JP6008395B2 - Lithium reagent composition, lithium reagent kit, and lithium ion measurement method. - Google Patents

Description

  The present invention relates to a lithium reagent composition, a lithium reagent kit, and a lithium ion measurement method used for quantitative measurement of lithium in an aqueous solution such as a biological sample and an environmental sample.

Conventionally, lithium-containing mood stabilizers and antidepressants have been widely used because they are effective, but it is necessary to control the lithium concentration in the serum within an appropriate range upon administration.
As a lithium reagent composition used for quantitative determination of lithium in liquid, lithium carbonate tablets (oral administration) are widely used as a treatment for bipolar disorder (manic depression) or as an anti-depressant and mood stabilizer. It is prescribed. Lithium carbonate (Li ₂ CO ₃ ) has the feature that the administration effect does not appear unless it is prescribed up to the blood concentration that causes lithium poisoning, and since the treatment area and the poisoning area are very close, drug blood concentration monitoring Is designated as a necessary item (TDM).

More specifically, it should always be adjusted so that the lithium concentration in the sample plasma of the patient is 0.6-1.2 mEq / L, which is too low when the lithium concentration in the serum is below 0.6 mEq / L. If the dose is too high, there will be no antidepressant effect, and conversely, the plasma concentration will generally exceed 1.5 mEq / L, and if administered excessively, the concentration may increase, causing lithium poisoning, and overdose may be fatal. Symptoms of addiction, including dysarthria, nystagmus, kidney damage, convulsions. If these potentially dangerous signs are seen, treatment must be discontinued, plasma concentrations remeasured, and measures taken to alleviate lithium intoxication.
Thus, although lithium salt antidepressants are effective in the treatment of depressed patients, etc., excessive doses can cause serious damage, so when lithium-containing antidepressants are administered, serum is always in the serum. It is essential to monitor the lithium concentration of 0.6 to 1.2 mEq / L.

For this reason, conventionally, quantitative measurement of lithium in serum is required, and development of a liquid reagent composition for clinical tests that enables colorimetric measurement of lithium has been underway.
As this prior art, Patent Document 1 discloses a reagent composition for measuring the concentration of lithium in a biological specimen using a primary color cryptandinophore.
Patent Document 2 discloses a macrocyclic compound having a pyrrole ring, which is an analytical reagent that reacts with lithium ions by bonding eight bromine (Br) atoms to the β-position of the pyrrole ring.

  Non-Patent Document 1 discloses that lithium ions can be detected and separated by a compound in which all hydrogen bonded to carbon of tetraphenylporphyrin is replaced with fluorine.

JP-A-7-113807 European Patent No. 1283986 (B1) Analytical Chemistry Vol.51, No.9, PP.803-807 (2002) [Synthesis of F28 tetraphenylporphyrin and its application to separation and detection of lithium ion] Kenji Koyanagi and Masaaki Tabata

Several conventional lithium reagent compositions are known, but the composition is poisonous or deleterious, the drug substance is unstable and expensive, and most drug substances do not dissolve in water or When dissolved, it deactivates and does not develop color, and the color development reaction is slow.
The technique disclosed in Patent Document 2 that is said to have overcome these problems enables a color development method, but because the color development sensitivity is too high, the specimen needs to be diluted and the specification of the reagent composition is pH 11 or higher. Therefore, it is easily altered by CO ₂ in the air, the measurement data is unstable, and when the pH is 11 or more, only a concentrated hydroxide solution such as sodium hydroxide or potassium hydroxide can be used. It cannot be maintained, and since these are deleterious objects, they are evasive for the user and are difficult to handle, and the actual storage requires a dedicated container rather than a general purpose. In order to make up for these defects, there was a problem that the mechanical equipment was large and dedicated equipment was required, and the versatility was lacking. For this reason, there is a problem that it is difficult to apply to on-site monitoring and POCT (Point Of Care Testing).

By the way, although the reagent composition for the purpose of quantifying lithium in Patent Document 1 uses a compound that is completely different from the present invention, it can be used only at pH 12, and as described above, it is pH 11 or higher. As a result, only concentrated hydroxide solutions such as sodium hydroxide and potassium hydroxide can be used. These are deleterious substances, which are difficult to handle for the user. There was a problem that equipment was necessary and lacked versatility.
In addition, Koyanagi et al., A non-patent document 1, discloses that F28 tetraphenylporphyrin can be used to separate and detect lithium ions. However, solvent extraction using chloroform, which is oily and poisonous. Otherwise, lithium could not be detected / separated. Above all, there is a problem that lithium in an aqueous solution cannot be directly quantified without complicated pretreatment, and in particular, lithium ions in serum cannot be measured quickly and quantitatively. As described above, it is difficult to detect lithium ions in an aqueous solution using F28 tetraphenylporphyrin, and it has been difficult to measure the concentration quantitatively.

  The present invention has been made in view of the above-described problems, and intends to provide a lithium reagent composition and a lithium reagent kit capable of measuring the concentration (quantitative) of lithium in an aqueous solution such as a biological sample or an environmental sample. In addition, an object of the present invention is to provide a method for measuring lithium ions by a lithium reagent composition that can measure the concentration of lithium immediately with a simple colorimeter and that can be visually judged for screening.

で表される化合物と、水に混合し得る有機溶剤と、ｐＨ調節剤と、トリエタノールアミンとを包含した水溶液とすることを特徴とするリチウム試薬組成物である。
請求項２の発明は、請求項１に記載のリチウム試薬組成物に、ポリオキシエチレンオクチルフェニルエーテル(商標登録：TritonX-100 )等の安定剤を含有したことを特徴とするリチウム試薬組成物であり、請求項３乃至請求項５は後述する具体的な安定剤を指定したものである。
In order to solve the above problems, the invention of claim 1 is a structural formula in which all hydrogen bonded to carbon of tetraphenylporphyrin is replaced with fluorine.

A lithium reagent composition characterized by comprising an aqueous solution containing an organic solvent that can be mixed with water, a pH adjuster, and triethanolamine .
The invention according to claim 2 is a lithium reagent composition characterized in that the lithium reagent composition according to claim 1 contains a stabilizer such as polyoxyethylene octylphenyl ether (trademark registration: TritonX-100). Yes, claims 3 to 5 specify specific stabilizers to be described later.

