JP3008719B2 - Method for stabilizing a solution containing ascorbic acid and / or a salt thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an ascorbic acid and / or
And a method for suppressing browning and deterioration of a solution containing a salt thereof and an iron or unsaturated metal in a test sample, wherein a solution containing ascorbic acid or / and a salt thereof stabilized by the method is used as a reducing agent solution. The present invention relates to a method for measuring iron binding ability.

[0002]

2. Description of the Related Art Ascorbic acid and / or a salt thereof have been used for a long time as a health drug because they are anti-scurvy factors. Recently, a large number of functional foods and cosmetics containing ascorbic acid or / and salts thereof have been manufactured and sold, and the market is expanding. However, ascorbic acid is easily oxidized and has a property of browning when oxidized, so that there are many problems in using it in the fields of foods and cosmetics.

[0003] On the other hand, serum iron or unsaturated iron binding ability (UIBC) in clinical tests is measured by iron deficiency anemia, aplastic anemia, pernicious anemia, chronic hemorrhagic anemia, polycythemia vera, infectious It has an important significance in distinguishing various anemias such as anemia, liver diseases such as acute hepatitis, chronic hepatitis, and cirrhosis, and has a very high clinical significance. Since all iron in serum exists in a form bound to transferrin, which is one of serum globulin, when measuring serum iron, the chelate of transferrin and iron is removed, and iron is measured as free iron. I do.
In addition, transferrin in serum is present with one-third of it bound to iron, but the remaining two-thirds is in a form not bound to iron, and its amount is called unsaturated iron binding ability (UIBC). I have. As a method for measuring unsaturated iron binding ability, a method is generally used in which a known amount of iron is added to serum, the amount of remaining iron bound to transferrin is measured, and the unsaturated iron binding ability is determined from the reduced amount of iron. well known.

[0004] As a method for measuring iron, for example, α, α'-dipyridyl, o-phenanthroline, bathophenanthroline,
2,4,6-tripyridyl-S-triazine, 3- (2-pyridyl) -5,
Coloring of iron such as 6-bis (4-sulfophenyl) -1,2,4-triazine and nitrosophenol derivatives such as 2-nitroso-5- (N-propyl-N-sulfopropyl) aminophenol Although colorimetric analysis methods using agents are generally used, since these are all divalent iron color formers, their use requires the use of a reducing agent for reducing trivalent iron. Known trivalent iron reducing agents include L-ascorbic acid, thioglycolic acid, hydroxylamine hydrochloride, hydroquinone, hydrosulfite, sodium sulfite, hydrazine sulfate, metabisulfite (pyrosulfite) and the like. However, among these, L-ascorbic acid and thioglycolic acid are said to exhibit sufficient reducing power for precision analysis. However, these two reducing agents also have very serious defects in practical use. In other words, thioglycolic acid is relatively stable even in a solution state on the acidic side and has a sufficient reducing power, but has a malodor peculiar to a mercapto group,
It is not desirable for use. On the other hand, ascorbic acid has a problem that the stability in powder is relatively good, but the stability is extremely poor in a solution state.

As a method for stabilizing a solution containing ascorbic acid, a method in which a cyclic trimetaphosphate coexists (Japanese Patent Publication No. 59-32468), dl-N-acetylhomocysteine thiolactone (or N-acetyl-L-cysteine). ) And a method of adding a sulfite (JP-A-49-92219). Although these methods certainly have an effect of stabilizing the ascorbic acid solution, the method using coexisting cyclic trimetaphosphate has a residual ratio of ascorbic acid of only about 80% after standing at room temperature for 4 weeks. In addition, in the method of adding dl-N-acetylhomocysteine thiolactone (or N-acetyl-L-cysteine) and sulfite, only a stabilizing effect upon storage for about 10 days is recognized, When sulfite alone is used, no stabilizing effect of ascorbic acid is observed.

