JP7210899B2 - Method for producing purified 4-aminoantipyrine and produced 4-aminoantipyrine - Google Patents

Description

The present invention relates to a method for producing purified 4-aminoantipyrine (hereinafter also referred to as 4AA) and the produced 4-aminoantipyrine. 4-Aminoantipyrine obtained by the production method of the present invention can be applied to a composition for measuring biological components and a method of analyzing biological components using the composition in clinical diagnosis. Furthermore, the 4-aminoantipyrine of the present invention can be applied to a composition for measuring a biological component using a redox reaction, a method of analyzing a biological component using the composition, and the like.

Conventionally, in clinical diagnosis, measurement of biological components by an enzymatic method has been performed, and in particular, a method using an oxidase-peroxidase-redox coloring reagent (hereinafter also referred to as a coloring agent) system, that is, a measurement target in a specimen. A widely used method is to cause an enzymatic reaction of a substance to generate hydrogen peroxide, which is then reacted with a coloring agent in the presence of peroxidase for colorimetric determination. (Non-Patent Document 1)

Examples of the redox coloring reagent system include a method using a hydrogen donor and a coupler. A typical example is the Trinder method in which a hydrogen donor and a coupler are oxidatively condensed with hydrogen peroxide in the presence of peroxidase to form a dye. As a coupler used in this method, for example, 4-aminoantipyrine (hereinafter also referred to as 4-aminoantipyrine) is known.

BUNSEKI KAGAKU Vol. 45, No. 2, pp. 111-124 (1996)

An object of the present invention is to obtain purified 4-aminoantipyrine. 4-Aminoantipyrine obtained by the production method of the present invention can be applied to a composition for measuring biological components, which has superior sensitivity in the method for measuring biological components. Furthermore, 4-aminoantipyrine obtained by the production method of the present invention can be applied to a composition for measuring biological components, which has superior sensitivity in measuring methods for measuring biological components using oxidation-reduction reactions.

The present inventor prepared an enzyme-peroxidase-chromogenic agent-based biocomponent measurement reagent using 4-aminoantipyrine and a hydrogen donor, and experienced a decrease in sensitivity of the reagent for unknown reasons when using it for biocomponent measurement. .
Since the degree of decrease in sensitivity varied depending on the lot of the reagent composition prepared for measurement, the present inventors conducted various studies on the reagent composition. As a result, it was unexpectedly found that 4-aminoantipyrine contained a very small amount of a substance called 4-hydroxyantipyrine (hereinafter also referred to as 4HA). When 4-hydroxyantipyrine is present in the reagent, structurally 4-hydroxyantipyrine does not undergo a coupling reaction with a hydrogen donor, but in the presence of hydrogen peroxide, 4-hydroxyantipyrine reacts with peroxidase, consuming hydrogen peroxide. be done. Since the hydrogen peroxide generated by reacting the substance to be measured in the sample with an enzyme is consumed by 4-hydroxyantipyrine, the amount of color developed by the reaction of the original 4-aminoantipyrine-hydrogen donor was found to decrease, resulting in decreased sensitivity. Then, by setting the concentration of 4-hydroxyantipyrine in the reagent composition to a certain level or less, the sensitivity is at the designed level in the measurement method for measuring biological components using an enzyme-peroxidase-coloring agent-based biological component measurement reagent. It was found that good measurement can be performed without lowering from

The present inventors further investigated a method for purifying 4-aminoantipyrine. As a result, if 4-aminoantipyrine purified by the method of the present invention is used, the enzyme-peroxidase-coloring agent system biological component measurement method using 4-aminoantipyrine and a hydrogen donor does not have the above problems. The present invention was completed by discovering that good measurement can be performed without

That is, the present invention consists of the following configurations.

(Section 1)
A method for producing purified 4-aminoantipyrine to be used as a reagent for measuring biological components in which sensitivity reduction in an enzyme-peroxidase-chromogenic agent system is suppressed, comprising the following steps:
(1) preparing a solution by dissolving crude 4-aminoantipyrine in a solvent;
(2) a step of separating and removing 4-hydroxyantipyrine from the dissolution solution using affinity for a ligand-bound resin;
A method comprising:

(Section 2)
In the step (2), 4-hydroxyantipyrine is added so that the concentration of 4-hydroxyantipyrine in the enzyme-peroxidase-coloring agent system coloring reaction solution using the biological component measurement reagent is 1.5 μg/ml or less. Item 1. The manufacturing method according to Item 1, wherein the is separated and removed.

