CN112763566A - Method for measuring iron content in serum - Google Patents
Method for measuring iron content in serum Download PDFInfo
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- CN112763566A CN112763566A CN201911057479.1A CN201911057479A CN112763566A CN 112763566 A CN112763566 A CN 112763566A CN 201911057479 A CN201911057479 A CN 201911057479A CN 112763566 A CN112763566 A CN 112763566A
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- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 title claims abstract description 110
- 210000002966 serum Anatomy 0.000 title claims abstract description 88
- 238000000034 method Methods 0.000 title claims abstract description 76
- 229910052742 iron Inorganic materials 0.000 title claims abstract description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 34
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 claims abstract description 29
- 238000011282 treatment Methods 0.000 claims abstract description 28
- 238000002360 preparation method Methods 0.000 claims abstract description 22
- 238000005259 measurement Methods 0.000 claims abstract description 18
- 239000002253 acid Substances 0.000 claims abstract description 9
- 239000012086 standard solution Substances 0.000 claims description 45
- 229910017604 nitric acid Inorganic materials 0.000 claims description 42
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 35
- 239000000243 solution Substances 0.000 claims description 26
- 239000003085 diluting agent Substances 0.000 claims description 25
- 238000010438 heat treatment Methods 0.000 claims description 23
- 238000007865 diluting Methods 0.000 claims description 22
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 18
- 239000012498 ultrapure water Substances 0.000 claims description 18
- 238000002156 mixing Methods 0.000 claims description 8
- 238000003556 assay Methods 0.000 claims description 6
- 239000012467 final product Substances 0.000 claims description 6
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims description 4
- 238000012545 processing Methods 0.000 claims description 4
- QZPSXPBJTPJTSZ-UHFFFAOYSA-N aqua regia Chemical compound Cl.O[N+]([O-])=O QZPSXPBJTPJTSZ-UHFFFAOYSA-N 0.000 claims description 2
- NYQDCVLCJXRDSK-UHFFFAOYSA-N Bromofos Chemical compound COP(=S)(OC)OC1=CC(Cl)=C(Br)C=C1Cl NYQDCVLCJXRDSK-UHFFFAOYSA-N 0.000 claims 1
- 239000006199 nebulizer Substances 0.000 claims 1
- 238000003113 dilution method Methods 0.000 abstract description 3
- 239000000523 sample Substances 0.000 description 66
- 239000010948 rhodium Substances 0.000 description 35
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 16
- 239000000203 mixture Substances 0.000 description 14
- 239000011159 matrix material Substances 0.000 description 13
- 238000004458 analytical method Methods 0.000 description 12
- 238000011835 investigation Methods 0.000 description 10
- 238000010790 dilution Methods 0.000 description 9
- 239000012895 dilution Substances 0.000 description 9
- 239000012491 analyte Substances 0.000 description 8
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 229940096339 ferric carboxymaltose injection Drugs 0.000 description 7
- MFBBZTDYOYZJGB-HAONTEFVSA-L (2s,3s,4s,5r)-4-[(2r,3r,4r,5s,6r)-5-[(2r,3r,4r,5s,6r)-3,4-dihydroxy-6-(hydroxymethyl)-5-[(2r,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-3,4-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-2,3,5,6-tetrahydroxyhexanoate;iron(3+);oxyg Chemical compound O.[OH-].[O-2].[Fe+3].O[C@@H]1[C@@H](O)[C@@H](O[C@@H]([C@H](O)CO)[C@@H](O)[C@H](O)C([O-])=O)O[C@H](CO)[C@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@@H](CO)O1 MFBBZTDYOYZJGB-HAONTEFVSA-L 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 229960004131 ferric carboxymaltose Drugs 0.000 description 5
- 230000002949 hemolytic effect Effects 0.000 description 5
- 241000282472 Canis lupus familiaris Species 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- -1 iron ions Chemical class 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 3
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000000120 microwave digestion Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- AACILMLPSLEQMF-UHFFFAOYSA-N 2,2-dichloroethenyl 2-ethylsulfinylethyl methyl phosphate Chemical compound CCS(=O)CCOP(=O)(OC)OC=C(Cl)Cl AACILMLPSLEQMF-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 230000008588 hemolysis Effects 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 2
- 239000004810 polytetrafluoroethylene Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 102000018832 Cytochromes Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 208000015710 Iron-Deficiency Anemia Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108091007187 Reductases Proteins 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- FWZTTZUKDVJDCM-CEJAUHOTSA-M disodium;(2r,3r,4s,5s,6r)-2-[(2s,3s,4s,5r)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol;iron(3+);oxygen(2-);hydroxide;trihydrate Chemical compound O.O.O.[OH-].[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[Na+].[Na+].[Fe+3].[Fe+3].[Fe+3].[Fe+3].[Fe+3].O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 FWZTTZUKDVJDCM-CEJAUHOTSA-M 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940125532 enzyme inhibitor Drugs 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000009616 inductively coupled plasma Methods 0.000 description 1
- 229940090044 injection Drugs 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 150000004698 iron complex Chemical class 0.000 description 1
- 150000002506 iron compounds Chemical class 0.000 description 1
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N iron oxide Inorganic materials [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 description 1