On the other hand, in the field of foods and the like, there is a method of adding fumaric acid (JP-A-4-352776).
The purpose is to improve the appearance and the flavor of the food and drink containing ascorbic acid due to browning, and not to increase the residual ratio of ascorbic acid itself after long-term storage. That is, according to the method, when the beverage containing ascorbic acid and fumaric acid is filled in a bottle and stored in a sealed state for 2 weeks (55 ° C.), the browning of the beverage can be prevented only. Therefore, the development of a more effective method for stabilizing a solution containing ascorbic acid has been desired.

[0007]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above circumstances, and provides an effective method for stabilizing a solution containing ascorbic acid or / and a salt thereof; It is an object of the present invention to provide a method for measuring iron or unsaturated iron binding ability (UIBC) in a test sample, using a solution containing as a reducing agent solution.

[0008]

SUMMARY OF THE INVENTION The present invention is a method for stabilizing a solution containing ascorbic acid or / and a salt thereof, wherein pyrosulfite is present in the solution.
Further, the present invention provides a solution containing ascorbic acid or / and a salt thereof (provided that an aminoketone derivative represented by the following general formula [I] is contained), characterized in that pyrosulfite coexists in the solution. Excluding cases where

Embedded image (Wherein, R ₁ represents a hydrogen atom or a C ₁ ~ ₄ alkyl group, R ₂ represents a phenyl group substituted by C ₁ ~ ₁₀ alkyl group in the para position, or R ₁ and R ₂ are C together X may form a ₃ to ₅ alkylene group, and X represents a monovalent anion or one equivalent of a polyvalent anion.) Further, the present invention relates to a method for measuring iron or UIBC using ascorbic acid or / and its salt as a reducing agent for trivalent iron, wherein pyrosulfite is added to a solution containing ascorbic acid or / and its salt. A method for measuring iron or UIBC in a test sample, characterized by using the resulting solution as a reducing agent solution. That is,
The inventors of the present invention have intensively studied a method for stabilizing a solution containing ascorbic acid or / and a salt thereof. As a result, pyrosulfite unexpectedly has a stabilizing effect on a solution containing ascorbic acid or / and / or a salt thereof. This led to the completion of the present invention.

As the salts of pyrosulfite used in the present invention, any of alkali metal salts, ammonium salts, and alkaline earth metal salts can be used as long as the required amount can be dissolved. Usually, sodium pyrosulfite and potassium pyrosulfite are preferably used because they are easy to put. The optimum concentration of pyrosulfite varies depending on pH, ascorbic acid concentration and the like, but is usually 0.001 w / v% or more, preferably 0.01 w / v% or more. Also, iron or U
In the measurement of IBC, when pyrosulfite is used as a stabilizer for ascorbic acid to be used as a reducing agent, it must be used at a low concentration that does not affect the effect of the reduction of pyrosulfite in the measurement system. Preferably, the concentration of pyrosulfite in that case is 0.01 to 0.
It is about 5w / v%. The pH of the solution containing ascorbic acid and / or a salt thereof according to the present invention is usually in the range of 2 to 8, preferably in the range of 2 to 5. In the solution containing ascorbic acid and / or a salt thereof according to the present invention, the solvent used is such that the reagents used are dissolved and the final pH
May be any one as long as it falls within the range of 2 to 8, and examples thereof include distilled water, ion-exchanged water, and various buffers having a buffering capacity within the above pH, but are not limited thereto. Absent. .

