Reduction of influence of hemoglobin in measurement of zinc in biological sample

Abstract

Provided is a method for reducing the influence of hemoglobin in a biological sample, when colorimetry is executed for zinc in the biological sample using a chelate color former. This method enables accurate quantification of zinc in a sample even if the sample is hemolyzed.　When colorimetry is executed for zinc in a biological sample using a chelate color former, a reagent containing at least one of guanidine or a salt thereof and urea is added, together with a masking agent, to the biological sample containing hemoglobin.

Description

Reducing the influence of hemoglobin on zinc measurement in biological samples.

[0001] The present invention relates to the colorimetric measurement of zinc in biological samples, and more specifically, to a method for reducing the influence of hemoglobin when measuring zinc in biological samples, a method for accurately measuring zinc in biological samples colorimetrically while reducing the influence of hemoglobin, and a kit and reagents for carrying out these methods.

[0002] It is known that various bodily functions, such as development, growth, tissue repair, bone maintenance, reproduction, sensation, appetite, and immune function, are maintained in dependence on zinc. Therefore, zinc concentration in biological samples is considered important information in the diagnosis of diseases related to these functions.

[0003] Methods for measuring zinc concentration in biological samples include atomic absorption spectrometry, ICP emission spectrometry, and colorimetric analysis. Of these, colorimetric analysis is widely used clinically because it allows for simple and rapid measurement of zinc concentration using automated analyzers.

[0004] In colorimetric methods for measuring zinc concentration, chelating chromogens are generally used. The method utilizes the property that the color tone or absorption wavelength changes when zinc in a biological sample forms a complex with the chelating chromogen, thereby measuring the zinc concentration. However, since chelating chromogens also form complexes with metals other than zinc, when using chelating chromogens to colorimetrically measure zinc in a biological sample, it is necessary to remove other metals present in the biological sample, such as iron, copper, and nickel. For this reason, when using chelating chromogens to colorimetrically measure zinc in a biological sample, it has been proposed to include various masking agents in the reagent to prevent the reaction between the chelating chromogen and other metals present in the biological sample (References 1-3).

[0005] Furthermore, in colorimetric methods for measuring zinc in biological samples using chelating chromogens, since much of the zinc in biological samples is bound to proteins, it is desirable to dissociate the zinc from the proteins before reacting the chelating chromogen with the zinc in the biological sample. In this regard, Reference 1 discloses that by adding a nonionic surfactant to the biological sample, zinc can be released from the proteins without the need for a deproteinizer and then used for reaction with the chelating chromogen.

[0006] However, these conventional colorimetric methods for measuring zinc in biological samples do not specifically address the influence of hemoglobin present in the samples. Being able to accurately measure zinc in biological samples while reducing the influence of hemoglobin is clinically important, and a method that can resolve this issue is desirable.

[0007] In this regard, a method has been proposed to reduce the influence of hemoglobin by adding a predetermined nitrogen-containing compound to a biological sample containing hemoglobin through hemolysis, thereby preventing the degradation of hemoglobin and preventing the elution of iron. In particular, pyridine, imidazole, and their derivatives have been shown to be the most effective in preventing the elution of iron (Patent Document 4). This document also discloses that the absorbance of hemoglobin at a wavelength of 415 nm in a Tris buffer containing predetermined components can be maintained constant by adding a predetermined nitrogen-containing compound.

[0008] However, this document discloses that in a reaction system for measuring iron in a biological sample, or in a Tris buffer containing specific components, the degradation of hemoglobin can be prevented to prevent iron elution. It does not disclose that the influence of hemoglobin can be similarly reduced in a reaction system for measuring zinc in a biological sample. In fact, as demonstrated in the examples described later, pyridine and imidazole, which are considered most effective in reducing the influence of hemoglobin in this document, cannot be used in a zinc measurement system in a biological sample because they significantly reduce the measurement sensitivity.

[0009] Japanese Patent No. 5360707, Japanese Unexamined Patent Publication No. 2009-210434, Japanese Unexamined Patent Publication No. 60-120249, Japanese Unexamined Patent Publication No. 60-35270

[0010] In view of the above prior art, the present invention aims to provide a method for reducing the influence of hemoglobin in a biological sample when colorimetrically measuring zinc in a biological sample using a chelating colorimetric agent, a method that enables accurate quantification of zinc in a sample even in hemolyzed biological samples, and a kit and reagents that enable these methods to be carried out.

[0011] In the colorimetric measurement of zinc in biological samples using a chelating chromogenic agent, we discovered that by adding a reagent containing at least one of guanidine or its salt and urea, along with a masking agent, to a hemoglobin-containing biological sample, and then adding a reagent containing a chelating chromogenic agent, we could suppress the influence of hemoglobin without affecting the reaction between zinc in the biological sample and the chelating chromogenic agent, thereby enabling accurate quantification of zinc in the biological sample. This led to the present invention.

[0012] In other words, the present invention provides the following kits, methods, and reagents in its embodiments: [1] A kit for measuring zinc in a biological sample, comprising a first reagent containing a masking agent and a second reagent containing a chelating colorant, wherein the first reagent further contains at least one hemoglobin-inhibiting agent selected from guanidine or a salt thereof and urea. [2] The kit according to [1], wherein the first reagent further contains a nonionic surfactant. [3] The kit according to [1] or [2], wherein the concentration of guanidine or a salt thereof in the first reagent is 400 to 4000 mM. [4] The kit according to [1] or [2], wherein the concentration of urea in the first reagent is 3 to 5 M. [5] The kit according to any one of [1] to [4], wherein the chelating colorant is 2-(5-bromo-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof, or 2-(5-nitro-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof. [6] The kit according to any one of [1] to [5], wherein the masking agent is at least one selected from bis-trispropane, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), tricine, trishydroxymethylaminomethane, deferoxamine or a salt thereof, and bicine. [7] The kit according to any one of [1] to [6], wherein the biological sample is plasma, serum, or urine. [8] A method for measuring zinc in a biological sample, comprising the steps of: adding a first reagent containing at least one hemoglobin-inhibiting agent selected from guanidine or a salt thereof and urea, together with a masking agent, to the biological sample; and adding a second reagent containing a chelating colorant to a mixture of the biological sample and the first reagent. [9] The method according to [8], wherein the first reagent further contains a nonionic surfactant.