- IEECXTSVVFWGSE-UHFFFAOYSA-M iron(3+);oxygen(2-);hydroxide Chemical group [OH-].[O-2].[Fe+3] IEECXTSVVFWGSE-UHFFFAOYSA-M 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- NDLPOXTZKUMGOV-UHFFFAOYSA-N oxo(oxoferriooxy)iron hydrate Chemical compound O.O=[Fe]O[Fe]=O NDLPOXTZKUMGOV-UHFFFAOYSA-N 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- MHOVAHRLVXNVSD-UHFFFAOYSA-N rhodium atom Chemical compound [Rh] MHOVAHRLVXNVSD-UHFFFAOYSA-N 0.000 description 1
- 238000005464 sample preparation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 229910003153 β-FeOOH Inorganic materials 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
-
- 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
-
- 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
- G01N1/38—Diluting, dispersing or mixing samples
-
- 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
- G01N1/44—Sample treatment involving radiation, e.g. heat
-
- 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
- G01N1/38—Diluting, dispersing or mixing samples
- G01N2001/386—Other diluting or mixing processes
Abstract
The invention provides a method for measuring the content of iron in serum, which comprises two steps of sample preparation and treatment and ICP-MS measurement. The method for measuring the iron content in the serum uses ICP-MS (inductively coupled plasma mass spectrometry) to measure the sample, adopts a direct dilution method after adding concentrated acid water bath in the serum sample treatment mode, and has the advantages of simple treatment process, accurate measurement result and high efficiency.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a method for measuring iron content in serum by ICP-MS.
Background
Iron is an important component of human hemoglobin, cytochrome enzyme and many reductases, participates in blood oxygen and carbon dioxide transportation, and oxidation, reduction and metabolism processes, and is an indispensable substance for normal physiological activities of the body. Insufficient iron intake consumes body iron stores, which in turn causes iron deficiency anemia.
In 2007, ferric carboxymaltose (ferrimaltose) comes into the market, meets the clinical requirement for large-dose rapid administration of iron, and becomes a representative drug of intravenous iron.
The ferric carboxymaltose is a macromolecular compound formed by poly 4R- ((1-4) -O-alpha-D-glucopyranosyl) -O-2R, 3S, 5R, 6-tetrahydroxyhexanoic acid wrapped by polynuclear beta-FeOOH, and the ferric carboxymaltose solid is dark brown amorphous powder, wherein the iron content is 24-32%, the polymaltose content is 25-50%, the relative molecular mass is about 15 ten thousand, the ferric carboxymaltose is soluble in water and insoluble in organic reagents such as ethanol. The ferric carboxymaltose has good stability, can endure high-temperature sterilization at 121 ℃, does not need to be added with a preservative in the preparation process, and the injection is a dark brown and physiological isotonic aqueous solution, has the pH of 5.0-7.0 and contains 50mg of elemental iron per milliliter. The carboxyl ferric maltose is a novel iron compound formed by wrapping ferric hydroxide core with carbohydrate shell, and after entering into the body through intravenous injection, the carboxyl ferric maltose can be phagocytized by macrophages in liver, spleen and bone marrow through distribution and can slowly release iron ions.
The method for measuring the content of the iron in the serum can be used for measuring by applying ICP-MS (inductively coupled plasma mass spectrometry), and has the advantages of high sensitivity, high measuring speed and high efficiency. The serum sample treatment method is a method of directly diluting serum with 50-100 times of pure water or low-concentration nitric acid and then measuring the content of iron element by ICP-MS (inductively coupled plasma-mass spectrometry), but the method is not suitable for measuring the content of iron in serum after the administration of the ferric carboxymaltose injection, because the method cannot completely destroy the structure of an iron complex. Meanwhile, in the research, the treatment method is found to have precipitation phenomenon after the standing time is prolonged, and the repeated measurement result after the treatment method is placed for a plurality of days has large fluctuation.
The microwave digestion method can completely decompose the sample and completely destroy the iron-sugar composite structure of the carboxyl ferric maltose, but the method has complex sample treatment process, the digestion amount is limited by the number of digestion tanks, and the efficiency is low.
Therefore, a method suitable for measuring the content of iron in serum after the administration of the ferric carboxymaltose injection is urgently needed to be established, and meanwhile, the sample treatment process is simple, the measurement result is accurate, and the efficiency is high.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provide the ICP-MS method for measuring the content of iron in serum, which has the advantages of simple sample treatment process, accurate measurement result and high efficiency.
The technical scheme of the invention is as follows:
a method for measuring the content of iron in serum comprises two steps of sample preparation and treatment and ICP-MS measurement.
Preferably, the sample is heated after adding concentrated acid in the sample preparation and treatment steps.
Preferably, the concentrated acid is 65-70 wt% concentrated nitric acid, aqua regia or perchloric acid.
More preferably, the concentrated acid is 65-68 wt% concentrated nitric acid.
Preferably, the heating treatment is water bath heating.