In order to carry out the present invention, pyrosulfite as a stabilizer according to the present invention only needs to coexist in a solution containing ascorbic acid and / or a salt thereof, and no special operation is required. . Ascorbic acid according to the present invention or /
And a solution containing a salt thereof, if necessary, in addition to pyrosulfite as a stabilizing agent, various preservatives, buffers, surfactants and other chemicals may coexist (but not necessarily Needless to say, a substance having reactivity with ascorbic acid and / or its salt or pyrosulfite cannot be present.) Further, in the method for measuring iron or UIBC according to the present invention, in which a solution containing ascorbic acid and / or a salt thereof and coexisting pyrosulfite is used as the reducing agent solution, a solution other than the reducing agent solution is used. Coloring reagents, buffers, various preservatives, surfactants, and other reagents are used in the per se known method of measuring iron or UIBC using a solution containing ascorbic acid or / and its salt as a reducing agent solution. All the reagents and the like to be used can be used as they are, and the concentration range and the like of the reagents and the like can be determined by appropriately selecting a concentration range and the like usually used in the measurement method known per se. In addition, the measurement operation and the like may be performed according to a method for measuring iron or UIBC known per se, except that the solution containing ascorbic acid or / and a salt thereof according to the present invention is used.

[0011] The stabilization method according to the present invention can be applied to various fields using ascorbic acid, such as clinical diagnostics, health pharmaceuticals
It can be widely used in functional foods, cosmetics, and other fields. Further, the method for measuring iron according to the present invention can be widely used not only in the field of clinical examination, but also in water quality examination and other various fields. Heretofore, there has been an example in which pyrosulfite has been used as a reducing agent in a reagent for measuring iron or UIBC, but this is extremely effective in stabilizing a solution containing ascorbic acid or / and a salt thereof. Or / and / or using a solution containing a salt thereof as a reducing agent solution
It was completely surprising that the method of measuring IBC can be effectively used for stabilizing a reducing agent solution.
The method for measuring iron or UIBC according to the present invention is not limited to the method used, but can be used for a measurement system using an automatic analyzer.

[0012]

Embodiment 1 [Preparation of ascorbic acid solution] Dissolve ascorbic acid to a concentration of 0.4% and sodium pyrosulfite to a concentration of 0.1% in a 0.4 M glycine buffer, and add hydrochloric acid or potassium hydroxide to the solution.
H was adjusted to 2.0-8.0. [Storage conditions] The prepared ascorbic acid solution was stored in an incubator at 25 ° C. [Measurement of Ascorbic Acid Concentration] Immediately after preparation, the ascorbic acid concentration of each of the ascorbic acid solutions after storage for one month and after storage for three months was measured by the following method. First, monoammonium phosphate is 15 g / dl (1.3 M / l), methanol is 7.5 ml / dl, acetylacetone is 0.15 ml / dl, catalase is 45,000 U / dl, and copper (II) sulfate pentahydrate is 2.5 mg. / dl
(0.1 mM / l) were dissolved in ion-exchanged water, and the pH was adjusted to 7.0 with sodium hydroxide to prepare a color reagent solution. Take 100 μl of each ascorbic acid solution, add 4.0 ml of the above coloring reagent solution, and heat up for 60 minutes in a 37 ° C constant temperature bath.
The absorbance at 410 nm was measured using the reagent blank as a control.
Standard solutions containing ascorbic acid of known concentration (10, 20,
A 100 μl portion of 30, 50, 80, 100 mg / dl) was taken, the absorbance was measured in the same manner, and a calibration curve was prepared from the concentration and the absorbance of the ascorbic acid standard solution. From this calibration curve, the ascorbic acid concentration in the ascorbic acid solution was calculated. With the ascorbic acid concentration immediately after preparation as 100%, the residual ratio of the ascorbic acid concentration in the ascorbic acid solution after storage for 1 month and after storage for 3 months was determined. Table 1 shows the results.

Comparative Example 1. Ascorbic acid was dissolved in 0.4M glycine buffer to a concentration of 0.4%, and the pH was adjusted to 2.0 to 8.0 with hydrochloric acid or potassium hydroxide. After that, it was stored in the same manner as in Example 1, and the ascorbic acid concentration immediately after the preparation was set to 100%. The residual ratio of the ascorbic acid concentration in the ascorbic acid solution after storage for 1 month and after storage for 3 months was the same as in Example 1. Asked by the way. The results are shown in Table 1.

