate sodium sucrose, chalybeate is added
It is 166.70 μ g/mL that amount is scaled serum iron, respectively in incubation time 10,20,30,45min, takes the addition of 100 μ l samples
Thiourea containing 115mM and 200mM Fructus Citri Limoniae aqueous acids (reagent I) 700ul, are subsequently adding ascorbic acid containing 150mM and 6mM are ferrous
The 350 μ L of aqueous solution (reagent II) of piperazine, after reaction 60min with spectrophotometer at the 562nm mensuration absorbance.
As a result calculate：With incubation time as abscissa x1, absorbance is vertical coordinate y1Linear regression is carried out, drafting draws song
Line equation is y1=0.0118x1+ 0.0869, r=0.9978.Thus obtain the time for 0 when absorbance be 0.0869, bring ferrum mark into
Directrix curve calculates unstable concentration of iron for 5.354 μ g/ml.
Take same batch glucose acid ferrum sodium formulation samples and repeat above-mentioned experimentation 5 times altogether, be measured and calculate with data,
As a result it is as shown in table 1.
Table 1
Embodiment 2：
The foundation of ferrum standard curve：With embodiment 1.
The detection method of unstable iron content in ferrous gluconate preparation of sodium：Similar embodiment 1, except for the difference that human serum with
When the compound composition injection of ferrous gluconate sodium sucrose is incubated, chalybeate addition is scaled serum iron for 100.0 μ g/mL.Take
Repeat experimentation 5 times altogether with batch glucose acid ferrum sodium formulation samples, be measured and calculate with data, as a result as shown in table 2.
Table 2
Embodiment 3：
The foundation of ferrum standard curve：Similar embodiment 1, except for the difference that replaces ascorbic acid with Sorbitol in reagent II.
The detection method of unstable iron content in ferrous gluconate preparation of sodium：Similar embodiment 1, except for the difference that human serum with
When the compound composition injection of ferrous gluconate sodium sucrose is incubated, chalybeate addition is scaled serum iron for 40.0 μ g/mL, reagent
Replace thiourea with CHAPS (Shanghai Hao Yang bio tech ltd) in I, in reagent II, replace ascorbic acid with Sorbitol.Take
Repeat experimentation 5 times altogether with batch glucose acid ferrum sodium formulation samples, be measured and calculate with data, as a result as shown in table 3.
Table 3
Embodiment 4：
The foundation of ferrum standard curve：With embodiment 1.
The detection method of unstable iron content in ferrous gluconate preparation of sodium：Similar embodiment 1, except for the difference that human serum with
When the compound composition injection of ferrous gluconate sodium sucrose is incubated, chalybeate addition is scaled serum iron for 100.0 μ g/mL, examination
Replace thiourea with dithiothreitol, DTT (Shanghai Lian Shuo bio tech ltd) in agent I.
Take same batch glucose acid ferrum sodium formulation samples and repeat experimentation 5 times altogether, be measured and calculate with data, as a result
As shown in table 4.
Table 4
Embodiment 5：
The foundation of ferrum standard curve：With embodiment 1.
The detection of unstable iron content in Astrafer：At room temperature, in 1.5mL EP pipes, human serum with it is right
Sugared acid anhydride rail injection liquid (Pharmacosmos A/S) incubation of rotation, it is 66.70 μ g/ml that chalybeate addition is scaled serum iron,
Respectively in incubation time 10,20,30,45min, take 100 μ l samples and add thiourea containing 115mM and 200mM Fructus Citri Limoniae aqueous acids
(reagent I) 700 μ l, are subsequently adding aqueous solution (reagent II) 350uL of ascorbic acid containing 150mM and 6mM ferrous iron piperazines, reaction
After 60min with spectrophotometer at the 562nm mensuration absorbance.
Data are calculated：With incubation time as abscissa x2, absorbance is vertical coordinate y2Linear regression is carried out, drafting draws song
Line equation is y2=0.000986x2+ 0.0180, r=0.9973.Thus obtain the time for 0 when absorbance be 0.0180, bring ferrum into
Standard curve calculates unstable concentration of iron for 0.994 μ g/ml.
Take same batch Astrafer sample and repeat above-mentioned experimentation 5 times, be measured and calculate with data, as a result
As shown in table 5.
Table 5
Embodiment 6：
The foundation of ferrum standard curve：With embodiment 1.