More preferably, the heating temperature is 70-85 ℃, and the heating time is 0.5-3 h.
Further, the method specifically comprises the following steps:
(1) sample preparation and processing
Preparation of standard curve sample: taking blank serum, an internal standard solution and standard solutions with different Fe element concentrations, adding concentrated nitric acid, heating, diluting with water, and shaking up to obtain the final product;
treating a serum sample to be detected: taking the serum to be detected and the internal standard solution, adding concentrated nitric acid, heating, diluting with water, and shaking up to obtain a serum sample;
the internal standard solution contains Rh element, the concentration of the Rh element is 5-50 mg/mL, and the concentration of the Rh element is preferably 10-30 mg/mL;
(2) measurement of ICP-MS
And (3) standard curve equation preparation: measuring the corresponding value of Fe element and the response value of Rh element of the standard curve sample by ICP-MS, and drawing a standard curve by taking the concentration of Fe element solution as an abscissa and the ratio of the response values of Fe element and Rh element as an ordinate to obtain a standard curve equation;
and (3) determination: and measuring the corresponding value of the Fe element and the response value of the Rh element of the serum sample by using ICP-MS, substituting the ratio of the response values of the Fe element and the Rh element into a standard curve equation, and calculating the content of the Fe element.
Preferably, the mass spectrometer of the assay is PerkinElmer NEXION 350X.
Preferably, the atomizer of the mass spectrometer is MicroMist, the temperature of the atomizing chamber is 3 ℃, and the operation mode is He Gas.
Preferably, 65-68 wt% of concentrated nitric acid is added in the step (1), and the mixture is heated in a water bath at 80 ℃.
Further, the preparation of the standard curve sample in the step (1) is specifically as follows: mixing blank serum 100 mul, internal standard solution 200 mul and standard diluent 100 mul with different Fe element concentrations in a centrifuge tube respectively, adding concentrated nitric acid 1-3 ml, heating in a water bath for 0.5-3 h, diluting with ultrapure water to 10ml, and shaking up to obtain the final product.
Further, the sample treatment in step (1) is specifically: and (3) putting 100 mul of serum to be detected and 200 mul of internal standard solution into a centrifuge tube, adding 1-3 ml of concentrated nitric acid, heating in a water bath for 0.5-3 h, diluting to 10ml with ultrapure water, and shaking up to obtain a serum sample.
The Rh element standard solution of the present invention can be purchased from commercial sources or prepared by itself. An exemplary preparation method of the Rh element standard solution with Rh concentration of 1000mg/L is as follows:
the method A comprises the following steps: 0.1000g of rhodium powder (mass fraction is more than or equal to 99.99%) is weighed, placed in a 50mL glass tube, added with 8mL of hydrochloric acid (density is 1.19g/mL, MOS grade) and 2mL of hydrogen peroxide (volume fraction is 30%, purity is not lower than super grade purity), and sealed. Dissolving at 150 deg.C for 48 hr, cooling, and opening tube. The tube sample was washed with hydrochloric acid (1+1)) into a 100mL volumetric flask and diluted to the mark with water. And (5) uniformly mixing. 1mL of this solution contained 1.0mg of Rh element. (commercially available 37% strength hydrochloric acid, density of about 1.19 g/mL).
The Rh element standard solution with Rh concentration of 1000mg/L can be further diluted with nitric acid (e.g. 1.0mol/L) or hydrochloric acid solution (e.g. 10% by mass), for example, diluted to a Rh element concentration of 1-900 mg/L, for example, diluted to Rh element concentrations of 100mg/L, 80mg/L, 60mg/L, 40mg/L, 30mg/L, 20mg/L, 10mg/L and 5mg/L, respectively.
The Fe element standard solution in the invention can be purchased from commercial sources or prepared by self. An exemplary preparation method of the Fe element standard solution with the Fe concentration of 1000mg/L is as follows:
the method A comprises the following steps: weighing 0.1000g of metallic iron powder (mass fraction is more than or equal to 99.99%), placing the metallic iron powder into a 100mL polytetrafluoroethylene beaker, adding 3mL of hydrochloric acid (density is 1.19g/mL, MOS grade), heating at low temperature to completely dissolve the metallic iron powder, transferring the metallic iron powder into a 100mL volumetric flask, adding 10mL of hydrochloric acid (density is 1.19g/mL, MOS grade), and diluting the solution to a scale with water. And (5) uniformly mixing. 1mL of this solution contained 1.0mg of Fe element.
The method B comprises the following steps: 0.1430g of ferric oxide (the purity is not lower than the super grade purity) is weighed and placed in a 100mL polytetrafluoroethylene beaker, 10mL of water and 10mL of hydrochloric acid (the density is 1.19g/mL, MOS grade) are added, the mixture is heated and dissolved completely at low temperature, the mixture is moved into a 100mL volumetric flask, and the mixture is diluted to the scale with water. And (5) uniformly mixing. 1mL of this solution contained 1.0mg of Fe element.