[10] The method according to [8] or [9], wherein the first reagent contains guanidine or a salt thereof, and the concentration of guanidine or a salt thereof in the mixture after the addition of the first reagent is 400 to 4000 mM.

[11] The method according to [8] or [9], wherein the first reagent contains urea, and the concentration of urea in the mixture after the addition of the first reagent is 3 to 5 M.

[12] The method according to any one of [8] to

[11] , wherein the second reagent is added when the change in absorbance of the mixture after the addition of the first reagent has substantially finished.

[13] The method according to any one of [8] to

[12] , wherein the chelating colorant is 2-(5-bromo-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof, or 2-(5-nitro-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof.

[14] The method according to any one of [8] to

[13] , wherein the masking agent is at least one selected from bis-trispropane, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), tricine, trishydroxymethylaminomethane, deferoxamine or a salt thereof, and bicine.

[15] The method according to any one of [8] to

[14] , further comprising the step of determining the zinc concentration in the biological sample by measuring the absorbance of the reaction solution before and after the addition of the chelating colorant at a predetermined wavelength.

[16] A method for reducing the influence of hemoglobin when measuring zinc in a biological sample using a masking agent and a chelating chromogenic agent, comprising the step of adding to the biological sample, together with the masking agent, a reagent containing at least one hemoglobin influence inhibitor selected from guanidine or a salt thereof and urea, before reacting the chelating chromogenic agent with the zinc in the biological sample.

[17] The method according to

[16] , wherein the reagent further contains a nonionic surfactant.

[18] The method according to

[16] or

[17] , wherein the reagent contains the guanidine or a salt thereof, and the concentration of the guanidine or a salt thereof in the mixture after the addition of the first reagent is 400 to 4000 mM.

[19] The method according to any one of

[16] to

[18] , wherein the chelating colorant is 2-(5-bromo-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof, or 2-(5-nitro-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof.

[20] The method according to any one of

[16] to

[19] , wherein the masking agent is at least one selected from bis-trispropane, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), tricine, trishydroxymethylaminomethane, deferoxamine or a salt thereof, and bicine.

[21] A reagent for reducing the influence of hemoglobin when measuring zinc in a biological sample using a masking agent and a chelating colorant, comprising, together with the masking agent, at least one selected from guanidine or a salt thereof and urea.

[22] The reagent according to

[21] , further comprising a nonionic surfactant.

[23] The reagent according to

[21] or

[22] , wherein the concentration of guanidine or a salt thereof in the reagent is 400 to 4000 mM.

[24] The reagent according to

[21] or

[22] , wherein the concentration of urea in the reagent is 3 to 5 M.

[25] The reagent according to any one of

[21] to

[24] , wherein the chelating colorant is 2-(5-bromo-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof, or 2-(5-nitro-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof.

[26] The reagent according to any one of

[21] to

[25] , wherein the masking agent is at least one selected from bis-trispropane, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), tricine, trishydroxymethylaminomethane, deferoxamine or a salt thereof, and bicine.

[0013] In measuring zinc in biological samples using a chelating chromogenic agent and a masking agent, treating the biological sample with a reagent containing guanidine or its salt or urea along with the masking agent, and then reacting the chelating chromogenic agent with the zinc in the biological sample, allows for accurate measurement of zinc concentration while suppressing the influence of hemoglobin.

[0014] Embodiments of the present invention will be described in detail below. However, the present invention should not be understood as being limited to the following embodiments. One embodiment of the present invention relates to a kit for measuring zinc in a biological sample, comprising a first reagent containing a masking agent and a second reagent containing a chelating chromogenic agent, wherein the first reagent further contains a hemoglobin effect inhibitor. Another embodiment of the present invention relates to a method for measuring zinc in a biological sample, comprising the steps of adding a hemoglobin effect inhibitor to a biological sample to reduce the effect of hemoglobin, and reacting the chelating chromogenic agent with the zinc in the biological sample after the reduction of the effect of hemoglobin. Yet another embodiment of the present invention relates to a reagent for reducing the effect of hemoglobin when measuring zinc in a biological sample with a chelating chromogenic agent. Each embodiment will be described in detail below.

[0015] 1. Kit for measuring zinc in biological samples The kit according to this embodiment is used for measuring zinc in biological samples and includes a first reagent containing a masking agent and a hemoglobin effect inhibitor, and a second reagent containing a chelating colorimetric agent.

[0016] In this embodiment, there are no particular restrictions on the biological sample used to measure zinc using the kit, and it can be used with a variety of biological samples. Examples of biological samples include blood, serum, plasma, urine, feces, semen, cerebrospinal fluid, saliva, sweat, tears, ascites, amniotic fluid, organs such as the brain, hair, skin, nails, tissues such as muscles and nerves, and cells from humans or other animals. Typical biological samples are plasma, serum, or urine. If the sample is not a liquid, it can be pre-treated by extraction or solubilization according to known methods and then used as a liquid sample for measurement.

[0017] In this embodiment, the hemoglobin-inhibiting agent contained in the first reagent is selected from guanidine or its salts and urea. These can be included in the first reagent individually or in combination. These compounds complete the absorbance change caused by hemoglobin before the chelating chromogenic agent reacts with zinc in the biological sample, without adversely affecting the zinc measurement system using the chelating chromogenic agent and masking agent. This reduces the influence of the hemoglobin absorbance change on the absorbance after the addition of the second reagent, and as a result, zinc can be accurately quantified even in biological samples where hemoglobin is present. Examples of guanidine salts include guanidine sulfate, guanidine phosphate, guanidine carbonate, guanidine thiocyanate, guanidine nitrate, guanidine sulfamate, guanidium iodide, guanidine hydrobromide, and guanidine hydrochloride, with guanidine hydrochloride being preferred.