[0014]

[Table 1]

As is clear from the results in Table 1, when sodium pyrosulfite coexists in the ascorbic acid solution, the residual ratio of ascorbic acid in the solution is remarkably improved.
It can be seen that in H2 to H5, even after storage for 3 months, there was almost no change compared to immediately after preparation.

Embodiment 2 FIG. [Preparation of ascorbic acid solution] 0.4M glycine buffer contains 0.4% of ascorbic acid and 0.001% of sodium pyrosulfite.
It was dissolved to a concentration of 1% and adjusted to pH 3.0 with hydrochloric acid. [Storage conditions] The prepared ascorbic acid solution was stored in an incubator at 25 ° C. [Measurement of Ascorbic Acid Concentration] Immediately after the preparation, the ascorbic acid solution was measured in the same manner as in Example 1 for each of the ascorbic acid solutions after storage for 10 days and after storage for 30 days. The ascorbic acid concentration immediately after preparation is 100%,
The residual ratio of ascorbic acid concentration in the ascorbic acid solution after storage for 30 days and after storage for 30 days was determined in the same manner as in Example 1. Table 2 shows the results.

Comparative Example 2. Ascorbic acid was dissolved in a 0.4 M glycine buffer to a concentration of 0.4%, and the pH was adjusted to pH 3 with hydrochloric acid.
Adjusted to 0. Then, it was stored in the same manner as in Example 2. The ascorbic acid concentration immediately after the preparation was set to 100%, and the residual ratio of the ascorbic acid concentration in the ascorbic acid solution after storage for 10 days and after storage for 30 days was the same as in Example 1. Asked by the way. The results are shown in Table 2.

[0018]

[Table 2]

As is clear from the results in Table 2, when sodium pyrosulfite coexists in the ascorbic acid solution,
The residual ratio of ascorbic acid in the solution is significantly improved, especially when the concentration of sodium pyrosulfite becomes 0.01% or more.
It turns out that it is extremely stable even after storage for days.

Example 3 Measurement of serum iron [Preparation of ascorbic acid solution] In a 0.4M glycine buffer, ascorbic acid was 0.4%, sodium pyrosulfite was 0.1%,
Triton X-100 (chemical name: polyoxyethylene (10) octyl phenyl ether, trade name of Rohm and Haas Company) is dissolved to a concentration of 0.7%, and the pH is adjusted to 3.0 with hydrochloric acid.
Was adjusted. [Preparation of Coloring Solution] In a 0.1 M glycine buffer, sodium bathophenanthroline sulfonate was dissolved to a concentration of 0.1%, and the pH was adjusted to 3.0 with hydrochloric acid. [Measurement method of serum iron] 300 μl of the ascorbic acid solution prepared above was added to 20 μl of a serum sample, heated at 37 ° C. for 5 minutes, 75 μl of a coloring solution was added, and after further heating for 5 minutes, the main wavelength was 546.
The two-wavelength absorbance at 600 nm and the auxiliary wavelength of 600 nm was measured. The same measurement was performed using distilled water instead of the sample, and the result was regarded as a reagent blind. In addition, instead of the sample, use a standard solution (iron 200μg / d
The measurement was similarly performed using l). The serum iron concentration was calculated from the following equation 1.

(Equation 1) Table 3 shows the results.

Comparative Example 3 Using thioglycolic acid as a reducing agent and a color reagent
Fe C-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.), a commercial kit for serum iron measurement using 2-nitroso-5- (N-propyl-N-sulfopropyl) aminophenol (Nitroso-PSAP)
The serum iron concentration of the same serum sample as in Example 3 was measured by the following method. 0.4 M acetate buffer (pH 6.2
5, contains surfactant and reducing agent thioglycolic acid)
And 2.0 ml of the sample were mixed, and then a color reagent (Nitros
o-PSAP) was added thereto, mixed well, and allowed to stand at room temperature for 5 minutes. Then, the absorbance at 750 nm was measured using a reagent blank as a control. The same measurement was performed using distilled water in place of the serum sample, and the result was regarded as reagent blind. In addition, the same measurement was performed using an iron standard solution (iron 200 μg / dl) instead of the serum sample. From Equation 1, the concentration of serum iron in the sample was calculated. The results are shown in Table 3.