(Section 3)
Item 3. The manufacturing method according to Item 1 or 2, wherein the biological component measuring reagent is either a creatinine measuring reagent or a glycated hemoglobin measuring reagent.

(Section 4)
Item 4-Aminoantipine produced by the production method according to any one of Items 1 to 3, wherein 4-hydroxy in an enzyme-peroxidase-coloring agent system coloring reaction solution using the biological component measurement reagent 4-aminoantipyrine, wherein the concentration of antipyrine is 1.5 μg/ml or less.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to obtain biocomponent measurement reagents without lowering the sensitivity from the designed level for each reagent manufacturing lot in biocomponent determination by an enzymatic method using an oxidase-peroxidase-coloring agent system. . In addition, good measurement was made possible using the reagent.

FIG. 4 shows HPLC fractions of 4-aminoantipyrine drug substance. FIG. 4 shows the structural formulas of 4-aminoantipyrine and 4-hydroxyantipyrine. FIG. 4 is a graph showing the relationship between the concentration of 4-hydroxyantipyrine in the reaction solution and sample measurement sensitivity.

The present invention provides a method for producing purified 4-aminoantipyrine used in a biological component measurement reagent in which sensitivity reduction in an enzyme-peroxidase-coloring agent system is suppressed, comprising the following steps (1) and (2 ) to produce purified 4-aminoantipyrine.
(1) A step of preparing a solution by dissolving crude 4-aminoantipyrine in a solvent (2) Separating and removing 4-hydroxyantipyrine from the solution using the affinity for the ligand-bound resin process to do.

The method for producing purified 4-aminoantipyrine of the present invention is not limited except that it includes steps (1) and (2). For example, in addition to step (1) and step (2), any purification method known in the art can be used in combination. Examples of such purification methods include, but are not limited to, filtration, distillation, fractional distillation, recrystallization, extraction, and sublimation.

Crude 4-aminoantipyrine used in the present invention may contain 4-hydroxyantipyrine and synthetic by-products other than 4-hydroxyantipyrine and is purified using the methods described above. As such crude 4-aminoantipyrine, commercially available products or those obtained by known synthetic methods may be used.

In the present invention, a solution of 4-aminoantipyrine crystals may be obtained by dissolving crude 4-aminoantipyrine crystals in a solvent. Examples of the solvent used here include, but are not limited to, alcohol, acetonitrile, methanol-phosphate buffer, methanol-acetic acid buffer, aqueous solutions thereof, and the like.

As a specific embodiment of the step (2) in the method for producing purified 4-aminoantipyrine of the present invention, the step of separating and removing 4-hydroxyantipyrine using affinity for a ligand-bound resin is , 4-aminoantipyrine may be separated using column chromatography such as HPLC.

When column chromatography is used as a means of removing 4-hydroxyantipyrine contained in 4-aminoantipyrine, the physicochemical principle of the separation is not particularly limited. Examples include various principles such as partition (normal phase/reverse phase), adsorption, molecular exclusion, and ion exchange.

When column chromatography is used, general reversed-phase column chromatography is preferably used. The carrier for reversed-phase column chromatography is not particularly limited, but silica gel, polymer-based carriers, and the like are preferably used, and porous silica gel is more preferably used. When silica gel is used as a carrier, there are no particular restrictions on the presence or absence of end-capping.

A column chromatography apparatus can be used as a low-pressure, medium-pressure, or high-pressure chromatography system by properly adjusting the conditions to suit the purpose.

The type of ligand that binds to the column chromatography carrier is also not particularly limited. As the ligand, besides the widely used octadecyl group (ODS), a phenyl group and an octyl group can also be selected by optimizing the conditions. Binding of ligands may be monomeric or polymeric. Any filler can be used by optimizing the separation conditions.

The mobile phase for column chromatography may be water and a water-soluble solvent such as methanol or acetonitrile. It may be adjusted to the acidic side, or a small amount of ion pair reagent may be added.

The mobile phase flow rate may be optimized according to the capabilities of the system used. Alternatively, separation may be performed by stepwise elution instead of linear gradient.