The Fe element standard solution can be further diluted with nitric acid (e.g. 1.0mol/L) or hydrochloric acid (e.g. 10% by mass), for example, to a standard dilution with Fe element concentration of 1-900 mg/L, for example, to a standard dilution with Fe element concentration of 500mg/L, 400mg/L, 300mg/L, 200mg/L, 100mg/L, 50mg/L, 30mg/L and 15mg/L, respectively.
Has the advantages that:
the method for measuring the iron content in the serum uses ICP-MS (inductively coupled plasma mass spectrometry) to measure the sample, adopts a direct dilution method after adding concentrated acid water bath in the serum sample treatment mode, and has the advantages of simple treatment process, accurate measurement result and high efficiency.
The invention establishes a novel method for measuring the content of iron in serum by using ICP-MS through optimization, and the method has the advantages of high sensitivity, high measurement speed and high efficiency.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.
Example 1
Materials and instruments:
inductively coupled plasma mass spectrometer: PerkinElmer NEXION 350X;
an atomizer: MicroMist, atomization chamber temperature: 3 ℃, operation mode: he Gas;
beagle dogs, purchased from beijing marts biotechnology limited.
Solution preparation:
fe element standard solution: the concentration of Fe is 1000 mg/L;
standard solution of Rh element: the concentration of Rh is 1000 mg/L;
standard diluent: measuring 5ml, 4ml, 3ml, 2ml and 1ml of Fe element standard solution, respectively placing the Fe element standard solution in a 10ml measuring flask, adding 1.0mol/L nitric acid solution to dilute to a scale, and shaking up to obtain Fe element solutions with the concentrations of 500mg/L, 400mg/L, 300mg/L, 200mg/L and 100 mg/L; then 5ml, 3ml and 1.5ml of Fe element solution with the concentration of 100mg/L are measured and respectively placed in a 10ml measuring flask, 1.0mol/L nitric acid solution is added for dilution to the scale, and the mixture is shaken up to obtain Fe element solutions with the concentrations of 50mg/L, 30mg/L and 15 mg/L;
internal standard solution: measuring 1ml of Rh element standard solution, placing the solution in a 50ml measuring flask, diluting the solution to a scale with 1.0mol/L nitric acid solution, shaking up to obtain Rh element solution with the concentration of 20mg/L, namely the internal standard solution.
The method comprises the following specific steps:
(1) sample preparation and processing
Preparation of standard curve sample: taking 100 mul of beagle blank serum, 200 mul of internal standard solution and 100 mul of standard diluent with different Fe element concentrations, respectively placing the beagle blank serum, the internal standard solution and the standard diluent into 15ml centrifuge tubes, adding 1ml of 65 wt% concentrated nitric acid, heating in 80 ℃ water bath for 1 hour, diluting the processed sample to 10ml with ultrapure water, and shaking up to obtain the beagle blank serum.
The blank group without the internal standard and the internal standard group only added with the internal standard are prepared according to the same method, two samples are prepared in each group, and the specific groups are shown in table 1.
TABLE 1 grouping of Standard curves
Treating a serum sample to be detected: taking 100 mul of beagle serum to be detected and 200 mul of internal standard solution, placing the beagle serum and the internal standard solution into a 15ml centrifuge tube, adding 1ml of 65 wt% concentrated nitric acid, heating in a water bath at 80 ℃ for 1 hour, diluting the processed sample to 10ml with ultrapure water, and shaking up to obtain the serum sample.
(2) Measurement of ICP-MS
And (3) standard curve equation preparation: placing a standard curve sample on an ICP-MS sample tray, sucking the sample to determine the response value of Fe element and Rh element, and drawing a standard curve by taking the concentration of the Fe element solution as an abscissa and the ratio of the response values of the Fe element and the Rh element as an ordinate to obtain a standard curve equation;
and (3) determination: placing a serum sample to be detected on an ICP-MS sample tray, sucking the sample to determine the response value of Fe element and Rh element, substituting the ratio of the response values of Fe element and Rh element into a standard curve equation, and calculating to obtain the content of Fe element.
Wherein the linear range of the standard curve is 15 mg/L-500 mg/L.
Example 2
(1) Method Selective investigation
The ICP-MS analysis was performed on LLOQ samples prepared from 6 blank sera from different sources and 1 haemolytic blank serum and the corresponding blank sera, and each sample was analyzed for 3 samples.
Blank serogroup: taking 100 mu l of beagle blank serum and HNO3(1.0mol/L)300 mu L of the mixture is placed in a 15ml centrifuge tube, 1ml of concentrated nitric acid (65-68 wt%) is added, the mixture is heated in a water bath at the temperature of 80 ℃ for 1 hour, and then diluted to 10ml by ultrapure water and shaken up to obtain the product. Blank sera from each source were prepared in 3 samples in the same manner.
Hemolytic blank serogroup: taking 98 mu l of beagle blank serum, 2ul of whole blood and HNO3(1.0mol/L) 300. mu.l was separated in 15mlAdding 1ml of concentrated nitric acid (65-68 wt%) into a core tube, heating in a water bath at 80 ℃ for 1 hour, diluting with ultrapure water to 10ml, and shaking up to obtain the product. 3 samples were prepared in the same manner.