A lithium compound in an aqueous solution such as a biological sample or an environmental sample develops a color as a chelating agent (coloring agent) obtained by replacing all lithium bonded to carbon of the lithium reagent composition, particularly tetraphenylporphyrin, with fluorine.
Although it is difficult to obtain a color change from yellow to red, which is a color reaction between the F28 tetraphenylporphyrin compound and lithium ions, the serum lithium concentration ranges from 0.6 mg / dL to 2.0 mg / dL (0.9 mM to 3 In the examples of the present invention, the concentration of the compound of F28 tetraphenylporphyrin is 0.1 to 1.0 g / in the above-described lithium concentration range. It has also been found that accurate measurement is possible when L is set to 0.5 g / L.
Regarding the pH adjuster of the present invention, on the acidic side of pH less than 5.0, the color changing agent (chelating agent) of the present invention, the F28 tetraphenylporphyrin compound and lithium ions do not bind, so no color change occurs. Quantification of lithium is difficult. Further, when the pH is between 5 and 7, the color former and lithium ions react specifically, but the color development rate is slow. On the other hand, at pH 8 to 11, the color former and lithium ion react quickly and a stable color complex can be obtained. On the alkaline side exceeding pH 11, the temporal stability of the color tone of the chelating agent and the generated color complex is poor. This is due to the fact that the pH tends to fluctuate due to absorption of carbon dioxide in the air. Therefore, the pH adjuster of the lithium reagent composition requires a pH adjuster having a pH in the range of 7 to 12, or a pH buffer as the pH adjuster, and more preferably a pH of 8 to 11. It is necessary to use a regulator and a pH buffer.
The pH adjusting agent includes sodium hydroxide, potassium hydroxide, an alkaline agent containing ammonia, acetic acid, phosphoric acid, citric acid, carbonic acid, bicarbonate, oxalic acid, hydrochloric acid, an acid agent containing nitric acid, and salts thereof. The pH adjusting agent may be a pH buffering agent selected from citric acid, carbonic acid, bicarbonate, phosphoric acid, succinic acid, phthalic acid, ammonium chloride, sodium hydroxide, potassium hydroxide, Good buffer. MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine, Bicine, TAPS, CHES, CAPSO, CAPS, and these Use one selected from salts.
With these inclusions, the lithium reagent composition can perform a specific color reaction with respect to lithium in the range of pH 5 to pH 12.

The solvent (polar solvent) of the present invention is essential to be an organic solvent that can be mixed with water, but the organic solvent is mainly used as long as it can be uniformly mixed with an aqueous solution such as serum, plasma, or eluate that is the subject. Or an aqueous solution to which an organic solvent has been added. This is because when the concentration of lithium in a specimen is measured with a general-purpose automatic analyzer or ultraviolet-visible spectrophotometer, the specimen is an aqueous solution, and the reagent composition is preferably an aqueous solution as well. is there.
The organic solvent is selected from dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and dimethylacetamide (DMA).

In the reagent composition of the present invention, a stabilizer is mixed in an actual product, but in the present invention, a surfactant is used as a stabilizer. This surfactant has the effect of enhancing the dispersibility of F28 tetraphenylporphyrin and preventing suspension from the sample during the color development reaction, so it is necessary to incorporate a stabilizer to obtain these effects. is there.
These stabilizers are nonionic surfactants or anionic surfactants, and nonionic surfactants include sorbitan fatty acid esters, pentaerythritol fatty acid partial esters, propylene glycol monofatty acid esters, glycerin fatty acid monoesters. , Polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene polyoxypropylene glycol, polyoxyethylene fatty acid partial ester, polyoxyethylene sorbitol fatty acid partial ester, polyoxyethylene fatty acid ester, fatty acid diethanolamide, fatty acid monoethanol Amides, polyoxyethylene fatty acid amides, polyoxyethylene octyl phenyl ether (trademark registration: TritonX-100), p-nonylphenoxypolyglycidol And those selected from these salts are used.
Preferred nonionic surfactants include polyoxyethylene octyl phenyl ether (such as Triton X-100 (registered trademark)), p-nonylphenoxypolyglycidol and the like.

  Examples of the anionic surfactant as a stabilizer include an alkyl sulfate ester salt, a polyoxyethylene alkyl ether sulfate ester salt, a polyoxyethylene phenyl ether sulfate ester salt, an alkylbenzene sulfonate, and an alkane sulfonate. Representative examples include sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, sodium polyoxyethylene alkylphenyl ether sulfate, and salts thereof.

The lithium reagent composition of the present invention avoids interfering with the measurement of lithium concentration by ions other than lithium coexisting in the sample, or suppresses oxidation of the reagent composition and imparts its storage stability. One kind or a plurality of kinds of masking agents may be contained in the composition. However, if there are few ions other than lithium, it is not necessarily included.
Triethanolamine is the most suitable masking agent to be added to these lithium reagent compositions, but ethylenediamine, N, N, N ′, N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), pyridine, 2,2 -Bipyridine, propylenediamine, diethylenetriamine, diethylenetriamine-N, N, N ', N ", N" -pentaacetic acid (DTPA), triethylenetetraamine, triethylenetetramine-N, N, N', N ", N"', N "'-hexaacetic acid (TTHA), 1,10-phenanthroline, dipotassium ethylenediaminetetraacetate (EDTA · 2K ), O, O'-bis (2-aminophenyl) ethylene glycol-N, N, N ' , N'-tetraacetic acid (BAPTA), N, N-bis (2-hydroxyethyl) glycine (Bicine), trans-1,2-diaminocyclohexane-N, N, N ', N'-tetraacetic acid (CyDTA) , O, O'-bis (2-aminoethyl) ethylene glycol-N, N, N ', N'-tetraacetic acid (EGTA), N- (2-hydro Sill) iminodiacetic acid (HIDA), iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), nitrilotris methyl phosphate (NTPO) and is believed to be possible those selected from the salts. However As mentioned above, preferably triethanolamine is optimal.

  The lithium reagent composition of the present invention may include a preservative to prevent degradation by microorganisms. An antiseptic | preservative is not specifically limited, For example, sodium azide, Procline (trademark) etc. can be used. The concentration of the preservative is not particularly limited, and when sodium azide is used, it may be a concentration generally used as a preservative, for example, about 0.1% by mass with respect to the reaction solution. However, preservatives are usually prescribed for products intended for long-term storage.

で表される化合物と、前記水に混合し得る有機溶剤と前記ｐＨ調節剤と、前記マスキング剤を第二試薬とし、これら両試薬をセパレートにして保存し、測定直前に両試薬を混合してリチウム試薬組成物として使用するリチウム測定試薬キットとすることができる。
In order to make it possible to preserve the function of the lithium reagent composition for a long period of time, the invention of claim 6 includes the pH regulator and the masking agent in the composition of the lithium reagent composition of claim 1 . One reagent,
Structural formula in which all hydrogen bonded to carbon of tetraphenylporphyrin is replaced with fluorine.

The compound represented by the above, the organic solvent that can be mixed in the water, the pH adjuster, the masking agent as a second reagent, both these reagents are stored separately, and both reagents are mixed immediately before the measurement. It can be set as the lithium measuring reagent kit used as a lithium reagent composition.

