JP2965563B2 - Component analysis method - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to a component that avoids the influence of nitrite ions, particularly to a method for analyzing the component in a biological sample. [Prior Art] Nitrite ion is a relatively unstable ion and has an oxidizing property and a reducing property, so that when it is present in a sample, it often has an adverse effect when measuring other components in the sample. Was seen. For example, when the residual chlorine is measured by the o-tolidine method or the iodine method, when the chloride ion is measured by the mercuric thiocyanate colorimetric method, when the nitrate ion is measured by the phenol disulfonic acid method, and the phosphate ion is the molybdenum blue method. At the time of measurement, when measuring sulfur by the N, N-dimethyl-p-phenyldiamine method, the Coomassie brilliant blue G-250 method for trace proteins, pyrogallol red-molybdate complex method, brompirogallol red-tungstate complex method, pyrocate At the time of measurement by the roll violet-tungstate complex method or the Coomassie brilliant blue R method, etc., the color formation is prevented or the blind value is increased to prevent the quantitative or qualitative reaction or to cause an error. Often there were times. Especially, in the measurement of trace protein in urine, the boundary between the normal value and the abnormal value is around 10 mg / d as protein concentration, so 4-6 mg / d by nitrite ion.
The occurrence of a negative error in d was a major problem in determining the patient's condition and disease state. Therefore, when performing the above-described various measurement methods, if coexistence of nitrite ion is considered in the sample, the blind value of the sample should be separately checked in advance, or the nitrite ion should be measured in advance, for example, by using hydrogen peroxide. In practice, it is not possible to say that an accurate value has been obtained unless measurement is performed using a sample that has been removed by oxidative decomposition with permanganate or the like. There is a resistance to adopting this method aggressively due to the increase in the number of steps in the measurement operation and the complexity of the operation, and the emergence of a new method that can more easily and more effectively avoid the effects of nitrite ions is strongly emerging. Was desired. [Object of the present invention] The object of the present invention is, in various measurement methods as described above,
It is an object of the present invention to provide a method for easily removing nitrite ions which affect a measured value without increasing the number of reaction operation steps or complicating the operation. [Configuration of the Invention] In order to achieve the above object, the present invention has the following configuration. In a method for analyzing a component that does not require nitrite ion and is affected by the presence of nitrite ion, coexisting nitrite ion can react with the target reaction before affecting it. A method for analyzing a component, wherein the measurement is performed in the presence of one or more nitrogen-containing organic compounds selected from the group consisting of (a) to (d). (A) Equation-1 (However, R ^{1 to} R ⁵ each independently represent a sulfonic acid group, a carboxyl group, a hydroxyl group, a sulfonylamino group, a halogen atom,
R ⁶ represents an alkyl group having 1 to 4 carbon atoms, a nitro group or a hydrogen atom, and R ⁶ represents an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group having 1 to 4 carbon atoms, an amino group or a hydrogen atom. )
And the soluble salts thereof. (B) Formula-2 R ⁷ —NH—R ⁸ [where R ⁷ and R ⁸ are each independently an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group having 1 to 4 carbon atoms, 1 carbon atom
To 4 aminoalkyl groups or hydrogen atoms (provided that R ⁷
Except when R and R ⁸ are both hydrogen atoms. ). And the soluble salts thereof. (C) A hydrazine derivative represented by the formula-3 R ⁹ -NHNH ₂ (where R ⁹ represents an alkyl group having 1 to 4 carbon atoms or a hydrogen atom) and soluble salts thereof. (D) Formula-4 (However, R ^{10 to} R ¹² each independently represent a hydrogen atom,
