Method for detecting nitrites

Abstract

To provide an inexpensive method for detecting nitrites that can reliably detect them. [Solution] A method for detecting nitrites, comprising: a first step of reacting a liquid sample with indole under hydrochloric acid conditions; and a second step of measuring the absorbance of the compound obtained in the first step in a wavelength range of 470 nm to 510 nm.

Description

Technical Field

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

Background Art

[0002] Conventionally, as one of the important measurement items in medical diagnosis, there is the measurement of nitrite in body fluids. Nitrite is produced, for example, by the nitrate reduction activity of bacteria such as Escherichia coli, Enterobacter aerogenes, Salmonella typhi, Salmonella paratyphi, Staphylococcus aureus, and Enterococcus faecalis. Therefore, detecting nitrite, particularly nitrite in urine, is very important for diagnosing urinary tract infections caused by pathogenic bacteria and microorganisms in the human body.

[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: for example, see Non-Patent Document 1). The concentration of this azo dye has a sufficient correlation with the concentration of nitrite, serving as a measure of the amount of nitrite.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

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

[0006] Therefore, the present invention aims to provide an inexpensive method for detecting nitrites that can reliably detect them. [Means for solving the problem]

[0007] To achieve the above objective, the detection method of the present invention is The first step involves reacting a liquid sample with indole under hydrochloric acid conditions, A second step involves measuring the absorbance of the compound obtained in the first step in the wavelength range of 470 nm to 510 nm, It has the characteristic of including [something]. [Effects of the Invention]

[0008] According to one embodiment of the present invention, it is possible to provide a reliable and inexpensive method for detecting nitrites. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 is a flowchart of an example of a nitrite detection method according to this embodiment. [Figure 2] Figure 2 shows an example of the absorption spectrum of a red pigment compound in the nitrite detection method according to this embodiment. [Figure 3] Figure 3 is a schematic diagram illustrating the correlation between nitrite concentration and absorbance in the nitrite detection method according to this embodiment. [Modes for carrying out 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) Figure 1 shows a flow chart of an example of a nitrite detection method according to this embodiment.

[0012] As shown in Figure 1, the method for detecting nitrite according to this embodiment is: The first step (S100) involves reacting the sample with indole under hydrochloric acid conditions, A second step (S200) is performed to measure the absorbance of the sample obtained in the first step in the wavelength range of 470 nm to 510 nm, Includes.

[0013] Furthermore, the method for detecting nitrite according to this embodiment is The process may also include a third step (S300) in which the amount of nitrite in the sample is quantified based on the absorbance measured in the second step.

[0014] Each step will be explained in more detail.

[0015] The first step involves reacting the sample with indole under hydrochloric acid conditions to color only the nitrite in the sample. Since commercially available indole is usually in powder form, it is preferable to use indole dissolved in an organic solvent such as ethanol or methanol. In this case, the concentration of indole added, i.e., the concentration of indole in the color-developing solution, is preferably within the range of 0.0015% by mass or more and 0.035% by mass or less.

[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 within the range of 0.015% by mass or more and 3.5% by mass or less. The concentration of indole and / or hydrochloric acid affects the degree of color development when detecting nitrite, but by setting each within the above range, the color development can be visually confirmed when detecting nitrite, and the absorbance in the wavelength range of 470 nm to 510 nm, as described later, can be reliably measured.

[0017] As will be explained in more detail in the examples described later, nitrite reacts with indole under hydrochloric acid conditions to produce a compound (2-indolyl-3-oxime-3H-indole; hereafter sometimes referred to as a red pigment compound) that has a maximum absorbance peak in the wavelength region of approximately 492 nm to 495 nm.

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

[0019] In addition to nitrite (nitrite ion), urine also contains urea, ammonia, sodium ion, potassium ion, and nitrate ion. In addition, albumin (for example, patients with reduced kidney function) and glucose (for example, diabetic patients) also exist. Although it will be described in more detail in the examples described later, in the detection method according to the present embodiment, the above components do not generate a chromogenic compound. Therefore, it can be said that this is a method capable of specifically coloring and detecting only nitrite.

[0020] Among the above components, ammonia, sodium ion, potassium ion, and nitrate ion also exist in water. Therefore, the detection method in the present embodiment can also be applied to a method for detecting nitrite in water, for example, in water quality inspection applications.