According to the seventh and eighth aspects of the present invention, in the method for measuring lithium ions of a lithium reagent composition, the serum and plasma test samples are obtained by replacing all hydrogen bonded to carbon of tetraphenylporphyrin with fluorine. a compound represented by the structural formula as a chelating agent, an organic solvent which can be mixed in water, and pH adjusting agent, an aqueous solution which includes a triethanolamine, to come in contact with serum and plasma test sample, serum and plasma lithium and the structural formula (formula 1) concentration or change in color tone of the color of the lithium complex which is more formed in the reaction with the compound represented by the test sample, in particular, coloration color change from yellow to red (color Lithium ion measurement method characterized by measuring the concentration of lithium ions in an aqueous solution mixed with serum and plasma test samples by reading the reaction) visually or with a colorimeter Is the law.
The lithium reagent composition of claims 1 to 6 measures the absorbance of the lithium complex and its spectrum, and similarly calculates the quantitative value of the unknown sample using that of the lithium standard sample of known concentration as the reference concentration. In particular, in the coloration of the lithium complex, and in its spectrum, the sensitivity is measured using a wavelength band of 530 nm to 560 nm in the wavelength range of 550 nm or in the vicinity thereof, or the wavelength of 565 nm in the vicinity of the wavelength of 570 nm or in the vicinity thereof. It is preferable to calculate the concentration of lithium by measuring the sensitivity in the wavelength band from 650 nm to 650 nm.

The measurement method is to come in contact with serum and lithium reagent composition of plasma test samples described above, color development of lithium complexes, and their absorbance, or by measuring the spectrum, wavelength 550nm at its spectrum or wavelength of near It is preferable that the sensitivity is measured using the wavelength band of 530 nm to 560 nm as a measurement wavelength, or the quantitative value of lithium is calculated by measuring the sensitivity of the wavelength band of 565 nm to 650 nm at or near the wavelength of 570 nm.
The linearity of the calibration curve is better in the wavelength band of 550 nm or in the vicinity of the wavelength range from 530 nm to 560 nm than in the case where the Soret band is set as the photometric wavelength, so that a simple colorimeter or spectrophotometer can be used. The density can be easily calculated, and the color tone changes vividly from yellow to red, so that the density level can be determined visually. Therefore, the conventional measurement of lithium concentration requires a large dedicated device. However, the lithium concentration can be measured with a portable colorimeter or a widely used ultraviolet-visible spectrophotometer, and configured as a POCT kit. You can also.

  According to the lithium reagent composition, the lithium reagent kit, and the lithium ion measurement method of the present invention, the concentration of lithium in an aqueous solution such as a biological sample or an environmental sample can be measured, and the calibration curve by the lithium reagent composition used in the present invention Is linear in a practical range where the lithium concentration is 0.6 to 1.2 mEq / L, and the concentration can be obtained by simple calculation using the numerical values of a colorimeter and an ultraviolet-visible spectrophotometer. Therefore, the lithium concentration of a serum specimen that is a biological sample can be quickly quantified with a popular spectrophotometer, and the information can be used as a management index for TDM treatment, for example. Moreover, it is possible to quantitatively analyze a large number of samples in a short time by applying to an automatic clinical chemistry analyzer.

  In addition, although the spectroscopic measurement is possible by adjusting the pH of the lithium reagent composition in the range of pH 5 to pH 12, the chelating agent of the present invention (F28 tetraphenylporphyrin) and lithium ions are bound on the acidic side below pH 5. No color change depending on the lithium concentration occurs. On the alkaline side above pH 12, the chelating agent and the color complex formed have poor color tone stability. Further, the pH tends to fluctuate due to absorption of carbon dioxide in the air, which also adversely affects the stability of the color tone. When the pH is between 5 and 7, the chelating agent and lithium ions bind and develop a specific color as a lithium complex, but the color development rate is slow, and between pH 8 and 11 the chelating agent and lithium ions bind quickly. Color is specific and stable. Therefore, the pH is more preferably 8-11.

Furthermore, the tetraphenylporphyrin metal complex has a typical spectral region in the vicinity of 380 nm to 460 nm at which the maximum sensitivity, which is called the Soret band, can be used as the photometric wavelength. Since the sensitivity is too large with respect to the concentration range, a sample dilution operation is necessary, which complicates the operation and increases the size of the measurement device due to the addition of a dilution device and the like.
On the other hand, in the present invention, the photometric wavelength is 550 nm, which is a wavelength several times lower in sensitivity than the Soret band wavelength, or a wavelength band from 530 nm to 560 nm in the vicinity thereof, so that it is optimal for the concentration contained in the specimen. By obtaining the sensitivity, a diluting operation or a complicated operation such as a diluting device and accompanying equipment are unnecessary. Furthermore, since the wavelength band of the present invention has better linearity of the calibration curve than the case where the Soray band is the photometric wavelength, the calculation of the concentration from the measured value by a simple colorimeter or ultraviolet-visible spectrophotometer Since the color tone vividly changes from yellow to red, it is possible to determine the density level visually.
In addition, when the Soret band is set to the photometric wavelength, there is a concern about the influence on the lithium quantitative value caused by other organic substances and coloring components overlapping with the color tone of the wavelength band, such as nitrate ion, creatinine, bilirubin, biliverdin, hemolyzed hemoglobin, etc. However, when the wavelength band according to the present invention is set to the photometric wavelength, the influence is small, and a more accurate lithium concentration can be obtained.
Therefore, the conventional measurement of the lithium concentration requires a large dedicated device. However, according to the present invention, the lithium concentration can be measured with a portable colorimeter and can be configured as a POCT kit.

It is a figure of the reference calculation table | surface of the appropriate amount density | concentration of F28 tetraporphyrin of this invention. The graph of the experimental result in the ultraviolet-visible spectrophotometer of Example 1 in this invention, The figure of the graph of the lithium concentration calibration curve according to photometric wavelength in Example 1 in this invention, The graph of the spectrum change (coloring reaction) of F28 tetraphenylporphyrin-lithium complex formation in Example 1 in the present invention, The graph of the correlation test result of the serum sample measured value of Example 1 and the atomic absorption method (conventional method) measured value in the present invention, [Table 1] of comparison of measured values by an automatic analyzer using control serum as a sample [Table 2] of visual lithium detection of the present invention, FIG. 4 is a graph of the absorbance spectrum in Example 1 of the present invention; [Table 3] for comparison of measured values due to differences in the organic solvent of the present invention, [Table 4] for comparison of measured values due to differences in stabilizers in the present invention It is a figure of [Table 5] of the comparison of the measured value by the difference in a masking agent in this invention.

The present inventors diligently studied a lithium reagent composition capable of more easily and quantitatively measuring the lithium concentration in serum and plasma, and obtained a macrocyclic compound having a pyrrole ring, whose production method was disclosed in Non-Patent Document 1 described above. The present invention was conceived by paying attention to the following structural formula (hereinafter referred to as F28 tetraphenylporphyrin) in which all hydrogen bonded to carbon of tetraphenylporphyrin was replaced with fluorine to make 28 fluorine atoms.