The detection of unstable iron content in Astrafer：With embodiment 5, except for the difference that human serum and dextran
When rail injection liquid is incubated, chalybeate addition is scaled serum iron for 100.00 μ g/mL.Take same batch Astrafer
Sample repeats experimentation 5 times altogether, is measured and calculates with data, as a result as shown in table 6.
Table 6
Embodiment 7：
The foundation of ferrum standard curve：Similar embodiment 1, except for the difference that replaces ascorbic acid with Sorbitol in reagent II.
The detection of unstable iron content in Astrafer：Similar embodiment 5, except for the difference that human serum and dextrose
When acid anhydride rail injection liquid is incubated, it is 200.0 μ g/mL that chalybeate addition is scaled serum iron, replaces sulfur with CHAPS in reagent I
Urea, replaces ascorbic acid with Sorbitol in reagent II.Take same batch Astrafer sample and repeat experimentation 5 times altogether,
It is measured and calculates with data, as a result as shown in table 7.
Table 7
Embodiment 8：
The foundation of ferrum standard curve：With embodiment 4.
The detection method of unstable iron content in Astrafer：Similar embodiment 5, except for the difference that human serum with it is right
When the sugared acid anhydride rail injection liquid of rotation is incubated, it is 100.0 μ g/mL that chalybeate addition is scaled serum iron, with two sulfur threoses in reagent I
Alcohol replaces thiourea.Take same batch Astrafer sample and repeat experimentation 5 times altogether, be measured and calculate with data, knot
Fruit is as shown in table 8.
Table 8
The experimental result of above-described embodiment shows, when human serum is incubated with iron-carbohydrate complex, chalybeate addition
It is 50～180 μ g/mL to be scaled serum iron, and protein denaturant is thiourea, when reducing agent is ascorbic acid, accuracy in detection
Height, error are little.
The minority embodiment of the present invention is provide only more than, not specified operating condition can be according to this area routine
Method is carried out.Some operating process, such as preparation of ferrum standard solution, the preparation of reagent I and II, the acquisition of curvilinear equation, number
Can carry out in accordance with conventional methods known in the art according to calculating etc., it is not necessary to the concrete restriction being confined in embodiment.Unless
Special instruction, the operating process are all carried out at room temperature, preferably 10～35 DEG C.Without departing from the essential and spiritual of the present invention
Under the premise of the improvement made or equivalence changes belong to the scope of protection of the invention.
Claims (14)
1. it is a kind of determine iron-carbohydrate complex in unstable ferric iron content method, it is characterised in that methods described
Comprise the steps：
Iron-carbohydrate complex is contacted with human serum, be ferrous irons by the ferric iron back combined with human serum；Will be also
The ferrous irons that original is obtained generate colored compound with developer effect, measure the absorbance of colored compound, with incorporation time are
Abscissa, absorbance are vertical coordinate, obtain curvilinear equation, the absorption values when time of trying to achieve is 0；With ferric iron titer Jing
Cross same reduction and chromogenic reaction processes the concentration of iron-absorbance standard curve for obtaining and is compared, be calculated ferrum-carbon aquation
Unstable ferric content in compound complex.
2. method according to claim 1, it is characterised in that methods described comprises the steps：
Make iron-carbohydrate complex mix with human serum, mixing is obtained respectively in more than three different time points of mixing
Thing, adds protein denaturant stopped reaction, then is ferrous irons by the ferric iron back combined with human serum；Then reduction is obtained
The effect of ferrous irons and developer generate colored compound, measure its absorbance, with incorporation time as abscissa, absorbance is vertical
Coordinate, obtains curvilinear equation, the absorption values when time of trying to achieve is 0, and the absorption values are substituted into by ferric iron titer
Concentration of iron-absorbance standard curve the equation for obtaining is processed through same reduction and chromogenic reaction, ferrum-carbon hydrate is calculated
Unstable ferric content in thing complex.
3. method according to claim 1, it is characterised in that methods described comprises the steps：
1) foundation of standard curve：
The ferric iron standard substance serial solution of more than three kinds variable concentrations is prepared, is gone back under reducing agent effect in acid medium
Originally it was ferrous irons, and developer was generated into colored compound with ferrous irons effect then, absorbance is measured, with ferric concentration is
Abscissa, absorbance are vertical coordinate, obtain calibration curve equation；
2) process of iron-carbohydrate complex sample：
Human serum and iron-carbohydrate complex sample mix are carried out into common incubation, in more than three different times of incubation
Point obtains a certain amount of sample respectively, adds protein denaturant stopped reaction, will be with people under reducing agent effect in acid medium
The ferrum that serum is combined is reduced to ferrous irons, and developer generates colored compound with ferrous irons effect, measures absorbance, during being incubated
Between be abscissa, absorbance is vertical coordinate, obtains curvilinear equation, the absorption values when time of trying to achieve is 0, by the extinction number of degrees
Value substitutes into calibration curve equation, is calculated unstable ferric content in iron-carbohydrate complex.