4 represents an alkyl group or a phenyl group. And a soluble salt thereof. "A reagent for measuring residual chlorine, chloride ions, nitrate ions, phosphate ions or trace proteins in a specimen in which nitrate ions coexist, and is selected from the group consisting of the following (a) to (d): A measuring reagent comprising one or more selected from nitrogen-containing organic compounds. (However, R ^{1 to} R ⁵ each independently represent a sulfonic acid group, a carboxyl group, a hydroxyl group, a sulfonylamino group, a halogen atom,
R ⁶ represents an alkyl group having 1 to 4 carbon atoms, a nitro group or a hydrogen atom, and R ⁶ represents an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group having 1 to 4 carbon atoms, an amino group or a hydrogen atom. )
And the soluble salts thereof. (B) Formula-2 R ⁷ —NH—R ⁸ [where R ⁷ and R ⁸ are each independently an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group having 1 to 4 carbon atoms, 1 carbon atom
To 4 aminoalkyl groups or hydrogen atoms (provided that R ⁷
Except when R and R ⁸ are both hydrogen atoms. ). And the soluble salts thereof. (C) A hydrazine derivative represented by the formula-3 R ⁹ -NHNH ₂ (where R ⁹ represents an alkyl group having 1 to 4 carbon atoms or a hydrogen atom) and soluble salts thereof. (D) Formula-4 (However, R ^{10 to} R ¹² each independently represent a hydrogen atom,
4 represents an alkyl group or a phenyl group. And a soluble salt thereof. That is, the present inventors add a specific aromatic or aliphatic primary or secondary amine, hydrazine derivative, or thiourea derivative to an analytical reagent (measurement reagent), and add it to a sample. When nitrite is removed by reacting with ions to generate diazo compounds, alcohols, nitrosamines, nitrogen, etc., nitrite ions are not affected at all and do not affect the measurement of the target component in the sample. The present inventors have found that it is possible to carry out the removal and the analysis at the same time, and have completed the present invention. In the present invention, the formula used for the removal of nitrite ions R ^{1 to} R ⁵ of the aniline derivative represented by each independently represent a sulfonic acid group, a carboxyl group, a hydroxyl group, a sulfonylamino group, for example, a halogen atom such as chlorine, bromine or iodine, for example, a methyl group, an ethyl group, a propyl group; butyl group such as an alkyl group having 1 to 4 carbon atoms, a nitro group or a hydrogen atom, R ⁶
Is an alkyl group having 1 to 4 carbon atoms such as a methyl group, an ethyl group, a propyl group, and a butyl group; for example, a hydroxyalkyl group having 1 to 4 carbon atoms such as a hydroxymethyl group, a hydroxyethyl group, a hydroxypropyl group, and a hydroxybutyl group; Indicates an amino group or a hydrogen atom. Examples of the soluble salts include sulfonic acid groups represented by R ^{1 to} R ⁵ , in addition to mineral salts (eg, hydrochloride, sulfate, etc.) relating to the aniline skeleton.
An alkali metal salt or an ammonium salt of a carboxyl group, or a sulfonylamino group represented by R ^{1 to} R ⁵ , or a mineral acid salt relating to the amino group represented by R ⁶ (for example, hydrochloride,
Sulfates). R ⁷ and R ⁸ of the aliphatic amines represented by R ⁷ —NH—R ^{8 according} to the present invention are each independently an alkyl having 1 to 4 carbon atoms such as a methyl group, an ethyl group, a propyl group and a butyl group. Groups such as hydroxymethyl, hydroxyethyl,
Hydroxypropyl group, hydroxybutyl group, etc.
To 4 hydroxyalkyl groups such as an aminomethyl group,
It represents an aminoalkyl group having 1 to 4 carbon atoms, such as an aminoethyl group, an aminopropyl group, an aminobutyl group, or a hydrogen atom (however, unless R ⁷ and R ⁸ are both hydrogen atoms).
Examples of the soluble salts include mineral salts (eg, hydrochloride, sulfate, etc.) relating to the —NH— portion of R ⁷ —NH—R ⁸ .