  Patent Documents 2 and 3 listed above as lithium reagent compositions using a macrocyclic compound having a pyrrole ring are macrocyclic compounds having a pyrrole ring, in which eight bromine (Br) atoms are bonded to the β-position of the pyrrole ring. An analytical reagent that reacts with lithium ions has been developed, but it is difficult to react with lithium ions unless it is alkaline at pH 11 or higher. However, if it is F28 tetraphenylporphyrin, it reacts even at pH 5 to pH 12. This F28 tetraphenylporphyrin is used as a chelating agent so that it can be used for quantitative measurement of lithium ions in an aqueous solution system. Examples of the lithium reagent composition of the present invention will be described below as a lithium quantitative measurement reagent. To do.

Example 1 (Sample 1)
In Example 1, a first reagent as a pH buffer solution was prepared, a second reagent was prepared as a coloring reagent, and both solutions were mixed immediately before measurement to prepare a lithium reagent composition. This is because both solutions may be prepared from the beginning, but the reagent is prevented from deteriorating due to long-term storage.
Here, a method for producing a reagent composition will be described.
First, a first reagent as a pH buffer solution is mainly prepared, and its composition is as follows.

[Example 1]
(1) First reagent (as stabilizer / buffer)
Chelating agent: None Organic solvent: None Stabilizer (dispersant: nonionic surfactant):
TritonX-100 (registered trademark)
(Polyoxyethylene octyl phenyl ether) 1.0% by weight
Masking agent: Triethanolamine 10 mM
In the above, 7% by weight of ammonium chloride was added to adjust the pH to 10, and 1 L with purified water was stored in a general-purpose storage container.
TritonX-100 (registered trademark) (polyoxyethylene octylphenyl ether) was adjusted to 1.0% by weight. However, if the amount is too small, turbidity is rarely generated at the time of measurement. Since it may occur or both factors may affect the reproducibility, the range of 0.1 to 5.0% by weight is good, and preferably 1.0% by weight.
The masking agent is triethanolamine (10 mM). However, if the amount is too small, the masking effect is reduced in a sample containing excessive impurities other than lithium ions, and if too large, the lithium ions themselves are masked. Since it may cause a measurement error, the range of 1.0 to 100 mM is good, preferably 10 mM.

Next, a second reagent is mainly prepared as a coloring reagent, and the composition thereof is as follows.
(2) Second reagent (as a color reagent)
Chelating agent: F28 tetraphenylporphyrin 0.5 g / L
Organic solvent: dimethyl sulfoxide (DMSO) 20% by weight
Stabilizer (dispersant: nonionic surfactant): TritonX-100 (registered trademark)
(Polyoxyethylene octyl phenyl ether) 1.0% by weight
Masking agent: Triethanolamine 10 mM
To this, MOPS (Good buffer) was added so that the concentration was 0.05 M (mol / L), the pH was adjusted to 7.0, and 1 L with purified water was stored in a general-purpose storage container.

By the way, in Example 1, although it is difficult to obtain a color development reaction of the F28 tetraphenylporphyrin compound, the concentration of lithium in serum in clinical examination is widely in the range of 0.6 mM to 3 mM. Accuracy is required. In the examples of the present invention, in the above-mentioned lithium concentration range, the concentration of the compound of F28 tetraphenylporphyrin is 0.1 to 1.0 g / L, preferably 0.5 g / L. It was also found that it can be measured accurately.
When the lithium concentration is in the range of 0.6 mM to 3 mM, the concentration of F28 tetraphenylporphyrin in the final reagent composition can be measured in the range of 0.1 to 1.0 g / L, preferably 0.5. g / L is good. If the amount is too small, the reaction between F28 tetraphenylporphyrin and lithium ions does not occur sufficiently. If the amount is too large, the absorbance of the blank derived from F28 tetraphenylporphyrin increases, so 0.5 g / L.
This point will be described in more detail. In this reaction, F28 tetraphenylporphyrin and lithium ions form a chelate complex having a molar ratio of 1: 1. Here, when a sample containing 3 mM of the lithium concentration in the sample is reacted under the conditions of Example 1 using this reagent composition, the lithium concentration in the reaction system is about 0.02 mM. Therefore, if the concentration of F28 tetraporphyrin that reacts 1: 1 is not more than 0.02 mM in the reaction system, lithium in the sample cannot be reacted without excess or deficiency.

In general, a complex formation reaction (coloring reaction) between a chelating agent and a metal ion requires a chelating agent (F28 tetraporphyrin) having a molar concentration of 10 to 10 times that of the target substance (lithium). As shown in the reference calculation table of the appropriate amount concentration of F28 tetraporphyrin, the reagent composition is configured so that the concentration of F28 tetraporphyrin at the time of reaction is from 10 to 10 times. In addition, the amount of reagent composition added and the amount of sample in the so-called dose at the time of measurement reaction vary slightly depending on the photometric model and target threshold value, so the chelating agent concentration in the reagent composition is 1X. A larger amount of 0.5 g / L, which is five times the amount of 0.1 g / L, can withstand wider measurement conditions. For example, in the case of measurement with a model having a small amount of sample, the amount of the sample is expected to be increased by about 2 to 5 times that of Example 1, so that the reagent as a 5-fold amount in advance is used. There is no shortage if the composition is 0.5 g / L. On the other hand, even if the chelating agent is charged in a molar ratio of 10 times or more, there is no kinetic significance given to the color development reaction, there is only a concern about an increase in the reagent blank value, and there is no advantage of adopting this.
As described above, it is only necessary to achieve the reaction molar ratio condition between the chelating agent and lithium. For example, when the concentration of the chelating agent (F28 tetraporphyrin) of the second reagent is 1.0 g / L, The amount of the second reagent added can be halved. Alternatively, when the amount of the sample is halved, the chelating agent concentration can be halved in the same manner.
As described above, in Example 1, F28 tetraphenylporphyrin was set to 0.5 g / L. As a result of considering that the reaction molar amount was satisfied and the reagent blank value was minimized, 0.1 to 0.1 was obtained. A range of 1.0 g / L is optimal.

Moreover, although dimethyl sulfoxide (DMSO) was 5 to 30 weight%, when too small, the dispersibility in the solution of F28 tetraphenylporphyrin will fall, and when too large, the ratio of the organic solvent in a reagent composition will increase. Therefore, it is preferably 20% by weight.
Here, F28 tetraphenylporphyrin in this example has the following structural formula in which all hydrogen bonded to carbon of tetraphenylporphyrin is replaced with fluorine.