[0018] The concentration of the hemoglobin inhibitor in the first reagent may vary depending on the type of hemoglobin inhibitor and the amount of the first reagent added to the biological sample. If guanidine or a salt thereof is contained, the concentration of guanidine or a salt thereof in the first reagent is preferably 350 mM to 4300 mM, more preferably 400 mM to 4000 mM, even more preferably 450 to 3000 mM, even more preferably 500 to 2000 mM, even more preferably 550 to 1500 mM, and particularly preferably 600 to 1000 mM. If urea is contained, the concentration of urea in the first reagent is preferably 2 to 5.5 M, more preferably 3 to 5 M, even more preferably 3.5 to 4.5 M, and particularly preferably 3.8 to 4.0 M. If the first reagent contains guanidine or a salt thereof and urea, the 1 M of guanidine or a salt thereof is converted to 2 M, preferably 6 M, more preferably 7 M, so that the overall concentration is preferably 2 to 5.5 M, more preferably 3 to 5 M, even more preferably 3.5 to 4.5 M, and particularly preferably 3.8 to 4.0 M.

[0019] In the zinc measurement kit according to this embodiment, the first reagent contains a masking agent to prevent the reaction of metals other than zinc in the biological sample with the chelating colorant. In this specification, "masking agent" means a compound that can prevent any metal other than zinc in the biological sample from reacting with the chelating colorant by binding to it. Examples of metal ions other than zinc in the biological sample include iron, copper, and nickel. Examples of masking agents include bis-trispropane, bis-tris, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), 2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid (TAPSO), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), tricine, trishydroxymethylaminomethane, deferoxamine or its salt, and N,N-bis(2-hydroxyethyl)-2-aminoethanesulfone Examples include acids (BES) and bicine, with bis-trispropane, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), 2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid (TAPSO), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), tricine, trishydroxymethylaminomethane, deferoxamine or its salts, or bicine being preferred, and bis-trispropane being particularly preferred.

[0020] The concentration of the masking agent in the first reagent can be adjusted according to the assumed concentrations of metals other than zinc, such as iron, copper, and nickel, in the biological sample, the ratio of the addition amount of the first reagent to the dispensed amount of the biological sample, and the type of the masking agent. Usually, in the first reagent, it may be in the range of 0.1 to 150 mM, preferably in the range of 0.2 to 100.0 mM, more preferably in the range of 0.3 to 50.0 mM, still more preferably in the range of 0.5 to 10.0 mM, and particularly preferably in the range of 1.0 to 3.0 mM. Also, even if the masking agent is contained in an amount exceeding 150 mM, there is no problem, but a sufficient effect can be obtained up to that amount. In particular, when deferoxamine or its salt is used as the masking agent, in the first reagent, the range of 0.05 to 10.0 mM is preferable, the range of 0.1 to 5.0 mM is more preferable, the range of 0.2 to 3.0 mM is still more preferable, and the range of 0.25 to 1.5 mM is particularly preferable. Also, when bis-tris propane or its salt is used as the masking agent, in the first reagent, the range of 5.0 to 65.0 mM is preferable, the range of 10.0 to 60.0 mM is more preferable, the range of 15.0 to 55.0 mM is still more preferable, and the range of 20.00 to 50.0 mM is particularly preferable.

[0021] In the zinc measurement kit according to the present embodiment, the first reagent and / or the second reagent may contain a surfactant. For example, it can include a nonionic surfactant, an anionic surfactant, a cationic surfactant, or an amphoteric surfactant. These surfactants can be selected from known surfactants used in test drugs.

[0022] In the zinc measurement kit according to a preferred aspect of the present embodiment, the first reagent and / or the second reagent, preferably at least the first reagent, contains a nonionic surfactant. The nonionic surfactant not only brings general characteristics as a surfactant, such as adjusting the surface tension of the reagent and improving the dispensing accuracy, but also prevents proteins present in the biological sample from binding to zinc ions, and can avoid the influence of proteins present in the biological sample without pretreating the biological sample with a protein removing agent or including a protein removing agent in the first reagent.

[0023] As nonionic surfactants, polyoxyalkylene ether compounds, polyhydric alcohol ester compounds, polyoxyethylenated polyhydric alcohol fatty acid esters, and amide or amine compounds can be mentioned. From the viewpoint of the effect of preventing proteins present in biological samples from binding to zinc ions, polyoxyalkylene ether compounds are preferred.

[0024] As polyoxyalkylene ether compounds, polyoxyethylene alkyl ethers (preferably, H-(O-CH ２ -CH ２ ) ３－１０ -O-C ３－２０ alkyl ethers), polyoxypropylene alkyl ethers (preferably, H-(O-CH ２ -CH ２ -CH ２ ) ３ -), １０ -O-C ３－２０ alkyl ethers), polyoxyethylene alkyl phenyl ethers (preferably, H-(O-CH ２ -CH ２ ) ３－１０ -O-C ３－２０ alkyl phenyl ethers), polyoxypropylene alkyl phenyl ethers (preferably, H-(O-CH ２ -CH ２ -CH ２ ) ３－１０ -O-C ３－２０ alkyl phenyl ethers), polyoxyethylene polystyryl phenyl ethers (preferably, H-(O-CH ２ -CH ２ ) ３－１０ -O-tri- or distyryl phenyl), polyoxyethylene polyoxypropylene glycols (preferably, H-(O-CH ２ -CH ２ ) ３－１０ -(O-CH ２ -CH ２ -CH ２ ) ３ -(O-CH ２ -CH ２ ) ３－１０-OH), polyoxyethylene sorbitan fatty acid esters (preferably, H-(O-CH ２ -CH ２ )) ３－１０ -sorbitan C １０－２５ fatty acid esters), and propylene oxide-ethylene oxide block copolymers are mentioned. Polyoxyethylene alkyl ethers and polyoxyethylene alkyl phenyl ethers are preferred, polyoxyethylene octyl phenyl ether and polyoxyethylene lauryl ether are more preferred, and H-(O-CH ２ -CH ２ )) ３－１０ -octyl phenyl ether) and H-(O-CH ２ -CH ２ )) ３－１０ -lauryl ether are particularly preferred. Commercially available products include Triton X100 (manufactured by Dow Chemical), Triton X405 (manufactured by Dow Chemical), Triton X114 (manufactured by Dow Chemical), Emulgen A-500 (Kao Corporation), Bridg 35 (Kishida Chemical), Brij35 (Sigma-Aldrich), Tween 20 (Biomedical Science). In a preferred embodiment, the first reagent contains polyoxyethylene lauryl ether, preferably H-(O-CH ２ -CH ２ )) ３－１０ -lauryl ether, and the second reagent contains polyoxyethylene octyl phenyl ether, preferably H-(O-CH ２ -CH ２ )) ３－１０ -octyl phenyl ether).