(Permissible concentration of 4-hydroxyantipyrine in the reaction solution)
The permissible concentration of 4-hydroxyantipyrine in the reaction solution in the biological component measurement of the present invention is preferably 1.5 μg/ml or less, more preferably 1.0 μg/ml or less, and still more preferably 0.5 μg/ml or less. , particularly preferably 0.3 μg/ml or less.

Using creatinine as an example of a biological component to be measured, the effect of 4-hydroxyantipyrine on lowering the sensitivity of biological component measurement and the permissible concentration of 4-hydroxyantipyrine in the reaction solution will be described below.

In recent years, eGFR calculation requires creatinine measurement accuracy to two decimal places, and the minimum detection sensitivity is required to be about 0.03 mg/dL in terms of creatinine concentration.

On the other hand, according to the present inventor's studies so far, the variation of the creatinine reagent blank is about σ = 0.045 to 0.114 mABS, which is generally considered to be the minimum detection sensitivity of in vitro diagnostic agents, 2.6σ (99 .5% normal distribution) is calculated to be about 0.117 to 0.296 mABS.
Therefore, if there is an absorbance of 0.296mABS, which is the maximum value of 2.6σ, that is, about 0.3mABS or more, it can be detected as a signal, and it is considered possible to determine the presence or absence of creatinine and to quantify creatinine.

That is, when the creatinine concentration is 0.03 mg/dL, a minimum detection sensitivity of about 0.3 mABS is required, which corresponds to a detection sensitivity of about 50 mABS when the creatinine concentration is 5 mg/dL. .

In Example 4, a 5 mg/dL creatinine aqueous solution was used to prepare measurement reagents to which various concentrations of 4-hydroxyantipyrine were added, and creatinine measurement sensitivity was measured. The results are shown in FIG.

From FIG. 3, it can be read that the concentration of 4-hydroxyantipyrine at a detection sensitivity of 50 mABS is approximately 1.5 μg/ml.

From the above, it can be seen that the permissible concentration of 4-hydroxyantipyrine in the reaction solution in the biological component measurement of the present invention is preferably 1.5 μg/ml or less.
In addition, when more sensitive measurement is required, the permissible concentration of 4-hydroxyantipyrine in the reaction solution in the biological component measurement of the present invention is more preferably 1.0 μg / ml or less, and more preferably 0 .5 μg/ml or less, particularly preferably 0.3 μg/ml or less.

The content ratio of 4-hydroxyantipyrine was obtained by dissolving a certain amount of purified 4-aminoantipyrine in PIPES-NaOH 50 mM (pH 7.4) to a concentration of 0.6 g/L, and then adding 4-hydroxyantipyrine described later. Defined by calculation from the amount of 4-hydroxyantipyrine measured using the quantitative method.

(Quantitative method for 4-hydroxyantipyrine)
Although the method for quantifying 4-hydroxyantipyrine is not particularly limited, the method used in the present specification will be described as a specific embodiment.

As used herein, 4-hydroxyantipyrine was quantified by the following HPLC method.
(HPLC analysis conditions)
(1) Column Imtakt Cadenza CD-C18 2.0 x 150 mm
(2) Mobile phase A: 0.1% formic acid, B: methanol (3) Gradient conditions 0 min (A95%, B5%) - (linear gradient during this time) -15 min (A2%, B98%) -25 min (A2%, B98%)
(4) Flow rate 0.2 mL/min
(5) Column temperature 40°C
(6) Sample injection volume 5 μL
(7) Detection wavelength UV250nm

Using 4-hydroxyantipyrine (Sigma) with a known concentration as a standard, confirming the coincidence of the detection position of the HPLC fraction peak with the object to be measured, and comparing the area of the fraction peak with the object to be measured. Quantification was performed using

Sigma's 4-Hydroxyantipyrine includes Cas. NO. 1672-63-5, product number 109428-5G, 99% pure product was used.

A sample used for quantification of 4-hydroxyantipyrine is preferably subjected to pretreatment such as dilution and deproteinization before being subjected to a quantification procedure such as HPLC.
For example, 4-aminoantipyrine is prepared in the form of a solution that is normally used for biological component measurement, and further diluted with a solvent that has good affinity with aqueous solutions and excellent solubility of 4-hydroxyantipyrine. It is preferable to use it for quantification of 4-hydroxyantipyrine. The dilution ratio is preferably about 4/3 to 5 times, more preferably about 2 times, so as not to affect the stability of the prepared measurement sample and the measurement sensitivity of HPLC.