LLOQ sample group prepared with blank serum: taking 100 mu L of blank serum, 200 mu L of Rh standard solution diluent (20mg/L) and 100 mu L of Fe diluent (15mg/L), placing the blank serum, 1ml of concentrated nitric acid (65-68 wt%) in a 15ml centrifuge tube, heating the mixture in a water bath at 80 ℃ for 1 hour, diluting the mixture to 10ml with ultrapure water, and shaking up to obtain the reagent. 3 samples were prepared in the same manner.
The results of the method selectivity study are shown in Table 2.
TABLE 2 results of the Selective examination
The ratios of the response values of the analyte in the blank serum prepared samples from six groups of different sources to the response value of the analyte in the LLOQ sample prepared from the blank serum are respectively 12%, 8.1%, 12%, 7.4%, 5.3% and 11%, and are not higher than 20.0%.
The ratio of the internal standard response value of the blank serum preparation samples from six groups of different sources to the internal standard response value of the LLOQ sample prepared from the blank serum is respectively 0.00624%, 0.0072%, 0.0063%, 0.0081%, 0.0042% and 0.0061%, and is not higher than 5%.
The ratio of the response value of the analyte in the sample prepared from the hemolytic serum containing 2% of whole blood to the response value of the analyte in the LLOQ sample prepared from the normal blank serum is 142% which is far higher than the required standard of 20%, because the iron content in the red blood cells is high and the hemolytic serum has great interference on the determination of the serum iron, so the hemolysis condition of the blood sample needs to be noticed in the blood sample collection, and for the hemolytic sample, the hemolysis condition needs to be recorded and processed.
The ratio of the response value of the internal standard in the hemolyzed serum prepared sample containing 2% of whole blood to the response value of the internal standard in the LLOQ sample prepared from normal blank serum was 0.0544%, which was not higher than 5%.
Experimental results show that the determination method has good selectivity, and can distinguish target analytes, internal standards and endogenous components of the matrix or other components.
(2) Method calibration standard curve inspection
The standard curve is designed into 8 points, and the serum concentrations of Fe are respectively as follows: 15. 30, 50, 100, 200, 300, 400, 500 mg/L. The method validation of three assay batches can be performed discontinuously, with two sample analyses performed for each concentration of each assay batch, two blank samples (without internal standard), two zero concentration samples (with internal standard) analyses performed simultaneously, and two standard curves at the beginning and end of the assay batch, respectively. Blank and zero concentration samples are used for judging whether endogenous interference exists or the samples are polluted, and do not participate in standard curve calculation. Taking the concentration of the object to be measured as an abscissa, taking the ratio of the response value of the object to be measured to the response value of the internal standard as an ordinate, and performing linear regression operation by using a weighted (weight is 1/SD multiplied by SD) least square method to obtain a linear regression equation which is a standard curve. Standard curve each non-zero sample is calculated from the standard curve and the deviation of the resulting concentration compared to the set concentration must fall within an acceptable range.
Samples were examined according to the standard curve prepared in table 1, two samples each. Three batches were prepared for examination at different days.
The method correction standard curve investigation experiment results are shown in tables 3-8.
TABLE 3 Standard Curve 1-1
The data are processed and calculated by mass spectrum software, and the curve equation is obtained, wherein Y is 0.0012X +0.0003, and r is 0.9998.
TABLE 4 Standard Curve 1-2
The data are processed and calculated by mass spectrum software, and the curve equation is obtained, wherein Y is 0.0012X +0.0012, and r is 0.9999.
TABLE 5 Standard Curve 2-1
The data are processed and calculated by mass spectrum software, and the curve equation is obtained, wherein Y is 0.001X +0.0015, and r is 0.9998.
TABLE 6 Standard Curve 2-2
The data are processed and calculated by mass spectrum software, and the curve equation is obtained, wherein Y is 0.0011X +0.0016, and r is 0.9999.
TABLE 7 Standard Curve 3-1
The data are processed and calculated by mass spectrum software, and the curve equation is obtained, wherein Y is 0.001X +0.0014, and r is 0.9999.
TABLE 8 Standard Curve 3-2
The data are processed and calculated by mass spectrum software, and the curve equation is obtained, wherein Y is 0.0011X +0.0019, and r is 0.9999.
In the experiment, three batches of standard curve samples are prepared, six standard curve equations are obtained, and the concentration of the substance to be measured is calculated back for each concentration point in the standard curve range.
All lower quantitation (LLOQ) samples have relative deviations (RE) within ± 20.0%; the relative deviation (RE) of the samples was within ± 15.0% for each concentration.
In the six standard curves, the ratio of the response value of the analyte of the STD0 sample to the response value of the analyte of the LLOQ sample is not higher than 20%, which meets the requirement of method selectivity, namely the response value of the analyte in the blank serum (STD0) is not higher than 20.0% of the response value of the analyte in the LLOQ prepared from the corresponding blank serum.
The squared values of the correlation coefficients (r2) for the six standard curves are each greater than 0.99.