[0025] Polyhydric alcohol ester compounds include glycerin fatty acid esters, sorbitan fatty acid esters, pentaerythritol fatty acid esters, propylene glycol mono-fatty acid esters, and sucrose fatty acid esters.

[0026] Examples of polyoxyethylene-modified polyhydric alcohol fatty acid esters include polyoxyethylene sorbitan fatty acid partial esters, polyoxyethylene sorbitol fatty acid partial esters, polyoxyethylene glycerin fatty acid partial esters, polyethylene glycol fatty acid esters, polyglycerin fatty acid partial esters, and polyoxyethylene-modified castor oil.

[0027] Examples of amide or amine compounds include fatty acid diethanolamide, N,N-bis-2-hydroxyalkylamine, polyoxyethylene alkylamine, triethanolamine fatty acid ester, and trialkylamine oxide.

[0028] The concentration of the surfactant, preferably a nonionic surfactant, in the first reagent may vary depending on the mixing ratio of the first reagent, the biological sample, and the second reagent, but is usually in the range of 0.01 to 10%, more preferably in the range of 0.1 to 5%, and particularly preferably in the range of 0.5 to 3%. Similarly, the concentration of the surfactant, preferably a nonionic surfactant, in the second reagent may also vary depending on the mixing ratio of the second reagent, the biological sample, and the first reagent, but is usually in the range of 0.005 to 5%, more preferably in the range of 0.05 to 3%, and preferably in the range of 0.1 to 2%.

[0029] In the zinc measurement kit according to this embodiment, the second reagent contains a chelating chromogenic agent. In this specification, "chelating chromogenic agent" means a compound that combines with zinc in the sample to form a complex, thereby changing its color or absorption wavelength. Such chelating chromogenic agents may also include compounds that react with other metals present in the sample, such as iron, copper, and nickel. In this case, the masking agent described above is used to remove the other metals.

[0030] Examples of chelating colorants include 2-(5-bromo-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol (5-Br-PAPS), 2-(5-nitro-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol (nitro-PAPS), 2-(5-bromo-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)aniline (5-Br-PSAA), bismuthiol II, BPA, calcein, calcein blue, and chromotropic acid. acid), Cu-1-(2-pyridylazo)-2-naphthol (Cu-PAN), diantipyrimethane, murexide, 2-nitroso-5-[N-n-propyl-N-(3-sulfopropyl)amino]phenol (nitroso-PSAP), o-phenanthroline, 1-(2-pyridylazo)-2-naphthol (PAN), 4-(2-pyridylazo)resorcinol (PAR), 3-(2-pyridyl)-5,6-bis(4-sulfophenyl)-1,2,4-triazine, disodium salt (PDTS), pyrogallol sulfonphthaley Examples include nitrate (PR), salicylideneamino-2-thiophenol (SATP), 1,2-dihydroxy-3,5-benzenesulfonic acid disodium monohydrate (Tiron), 5,10,15,20-tetraphenyl-21H,23H-porfinetetrasulfonic acid disulfate tetrahydrate (TPPS), 2,4,6-tris(2-pyridyl)-1,3,5-triazine (TPTZ), 3,3'-bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonphthalein disodium salt (XO), or zincon. Among these, 5-Br-PAPS or nitro-PAPS are preferred.

[0031] The concentration of the chelating chromogen in the second reagent can be adjusted according to the reaction protocol and the expected zinc concentration in the biological sample to be measured. When using an automated analyzer, the concentration of the chelating chromogen in the second reagent is usually designed to be in the range of 10 to 500 μM in the reaction solution, and particularly preferably in the range of 20 to 100 μM.

[0032] In the zinc measurement kit according to this embodiment, the pH of the first reagent and the second reagent are set so that the pH of the reaction solution is within a range in which the chelating chromogenic agent and the zinc in the biological sample can react to form a complex. The pH of the first reagent and the second reagent are adjusted so that the pH of the reaction solution is within a range suitable for complex formation, depending on the reaction protocol, specifically the ratio of the amounts of the first reagent and the second reagent added, and the type of chelating chromogenic agent. The pH of the first reagent is usually in the range of 6 to 11, preferably in the range of 7 to 10, and more preferably in the range of 8 to 10. Similarly, the pH of the second reagent is usually in the range of 6 to 11, preferably in the range of 7 to 10, and more preferably in the range of 8 to 10.

[0033] To maintain such a pH range, it is preferable that the first and second reagents contain a buffering agent that has buffering capacity within the selected pH range. Examples of buffering agents include carbonic acid, boric acid, phosphoric acid, trishydroxymethylaminomethane, imidazole, glycylglycine, 2-morpholinoethanesulfonic acid (MES), bis-tris, N-(2-acetamide)iminodiacetic acid (ADA), N-(2-acetamide)-2-aminoethanesulfonic acid (ACES), bis-trispropane, piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), and 2-hydroxy-3-morpholinopropane. N-sulfonic acid (MOPSO), 3-morpholinopropanesulfonic acid (MOPS), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIP SO), 2-hydroxy-N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPSO), piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) dihydrate (POPSO), 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid, monohydrate (HEPPS), 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid, monohydrate Examples of buffering agents include (HEPPSO), tricine, bicine, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), or salts thereof, and these should be selected according to the desired pH range.

[0034] In the zinc measurement kit according to this embodiment, the first reagent and / or the second reagent may contain other components such as preservatives and stabilizers as needed. These other components can be appropriately selected from known components used in biochemical diagnostic reagents.

[0035] The above description pertains to a two-reagent kit containing a first reagent and a second reagent, but the present invention is also applicable to one-reagent and three-reagent systems. For example, the first reagent described above can be divided into a reagent containing a masking agent and a reagent not containing a masking agent to create a three-reagent system. Alternatively, the first and second reagents described above can be combined into a single reagent to create a one-reagent system. In the case of a one-reagent system, the concentrations of each component described above should correspond to the concentrations obtained when the first and second reagents of the aforementioned one-reagent system are mixed, and the mixing ratio of the first and second reagents will be described later.

