Nitrite detection methods

Abstract

To provide a method for detecting nitrite reliably and inexpensively. A method for detecting nitrite includes a first step of reacting a liquid sample with indole under acidic conditions with hydrochloric acid, and a second step of measuring the absorbance of the compound obtained in the first step in a wavelength region ranging from 470 nm to 510 nm.

Description

[Technical Field]

[0001] The present invention relates to a method for detecting nitrite in a sample. [Background technology]

[0002] Measurement of nitrite in body fluids has long been an important measurement item in medical diagnosis. Nitrite is produced by the nitrate-reducing activity of bacteria such as Escherichia coli, Escherichia coli paracoli, Salmonella typhi, Salmonella paratyphi, Staphylococcus aureus, and Enterococcus faecalis. Therefore, detecting nitrite, especially nitrite in urine, is extremely important for diagnosing urinary tract infections caused by pathogenic bacteria and microorganisms.

[0003] Currently, common reagents for detecting nitrite include sulfanilamide and naphthylethylenediamine. Sulfanilamide reacts with nitrite under acidic conditions to produce a diazo compound, which reacts with naphthylethylenediamine to produce a pink azo dye (detection signal) (Griess method: see, for example, Non-Patent Document 1). The concentration of this azo dye has a good correlation with the concentration of nitrite, and can therefore be used as a measure of the amount of nitrite. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Toshiro Yamada, Fujio Egami, et al. (eds.): "Standard Biochemical Experiments", Bunkodo, Tokyo, p. 348 (1953) Summary of the Invention [Problem to be solved by the invention]

[0005] However, naphthylethylenediamine, the reagent used in the Griess method, is a very expensive reagent, and the cost of the test is also high.

[0006] Therefore, an object of the present invention is to provide a method for detecting nitrite reliably and inexpensively. [Means for solving the problem]

[0007] In order to achieve the above object, the detection method of the present invention comprises: A first step of reacting a liquid sample with indole under acidic conditions with hydrochloric acid; a second step of measuring the absorbance of the compound obtained in the first step in a wavelength region ranging from 470 nm to 510 nm; It has the characteristic of including [Effects of the Invention]

[0008] According to one embodiment of the present invention, it is possible to provide a method for detecting nitrite reliably and inexpensively. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a flow diagram of an example of a method for detecting nitrite according to this embodiment. [Figure 2] FIG. 2 shows an example of the absorption spectrum of a red pigment compound in the method for detecting nitrite according to this embodiment. [Figure 3] FIG. 3 is a schematic diagram for explaining the correlation between the concentration of nitrite and absorbance in the method for detecting nitrite according to this embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0010] A detection method according to one embodiment of the present invention will be described below with reference to the drawings.

[0011] (Detection method) FIG. 1 shows a flow diagram of an example of a method for detecting nitrite according to this embodiment.

[0012] As shown in FIG. 1, the method for detecting nitrite according to this embodiment includes the following steps: A first step (S100) of reacting a sample with indole under acidic conditions with hydrochloric acid; a second step (S200) of measuring the absorbance of the sample obtained in the first step in a wavelength region ranging from 470 nm to 510 nm; Includes.

[0013] In addition, the method for detecting nitrite according to this embodiment includes the steps of: The method may include a third step (S300) of quantifying the amount of nitrite in the sample based on the absorbance measured in the second step.

[0014] Each step will now be described in more detail.

[0015] The first step involves reacting a sample with indole under acidic conditions using hydrochloric acid to color only the nitrite in the sample. Commercially available indole is usually in powder form, so it is preferable to use powdered indole dissolved in an organic solvent such as ethanol or methanol. The concentration of indole added, i.e., the concentration of indole in the color-developing solution, is preferably in the range of 0.0015% by mass to 0.035% by mass.

[0016] Furthermore, the concentration of hydrochloric acid added in the first step, i.e., the concentration of hydrochloric acid in the color-developing solution, is preferably in the range of 0.015% by mass to 3.5% by mass. Although the indole concentration and / or the hydrochloric acid concentration affect the degree of color development during nitrite detection, by setting each concentration within the above range, color development during nitrite detection can be confirmed visually and absorbance in the wavelength range of 470 nm to 510 nm, as described below, can be reliably measured.