(3) Method accuracy survey
The method verifies that each of the three analysis batches (completed in at least two days) was subjected to 6 sample analysis for each concentration of QC samples (Fe serum concentrations of 15, 45, 250, 375mg/L, respectively) at the lower, medium, and upper concentrations, and the intra-batch precision (CV%), inter-batch precision (CV%) and relative deviation (RE%) were calculated.
Taking 100 mu L of blank serum, 200 mu L of Rh standard solution diluent (20mg/L) and 100 mu L of Fe diluent, putting the blank serum, the Rh standard solution diluent and the Fe diluent into a 15ml centrifuge tube, adding 1ml of concentrated nitric acid (65-68 wt%), heating the mixture in a water bath at 80 ℃ for 1 hour, diluting the mixture to 10ml with ultrapure water, and shaking the mixture uniformly to obtain the reagent.
Samples with the lower limit of quantitation, four concentrations in the lower and the upper range were obtained and arranged in parallel in 6 portions.
Table 9 method accuracy survey group
Three total approved accuracy samples were prepared on three days, and the method was investigated for intra-and inter-batch accuracy.
The experimental results of the method accuracy investigation are shown in tables 10-12.
(4) Investigation of method precision
The sample preparation method examines samples with the same accuracy, three batches of samples are prepared in three days, and the intra-batch precision and the inter-batch precision of the method are examined.
The experimental results of the precision investigation of the method are shown in tables 10-12.
TABLE 10 accuracy precision investigation-1
TABLE 11 accuracy precision investigation-2
TABLE 12 accuracy precision investigation-3
The three approved precision sample investigation results all meet the following requirements:
internal precision: the method verifies that the precision of the measured value of each analysis batch of the quantitative lower limit sample is less than or equal to 20.0 percent; the precision of the measured value of other concentration level QC samples is less than or equal to 15.0 percent;
batch precision: the method verifies three analysis batches, and the precision of the measured value of the quantitative lower limit sample is less than or equal to 20.0 percent; the precision of the measured value of other concentration level QC samples is less than or equal to 15.0 percent;
in-batch accuracy: the method verifies that the relative deviation of the average value of the measured values of the quantitative lower limit samples and the theoretical value is within +/-20.0% in each analysis batch; the relative deviation of the measured value mean value of other QC samples with the same concentration level and the theoretical value is within +/-15.0%, and the relative deviation of at least 4 samples is within +/-15.0%;
batch-to-batch accuracy: the method verifies three analysis batches, and the relative deviation of the average value of the measured values of the quantitative lower limit samples and the theoretical value is within +/-20.0 percent; the measured mean values of other QC samples at the same concentration level are within + -15.0% of the theoretical values, and at least a total number of 2/3 samples in QC samples at the same concentration level are within + -15.0% of the relative deviation.
(5) Dilution reliability investigation
And (4) considering whether to perform a dilution test and a dilution multiple according to the actual concentration of the object to be tested and the linear range of the method. A diluted QC sample with a serum concentration of 1000mg/L containing Fe was prepared, diluted 5-fold with a beagle blank serum, and subjected to 6-sample analysis to calculate precision (CV%) and relative deviation (RE%).
The results of the dilution reliability test are shown in Table 13.
TABLE 13 dilution reliability examination
The experimental result shows that the precision of the measured value of 6 samples is less than or equal to 15.0 percent; the relative deviation of the mean values of the measurements of the 6 samples was within. + -. 15.0%.
(6) Matrix effect investigation
And (5) inspecting the matrix effect of low and high QC concentrations. Taking 6 parts of different sources of fasting blank serum, carrying out the same treatment except that no internal standard is added, adding a certain concentration of control solution into the obtained supernatant to ensure that the final concentrations of the substance to be detected and the internal standard are respectively the same as the theoretical concentrations of the low and high QC samples after treatment, and carrying out 3-sample analysis on each source serum sample at each concentration level. Meanwhile, water is used to replace the blank serum, and 3 samples are analyzed after the same operation. And respectively calculating respective matrix factors of the object to be measured and the internal standard under the two processing modes, and calculating the matrix factor after internal standard normalization according to the matrix factors. The matrix effect of the assay was evaluated by normalizing the degree of variation of the matrix factor.
Taking Rh standard solution diluent (20mg/L), sucking 4ml, placing in a 10ml measuring flask, and adding 1.0mol/L HNO3Diluting to scale to obtain Rh diluent with concentration of 8 mg/L.
Taking the diluted solution (100mg/L) of Fe standard solution, sucking 7.5ml, placing in a 10ml measuring flask, and adding 1.0mol/L HNO3Diluting to scale to obtain Fe diluent with concentration of 75 mg/L; taking Fe standard solution diluent (10mg/L), sucking 9ml, placing in a 10ml measuring flask, and adding 1.0mol/L HNO3Diluting to scale to obtain Fe diluent with concentration of 9 mg/L.
Taking 1ml of Rh standard solution diluent (8mg/L) and 1ml of Fe standard solution diluent (75mg/L), placing in a 10ml measuring flask, and adding 1.0mol/L HNO3Diluting to scale to obtain high concentration contrast solution; taking 1ml of Rh standard solution diluent (8mg/L) and 1ml of Fe standard solution diluent (9mg/L), placing in a 10ml measuring flask, and adding 1.0mol/L HNO3Diluting to scale to obtain low concentration control solution.