[0036] In the case of a single-reagent system, the pH of the reagent should be within the same pH range as the reaction solution suitable for complex formation, depending on the type of chelating chromogen. For example, if the chelating chromogen is 2-(5-bromo-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof, or 2-(5-nitro-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof, the pH of the reagent is usually in the range of 5 to 11, preferably in the range of 7 to 11, and more preferably in the range of 8 to 10.

[0037] 2. Method for reducing the influence of hemoglobin in the measurement of zinc using a chelating chromogenic agent, and method for measuring zinc in a biological sample. This embodiment relates to a method for reducing the influence of hemoglobin in the measurement of zinc using a chelating chromogenic agent, which includes adding a hemoglobin influence inhibitor to a biological sample, and a method for measuring zinc in a biological sample, which includes the step of reacting the chelating chromogenic agent with the zinc in the biological sample after the reduction of the hemoglobin influence.

[0038] In a method to reduce the influence of hemoglobin, a reagent containing a hemoglobin inhibitor (the first reagent in a two-reagent system) is added to the biological sample along with a masking agent before reacting the chelate chromogen with zinc in the biological sample, or the reagent containing the hemoglobin inhibitor is added together with the reagent containing the masking agent, or before or after adding the reagent containing the masking agent, and then mixed. When a reagent containing a masking agent is added to a hemoglobin-containing sample, the hemoglobin is broken down and the absorbance changes over time. However, by adding a hemoglobin inhibitor, this change is accelerated, and when the absorbance is measured by reacting the chelate chromogen with zinc in the sample, the absorbance can be stabilized and the influence of hemoglobin can be avoided.

[0039] The types of biological samples, hemoglobin inhibitors, and masking agents, as well as the concentrations of hemoglobin inhibitors and masking agents in the reagents, are as described in the embodiments relating to the kit.

[0040] There are no particular restrictions on the amount of biological sample dispensed, and it can be various amounts depending on the reaction conditions. For example, when using an automated analyzer, the amount is usually small, but considering the measurement sensitivity, it is usually set in the range of 0.5 to 20.0 μL, preferably in the range of 1.0 to 15.0 μL. Furthermore, the amount of reagent containing a hemoglobin effect inhibitor together with the masking agent (the first reagent in the case of a two-reagent system), or the total amount of reagents containing the masking agent and the hemoglobin effect inhibitor, should preferably be an amount that allows the absorbance change caused by the reaction of hemoglobin in the biological sample with the masking agent to proceed rapidly. From this viewpoint, when using an automated analyzer, these amounts are usually 50 to 300 μL, preferably 80 to 200 μL.

[0041] In a mixture of a biological sample and a reagent containing a hemoglobin inhibitor together with a masking agent (the first reagent in the case of a two-reagent system), or in a mixture of a biological sample and a reagent containing a masking agent and a reagent containing a hemoglobin inhibitor, the concentration of the hemoglobin inhibitor is preferably 350 mM to 4300 mM, more preferably 400 mM to 4000 mM, even more preferably 450 to 3000 mM, even more preferably 500 to 2000 mM, even more preferably 550 to 1500 mM, and particularly preferably 600 to 1000 mM in the case of guanidine or its salt. In the case of urea, the concentration in the mixture is preferably 2 to 5.5 M, more preferably 3 to 5 M, even more preferably 3.5 to 4.5 M, and particularly preferably 3.8 to 4.0 M.

[0042] Furthermore, the reagent containing the hemoglobin-inhibiting agent and / or the masking agent (the first reagent in the case of a two-reagent system) preferably further contains a nonionic surfactant, preferably a polyoxyalkylene ether compound. In such cases, the concentration of the nonionic surfactant in the mixture of the biological sample and the reagent containing the hemoglobin-inhibiting agent and / or the masking agent (the first reagent in the case of a two-reagent system) is preferably in the range of 0.01 to 10% by mass, more preferably in the range of 0.1 to 5% by mass, and particularly preferably in the range of 0.5 to 3% by mass, from the viewpoint of exhibiting the effect of dissociating zinc from proteins.

[0043] To measure zinc in a biological sample, a mixture containing the biological sample, a hemoglobin inhibitor, a masking agent, and optionally a nonionic surfactant is mixed with a reagent containing a chelating chromogenic agent (the second reagent in the case of a two-reagent system). The mixture is then mixed, and the zinc in the sample reacts with the chelating chromogenic agent to form a complex. Once the complex is formed, the absorption wavelength changes. By measuring the absorbance of the reaction solution before and after color development at a predetermined wavelength, the concentration of zinc in the sample can be determined from the difference in absorbance. Typically, a calibration curve showing the relationship between concentration and absorbance is prepared in advance using a standard solution with a known zinc concentration.

[0044] The amount of reagent containing the chelating chromogen (the second reagent in the case of a two-reagent system) added is determined according to the amount of the other reagent (the first reagent in the case of a two-reagent system) added and the assumed zinc concentration in the biological sample. Typically, the ratio of the other reagent (the first reagent in the case of a two-reagent system): the reagent containing the chelating chromogen (the second reagent in the case of a two-reagent system) is 10:1 to 1:3, preferably 8:1 to 1:1, and particularly preferably 5:1 to 2:1. When using an automated analyzer, the amount of reagent containing the chelating chromogen (the second reagent in the case of a two-reagent system) added is usually 5 to 100 μL, preferably 20 to 60 μL.

[0045] The concentration of the chelating colorant in the reaction solution is usually 10 to 500 μM, preferably 10 to 50 μM. Furthermore, if either reagent (in the case of a two-reagent system, the first reagent and / or the second reagent) further contains a nonionic surfactant, the concentration of the nonionic surfactant in the reaction solution is preferably in the range of 0.02 to 12% by mass, more preferably in the range of 0.15 to 6% by mass, and particularly preferably in the range of 0.7 to 3.5% by mass, from the viewpoint of exhibiting the effect of dissociating zinc from protein.