[0017] As will be explained in more detail in the Examples below, when nitrite is reacted with indole under acidic conditions using hydrochloric acid, a compound (2-indolyl-3-oxime-3H-indole; hereinafter, sometimes referred to as a red pigment compound) having a maximum absorbance peak in the wavelength region of approximately 492 nm to 495 nm is produced.

[0018] Therefore, in the second step, the presence or absence of nitrite in the sample can be confirmed by measuring the absorbance of the liquid obtained in the first step in the wavelength region ranging from 470 nm to 510 nm or by visually confirming the color development of the liquid.

[0019] In addition to nitrite (nitrite ion), urine also contains urea, ammonia, sodium ions, potassium ions, and nitrate ions, as well as albumin (e.g., in patients with impaired renal function) and glucose (e.g., in patients with diabetes). As will be explained in more detail in the Examples below, in the detection method according to this embodiment, the above components do not produce color-developing compounds. Therefore, it can be said that this method can specifically detect only nitrite by color development.

[0020] Furthermore, since ammonia, sodium ions, potassium ions, and nitrate ions are also present in water, the detection method of this embodiment can also be applied to detecting nitrite in water, for example, in water quality testing applications.

[0021] The red pigment compound produced in step 1 preferably has a nitrite concentration of 0.1 mg / L or higher, considering factors such as the lower limit of quantitation of absorbance. Furthermore, as will be described in detail later, this red pigment compound exhibits a sufficient correlation with nitrite concentrations in the range of 0.1 mg / L to 25 mg / L. Therefore, in step 3, the amount of nitrite in the sample can be quantified by appropriately diluting and measuring the sample based on the absorbance measured in step 2.

[0022] The nitrite detection method according to this embodiment uses indole and hydrochloric acid as detection reagents, and compared to the conventional Griess method using sulfanilamide and sulfanilamide, the detection method according to this embodiment can reduce the cost of reagents and can therefore be said to be an inexpensive detection method.

[0023] The method for detecting nitrite according to this embodiment will be described in detail below with reference to examples.

[0024] Example 1 An example in which the red pigment compound produced in the first step according to this embodiment was identified will be described.

[0025] 300 μL of 0.01% indole (solvent: ethanol) was added to 300 μL of a 100 mg / L nitrite ion standard solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product code 146-06453, diluted 10-fold with purified water), mixed, and then 300 μL of 10% hydrochloric acid was added and mixed, followed by allowing to stand at room temperature for 5 minutes to obtain the sample of Example 1. The indole, ethanol, and hydrochloric acid used were indole (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product code 092-00162), ethanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product code 057-00456), and 10% hydrochloric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product code 085-07535). The resulting sample was extracted with chloroform, concentrated, and then separated by thin-layer chromatography (TLC). The separated sample was subjected to imaging mass spectrometry using a laser desorption ionization mass spectrometry (LDI-MS: SolariX (Bruker Daltonics Inc.) magnet: 9.4 T) in positive ionization mode.

[0026] The obtained spectrum had a mass spectrum consistent with that of 2-indolyl-3-oxime-3H-indole, and this example clarified the structural formula of the red pigment obtained in Step 1. That is, it was found that in Step 1, nitrite and indole react in the main reaction according to the following formula (1) to produce 2-indolyl-3-oxime-3H-indole.

[0027] [ka]

[0028] Furthermore, the absorption spectrum of the red pigment compound in the sample of Example 1 was measured using a spectrophotometer U-2900 (manufactured by Hitachi High-Technologies Corporation). Figure 2 shows an example of the absorption spectrum of the red pigment compound in the nitrite detection method according to this embodiment. In addition, in Figure 2, the vertical line indicating the wavelength of 490 nm is shown as a dashed line.

[0029] As shown in FIG. 2, the absorption curve of the red pigment compound obtained after the first step was found to have an absorbance peak in the wavelength region of approximately 492 nm to 495 nm.

[0030] Example 2 Next, an example will be described in which it was confirmed that the nitrite detection method according to this embodiment does not produce coloration with other components in urine (or water).

[0031] Samples of Comparative Examples 1 to 7 were obtained in the same manner as in Example 1, except that the following standard solutions or aqueous solutions were used instead of the 100 mg / L nitrite ion standard solution.