The diluent solvent for sample preparation used for quantification of 4-aminoantipyrine used in the present invention is not particularly limited, and low-molecular-weight alcohols and mobile phase solvents used in HPLC methods are used, but in particular methanol and ethanol are used. Affinity with aqueous solution and solubility of 4-hydroxyantipyrine are good, so it is preferably used.

Although the method of protein removal is not limited, for example, a method of removing protein by ultrafiltration can be mentioned. Specifically, protein can be removed by centrifuging at a centrifugal acceleration of 2900 g for about 20 minutes and ultrafiltrating using an Amicon Ultra 30K filter (MILLIPORE).

(Biological component measurement method)
4-Aminoantipyrine purified by the method of the present invention can be applied to a biological component measuring method including the following steps (1) to (3).
(1) A step of generating hydrogen peroxide from the substrate of the enzyme by the action of an oxidase capable of generating hydrogen peroxide.
(2) A step of reacting the hydrogen peroxide generated in the step (1) with an oxidation-reduction coloring reagent that reacts with hydrogen peroxide to develop a color by the action of peroxidase.
(3) A step of colorimetrically quantifying the reaction product colored in step (2).

The biological component measuring method to which 4-aminoantipyrine purified by the method of the present invention is applied is a biological component measuring method by an enzymatic method, particularly a method using an oxidase-peroxidase-coloring agent system, that is, measurement in a specimen. In this method, a target substance is subjected to an enzymatic reaction to generate hydrogen peroxide, which is then reacted with a coloring agent in the presence of peroxidase for colorimetric determination.

A biological component measuring method using this principle has already been established in the technical field. Therefore, the knowledge can be applied to the present invention to measure the amount or concentration of biological components in various samples, and the mode is not particularly limited.

Specific aspects of the biological component measurement method will be described below using uric acid (UA), creatinine (CRE), triglyceride (TG), cholesterol (CHO), and glycated hemoglobin (HbA1c) as examples.

When measuring urea (UA), hydrogen peroxide produced by the reaction of uricase (oxidase) with UA as a substrate can be quantified using a peroxidase-coloring agent system.

When creatinine (CRE) is measured, hydrogen peroxide is not directly produced in the reaction of creatinine amidinohydrolase with CRE as a substrate, so creatine amidohydrolase and creatine amidohydrolase, to which the creatine produced in the reaction of creatinine amidinohydrolase was previously added to the reagent, were used. By designing a so-called coupling reaction in which sarcosine is produced by reacting and further producing hydrogen peroxide using sarcosine oxidase (oxidase) to which sarcosine has been added in advance to the reagent, the CRE concentration by the peroxidase-chromogenic agent system can be quantified.

When measuring triglyceride (TG), lipoprotein lipase using TG as a substrate and glycerol kinase and glycerol-3-phosphate oxidase (oxidase) as coupling enzymes are used to generate hydrogen peroxide, thereby producing peroxidase- The chromogen system allows quantitation of TG concentration.

When measuring glycated hemoglobin (HbA1c), hydrogen peroxide produced by the reaction of glycated hemoglobin oxidase (for example, fructosyl amino acid oxidase) using glycated hemoglobin as a substrate can be quantified in a peroxidase-coloring agent system.

In this way, even if there is no suitable enzyme that catalyzes the reaction that directly oxidizes the measurement target to generate hydrogen peroxide, a reaction that can convert the measurement target into a substrate for an oxidase that can generate hydrogen peroxide. (It is also possible to measure the concentration or amount of biological components other than the above by appropriately designing a coupled reaction that combines an enzyme that catalyzes (which may involve several steps of enzymatic reactions) and the oxidase. It is possible.

A sample containing a biological component to be measured is not particularly limited, and examples thereof include blood, urine, beverages, food, and the like. Among them, human body fluids (for example, blood-derived samples such as serum and plasma, and urine-derived samples) are preferably measured.

(oxidase)
The oxidase used in the biological component measurement method to which 4-aminoantipyrine purified by the method of the present invention is applied can be any enzyme that can generate hydrogen peroxide from a substrate, depending on the target measurement target. Can be used without restrictions. Specific examples include uricase, sarcosine oxidase, glycerol-3-phosphate oxidase, and the like, as described above. As commercially available products, UAO-211 (manufactured by Toyobo), SAO-351 (manufactured by Toyobo), G3O-311 (manufactured by Toyobo) and the like are preferably used. There are no particular restrictions on the amount used or the form of addition.