[0046] The appropriate pH of the reaction solution varies depending on the chelating chromogen used, and it is preferable to set the pH to an appropriate level depending on the chelating chromogen used. For example, when the chelating chromogen is 2-(5-bromo-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof, or 2-(5-nitro-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof, the pH of the reagent is usually in the range of 5 to 11, preferably in the range of 7 to 11, and more preferably in the range of 8 to 10.

[0047] In a two-reagent or three-reagent system, it is preferable to add the reagent containing the chelating chromogenic agent (the second reagent in the case of a two-reagent system) after adding the reagent containing the hemoglobin effect inhibitor (the first reagent in the case of a two-reagent system) when the change in absorbance of the mixture has substantially finished. Adding the reagent containing the hemoglobin effect inhibitor to a biological sample allows the change in absorbance that occurs when hemoglobin is present in the biological sample to be completed quickly, and by adding the second reagent at this point, the influence of hemoglobin can be reduced. The change in absorbance over time after the addition of the first reagent due to the presence of hemoglobin may differ depending on the type of masking agent and the concentrations of hemoglobin and the hemoglobin effect inhibitor in the reaction solution. Therefore, it is preferable to determine the timing of adding the second reagent according to the type and concentration of masking agent used, assuming a highly hemolyzed sample.

[0048] In one reagent system, after adding a reagent containing a hemoglobin-inhibiting agent along with a chelating chromogen and a masking agent to a biological sample, the absorbance at a predetermined wavelength increases sharply as a complex is formed. However, in the case of a biological sample containing hemoglobin, the absorbance then gradually decreases as hemoglobin decomposes. Therefore, to avoid this change in absorbance due to hemoglobin decomposition, it is preferable to measure the absorbance after the change in absorbance has finished to determine the zinc concentration.

[0049] Measurement is preferably performed using the endpoint method, and particularly preferably using the two-point endpoint method. The measurement wavelength should generally be selected near the maximum absorption wavelength, depending on the chelating chromogen used. To avoid the influence of impurities such as chyle, measurements may be performed using two wavelengths (a primary wavelength near the maximum absorption wavelength and a secondary wavelength shifted to a longer wavelength within a certain range from the primary wavelength). The choice of wavelength for a known chelating chromogen can be determined by referring to prior art that measures zinc using that chelating chromogen. Alternatively, the wavelength can be determined by analyzing the absorption spectra of the chelating chromogen and its complex with zinc, and selecting a wavelength near the maximum absorption wavelength of the complex.

[0050] 3. Reagents for reducing the influence of hemoglobin when measuring zinc in a biological sample with a chelate colorimetric agent The first reagent constituting the kit described above, or a reagent containing a hemoglobin influence inhibitor without a masking agent, can be commercially available separately from the kit described above. Accordingly, another embodiment of the present invention relates to a reagent for reducing the influence of hemoglobin when measuring zinc in a biological sample with a chelate colorimetric agent, which contains a hemoglobin influence inhibitor. The hemoglobin influence inhibitor and its concentration are as described in the embodiment relating to the kit. This reagent may be the hemoglobin influence inhibitor itself, or it may be an aqueous solution in which the hemoglobin influence inhibitor is dissolved in a buffer solution of a predetermined pH. Furthermore, this aqueous solution may also contain other components such as preservatives and stabilizers. The buffer, the pH of the aqueous solution, and other components such as preservatives and stabilizers are as described in the first reagent of the embodiment relating to the kit.

[0051] This reagent preferably contains a masking agent, and more preferably further contains a nonionic surfactant. The concentrations of the masking agent, the nonionic surfactant, and these components are as described in the first reagent of the embodiment relating to the kit.

[0052] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0053] Test I 1. Reagent Preparation A first reagent containing deferoxamine mesylate as a masking agent, Bridge 35 as a nonionic surfactant, and various compounds as hemoglobin effect inhibitors was prepared, and a second reagent containing 5Br-PAPS as a chelating colorant was prepared. More specifically, the following:

[0054] [Example 1] (1) Preparation of the first reagent In an aqueous sodium carbonate solution, deferoxamine mesylate as a masking agent, guanidine hydrochloride as a hemoglobin effect inhibitor, and Brij35 (Sigma-Aldrich) as a nonionic surfactant were dissolved at 25°C at the following concentrations to prepare the first reagent with a pH of 9.4. First reagent: pH 9.4 (25°C) Deferoxamine mesylate 1.5 mM Brij35 1.0% Sodium carbonate 200 mM Guanidine hydrochloride 400 mM

[0055] (2) Preparation of the second reagent A second reagent with a pH of 9.4 was prepared by dissolving 5Br-PAPS as a chelating colorant and Triton X100 as a nonionic surfactant in an aqueous sodium carbonate solution at 25°C to the following concentrations. Second reagent: pH 9.4 (25°C) 5Br-PAPS 0.2 mM Sodium carbonate 200 mM Triton X100 0.5%

[0056] [Control Reagent] A first reagent with the same composition as the first reagent in Example, except that it did not contain guanidine hydrochloride and did not contain a hemoglobin inhibitor, and a second reagent identical to the second reagent in Example 1 were prepared. First reagent: pH 9.4 (25°C) Deferoxamine mesylate 1.5 mM Brij35 1.0% Sodium carbonate 200 mM Second reagent: pH 9.4 (25°C) 5Br-PAPS 0.2 mM Sodium carbonate 200 mM Triton X100 0.5%

[0057] [Examples 2-6] Reagent 1 was prepared with the same composition as Reagent 1 of Example 1, except that guanidine hydrochloride was dissolved in an aqueous sodium carbonate solution at concentrations of 500, 550, 600, 750, and 3000 mM, respectively, as a hemoglobin inhibitor. Reagent 2 was prepared with the same composition as Reagent 2 of Example 1. Reagent 1: pH 9.4 (25°C) Deferoxamine mesylate 1.5 mM Brij35 1.0% Sodium carbonate 200 mM Guanidine hydrochloride 500, 550, 600, 750 or 3000 mM Reagent 2: pH 9.4 (25°C) 5Br-PAPS 0.2 mM Sodium carbonate 200 mM Triton X100 0.5%