(3) The preparation of a calibration curve using a lithium concentration known sample with a lithium reagent in which the first reagent and the second reagent are mixed will be described.
In Example 1, 720 μL of the first reagent (buffer solution) and 240 μL of the second reagent (coloring reagent) were added to 6 μL of the sample. In this case, the first reagent has a buffer capacity at pH 10, and the pH of the test solution when the first reagent, the second reagent, and the sample are mixed is approximately pH = 10.
In this way, by using F28 tetraphenylporphyrin as a chelating agent, it is possible to achieve a color development reaction in the range of pH 5 to 10, so that it constitutes a lithium measurement reagent having a strong pH buffering action of pH 10 or less. PH fluctuations due to absorption of CO _{2 in} the air can be reduced, and as a result, adverse effects on measured values can be avoided. Moreover, it became possible to preserve | save in a general purpose container by this.
The first reagent and the second reagent may be mixed at the same ratio immediately before use, and the mixed solution may be added to the sample in the same volume. In this case, 940 μL of the mixed solution is added to 6 μL of the sample, A test solution may be used.

After reacting the test solution of pH 10 obtained by adding the sample to the mixed reagent at room temperature for 10 minutes, the absorbance at 550 nm was measured using a UV-visible spectrophotometer (Hitachi U-3900 type) with the reagent blank as a control. The results are a graph of Li concentration mg / dL and absorbance in FIG. 2, and a graph of visible spectrum change in F28 tetraphenylporphyrin-lithium complex formation in FIG.
It is not the wavelength that gives the maximum sensitivity, which is the typical Soray band (near 380 nm to 460 nm) for tetraphenylporphyrin metal complexes, but the wavelength that gives the optimum sensitivity for the lithium concentration range in the serum sample, or its vicinity. By setting the wavelength range of 530 nm to 560 nm as the photometric wavelength, a diluting operation or a complicated operation such as a diluting device and accompanying equipment are not required.
Furthermore, as shown in the graph of Li concentration mg / dL and absorbance (light wavelength * 405 nm, × 415 nm, ● 550 nm) in FIG. 3, the above-mentioned wavelength band of 550 nm or a wavelength band of 530 nm to 560 nm in the vicinity thereof is Since the linearity of the calibration curve is better than when the band is the photometric wavelength, it is easy to calculate the density with a simple colorimeter or spectrophotometer, and the color tone changes vividly from yellow to red. Further, the density level can be determined visually. Therefore, the conventional lithium concentration measurement requires a large dedicated device, but according to the present invention, the lithium concentration can be measured with a portable colorimeter or a widely used ultraviolet-visible spectrophotometer. It can also be configured as.
However, in the graph of FIG. 3, the wavelength ● 550 nm is Example 1 itself, and the photometry wavelengths of the Soray band are * 405 nm and × 415 nm, the first reagent and the second reagent are added as in Example 1, but the sensitivity is Was too high, the sample was diluted 5 times and the measurement reaction was carried out using wavelengths of 405 nm and 415 nm. As can be seen from the graph of FIG. 3, calibration curves with wavelengths of 405 nm and 415 nm are not straight lines, but when 550 nm in this example is used as a photometric wavelength, a calibration curve with good linearity can be obtained.

Further, as shown in the graph of the spectrum change of F28 tetraphenylporphyrin-lithium complex formation shown in FIG. 4, the lithium concentration is 0.6 mg / dL, 1.2 mg / dL, 1.8 mg / dL, 2.4 mg / dL, It can be confirmed fairly clearly that the absorbance increases linearly with 3.0 mg / dL. In proportion to the lithium concentration, the peak at 415 nm (Sorley band) typical for porphyrin-metal complexes and the absorption peak at 550 nm shown in the figure increase and the absorption peak at 570 nm decreases. As described above, it is preferable to set the photometric wavelength to 550 nm because a calibration curve with good linearity can be obtained as described above.
Of course, although the absorbance is 550 nm in the first embodiment, the wavelength range from 540 nm to 560 nm may be used as the photometric range. Depending on the measuring instrument, there may be no photometric filter of 550 nm. In this case, the photometric wavelength may be set to 540 nm or 560 nm where sensitivity is generated in the vicinity thereof. As shown in the graph of absorbance due to lithium concentration in Example 1 of FIG. 4, the decrease in sensitivity at 570 nm is also quantitative with respect to the lithium concentration. Therefore, the absorbance difference (ΔAbs) can also be obtained using the reagent blank as a control. It can be used as a photometric wavelength.
Furthermore, depending on the sample of the patient specimen, a contaminant that interferes with the wavelength of 550 nm is generated, and if the error occurs in the data at the wavelength of 550 nm, the wavelength of 570 nm, or in the vicinity of 565 nm to 650 nm, is avoided. The photometric wavelength may be selected from the range, and the lithium concentration may be calculated using the decrease in sensitivity as the absorbance difference.

The following experimental results demonstrate that the lithium concentration can be measured almost accurately with the lithium reagent of Example 1 of the present invention.
[Experimental result of UV-visible spectrophotometer (Hitachi U-3900)]
The graph of FIG. 2 is a measurement test result with an ultraviolet-visible spectrophotometer (Hitachi U-3900 type). The abscissa X is a known lithium ion concentration (Li concentration mg / dL) prepared in advance, and the ordinate Y is a result of linear regression by plotting an absorbance difference at 550 nm by an ultraviolet-visible spectrophotometer.
It can be seen from the graph of FIG. 2 that the obtained absorbance is proportional to the lithium concentration, and a calibration curve with good linearity is drawn.

[Results of correlation test between the method of the present invention and the atomic absorption method using serum as a sample]
The graph of FIG. 5 shows the results of a correlation test between the measurement method in Example 1 described in FIG. 2 and the measured value of lithium concentration in the conventional atomic absorption method (conventional method) using the same serum as a sample. The vertical axis Y is a measured value of lithium concentration according to the present invention, and the horizontal axis X is a measured value of lithium concentration by atomic absorption method (conventional method). From the regression line shown in FIG. Correlation was shown. Therefore, it was shown that lithium in the serum sample could be correctly quantified by the ultraviolet-visible spectrophotometric determination method using the reagent composition of the present invention for the same serum sample.

[Comparison of measured values by automated analysis using control serum as a sample]
As control sera for which the lithium concentration was given, Precinorm U (Roche), Precipath U (Roche), Pasonorm H (SERO AS), Auto norm (SERO AS) Measured by a one-point end method using a biochemical automatic analyzer (Hitachi H-7700 type) as a photometric wavelength at 546 nm (wavelength mounted on this unit with a wavelength close to 550 nm).

(Device parameter)
Reagent: 0.24 mL
Sample: 0.005 mL
Photometric wavelength (main / sub): 546 nm / 700 nm
Metering time: 10 minutes Temperature: 37 ° C
1-point end / increase method

The experimental results under the above setting conditions are shown in [Table 1] in FIG. 6. The measured values in the examples of the present invention are in good agreement with the guaranteed values. It turns out that serum lithium can be measured sufficiently.