Mineral salts (eg, hydrochloride, sulfate, etc.) relating to the aminoalkyl group represented by R ⁷ or R ⁸ are also included. R ⁹ of the hydrazine derivative represented by R ⁹ -NHNH ₂ according to the present invention show a methyl group, an ethyl group, a propyl group, a butyl group such as an alkyl group or a hydrogen atom having 1 to 4 carbon atoms. The soluble salts are those which form a salt with a mineral acid (for example, hydrochloric acid, sulfuric acid, etc.) at the hydrazino group of R ⁹ —NHNH ₂ . According to the present invention R ^{10 to} R ¹² in the thiourea derivative represented by each independently represent a hydrogen atom, for example, an alkyl group having 1 to 4 carbon atoms such as a methyl group, an ethyl group, a propyl group or a butyl group, or a phenyl group. Further, the soluble salts refer to mineral salts such as hydrochlorides and sulfates thereof. Specific examples of these nitrogen-containing organic compounds according to the present invention include, for example, sodium m-aminobenzoate, p-aminosalicylic acid, sulfanilic acid, sulfanilamide, p-aminophenol, N-methyl-N-ethanolamine, phenyl Examples include hydrazine, hydrazine hydrochloride, thiourea, phenylthiourea, and the like, but are not particularly limited thereto. The analysis method of the present invention is known per se according to the measurement (analysis) target except for adding the above-mentioned compounds (a) to (d) to the analysis reagent (measurement reagent) for the purpose of eliminating the influence of nitrite ions. It is sufficient to perform measurement (analysis) according to a measurement method (analytical method) known per se using the measurement reagent described above. The compounds (a) to (d) used for the purpose of eliminating the influence of nitrite ions are usually contained in the measuring reagent in an amount of 0.001 to 5.0%, preferably 0.01 to 5.0%.
0.50.5% is added. The compounds described above can be used in combination of two or more as long as they do not inhibit the reaction relating to the measurement. In addition, although the number of steps increases, there is no problem even if the above-mentioned compound is prepared as an aqueous solution, added to a sample in advance, and treated with nitrite ions, and then measured according to a normal measurement method using a normal measurement reagent. Needless to say. Examples of the measurement object that can be measured (analyzed) by the method of the present invention include, for example, residual chlorine, chloride ions, nitrate ions, phosphate ions, trace proteins, and the like, but needless to say, the present invention is not limited to these. . The analysis method of the present invention can be applied to both analysis using a method and instrument analysis. Further, the method of the present invention can be applied to a simple test paper method and a so-called dry quantitative method using a multilayer analysis sheet (multilayer integrated quantitative analysis film) containing a reaction reagent. Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto. [Examples] Experimental example 1. Investigation of reactivity with nitrite ion <Examination method-1> 3.0 ml of a reagent obtained by dissolving 0.1% of the compound according to the present invention in 0.1 M sodium chloride / hydrochloric acid buffer (pH 2.0) Then, 50 μl of 0.1% sodium nitrite solution was added, and the solution was allowed to stand at room temperature for a certain period of time, and the coloring of the solution was examined. -Results The results are shown in Table 1. In addition, for the colored ones, the measurement wavelengths which are not affected by the coloring when used for actual measurement are also shown. <Examination method-2>-Reagents Pyrogallol red 25 mg Ammonium molybdate 30 mg Gum arabic 20 g Tartaric acid 1 g The above substance was dissolved in 0.1 M glycine buffer 1, and the compound of the present invention was adjusted to 0.1 w / v%. And adjusted to pH 2.2 with hydrochloric acid to obtain a reagent.・ Sample Prepare a solution containing 75 mg / d of albumin, divide it into two parts, add sodium nitrite to only one side so that the concentration becomes 0.1%, and mix it with a sample containing nitrite ions and a sample containing no nitrite ions. did.・ Operation method Add 50 μL of sample to 3.0 ml of reagent, mix well, and mix at room temperature.