Taking 100 mu L of fasting serum blank of beagle dog and 1.0mol/L HNO3And (3) putting 300 mu l of the solution into a 15ml centrifuge tube, adding 1ml of concentrated nitric acid (65-68 wt%), and treating in a water bath at 80 ℃ for 1 hour. Then, ultrapure water was added thereto to a volume of 10ml, followed by shaking. And taking 1ml of the supernatant, and adding 1ml of the prepared control solution with different concentrations to obtain matrix extraction sample adding samples with different concentrations. Blank sera were derived from 6 different beagle dogs, with two higher and two lower samples prepared for each serum and three samples prepared in parallel for each concentration.
Taking 100 mu L of ultrapure water and 1.0mol/L of HNO3And (3) putting 300 mu l of the solution into a 15ml centrifuge tube, adding 1ml of concentrated nitric acid (65-68 wt%), and treating in a water bath at 80 ℃ for 1 hour. Then, ultrapure water was added thereto to a volume of 10ml, followed by shaking. Taking 1ml of the supernatant, and adding 1ml of the control solution with different concentrations prepared above to obtain standard solution samples with different concentrations. Three samples were prepared in parallel for each concentration.
The results of the matrix effect investigation experiments are shown in tables 14-15.
TABLE 14 investigation of matrix Effect-Low concentration samples
Table 15 matrix effect investigation-high concentration samples
The experimental results show that the precision (CV%) of the average value of the internal standard normalized matrix factor of 6 different sources is less than or equal to 15.0 percent under two concentrations.
Example 3
And (3) taking the serum to be detected of the beagle dog, respectively treating the serum by using a microwave digestion method and the method, then measuring the concentration of the iron element in the sample by using ICP-MS (inductively coupled plasma-mass spectrometry), and calculating the difference of the measurement results of the two treatment methods.
The experimental method is as follows:
the sample is obtained by taking blood at different time points after the intravenous administration of the beagle, namely the same batch of serum samples, is treated by different treatment methods, is measured under the same measuring method, and is compared and analyzed. The administration concentration of the ferric carboxymaltose injection is 20mg/kg, and the used ferric carboxymaltose injection is sourced from Jiangsu Osaikang pharmaceutical industry Co.
Taking 100 mu L of serum to be detected, 100 mu L of Rh standard solution diluent (20mg/L) and 200 mu L of ultrapure water, placing the mixture into a digestion tank, adding 700 mu L of concentrated nitric acid (65-68 wt%) into the digestion tank, and digesting. And (3) taking 1ml of the digested sample, placing the digested sample into a 15ml centrifuge tube, adding ultrapure water to 10ml, and shaking up to obtain the biological enzyme inhibitor.
Taking 100 μ L of the serum to be detected, 200 μ L of Rh standard solution diluent (20mg/L) and HNO3(1.0mol/L)100 mul, placing in a 15ml centrifuge tube, adding 1ml of concentrated nitric acid (65-68 wt%), heating in a water bath at 80 ℃ for 1 hour, diluting to 10ml with ultrapure water, and shaking up to obtain the product.
The above samples were taken to determine serum iron concentration, and the results are shown in Table 16.
TABLE 16 comparison of microwave digestion with direct dilution after Water bath
The experimental results show that the relative deviation of the experimental results obtained by the two treatment methods is lower than 10%. Where the 3.5h and 4h groups in Table 16 had determined that the results were below the lower limit of quantitation, the concentrations were normalized to 0. The method of the invention has more convenient and rapid treatment and high efficiency.
Example 4
The stability of the treatment method is considered, and the used ferric carboxymaltose injection is sourced from Jiangsu Osaikang pharmaceutical industry Co.
(1) Freeze thaw stability
Mixing 700 mul of beagle blank serum with 700 mul of diluted ferric carboxymaltose injection containing different iron concentrations to prepare low and high QC concentration serum samples, freezing the serum samples in a refrigerator at the temperature of-20 ℃ (the set temperature) for more than 24 hours, completely thawing the serum samples at room temperature, mixing the serum samples by vortex, and then transferring the serum samples to the original condition for freezing for not less than 12 hours. The freezing-unfreezing cycle is repeated for at least more than 4 times, 200 mu L of mixed solution and 200 mu L of Rh standard solution diluent (20mg/L) are taken after the last unfreezing, the mixed solution and the Rh standard solution diluent are placed in a 15ml centrifuge tube, 1ml of concentrated nitric acid (65-68 wt%) is added, and the mixture is treated in a water bath at 80 ℃ for 1 hour. Adding ultrapure water to 10ml, and shaking up to obtain the final product.
6 samples were prepared for each concentration in the same manner and the results are shown in Table 17.
TABLE 17 Freeze thaw stability Studies
The experimental results show that the mean precision (CV%) of the measured values at each concentration level is less than or equal to 15.0%, and the relative deviation of the mean values is within +/-15.0%.