[Visual lithium detection method]
The results of visual observation of the test solution to be measured are shown in [Table 2] in FIG.
To 8 μL of the sample, 920 μL of a coloring reagent solution obtained by mixing the first reagent and the second reagent was added, and after reacting at room temperature for 10 minutes, the coloration was visually observed. Samples were compared using a standard color lithium standard solution and a control serum for each concentration level as color samples.
In each concentration range, a color change from yellow to red was confirmed, and the color of the control serum was in good agreement with the color sample. It can be seen that the lithium concentration in serum can be determined quickly and easily without using a special apparatus.
As described above, it can be seen that the lithium concentration can be measured almost accurately with the lithium reagent of Example 1 of the present invention.

Next, the composition is the same as in Example 1 and the preparation method is basically the same. However, in the lithium reagent composition, MOPS is added so that the first reagent is 0.1 M (mol / L), and the pH is 8.0. Prepared to 1 L with purified water. That is, the pH of the test solution in which the first reagent, the second reagent, and the reagent were mixed during the test was adjusted to be approximately pH = 8.
[Example 2]
(1) First reagent (as stabilizer / buffer)
Chelating agent: None Organic solvent: None Stabilizer (dispersing agent: nonionic surfactant): TritonX-100 (registered trademark)
(Polyoxyethylene octyl phenyl ether) 1.0% by weight
Masking agent: Triethanolamine 10 mM
Above, MOPS was added so that it might become 0.1M, it adjusted to pH8, and it was stored in the general purpose storage container as 1L with purified water.

(2) Second reagent (as a color reagent)
Chelating agent: F28 tetraphenylporphyrin 0.5 g / L
Organic solvent: dimethyl sulfoxide (DMSO) 20% by weight
Stabilizer (dispersing agent: nonionic surfactant): TritonX-100
(Registered trademark) (polyoxyethylene octylphenyl ether) 1.0 wt%
Masking agent: Triethanolamine 10 mM
To this, MOPS (buffering agent) was added so as to be 0.05 M, adjusted to pH 7.0, and stored in a general-purpose storage container as 1 L with purified water.

  In the same manner as in Example 1, the lithium concentration was measured by adding 720 μL of the first reagent (buffer solution) and 240 μL of the second reagent (coloring reagent) to 6 μL of the sample, reacting at room temperature for 10 minutes, and then observing an ultraviolet-visible spectrophotometer ( The absorbance at a wavelength of 550 nm was measured using a reagent blank as a control.

[Experimental result of UV-visible spectrophotometer (Hitachi U-3900)]
The graph of FIG. 8 is an experimental result with an ultraviolet-visible spectrophotometer (Hitachi U-3900 type). The abscissa X indicates a known lithium ion concentration (Li concentration mg / dL) prepared in advance, and the ordinate Y indicates It is the result of plotting the absorbance difference at 550 nm of the UV-visible spectrophotometer and performing linear regression.
It can be seen from the graph of FIG. 8 that the reagent composition constituted at pH 8, that is, the absorbance difference obtained even under the measurement condition of pH 8, is proportional to the lithium concentration and becomes a calibration curve with good linearity. It is that.

However, the reaction rate is somewhat slow under the measurement condition of pH 8, and quantitatively stabilizes in about 10 to 20 minutes. On the other hand, when the pH is 10, the reaction is completed within 10 minutes. Therefore, when the pH of the buffer system of the lithium reagent composition of the present invention is in the range of 5 to 10, a concentrated hydroxide solution such as sodium hydroxide or potassium hydroxide is mainly used as in the case of pH 11 or more. It is not necessary to use a buffered system, and handling is simple. Although the pH is set according to the needs of the user, it is possible to use a Good buffer, an ammonium chloride system, and a carbonic acid system that have a fast reaction rate and can maintain a sufficient buffering power when the pH is 10. In consideration of practicality, it is preferable to carry out the reaction in the pH 10 buffer system of Example 1 which reacts quickly and accurately.
As described above, the pH adjuster of the lithium reagent composition of the present invention requires a pH adjuster having a pH in the range of 7 to 12, or a pH buffer as a pH adjuster, and more preferably pH 8 to 11 is used, and more preferably, the pH adjuster and the pH buffer are used so that the pH is about 10.

Next, the selection of an organic solvent that can be mixed with water will be described. However, it is important that the solvent of the present invention is an organic solvent that can be mixed with water. Since it is an aqueous solution, it may be a liquid mainly composed of an organic solvent or an aqueous solution to which an organic solvent is added, as long as the components in the reagent composition are stabilized as an aqueous solution. In particular, when the lithium concentration in a specimen is measured with a general-purpose automatic analyzer or ultraviolet-visible spectrophotometer, basically, an aqueous solution to which an organic solvent is added is desirable.
Therefore, an organic solvent that can be mixed in water different from those in Examples 1 and 3 will be described in Example 3. In Example 3, the preparation method is basically the same as in Example 1, but the organic solvent of the second reagent in the lithium reagent is not dimethylsulfoxide (DMSO) (20 wt%) but dimethylformamide (DMF) as described below. ) (20% by weight).

[Example 3]
(1) First reagent (as buffer)
Chelating agent: None Organic solvent: None Stabilizer (dispersant: nonionic surfactant): TritonX-100
(Registered trademark) (polyoxyethylene octylphenyl ether) 1.0 wt%
Masking agent: Triethanolamine 10 mM
In the above, 7% by weight of ammonium chloride was added to adjust the pH to 10, and 1 L with purified water was stored in a general-purpose storage container.

(2) Second reagent (as a color reagent)
Chelating agent: F28 tetraphenylporphyrin 0.5 g / L
Organic solvent: dimethylformamide (DMF) 20% by weight
Stabilizer (dispersant: nonionic surfactant): TritonX-100
(Registered trademark) (polyoxyethylene octyl phenyl ether) 1.0 wt%
Masking agent: Triethanolamine 10 mM
To this, MOPS (buffering agent) was added so as to be 0.05 M, adjusted to pH 7.0, and stored in a general-purpose storage container as 1 L with purified water.

The organic solvent that can be mixed with water in Example 1 was dimethyl sulfoxide (DMSO) (20% by weight), and in Example 3, dimethylformamide (DMF) (20% by weight). The lithium ion concentration was measured as a lithium reagent composition in which dimethylacetamide (DMA) (20% by weight) was mixed as an organic solvent that could be mixed.
[Example 4]
(1) First reagent (as buffer)
Chelating agent: None Organic solvent: None Stabilizer (dispersant: nonionic surfactant): TritonX-100
(Registered trademark) (polyoxyethylene octyl phenyl ether) 1.0 wt%
Masking agent: Triethanolamine 10 mM
In addition, 7% by weight of ammonium chloride was added to adjust the pH to 10, and 1 L of purified water was stored in a general-purpose storage container.