(peroxidase)
Any kind of enzyme can be used as the peroxidase to be used in the biological component measurement method to which 4-aminoantipyrine purified by the method of the present invention is applied, as long as it is an enzyme that catalyzes the reaction between hydrogen peroxide and a redox coloring reagent. may be used, and examples thereof include plant-derived, bacterial-derived, and basidiomycete-derived peroxidases. Among these, horseradish, rice, and soybean-derived peroxidases are preferred, and horseradish-derived peroxidases are more preferred, for the reasons of purity, availability, price, and the like. As commercially available products, PEO-131 (manufactured by Toyobo), PEO-301 (manufactured by Toyobo), PEO-302 (manufactured by Toyobo) and the like are preferably used. There are no particular restrictions on the amount used or the form of addition.

Peroxidase activity is defined in the following manner.
14 mL of distilled water, 2 mL of 5% (W/V) pyrogallol aqueous solution, 1 mL of 0.147 M hydrogen peroxide solution and 2 mL of 100 mM phosphate buffer (pH 6.0) were mixed in order, and then preliminarily adjusted at 20 ° C. for 5 minutes. and add 1 mL of the sample solution to initiate the enzymatic reaction.
After reacting for 20 seconds, 1 mL of 2N aqueous sulfuric acid solution is added to stop the reaction, and the produced purpurogallin is extracted 5 times with 15 mL of ether.
After combining the extracts, the total volume is adjusted to 100 mL, and absorbance at a wavelength of 420 nm is measured (ΔOD _test ).
On the other hand, in the blind test, 14 mL of distilled water, 2 mL of 5% pyrogallol aqueous solution, 1 mL of 0.147 M hydrogen peroxide solution and 2 mL of 100 mM phosphate buffer (pH 6.0) were sequentially mixed, and then 1 mL of 2N sulfuric acid aqueous solution was added and mixed. , then add 1 mL of sample solution to prepare.
This liquid is subjected to ether extraction in the same manner as above, and the absorbance is measured (ΔOD _blank ).
The amount of purpurogallin produced is calculated from the difference in absorbance between ΔOD _test and ΔOD _blank , and the peroxidase activity is calculated.
The amount of enzyme that produces 1.0 mg of purpurogallin in 20 seconds under the above conditions is defined as 1 purpurogallin unit (U). The calculation formula is as shown below.
U/mL
= {ΔOD (OD _test -OD _blank ) × dilution ratio} / {0.117 × 1 (mL))
= ΔOD × 8.547 × dilution ratio U / mg = {U / mL} × 1 / C
0.117: Absorbance at 420 nm of 1 mg% purpurogallin ether solution C: Enzyme concentration at dissolution (c mg/mL)
(One propurgarine unit corresponds to 13.5 international units (using o-dianisidine as a substrate under reaction conditions of 25°C).)
In the above measurement, the sample solution was dissolved in ice-cooled 0.1 M phosphate buffer pH 6.0 in advance, and diluted with the same buffer to 3.0 to 6.0 purpurogallin units (U)/mL. It is preferable to use it for measurement.

(Redox coloring reagent)
Any kind of dye can be used as the redox coloring reagent used in the biological component measurement method to which 4-aminoantipyrine purified by the method of the present invention is applied, as long as it reacts with hydrogen peroxide to develop a color. may also include, for example, combinations of hydrogen donors and couplers. There are no particular restrictions on the amount used or the form of addition. All of these can be obtained as commercially available products.

A representative example using a hydrogen donor and a coupler is the Trinder method in which the hydrogen donor and coupler are oxidatively condensed with hydrogen peroxide in the presence of peroxidase to form a dye.

The hydrogen donor is not particularly limited as long as it has the property of donating hydrogen, as long as the effects of the present invention are exhibited. Phenol, phenol derivatives, aniline derivatives, naphthol, naphthol derivatives, naphthylamine, naphthylamine derivatives and the like are known as hydrogen donors used in the Trinder method and the like. Such hydrogen donors are also called oxidative coloring reagents and the like.