After standing for 0 minutes, the absorbance at 600 nm was measured. The absorbance of the sample containing no nitrite ion was E _S1, and the absorbance of the sample containing nitrite ion was E _S2 . Further, the absorbance obtained by the same operation using purified water instead of the sample was E _B. Using these values, a determination value V was obtained according to the following equation. V = [(E _S2 −E _B ) − (E _S1 −E _B ) × 100] Symbols are indicated as follows according to the value of V. ○: 90 ≦ V Δ: 50 ≦ V <90 ×: 50 > V Results The results are shown in Table 1. Comparative Experimental Example Instead of the engaging member according to the present invention in Experimental Example 1, a nitrogen-containing organic compound which does not fall under the scope of the present invention was used. The reaction with nitrite ion was examined in exactly the same manner, and the results are shown in Table 1. The results when no nitrogen-containing organic compound was added are also shown in Table 1. As described above, the compound according to the present invention can effectively remove nitrite ions, but the same nitrogen-containing organic compound which does not fall within the scope of the present invention has no effect at all. Some of the compounds are slightly colored due to the reaction with nitrite ions, as described in the table. People no practical problem at all be carried out measurements at more than a certain wavelength. Experimental Example 2. Examination of Required Concentration Sulfanilic acid was used as the nitrogen-containing organic compound according to the present invention, and the necessary concentration was examined when albumin was measured. <Examination method>-Reagent for pyrogallol red-molybdate complex method Pyrogallol red 25 mg Ammonium molybdate 30 mg Arabic gum 20 g Tartaric acid 1 g The above substance was dissolved in 0.1 M glycine buffer solution 1 and sulfanilic acid was further added to a predetermined concentration. PH with hydrochloric acid
Was adjusted to 2.2 to obtain a reagent. Reagent for Coomassie Brilliant Blue Method Coomassie Brilliant Blue G-250 100 mg Oxalic acid 80 g The above substance was dissolved in purified water 1 and sulfanilic acid was added to a predetermined concentration to prepare a reagent. -Sample A solution containing albumin at 75 mg / d was prepared, and sodium nitrite was added to the solution to a concentration of 0.1% to prepare a sample.・ Operation method Add 50 μL of sample to 3.0 ml of reagent, mix well, and mix at room temperature.
After standing for 0 minutes, the absorbance at 600 nm was measured and the absorbance E _S
(However, the values obtained by the reagent of sulfanilic acid no addition was E _SO.) Was obtained. Solution of purified water and albumin containing 75 mg / d in place of the sample (nitrite ion contained no.) Was E _B and E _STD to perform the absorbance obtained the same operation using a. Using these values, the nitrite ion removal rate J was determined according to the following equation. . J = illustrating a _{[(E S -E SO) ÷} (E STD -E B] × 100 · Results The results in Table 2. As apparent from the results, pyrogallol red -
It was found that the addition of 0.03% in the molybdic acid complex method and 0.008% in the Coomassie brilliant blue method can almost completely eliminate the negative error due to the influence of nitrite ions. Experimental Example 3 Investigation on the Influence on the Calibration Curve The effect of sulfanilic acid on the calibration curve when albumin was measured by the pyrogallol red-molybdate complex method using sulfanilic acid as the nitrogen-containing organic compound according to the present invention The presence or absence was checked.・ Reagent Drug Pyrogallol Red 25mg Ammonium molybdate 30mg Arabic gum 20g Tartaric acid 1g Dissolve the above substance in 0.1M glycine buffer 1 and add hydrochloric acid
After adjusting the pH to 2.2, bisect the mixture, and add 1 g of sulfanilic acid to one.
Was added to obtain reagents with and without sulfanilic acid. -Sample A solution containing albumin at a concentration of 350 mg / d was diluted by a predetermined factor to obtain a sample.・ Operation method Add 50 μL of sample to 3.0 ml of reagent, mix well, and mix at room temperature.