4. method as claimed in claim 2 or claim 3, wherein, the protein denaturant is selected from carbamide, thiourea, 3- [3- (gallbladder amide
Propyl group) dimethylamino] propane sulfonic acid inner salt, dithiothreitol, DTT, one or more in IPG buffer.
5. method as claimed in claim 3, wherein, the acid medium is citric acid.
6. the method as described in any one of claim 1-3, wherein, the reduction is carried out in the presence of a reducing agent, the reduction
Agent is selected from reproducibility flavin mononucleotide (FMN), dithionite, hydroxyl acetate, hydroquinone, lactic acid or lactate, citric acid or lemon
In lemon hydrochlorate, bicarbonate, pyruvate, succinic acid or succinate, Fructose, cysteine, Sorbitol or ascorbic acid
One or more.
7. the method as described in any one of claim 1-3, wherein the developer is ferrous piperazine.
8. method as claimed in claim 3, wherein the acid medium and protein denaturant are added simultaneously, then developer with
Reducing agent is simultaneously added, and makes the ferrous irons that reduction is produced react with developer immediately generation colored compound.
9. the method as described in any one of claim 8, wherein, the addition of the acid medium make system pH be controlled to 1～
4；The reducing agent consumption is relative at least 10 times excess of iron-carbohydrate complex.
10. the method as described in any one of claim 1-3, wherein the colored compound be with spectrophotometer in wavelength
Mensuration absorbance at 562nm.
Method described in 11. any one of claim 1-3, including step：
1) foundation of standard curve：
At room temperature, the ferrum standard substance serial solution of 0.1～100 μ g/mL more than 3 kinds variable concentrations of scope is prepared, respectively in institute
Add containing Fructus Citri Limoniae aqueous acid in stating ferrum standard substance serial solution, add the aqueous solution containing ascorbic acid and ferrous piperazine,
After reaction with spectrophotometer in 562nm locate mensuration absorbance, with concentration of iron as abscissa X, absorbance be vertical coordinate Y, obtain
Calibration curve equation Y=aX+b；
2) measure of the unstable iron content of iron-carbohydrate complex sample：
At room temperature, human serum is incubated with iron-carbohydrate complex sample mix, wherein in sample, ferrum always contains
Amount is scaled serum iron for 50～180 μ g/mL, respectively the different time points sampling of three during incubation or more
Product, add containing thiourea and Fructus Citri Limoniae aqueous acid, be subsequently adding the aqueous solution containing ascorbic acid and ferrous piperazine, light splitting is used after reaction
Photometer mensuration absorbance at the 562nm, with incubation time as abscissa X1, absorbance is vertical coordinate Y1Linear regression is carried out, is obtained
To equation Y1=a1X1+b1, it is b thus to obtain absorption values of time when being 01；By sample absorbance numerical value b1Substitution standard
Curvilinear equation, is calculated unstable ferric content in iron-carbohydrate complex.
12. methods as claimed in claim 11, wherein, the standard substance used in the ferrum standard substance serial solution are crystallization
Ammonium ferric sulfate；The concentration of the ferrum standard substance serial solution is 5～80 μ g/m l；The iron-carbohydrate complex and people
The ratio of serum mixing is that the total content of ferrum is calculated as 65～170 μ g/ with human serum concentration of iron in iron-carbohydrate complex
ml。
13. methods as described in any one of claim 1-3, wherein the carbon hydrate in the iron-carbohydrate complex
One or more in gluconate, sucrose, dextran, dextrin or Sorbitol of thing.
14. methods as described in any one of claim 1-3, wherein the iron-carbohydrate complex and human serum are total to
Incubation time is 10～60min.
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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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| 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 |
| CN102967649A (en) * | 2012-05-24 | 2013-03-13 | 新疆科丽生物技术有限公司 | Application of inductively coupled plasma mass spectrometry in drug testing of hemin |
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