For example, N-ethyl-N-sulfopropyl-3-methoxyaniline, N-ethyl-N-sulfopropylaniline, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline, N-sulfopropyl-3,5 -dimethoxyaniline, N-ethyl-N-sulfopropyl-3,5-dimethylaniline, N-ethyl-N-sulfopropyl-3-methylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl) -3-methoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N- (2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline, N-ethyl-N-(2 -hydroxy-3-sulfopropyl)-3-methoxyaniline, N-sulfopropylaniline, N-(2-hydroxy-3-sulfopropyl)-2,5-dimethylaniline, N-ethyl-N-(3-methyl phenyl)-N'-succinylethylenediamine, N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine and the like. Also, these hydrogen donors can be used in combination with couplers.

In addition, as a coupler, aminoantipyrine compounds such as 4-aminoantipyrine (4AA) and aminoantipyrine derivatives; vanillindiaminesulfonic acid compounds such as vanillindiaminesulfonic acid; methylbenzthiazolinone hydrazone (MBTH) and sulfonated methylbenz. Methylbenzthiazolinone hydrazone compounds such as thiazolinone hydrazone (SMBTH) are known. It is envisioned that aminoantipyrine-based compounds may contain 4-hydroxyantipyrine as a contaminant.

(Biocomponent measuring reagent)
4-Aminoantipyrine purified by the method of the present invention can be applied to biological component measurement reagents that satisfy the following requirements (a) to (d).
(a) contains an oxidase capable of generating hydrogen peroxide;
(b) contains peroxidase;
(c) contains a redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop a color;
(d) The concentration of 4-hydroxyantipyrine during the color reaction is 1.23×10 ⁻⁴ % or less.
Here, (d) the concentration of 4-hydroxyantipyrine during the color reaction is preferably 6.15×10 ⁻⁵ % or less, more preferably 6.15×10 ⁻⁶ % or less.

The oxidase, peroxidase, redox coloring reagent, etc. used in the biological component measuring reagent to which 4-aminoantipyrine purified by the method of the present invention is applied are the same as those described in the above description of the biological component measuring method. can be used. In addition, the method for quantifying the concentration of 4-hydroxyantipyrine in the reagent of the present invention and the method for reducing the concentration can also be applied in the same manner as the method described in the above description of the biological component measurement method.

A general-purpose automatic analyzer (for example, Hitachi Model 7180 automatic analyzer) can be used as a means for carrying out a biological component measurement method to which 4-aminoantipyrine purified by the method of the present invention is applied. The biological component measurement reagent of the present invention may be configured so as to be applicable to such an automatic analyzer. The embodiment is not particularly limited, for example, the form of a liquid reagent (or kit), the form of a reagent (or kit) composed of a combination of a dry formulation and a solution prepared by means of freeze-drying, a suitable carrier Various forms can be exemplified, such as a form using a kit or a sensor called a so-called dry system in which an enzyme or the like is carried on the surface.

A preferred form is a liquid reagent in which the reagent is divided into two (hereinafter also referred to as a two-reagent liquid reagent). In the method of analyzing with an automatic analyzer using this form of reagent, first, the first type of reagent (hereinafter also referred to as the first reagent or R1) is added to the sample and reacted for a certain period of time, and then the second type (hereinafter also referred to as a second reagent or R2) is further added and reacted, and the change in absorbance during this reaction is measured to quantify the target component.

In addition, when the biological component measuring reagent to which 4-aminoantipyrine purified by the method of the present invention is applied is divided into two or more and supplied, for example, in consideration of application to an automatic analyzer as described above. , the concentration of 4-hydroxyantipyrine in each packaged reagent may differ from the concentration during the color reaction. In that case, according to a predesigned method of use (for example, the method described in the instruction manual, etc.), from the concentration of 4-hydroxyantipyrine during the color reaction of each packaged reagent, 4- Determine whether the concentration of hydroxyantipyrine is 1.23×10 ⁻⁴ % or less.

It is preferable to incorporate a buffer component into the biological component measurement reagent to which 4-aminoantipyrine purified by the method of the present invention is applied. In addition, ascorbic acid oxidase, preservatives, salts, enzyme stabilizers, chromogen stabilizers, and the like may be added to the biological component measurement reagent of the present invention as long as they do not affect the reaction.

Examples of buffer components that can be contained in the biological component measurement reagent to which 4-aminoantipyrine purified by the method of the present invention is applied include Tris buffer, phosphate buffer, borate buffer, carbonate buffer, GOOD buffer and the like. The amount used, the set pH, the mode of addition, etc. are not particularly limited. All of these can be obtained as commercially available products.