[0021] From the viewpoint of the lower limit of quantitative absorbance, etc., it is preferable that the concentration of nitrite is 0.1 mg / L or more for the red pigment compound generated in the first step. Although it will be described in detail later, this red pigment compound has a sufficient correlation within the range where the concentration of nitrite is 0.1 mg / L or more and 25 mg / L or less. Therefore, in the third step, it is also possible to quantify the amount of nitrite in the sample by appropriately diluting and measuring based on the absorbance measured in the second step.

[0022] The detection method of nitrite according to the present embodiment uses indole and hydrochloric acid as detection reagents. Compared with the conventional Griess method using sulfanilamide and sulfanilamide, the detection method according to the present embodiment can suppress the cost related to the reagents, so it can be said that it is an inexpensive detection method.

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

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

[0025] Sample for Example 1 was obtained by adding 300 μL of 0.01% indole (solvent: ethanol) to 300 μL of 100 mg / L nitrite ion standard solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: product code 146-06453, diluted 10 times with pure water), mixing, and then adding 300 μL of 10% hydrochloric acid and mixing, and letting it stand at room temperature for 5 minutes. 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 obtained samples were extracted with chloroform, then concentrated, and separated by thin-layer chromatography (TLC). The separated samples were then subjected to imaging mass spectrometry in positive ionization mode using Laser Desorption Ionization Mass Spectrometry (LDI-MS: SolariX (Bruker Daltonics Inc.), magnet: 9.4T).

[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 the first step. Specifically, it was found that in the first step, nitrite and indole reacted according to the following formula (1) as the main reaction to obtain 2-indolyl-3-oxime-3H-indole.

[0027] [ka]

[0028] Furthermore, the absorption spectrum of the red pigment compound was measured for the sample from Example 1 using a U-2900 spectrophotometer (manufactured by Hitachi High-Tech 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 Figure 2, the vertical line indicating a wavelength of 490 nm is shown as a dashed line.

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

[0030] (Example 2) Next, we will describe an example in which it was confirmed that the nitrite detection method according to this embodiment does not produce color when other components are present 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 a 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 aqueous solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: product code 213-00173), Comparative Example 5: 1 g / dL albumin aqueous solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: product code 015-27053), 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 aqueous solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: product code 049-31165), I used it.

[0033] Upon visual inspection of the color development of the obtained sample from Example 1 and the samples from each comparative example, it was found that none of the samples other than the sample from Example 1, which used nitrite, developed color.

[0034] In other words, the detection method according to this embodiment is capable of coloring only nitrites in urine or water, and is capable of detecting only nitrites.

[0035] (Example 3) Next, we will describe an example that confirms 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 the nitrite ion standard solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: product code 146-06453) was diluted with pure water to the specified concentrations (0.1 mg / L, 0.5 mg / L, 1 mg / L, 5 mg / L, 10 mg / L, 25 mg / L).

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

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

[0039] As is clear from Figure 3, it was found that there is a sufficient correlation (proportional relationship) between nitrite concentration and absorbance within 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 color, making nitrite detection possible. However, when the nitrite concentration exceeds 25 mg / L, there is no proportional relationship between absorbance and nitrite concentration. Therefore, the amount of nitrite (or nitrite concentration) in the sample can be quantified by appropriately diluting the sample based on 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 Grease method which uses naphthylethylenediamine and sulfanilamide.

[0041] Furthermore, since the nitrite detection method according to this embodiment does not produce color 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 and the like.

[0042] Furthermore, the nitrite detection method according to this embodiment is also applicable to the quantitative determination of nitrites because there is a sufficient correlation between the absorbance in the wavelength range of 470 nm to 510 nm and the concentration of nitrites.

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

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

Claims

1. The first step involves reacting a liquid sample with indole under hydrochloric acid conditions, A second step involves measuring the absorbance of the compound obtained in the first step in the wavelength range of 470 nm to 510 nm, A method for detecting nitrites, including [specific type of nitrite].

2. The method further includes 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.

3. The method for detecting nitrite according to claim 1, wherein the concentration of indole in the compound obtained in the first step is in the range of 0.0015% by mass or more and 0.035% by mass or less, and the concentration of hydrochloric acid is in the range of 0.015% or more and 3.5% or less.

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