[0058] [Examples 7-9] Reagent 1 was prepared with the same composition as Reagent 1 of Example 1, except that in Reagent 1, urea was dissolved in an aqueous sodium carbonate solution at concentrations of 3, 4, and 5 M, respectively, instead of guanidine hydrochloride as a hemoglobin inhibitor. Reagent 2 was prepared with the same composition as Reagent 2 of Example 1. Reagent 1: pH 9.4 (25°C) Deferoxamine mesylate 1.5 mM Brij35 1.0% Sodium carbonate 200 mM Urea 3, 4, or 5 M Reagent 2: pH 9.4 (25°C) 5Br-PAPS 0.2 mM Sodium carbonate 200 mM Triton X100 0.5%

[0059] [Comparative Examples 1-3] Reagent 1 was prepared with the same composition as Reagent 1 of Example 1, except that in Reagent 1, sodium dodecyl sulfate (SDS) was dissolved in an aqueous sodium carbonate solution at concentrations of 0.05, 0.25, and 0.50% by mass, respectively, as a hemoglobin inhibitor, instead of guanidine hydrochloride. Reagent 2 was prepared with the same composition as Reagent 2 of Example 1. Reagent 1: pH 9.4 (25°C) Deferoxamine mesylate 1.5 mM Brij35 1.0% Sodium carbonate 200 mM SDS 0.05, 0.25, and 0.50% by mass Reagent 2: pH 9.4 (25°C) 5Br-PAPS 0.2 mM Sodium carbonate 200 mM Triton X100 0.5%

[0060] [Comparative Examples 4 and 5] A first reagent with the same composition as the first reagent of Example 1 was prepared, except that in the first reagent, 100 mM imidazole or 100 mM pyridine was dissolved in an aqueous sodium carbonate solution as a hemoglobin inhibitor instead of guanidine hydrochloride. A second reagent identical to the second reagent of Example 1 was also prepared. First reagent: pH 9.4 (25°C) Deferoxamine mesylate 1.5 mM Brij35 1.0% Sodium carbonate 200 mM Imidazole or pyridine 100 mM (2) Preparation of the second reagent Second reagent: pH 9.4 (25°C) 5Br-PAPS 0.2 mM Sodium carbonate 200 mM Triton X100 0.5%

[0061] 2. Sample Preparation: As samples, nine volumes of control serum (QAP Trol I, Sysmex Corporation) and one volume of hemoglobin set (hemoglobin concentrations of 5000 mg / dL and 0 mg / dL) from the Interference Check A Plus (Sysmex Corporation) kit were mixed to create a dilution series. This resulted in the preparation of serum samples with hemoglobin concentrations of 0, 125, 250, 375, or 500 mg / dL, respectively.

[0062] 3. Measurement of Zinc in Samples Using a TBA-120FR (Canon Medical), calibration was performed with physiological saline as the blank and a 200 μg / dL zinc aqueous solution as the calibrator, and the zinc concentration in each sample was measured. Sample dispensing volume: 4.0 μL Amount of first reagent added: 120 μL Amount of second reagent added: 40 μL Measurement wavelength (primary) / (secondary): 548 nm / 700 nm Analytical method: 2-point end method Photometric points: 16-33

[0063] 4. Measurement Results The following shows the results of measuring the zinc concentration in serum samples at each hemoglobin concentration using the reagents of each example or comparative example. In the table, the upper row of each value shows the measured zinc concentration, and the number in parentheses in the lower row shows the ratio (%) of the measured zinc concentration in serum samples containing hemoglobin at each concentration to the measured zinc concentration in serum samples with a hemoglobin concentration of 0.

[0064]

[0065] As described above, with the control reagent, the zinc concentration in serum samples with hemoglobin added was measured to be 6% to 33% lower than that of serum samples without hemoglobin, depending on the hemoglobin concentration. In contrast, the reagents of Examples 1 to 9, which use the first reagent with guanidine hydrochloride or urea added, showed a significant improvement in this effect of hemoglobin. On the other hand, the reagents of Comparative Examples 1 to 3, which use the first reagent with SDS added, did not show any improvement in the effect of hemoglobin. Furthermore, the reagents of Comparative Examples 4 and 5, which use the first reagent with imidazole or pyridine added, showed extremely low measurement sensitivity, making it impossible to perform proper calibration and thus impossible to measure the zinc in the sample. The difference in absorbance when physiological saline and calibrator (200 μg / dL zinc aqueous solution) were measured using the two-point end method with the control reagent and the reagents of Comparative Examples 4 and 5 is shown below (n=2).

[0066]

[0067] Test II 1. Reagent Preparation A first reagent containing bis-trispropane as a masking agent, Bridge 35 as a nonionic surfactant, and guanidine hydrochloride as a hemoglobin effect inhibitor was prepared, and a second reagent containing 5Br-PAPS as a chelating colorant was prepared. More specifically, the following is described.

[0068] [Example 10] (1) Preparation of the first reagent In an aqueous sodium carbonate solution, bistrispropane as a masking agent, guanidine hydrochloride as a hemoglobin effect inhibitor, and Brij35 (Sigma-Aldrich) as a nonionic surfactant were dissolved at 25°C at the following concentrations to prepare the first reagent with a pH of 9.4. First reagent: pH 9.4 (25°C) Bistrispropane 30 mM Brij35 1.0% Sodium carbonate 200 mM Guanidine hydrochloride 400 mM

[0069] (2) Preparation of the second reagent A second reagent with a pH of 9.4 was prepared by dissolving 5Br-PAPS as a chelating colorant and Triton X100 as a nonionic surfactant in an aqueous sodium carbonate solution at 25°C to the following concentrations: Second reagent: pH 9.4 (25°C) 5Br-PAPS 0.15 mM Sodium carbonate 200 mM Triton X100 0.5%

[0070] [Control Reagent] A first reagent with the same composition as the first reagent in Example, except that it did not contain guanidine hydrochloride and did not contain a hemoglobin inhibitor, and a second reagent identical to the second reagent in Example 1 were prepared. First reagent: pH 9.4 (25°C) Bis-trispropane 30 mM Brij35 1.0% Sodium carbonate 200 mM Second reagent: pH 9.4 (25°C) 5Br-PAPS 0.15 mM Sodium carbonate 200 mM Triton X100 0.5%

[0071] [Examples 11-23] Reagent 1 was prepared with the same composition as Reagent 1 of Example 1, except that guanidine hydrochloride was dissolved in an aqueous sodium carbonate solution as a hemoglobin inhibitor at concentrations of 500, 550, 600, 750, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, and 4000 mM, respectively, as Reagent 1. Reagent 2 was prepared with the same composition as Reagent 2 of Example 1. Reagent 1: pH 9.4 (25°C) Bis-trispropane 30 mM Brij35 1.0% Sodium carbonate 200 mM Guanidine hydrochloride 500-4000 mM Reagent 2: pH 9.4 (25°C) 5Br-PAPS 0.15 mM Sodium carbonate 200 mM Triton X100 0.5%

[0072] 2. Sample Preparation: A serum sample prepared in the same manner as in Test I was used as the sample.