GOOD buffers include N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N-cyclohexyl- 2-aminoethanesulfonic acid (CHES), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 2-morpholinoethanesulfonic acid (MES), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), N-tris(hydroxymethyl)methyl-2-aminomethanesulfonic acid (TES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), N-cyclohexyl-2- Hydroxy-3-aminopropanesulfonic acid (CAPSO), 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), 3-[4-(2-hydroxyethyl) -1-piperazinyl]propanesulfonic acid (EPPS), 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPSO), 3-morpholinopropanesulfonic acid (MOPS), 2 - hydroxy-3-morpholinopropanesulfonic acid (MOPSO), piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) (POPSO), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPSO), N-(2-acetamido)iminodiacetic acid (ADA), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-[tris(hydroxymethyl)methyl]glycine (Tricine), etc. exemplified.

In the biological component measurement reagent to which 4-aminoantipyrine purified by the method of the present invention is applied, the amounts and forms of addition of ascorbic acid oxidase, preservatives, salts, enzyme stabilizers, chromogen stabilizers, etc. etc. is not particularly limited. All of these can be obtained as commercially available products.

Antiseptics include Proclin 150, Proclin 200, Proclin 300, Proclin 950, azides, chelating agents, antibiotics, antibacterial agents, and the like.

Chelating agents include ethylenediaminetetraacetic acid and salts thereof.
Antibiotics include gentamicin, kanamycin, chloramphenicol and the like.

Antibacterial agents include methylisothiazolinone, imidazolidinyl urea, and the like.

Salts include sodium chloride, potassium chloride, aluminum chloride and the like.

Enzyme stabilizers include sucrose, trehalose, cyclodextrin, gluconate, amino acids and the like.

Chromogenic stabilizers include chelating agents such as ethylenediaminetetraacetic acid and salts thereof, cyclodextrin, and the like.

EXAMPLES The present invention will be specifically described below with reference to Examples, but the present invention is not limited to these.

(Example 1)
Since it was found that the degree of decreased sensitivity of the reagent for measuring biological components is due to lot differences in 4-aminoantipyrine bulk, it was found that the amount of trace impurities contained in 4-aminoantipyrine bulk varied from lot to lot. Assumed, impurities were detected by the quantitative method (HPLC method) for 4-hydroxyantipyrine described above. The purity of the 4-aminoantipyrine used for examining lot differences was 98.0% or higher according to JIS-K8048.

HPLC fractions of the 4-aminoantipyrine drug substance are shown in FIG. The peak at the elution time of 7 to 8 minutes on the horizontal axis of the graph is the peak of 4-aminoantipyrine. Besides this peak, three kinds of impurities were confirmed.

(Example 2)
When the contents of the three types of impurities (a, b, c) seen in FIG. 1 of Example 1 were examined in each lot, it was found that only the impurity b content varied greatly from lot to lot.
Therefore, the molecular weights and structures of these three substances were analyzed by mass spectrometry (MS spectrum method).

As a result, impurity b was identified as 4-hydroxyantipyrine.
FIG. 2 shows the structural formulas of 4-aminoantipyrine and 4-hydroxyantipyrine.

When HPLC was performed under the same conditions as in Example 1 using 4-hydroxyantipyrine (Sigma) with a known concentration as a standard, the elution times of the fraction peaks of 4-hydroxyantipyrine and impurity b matched.

(Example 3) Purification of 4-aminoantipyrine A commercially available bulk of 4-aminoantipyrine was purified by recrystallization according to a conventional method.
Methanol was added to 4-aminoantipyrine and heated to 62° C. to dissolve 4-aminoantipyrine. After removing the residue insoluble in hot alcohol with a glass filter, the solution was gradually cooled to precipitate crystals. The precipitated crystals were collected, dissolved in hot methanol in the same manner as above, cooled, recrystallized and collected.
Since the obtained crystals of 4-aminoantipyrine still contained many impurities and were strongly colored, the colored substance was removed with activated charcoal.
After dissolving the crystals again in a sufficient amount of methanol at room temperature, activated carbon was added and gently stirred to adsorb colored substances. After confirming that the solution was decolorized, the activated carbon was separated.
Since the methanol container of 4-aminoantipyrine from which the activated charcoal has been removed has a low concentration, it is heated to 50° C. in an evaporator while concentrating to halve the liquid volume, pure water is added to this concentrated liquid, and it is gradually cooled. It was allowed to stand still to grow crystals.
The obtained crystals were collected on a glass filter and dried under reduced pressure to obtain a crystal powder of 4-aminoantipyrine.