After standing 0 minutes to obtain the absorbance E _S measures the absorbance at 600 nm. The absorbance obtained by the same operation using purified water instead of the sample was E _B. -Results Table 3 shows the measurement results. As is clear from Table-3, the addition of sulfanilic acid did not affect the calibration curve. It should be noted that completely the same results were obtained when sulfanilamide was used instead of sulfanilic acid. Except that the albumin Experimental Example 4. reproducibility sample was a solution containing 75 mg / d are using the same reagents as in Experimental Example 3, was determined absorbance E _S in the same manner as in Experimental Example 3. Purified water and albumin content 10
0 mg / d of solution was determined each absorbance E _B and E _STD perform the same operation as a sample. From these values, the albumin content (mg / d) in the sample was determined according to the following equation. Albumin content (mg / d) = clear from _{[(E S -E B) ÷} (E STD -E B] × 100 · Results The results of measurement repeated 10 times per same sample are shown in Table -4. Table -4 No effect on reproducibility was observed due to the addition of sulfanilic acid, and the same results were obtained when thiourea was used instead of sulfanilic acid. Experimental Example 5. Examination of the coloring ratio between albumin and globulin The absorbance E _S and the absorbance E _S were measured in exactly the same manner as in Experimental Example 3 except that the sample was a solution containing albumin and / or globulin at a predetermined concentration. the E _B was measured. Only the albumin was determined color ratio G / A of albumin and globulin by the following equation E _S obtained by a solution containing 100 mg / d as E _{S100. G / A = [(E S} -E B) ÷ (E S100 -E B] a × 100 · RESULTS Measurement results As is clear from. Table -5 shown in Table 5, albumin and globulin by addition of sulfanilic acid No effect on the color development ratio was observed.When phenylhydrazine was used instead of sulfanilic acid, exactly the same results were obtained. Reference Example 1. Influence on measured values in the presence of bilirubin The same reagents as in Example 4 were used except that the sample was a 100 mg / d solution of albumin containing a predetermined concentration of bilirubin and / or sodium nitrite. The apparent albumin content (mg / d) in the sample was determined in exactly the same manner. -Results Table 6 shows the measurement results. As is clear from Table 6, it was found that sulfanilic acid can avoid not only the effect of nitrite ions but also the effect of bilirubin. Example 1 Quantification and Reagent of Trace Proteins in Human Urine Pyrogallol Red 25 mg Ammonium molybdate 30 mg Arabic gum 20 g Tartaric acid 1 g The above substance was dissolved in 0.1 M glycine buffer 1 and added with hydrochloric acid.
After adjusting the pH to 2.2, 2 g of sulfanilic acid was added to obtain a reagent. -Samples 15 samples of nitrite-negative human urine (samples Nos. 1 to 15) and 15 samples of nitrite-positive human urine (samples Nos. 16 to 30) were used as samples.・ Operation method Add 50 μL of sample to 3.0 ml of reagent, mix well, and mix at room temperature.
After standing 0 minutes to obtain the absorbance E _S measures the absorbance at 600 nm. Also, instead of the sample, purified water and albumin content 100m
g / d of the solution absorbance obtained by the same operation using the respective E _B and E _STD, was determined protein content in the sample by the following equation (a value in terms of albumin, mg / d). Protein content (value in terms of albumin, mg / d) = a _{[(E S -E B) ÷} (E STD -E B] × 100 · Results The results are shown in Table 7. and Table 8. Incidentally, Table In the table, measured values of nitrite (as sodium nitrite) and pH value by Pretest 9A (manufactured by Wako Pure Chemical Industries, Ltd.) are also shown.
However, the measured value of nitrite is 1.0 mg / d or more and 0.5
Amounts around mg / d and-represent 0.1 mg / d or less, respectively. Comparative Example 1.-Reagent A reagent obtained by removing sulfanilic acid from the reagent used in Example 1 was used as a reagent. -Sample Same as Example 1. -Operation method Same as Example 1. -Results The results are shown in Tables 7 and 8 together with the results of Example 1. As is clear from Tables 7 and 8, when a conventional reagent containing no sulfanilic acid was used, a negative error was caused by the influence of nitrite ions when measuring a nitrite-positive sample. When the reagent to which sulfanilic acid was added was used, accurate measurement values were obtained for both the nitrite-positive sample and the nitrite-negative sample. Example 2. Reagent Same as in Example 1. -Samples 15 samples of nitrite negative human urine of Example 1 (Sample Nos. 1 to 15)
A sample was prepared by adding sodium nitrite to each sample to a concentration of 0.1%. -Operation method Same as Example 1. -Results The results are shown in Table-9. Comparative Example 2. Reagent Same as Comparative Example 1. -Sample Same as Example 2. -Operation method Same as Example 1. -Results The results are shown in Table 9 together with the results of Example 2. As is clear from Table-9, when a conventional reagent containing no sulfanilic acid was used, a negative error clearly occurred due to the influence of nitrite ions when measuring a sample containing nitrite.