(2) Second reagent (as a color reagent)
Chelating agent: F28 tetraphenylporphyrin 0.5 g / L
Organic solvent: Dimethylacetamide (DMA) 20% by weight
Stabilizer (dispersant: nonionic surfactant): TritonX-100
(Registered trademark) (polyoxyethylene octyl phenyl ether) 1.0 wt%
Masking agent: Triethanolamine 10 mM
To this, MOPS (buffering agent) was added so as to be 0.05 M, adjusted to pH 7.0, and stored in a general-purpose storage container as 1 L with purified water.

As described above, a reagent composition was prepared by changing dimethyl sulfoxide in the lithium reagent of Example 1 to dimethylformamide (DMF) in Example 3 and changing to dimethylacetamide (DMA) in Example 4. As shown in FIG. 9, lithium in the control serum sample was quantified by the same procedure as in Example 1. As shown in [Table 3] of FIG. 9, the measured values were measured using this measurement method and the conventional measurement method. Compared.
From the results of [Table 3] in [Comparison of measured values due to differences in organic solvent of the present invention] in FIG. The value is 0.83 mM (mmol / L), and the measured value according to the composition of 20% by weight of dimethylformamide (DMF), which is an organic solvent that can be mixed in water of Example 3, is 0.81 mM. The measured value by (DMF) 20% by weight composition is 0.82 mM, which is 95% or more consistent with the measured value by atomic absorption photometry of 0.82 mM. Therefore, F28 tetraphenylporphyrin can be uniformly dispersed with these organic solvents to form a liquid reagent composition, and lithium in an aqueous solution sample such as serum can be accurately quantified.

Next, selection of a stabilizer for the lithium reagent composition will be described based on experimental results. In Examples 5 to 7, the stabilizer of the lithium reagent group is basically the same as the preparation method of Example 1, but the stabilizer is a nonionic surfactant (Example 5) as described below. Only, anionic surfactant (Example 6) alone, or a combination of both (Example 7) was tested.
First, the composition of Example 5 in the case of only the nonionic surfactant (TritonX-100 (registered trademark) (polyoxyethylene octyl phenyl ether) (Example 5, the composition itself is the same as Example 1) will be described. .

[Example 5]
(1) First reagent (as buffer)
Chelating agent: None Organic solvent: None Stabilizer (dispersing agent: nonionic surfactant): TritonX-100
(Registered trademark) (polyoxyethylene octylphenyl ether) 1.0% by weight
Masking agent: Triethanolamine 10 mM
7% by weight of ammonium chloride was added to this to adjust to pH 10, and 1 L with purified water was stored in a general-purpose storage container.

(2) Second reagent (as a color reagent)
Chelating agent: F28 tetraphenylporphyrin 0.5 g / L
Organic solvent: dimethyl sulfoxide (DMSO) 20% by weight
Stabilizer (dispersant: nonionic surfactant): TritonX-100
(Registered trademark) (polyoxyethylene octyl phenyl ether) 1.0 wt%
Masking agent: Triethanolamine 10 mM
To this, MOPS (buffering agent) was added so as to be 0.05 M, adjusted to pH 7.0, and stored in a general-purpose storage container as 1 L with purified water.

Example 6 describes the composition in the case of only an anionic surfactant (sodium dodecyl sulfate (manufactured by Wako Pure Chemical Industries)) (Example 6).
[Example 6]
(1) First reagent (as buffer)
Chelating agent: None Organic solvent: None Stabilizer (dispersing agent: anionic surfactant only):
Sodium dodecyl sulfate (Wako Pure Chemical Industries) 1.0 wt%
Masking agent: Triethanolamine 10 mM
7% by weight of ammonium chloride was added to this to adjust the pH to 10, and 1 L of purified water was stored in a general-purpose storage container.

(2) Second reagent (as a color reagent)
Chelating agent: F28 tetraphenylporphyrin 0.5 g / L
Organic solvent: dimethyl sulfoxide (DMSO) 20% by weight
Stabilizer (dispersant: anionic surfactant)
Sodium dodecyl sulfate (Wako Pure Chemical Industries) 1.0 wt%
Masking agent: Triethanolamine 10 mM
To this, MOPS (buffering agent) was added so as to be 0.05 M, adjusted to pH 7.0, and stored in a general-purpose storage container as 1 L with purified water.

Example 7 describes the composition when the stabilizer of the lithium reagent composition is a combination of both a nonionic surfactant and an anionic surfactant (Example 7).
[Example 7]
(1) First reagent (as buffer)
Chelating agent: None Organic solvent: None Stabilizer (dispersant: nonionic surfactant and anionic surfactant):
(a) Nonionic surfactant: Triton-X100 (registered trademark)
(Polyoxyethylene octyl phenyl ether) 1.0% by weight
(b) Anionic surfactant: sodium dodecyl sulfate (manufactured by Wako Pure Chemical Industries) 1.0 wt%
Masking agent: Triethanolamine 10 mM
7% by weight of ammonium chloride was added to this to adjust to pH 10, and 1 L with purified water was stored in a general-purpose storage container.

(2) Second reagent (as a color reagent)
Chelating agent: F28 tetraphenylporphyrin 0.5 g / L
Organic solvent: dimethyl sulfoxide (DMSO) 20% by weight
Stabilizer (dispersant: nonionic surfactant and anionic surfactant):
(a) Nonionic surfactant: Triton-X100 (registered trademark)
(Polyoxyethylene octyl phenyl ether) 1.0% by weight
(b) Anionic surfactant: sodium dodecyl sulfate (manufactured by Wako Pure Chemical Industries) 1.0 wt%
Masking agent: Triethanolamine 10 mM
To this, MOPS (buffering agent) was added so as to be 0.05 M, adjusted to pH 7.0, and stored in a general-purpose storage container as 1 L with purified water.

Using the above Example 5, Example 6, and Example 7, the lithium concentration in the control serum sample was quantified in the same procedure as in Example 1. The experimental results are shown in [Table 4] of [Comparison of measured values due to differences in stabilizers in the present invention] of FIG.
From the experimental results shown in [Table 4] in FIG. 10, the measured value of only the nonionic surfactant (0.82 mM), the measured value of only the anionic surfactant (0.83 mM), and both stabilizers are used in combination. Measured value (0.82 mM) is 95% or more, and the measured value is almost the same regardless of the type of surfactant and how to use it together. In this case, it can be seen that these surfactants may be used in combination.

Next, selection of the masking agent for the lithium reagent composition will be described.
As a masking agent for the lithium reagent composition, the above-described examples have described the case of using only triethanolamine, but it will be described that ethylenediaminetetraacetic acid (EDTA) can also be used.
In the case of only triethanolamine as a comparison target, the lithium reagent composition of Example 5 was used, and only when ethylenediaminetetraacetic acid (EDTA) was used (Example 8) and when both masking agents were used together (Example 9). The composition will be described.