The recrystallized 4-aminoantipyrine was dissolved in methanol, and the 4-aminoantipyrine fraction was collected by reversed-phase column chromatography using a silica gel ODS column.
When the 4-aminoantipyrine obtained by the above method was analyzed, the content of 4-hydroxyantipyrine was below the detection limit.

(Example 4) Relationship between 4-hydroxyantipyrine concentration (concentration in reaction solution) and creatinine measurement sensitivity In the second reagent of the following creatinine measurement reagent, 4-hydroxyantipyrine was added at a final concentration of 0.13 to 8.75 μg in the reagent. /ml to prepare each measurement reagent.

As a control, 4-aminoantipyrine containing no 4-hydroxyantipyrine purified in Example 3 was used to prepare a reagent kit for measuring creatinine, and measurement was performed in the same manner as described above.

A 5 mg/dL creatinine aqueous solution was used as a sample.

[Preparation of reagent]
A creatinine measuring reagent having the following composition was prepared.
First reagent PIPES-NaOH 50 mM pH 7.4
Ascorbic acid oxidase (Toyobo ASO-311) 3U/mL
Sarcosine oxidase (Toyobo SAO-351) 10 U/mL
Creatine amidinohydrolase (Toyobo CRH-229) 40U/mL
Catalase (Toyobo CAO-509) 130U/mL
N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline 0.14g/L
Second reagent PIPES-NaOH 50 mM pH 7.4
Creatinine amidohydrolase (CNH-311 manufactured by Toyobo) 400U/mL
Peroxidase (Toyobo PEO-302) 10 U/mL
4-aminoantipyrine 0.6g/L

[Measurement method]
Hitachi 7180 type automatic analyzer was used. 120 μL of the first reagent was added to 2.7 μL of the sample and incubated at 37° C. for 5 minutes to obtain the first reaction. After that, 40 μL of the second reagent was added and incubated for 5 minutes to obtain the second reaction. Absorbance at 546 nm (main wavelength) and absorbance at 800 nm (sub-wavelength) were measured by a two-point end method in which the absorbances of the first reaction and the second reaction were corrected for liquid volume and the difference was taken. It was obtained by calculating the absorbance obtained by subtracting the sub-wavelength from the main wavelength.

The concentration of 4-hydroxyantipyrine in the reaction solution under these measurement conditions is 0.03 to 2.14 μg/ml.

Results are shown in Table 1 and FIG.
It was confirmed that the higher the concentration of 4-hydroxyantipyrine in the reaction solution, the lower the sample measurement sensitivity.
The concentration of 4-hydroxyantipyrine in the reaction solution is preferably 1.5 μg/ml or less, more preferably 1.0 μg/ml or less, even more preferably 0.5 μg/ml or less, and 0.5 μg/ml or less. A particularly favorable result was obtained when the concentration was 3 μg/ml or less.

The present invention can be applied to measurement methods for measuring biological components using redox reactions, and reagents and compositions used in the methods.

Claims (3)

A method for producing purified 4-aminoantipyrine to be used as a reagent for measuring biological components in which sensitivity reduction in an enzyme-peroxidase-chromogenic agent system is suppressed, comprising the following steps:
(1) preparing a solution by dissolving crude 4-aminoantipyrine in methanol;
(2) a step of cooling the solution to precipitate crystals;
(3) a step of dissolving the crystals obtained in step (2) again in methanol, and then adding activated carbon to remove colored substances;
(4) adding water after removing the activated carbon from the solution in step (3);
(5) a step of cooling the dissolution solution to which water has been added in step (4) to precipitate crystals; A step of separating and removing 4-hydroxyantipyrine by purifying with
A method comprising: 4 so that the concentration of 4-hydroxyantipyrine during the coloring reaction of the enzyme-peroxidase-coloring agent system using the biological component measurement reagent is 1.5 μg/ml or less by the steps (2) to (6). - The production method according to claim 1, wherein hydroxyantipyrine is separated and removed. The manufacturing method according to claim 1 or 2, wherein the biological component measuring reagent is either a creatinine measuring reagent or a glycated hemoglobin measuring reagent.

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