[0032] The standard solution or aqueous solution used was: Comparative Example 1: 100 mg / L nitrate ion standard solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: product code 149-06443 diluted 10 times with pure water), Comparative Example 2: 1000 mg / L sodium ion standard solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: product code 193-09621), Comparative Example 3: 1000 mg / L potassium ion standard solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: product code 163-13991), Comparative Example 4: 1 g / dL urea (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: product code 213-00173) aqueous solution, Comparative Example 5: 1 g / dL albumin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: product code 015-27053) aqueous solution, Comparative Example 6: 7% aqueous ammonia (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: product code 016-03146 ​​diluted 4 times with pure water), Comparative Example 7: 1 g / dL glucose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: product code 049-31165) aqueous solution, was used.

[0033] The color development state of the obtained sample of Example 1 and the samples of each comparative example was visually confirmed, and it was found that no color was developed in any of the samples other than the sample of Example 1 in which nitrite was used.

[0034] That is, it was found that the detection method according to this embodiment is a method that can color only nitrite in urine or water and can detect only nitrite.

[0035] Example 3 Next, an example will be described in which it was confirmed that the nitrite detection method according to this embodiment can quantify the concentration of nitrite in a sample.

[0036] Each sample was prepared in the same manner as in Example 1, except that a nitrite ion standard solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: product code 146-06453) was diluted with pure water to a predetermined concentration (0.1 mg / L, 0.5 mg / L, 1 mg / L, 5 mg / L, 10 mg / L, 25 mg / L).

[0037] The absorbance of each of the obtained samples was measured at a wavelength of 500 nm using a spectrophotometer U-2900 (manufactured by Hitachi High-Technologies Corporation).

[0038] Fig. 3 is a schematic diagram illustrating the correlation between nitrite concentration and absorbance in the nitrite detection method according to this embodiment. The vertical axis in Fig. 3 represents absorbance at 500 nm, and the horizontal axis represents the concentration of nitrite ions. Fig. 3 also shows an approximate line in dotted lines to illustrate the correlation between nitrite concentration and absorbance.

[0039] As shown in Figure 3, there is a sufficient correlation (proportionality) between the nitrite concentration and absorbance when the nitrite concentration is in the range of 0.1 mg / L to 25 mg / L. Even when the nitrite concentration exceeds 25 mg / L, the red compound obtained in the first step develops a color, making it possible to detect nitrite. However, when the nitrite concentration exceeds 25 mg / L, there is no proportional relationship between the absorbance and the nitrite concentration. Therefore, the amount of nitrite in the sample (or the nitrite concentration) can be quantified by appropriately diluting and measuring the absorbance measured in the second step.

[0040] As described above, the nitrite detection method according to this embodiment can detect nitrite using indole and hydrochloric acid, and is therefore a less expensive detection method compared to the Griess method, which uses naphthylethylenediamine and sulfanilamide.

[0041] Furthermore, since the nitrite detection method according to this embodiment does not produce color with other components in urine or water, it can be applied not only to the detection of nitrite in urine tests but also to the detection of nitrite in water quality tests, etc.

[0042] Furthermore, the nitrite detection method according to this embodiment can also be applied to a method for quantifying nitrite, since there is a sufficient correlation between the absorbance in the wavelength range of 470 nm or more and 510 nm or less and the concentration of nitrite.

[0043] Although the embodiments of the present invention have been described above with reference to the drawings, the specific configurations should not be considered to be limited to these embodiments. The scope of the present invention is defined not only by the description of the above embodiments but also by the claims, and further includes all modifications within the meaning and scope of the claims. [Explanation of symbols]

[0044] S100 1st process S200 2nd process S300 3rd process

Claims

1. a first step of reacting a liquid sample with indole under acidic conditions with hydrochloric acid; a second step of measuring the absorbance of the compound obtained in the first step in a wavelength region ranging from 470 nm to 510 nm; Including, the concentration of the indole in the compound obtained in the first step is within a range of 0.0015% by mass or more and 0.035% by mass or less, and the concentration of the hydrochloric acid is within a range of 0.015% by mass or more and 3.5% or less; Methods for detecting nitrite.

2. The method further comprises a third step of quantifying the amount of nitrite in the liquid sample based on the absorbance measured in the second step. The method for detecting nitrite according to claim 1.

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