(2) Stability at room temperature
Mixing 700 mu L of beagle blank serum with 700 mu L of diluted ferric carboxymaltose injection containing different iron concentrations to prepare low and high QC concentration serum samples, standing at room temperature for not less than 24 hours, taking 200 mu L of mixed solution and 200 mu L of Rh standard solution diluent (20mg/L), placing the mixed solution in a 15ml centrifuge tube, adding 1ml of concentrated nitric acid (65-68 wt%), and treating in 80 ℃ water bath for 1 hour. Adding ultrapure water to 10ml, and shaking up to obtain the final product.
6 samples were prepared for each concentration in the same manner and the results are shown in Table 18.
TABLE 18 Room temperature stability Studies
The experimental results show that the mean precision (CV%) of the measured values at each concentration level is less than or equal to 15.0%, and the relative deviation of the mean values is within +/-15.0%.
(3) Sample stability after preparation
The low and high QC samples that passed the analytical test were re-measured after 10 days at room temperature and quantified using a freshly prepared and analyzed standard curve.
For each 6 samples concentration, the results are shown in Table 19.
TABLE 19 post-preparation sample stability
The experimental result shows that the average precision (CV%) of the measured value of each concentration level is less than or equal to 15.0%, the relative deviation of the average value is within +/-15.0%, and the measurement result of the method of the invention is stable and has small fluctuation when the sample is placed at room temperature for 10 days.
In the determination method of the present invention, the serum can be collected from animals or humans, and the bath time can be appropriately shortened according to the rise of the bath temperature.
The method for measuring the iron content in the serum uses ICP-MS (inductively coupled plasma mass spectrometry) to measure the sample, adopts a direct dilution method after adding concentrated acid water bath in the serum sample treatment mode, and has the advantages of simple treatment process, accurate measurement result and high efficiency; meanwhile, the method has stable measurement result and small fluctuation within 48 hours of placing the sample at room temperature.
The invention establishes a novel method for measuring the content of iron in serum by using ICP-MS through optimization, and the method has the advantages of high sensitivity, high measurement speed and high efficiency.
Claims (10)
1. A method for measuring the content of iron in serum is characterized by comprising two steps of sample preparation and treatment and ICP-MS measurement.
2. The method of claim 1, wherein the sample is heated after the step of preparing and treating the sample by adding a concentrated acid.
3. The method according to claim 2, wherein the concentrated acid is 65 to 70 wt% concentrated nitric acid, aqua regia or perchloric acid, preferably 65 to 68 wt% concentrated nitric acid.
4. The method according to claim 2, wherein the heating treatment is water bath heating, the temperature is 70-85 ℃, and the heating time is 0.5-3 h.
5. The method according to any one of claims 1 to 4, comprising in particular the steps of:
(1) sample preparation and processing
Preparation of standard curve sample: taking blank serum, an internal standard solution and standard solutions with different Fe element concentrations, adding concentrated nitric acid, heating, diluting with water, and shaking up to obtain the final product;
treating a serum sample to be detected: taking the serum to be detected and the internal standard solution, adding concentrated nitric acid, heating, diluting with water, and shaking up to obtain a serum sample;
the internal standard solution contains Rh element, the concentration of the Rh element is 5-50 mg/mL, and the concentration of the Rh element is preferably 10-30 mg/mL;
(2) measurement of ICP-MS
And (3) standard curve equation preparation: measuring the corresponding value of Fe element and the response value of Rh element of the standard curve sample by ICP-MS, and drawing a standard curve by taking the concentration of Fe element solution as an abscissa and the ratio of the response values of Fe element and Rh element as an ordinate to obtain a standard curve equation;
and (3) determination: and measuring the corresponding value of the Fe element and the response value of the Rh element of the serum sample by using ICP-MS, substituting the ratio of the response values of the Fe element and the Rh element into a standard curve equation, and calculating the content of the Fe element.
6. The method of claim 5, wherein the mass spectrometer of the assay is Perkinelmer NEXION 350X.
7. The method of claim 6, wherein the mass spectrometer has a MicroMist nebulizer, a chamber temperature of 3 ℃, and a He Gas mode of operation.
8. The method of claim 5, wherein in the step (1), 65-68 wt% of concentrated nitric acid is added and is subjected to heating treatment in a water bath at 80 ℃.
9. The method of claim 5, wherein the preparation of the standard curve sample in step (1) is specifically: mixing blank serum 100 mul, internal standard solution 200 mul and standard diluent 100 mul with different Fe element concentrations in a centrifuge tube respectively, adding concentrated nitric acid 1-3 ml, heating in a water bath for 0.5-3 h, diluting with ultrapure water to 10ml, and shaking up to obtain the final product.
10. The method according to claim 5, wherein the sample treatment in step (1) is specifically: and (3) putting 100 mul of serum to be detected and 200 mul of internal standard solution into a centrifuge tube, adding 1-3 ml of concentrated nitric acid, heating in a water bath for 0.5-3 h, diluting to 10ml with ultrapure water, and shaking up to obtain a serum sample.
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