[0073] 3. Measurement of Zinc in Samples Using a TBA-120FR (Canon Medical), the zinc concentration in each sample was measured using the following measurement parameters. Calibration was performed with physiological saline as the blank and a 200 μg / dL zinc aqueous solution as the calibrator. Measurements were performed twice for each sample, and the average value was used as the measurement result. Sample dispensing volume: 12.0 μL First reagent addition volume: 120 μL Second reagent addition volume: 40 μL Measurement wavelength (primary) / (secondary): 548 nm / 700 nm Analytical method: 2-point end method Photometric points: 16-33

[0074] 4. Measurement Results The following shows the results of measuring the zinc concentration in serum samples at each hemoglobin concentration using the reagents of each example or comparative example. In the table, the upper row of each value shows the measured zinc concentration, and the number in parentheses in the lower row shows the ratio (%) of the measured zinc concentration in serum samples containing hemoglobin at each concentration to the measured zinc concentration in serum samples with a hemoglobin concentration of 0.

[0075]

[0076] As described above, in the control reagent, the zinc concentration in serum samples with hemoglobin added was measured to be 2% to 23% lower than that of serum samples without hemoglobin, depending on the hemoglobin concentration. In contrast, in the reagents of Examples 10 to 23, which used the first reagent with 400 to 4000 mM guanidine hydrochloride added, this effect of hemoglobin was significantly improved.

Claims

1. A kit for measuring zinc in a biological sample, comprising a first reagent containing a masking agent and a second reagent containing a chelating colorant, wherein the first reagent further contains at least one hemoglobin-inhibiting agent selected from guanidine, its salts, and urea.

2. The kit according to claim 1, wherein the first reagent further contains a nonionic surfactant.

3. The kit according to claim 1, wherein the concentration of guanidine or its salt in the first reagent is 400 to 4000 mM.

4. The kit according to claim 1, wherein the concentration of urea in the first reagent is 3 to 5 M.

5. The kit according to claim 1, wherein the chelating colorant is 2-(5-bromo-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof, or 2-(5-nitro-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof.

6. The kit according to claim 1, wherein the masking agent is at least one selected from bis-trispropane, TAPS, TES, tricine, trishydroxymethylaminomethane, deferoxamine or a salt thereof, and bicine.

7. The kit according to any one of claims 1 to 6, wherein the biological sample is plasma, serum, or urine.

8. A method for measuring zinc in a biological sample, comprising the steps of: adding a first reagent containing at least one hemoglobin-inhibiting agent selected from guanidine, its salts, and urea, together with a masking agent, to the biological sample; and adding a second reagent containing a chelating colorant to a mixture of the biological sample and the first reagent.

9. The method according to claim 8, wherein the first reagent further contains a nonionic surfactant.

10. The method according to claim 8, wherein the first reagent contains guanidine or a salt thereof, and the concentration of guanidine or a salt thereof in the mixed solution after the addition of the first reagent is 400 to 4000 mM.

11. The method according to claim 8, wherein the first reagent contains urea, and the concentration of urea in the mixed solution after the addition of the first reagent is 3 to 5 M.

12. The method according to claim 8, wherein the second reagent is added when the change in absorbance of the mixture after the addition of the first reagent has substantially finished.

13. The method according to claim 8, wherein the chelating colorant is 2-(5-bromo-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof, or 2-(5-nitro-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof.

14. The method according to claim 8, wherein the masking agent is at least one selected from bis-trispropane, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), tricine, trishydroxymethylaminomethane, deferoxamine or a salt thereof, and bicine.

15. A method for reducing the influence of hemoglobin when measuring zinc in a biological sample using a masking agent and a chelating chromogenic agent, comprising the step of adding to the biological sample, together with the masking agent, a reagent containing at least one hemoglobin-inhibiting agent selected from guanidine or its salt and urea, before reacting the chelating chromogenic agent with the zinc in the biological sample.

16. The method according to claim 15, wherein the reagent further contains a nonionic surfactant.

17. The method according to claim 15, wherein the reagent contains the guanidine or a salt thereof, and the concentration of the guanidine or a salt thereof in the mixed solution after the addition of the first reagent is 400 to 4000 mM.

18. The method according to claim 15, wherein the chelating colorant is 2-(5-bromo-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof, or 2-(5-nitro-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof.

19. The method according to claim 15, wherein the masking agent is at least one selected from bis-trispropane, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), tricine, trishydroxymethylaminomethane, deferoxamine or a salt thereof, and bicine.

20. A reagent for reducing the influence of hemoglobin when measuring zinc in a biological sample using a masking agent and a chelating colorimetric agent, wherein the reagent contains, together with the masking agent, at least one selected from guanidine, its salts, and urea.

21. The reagent according to claim 20, further comprising a nonionic surfactant.

22. The reagent according to claim 20, wherein the concentration of guanidine or its salt in the reagent is 400 to 4000 mM.

23. The reagent according to claim 20, wherein the concentration of urea in the reagent is 3 to 5 M.

24. The reagent according to claim 20, wherein the chelating colorant is 2-(5-bromo-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof, or 2-(5-nitro-2-pyridylazo)-5(N-propyl-N-3-sulfopropylamino)phenol or a salt thereof.

25. The reagent according to claim 20, wherein the masking agent is at least one selected from bis-trispropane, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), tricine, trishydroxymethylaminomethane, deferoxamine or a salt thereof, and bicine.

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