It can be seen that when the reagent to which the sulfanilic acid according to the present invention was added was used, the same value as that of the sample without nitrous acid was obtained even with the sample with nitrous acid added. [Effects of the Invention] As described above, the present invention provides a simple method for removing nitrite ions that affect various reactions without increasing the number of reaction operation steps and complicating the operation, and the method for removing nitrite ions. An object of the present invention is to provide a method for analyzing a component in which the influence of ions is avoided, and the invention contributes greatly to the present invention.

Claims (1)

(57) [Claims] In a method for analyzing a component that does not require nitrite ion and is affected by the presence of nitrite ion, coexisting nitrite ion can react with the target reaction before affecting it. A method for analyzing a component, wherein the measurement is performed in the presence of one or more nitrogen-containing organic compounds selected from the group consisting of (a) to (d). (A) Equation-1 (However, R ^{1 to} R ⁵ each independently represent a sulfonic acid group, a carboxyl group, a hydroxyl group, a sulfonylamino group, a halogen atom,
R ⁶ represents an alkyl group having 1 to 4 carbon atoms, a nitro group or a hydrogen atom, and R ⁶ represents an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group having 1 to 4 carbon atoms, an amino group or a hydrogen atom. )
And the soluble salts thereof. (B) Formula-2 R ⁷ -NH-R ⁸ [where R ⁷ and R ⁸ are each independently an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group having 1 to 4 carbon atoms, and 1 carbon atom
To 4 aminoalkyl groups or hydrogen atoms (provided that R ⁷
Except when R and R ⁸ are both hydrogen atoms. ). And the soluble salts thereof. (C) A hydrazine derivative represented by the formula-3 R ⁹ -NHNH ₂ (where R ⁹ represents an alkyl group having 1 to 4 carbon atoms or a hydrogen atom) and soluble salts thereof. (D) Formula-4 (However, R ^{10 to} R ¹² each independently represent a hydrogen atom,
4 represents an alkyl group or a phenyl group. And a soluble salt thereof. 2. The analysis method according to claim 1, wherein the method for analyzing components is a method for analyzing a trace amount of protein. 3. A reagent for measuring residual chlorine, chloride ions, nitrate ions, phosphate ions or trace proteins in a sample in which nitrite ions coexist, and is a reagent selected from the group consisting of the following (a) to (d): A measurement reagent comprising a species or two or more types of nitrogen-containing organic compounds. (A) Equation-1 (However, R ^{1 to} R ⁵ each independently represent a sulfonic acid group, a carboxyl group, a hydroxyl group, a sulfonylamino group, a halogen atom,
R ⁶ represents an alkyl group having 1 to 4 carbon atoms, a nitro group or a hydrogen atom, and R ⁶ represents an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group having 1 to 4 carbon atoms, an amino group or a hydrogen atom. )
And the soluble salts thereof. (B) Formula-2 R ⁷ —NH—R ⁸ [where R ⁷ and R ⁸ are each independently an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group having 1 to 4 carbon atoms, 1 carbon atom
To 4 aminoalkyl groups or hydrogen atoms (provided that R ⁷
Except when R and R ⁸ are both hydrogen atoms. ). And the soluble salts thereof. (C) A hydrazine derivative represented by the formula-3 R ⁹ -NHNH ₂ (where R ⁹ represents an alkyl group having 1 to 4 carbon atoms or a hydrogen atom) and soluble salts thereof. (D) Formula-4 (However, R ^{10 to} R ¹² each independently represent a hydrogen atom,
4 represents an alkyl group or a phenyl group. And a soluble salt thereof.