[Example 8]
When only dipotassium ethylenediaminetetraacetate (EDTA · 2K (potassium)) is used as a masking agent.
(1) First reagent (as buffer)
Chelating agent: None Organic solvent: None Stabilizer: Triton-X100 (registered trademark)
(Polyoxyethylene octyl phenyl ether) 1.0% by weight
Masking agent: Dipotassium ethylenediaminetetraacetate (EDTA / 2K manufactured by Dojin Chemical) 10 mM
7% by weight of ammonium chloride was added to this to adjust the pH to 10, and 1 L of purified water was stored in a general-purpose storage container.

(2) Second reagent (as a color reagent)
Chelating agent: F28 tetraphenylporphyrin 0.5 g / L
Organic solvent: dimethyl sulfoxide (DMSO) 20% by weight
Stabilizer: Triton-X100 (registered trademark)
(Polyoxyethylene octyl phenyl ether) 1.0% by weight
Masking agent: Dipotassium ethylenediaminetetraacetate (EDTA / 2K manufactured by Dojin Chemical) 10 mM
To this, MOPS (buffering agent) was added so as to be 0.05 M, adjusted to pH 7.0, and stored in a general-purpose storage container as 1 L with purified water.

[Example 9]
When combined with triethanolamine and dipotassium ethylenediaminetetraacetate (EDTA 2K (potassium)) as masking agents.
(1) First reagent (as buffer)
Chelating agent: None Organic solvent: None Stabilizer: Triton-X100 (registered trademark)
(Polyoxyethylene octyl phenyl ether) 1.0% by weight
Masking agent:
Triethanolamine 10 mM
Ethylenediaminetetraacetic acid dipotassium (EDTA / 2K manufactured by Dojin Chemical) 0.1 mM
7% by weight of ammonium chloride was added to this to adjust to pH 10, and 1 L with purified water was stored in a general-purpose storage container.

(2) Second reagent (as a color reagent)
Chelating agent: F28 tetraphenylporphyrin 0.5 g / L
Organic solvent: dimethyl sulfoxide (DMSO) 20% by weight
Stabilizer: Triton-X100 (registered trademark)
(Polyoxyethylene octyl phenyl ether) 1.0% by weight
Masking agent:
Triethanolamine 10 mM
Ethylenediaminetetraacetic acid dipotassium (EDTA / 2K manufactured by Dojin Chemical) 0.1 mM
To this, MOPS (buffering agent) was added so as to be 0.05 M, adjusted to pH 7.0, and stored in a general-purpose storage container as 1 L with purified water.

Using Example 8 and Example 9 described above, the lithium concentration in the control serum sample was quantified in the same procedure as in Example 1. The experimental results are shown and described in [Table 5] of [Comparison of measured values due to differences in masking agent in the present invention] of FIG.
From the experimental results shown in [Table 5] in FIG. 11, the measured value of triethanolamine only (0.83 mM), the measured value of only dipotassium ethylenediaminetetraacetate (EDTA · 2K manufactured by Dojindo Chemical) (0.83 mM), both It can be seen that the measured value (0.82 mM) when masking agent is used in combination is 95% or more, and the measured value is almost the same regardless of the type of main masking agent and how to use it. It can be seen that these masking agents can be used as appropriate in order to prevent the deterioration of the reagent caused by a slight amount of mixed metal ions and to deal with the case where the sample contains an excessive amount of contaminating ions.

As described above, according to each embodiment of the present invention, the determination of lithium in an aqueous solution such as a biological sample or an environmental sample can be immediately measured with a simple colorimeter, and visually determined. Is possible.
Of course, the present invention is not limited to the above-described embodiments as long as the features of the present invention are not impaired. For example, in Example 1 to Example 9, the reagent composition for measuring the lithium concentration was separated from the first reagent and the second reagent to enable long-term storage. Of course, a single reagent in which the first reagent and the second reagent are mixed may be prepared and used.

Claims (8)

Structural formula in which all hydrogen bonded to carbon of tetraphenylporphyrin is replaced with fluorine.

Lithium reagent composition characterized by making it the aqueous solution containing the organic solvent which can be mixed with water, a pH adjuster, and a triethanolamine.   The lithium reagent composition according to claim 1, wherein a stabilizer is contained in the lithium reagent composition according to claim 1.   3. The lithium reagent composition according to claim 2, wherein the stabilizer in claim 2 is a nonionic surfactant and / or an anionic surfactant. The nonionic surfactant includes sorbitan fatty acid ester, pentaerythritol fatty acid partial ester, propylene glycol monofatty acid ester, glycerin fatty acid monoester, polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene polyoxypropylene glycol. , Polyoxyethylene fatty acid partial ester, polyoxyethylene sorbitol fatty acid partial ester, polyoxyethylene fatty acid ester, fatty acid diethanolamide, fatty acid monoethanolamide, polyoxyethylene fatty acid amide, polyoxyethylene octylphenyl ether (trademark registration: TritonX-100 ), p-nonylphenoxy polyglycidol and re according to claim 3, characterized in that it is selected from these salts Tium reagent composition. The anionic surfactant includes an alkyl sulfate ester salt containing sodium dodecyl sulfate, a polyoxyethylene alkyl ether sulfate ester salt, a polyoxyethylene phenyl ether sulfate sodium salt containing sodium polyoxyethylene alkylphenyl ether sulfate, and dodecylbenzene. 4. The lithium reagent composition according to claim 3 , wherein the composition is selected from alkylbenzene sulfonates and alkane sulfonates containing sodium sulfonate. Of the composition of the lithium reagent composition according to claim 1, an aqueous solution containing the pH adjuster, the masking agent, and the stabilizer is used as a first reagent,
Structural formula in which all hydrogen bonded to carbon of tetraphenylporphyrin is replaced with fluorine.

An aqueous solution containing a compound represented by: an organic solvent that can be mixed with water, the pH adjuster, the triethanolamine, and the stabilizer, as a second reagent,
A lithium reagent kit characterized in that both of these reagents are stored separately, and both reagents are mixed immediately before measurement and used as the lithium reagent composition according to claim 1. Structural formula in which all hydrogen bonded to carbon of tetraphenylporphyrin is replaced with fluorine in serum and plasma test samples

A compound in represented by an organic solvent which can be mixed into water, and p H adjusting agent, an aqueous solution which includes a triethanolamine, a serum and plasma test sample against corrosion, lithium serum and plasma test sample An aqueous solution mixed with serum and plasma test samples by visual or colorimetric reading of changes in color density or color tone of the lithium complex formed by the reaction of the compound represented by the structural formula (Chemical Formula 1) A method for measuring lithium ions, comprising: measuring a concentration of lithium ions contained in the solution. Changes in color in the lithium complexes, lithium ion measuring method according to claim 7, characterized in that the color change from yellow to red.

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