[0022]

[Table 3]

As is clear from the results shown in Table 3, serum iron was measured using the ascorbic acid solution containing sodium pyrosulfite according to the present invention as a reducing agent solution, and a conventional reagent for measuring serum iron. When compared with the case where the measurement was carried out using, there is no difference in the measured values, and it can be seen that the addition of pyrosulfite to the measurement system does not affect the measured value of serum iron.

Embodiment 4 Measurement of serum unsaturated iron binding ability (UIBC) [Preparation of buffer solution] Ammonium ferrous sulfate was dissolved in a 0.4 M glycine buffer solution (pH 8.6) to a concentration of 80 µg / dl as iron. [Preparation of ascorbic acid solution] Ascorbic acid was dissolved in distilled water to a concentration of 0.5%, sodium pyrosulfite to 0.1%, and sodium bathophenanthroline sulfonate to a concentration of 0.1%, and the pH was adjusted to 3.0. [Measurement method of UIBC] 300 µl of the above buffer solution was added to 20 µl of the sample.
After heating at 37 ° C. for 5 minutes, 75 μl of the above ascorbic acid solution was added, and after further heating for 5 minutes, two-wavelength absorbance at a main wavelength of 546 nm and a sub-wavelength of 600 nm was measured. Similarly, reagent blinding was measured, and UIBC was determined from the amount of reduced iron. Table 4 shows the results.

Comparative Example 4 Using sodium metabisulfite (sodium pyrosulfite) as a reducing agent and 2-nitroso-5- (N-propyl-N-sulfopropyl) aminophenol (Nitroso-PS) as a coloring reagent
UI, a reagent kit for measuring unsaturated iron binding ability using AP)
Using BC-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.), UIBC was measured for the same serum as in Example 4 by the following method. Buffer used (0.16M Tris buffer pH 8.6, reducing agent sodium metabisulfite, ammonium ferrous sulfate
^Contains 100 μg / dl as e ²⁺ . ) 2.0ml and serum sample 0.2ml
And left at room temperature for 30 minutes. Next, one drop of a color reagent solution (Nitroso-PSAP) was added and mixed well, and the mixture was allowed to stand at room temperature for 15 minutes, and the absorbance at 750 nm was measured using distilled water as a control. The measurement was similarly performed using distilled water instead of the serum sample, and the reagent was blinded. UIBC was calculated by the following equation.

(Equation 2) The results are shown in Table 4.

[0026]

[Table 4]

As is clear from the results in Table 4, UIBC was measured using an ascorbic acid solution containing sodium pyrosulfite according to the present invention as a reducing agent solution, and a conventional reagent for UIBC measurement was used. In comparison with the case where the measurement was carried out, no difference was observed in the measured values, and it was found that addition of pyrosulfite to the measurement system did not affect the measured values of UIBC.

Embodiment 5 FIG. Measurement of serum iron [Preparation of ascorbic acid solution] 0.4% ascorbic acid, 0.1% sodium pyrosulfite in 0.4M glycine buffer,
Triton X-100 (chemical name: polyoxyethylene (10) octyl phenyl ether, trade name of Rohm and Haas Company) is dissolved to a concentration of 0.7%, and the pH is adjusted to 3.0 with hydrochloric acid.
Was adjusted. After the preparation, it was stored in a 25 ° C. incubator for 3 months. [Preparation of Coloring Solution] In a 0.1 M glycine buffer, sodium bathophenanthroline sulfonate was dissolved to a concentration of 0.1%, and the pH was adjusted to 3.0 with hydrochloric acid. [Measurement method of serum iron] Using the above ascorbic acid solution immediately after preparation and after storage for 3 months, according to the method of Example 3,
Each was measured for serum iron. Table 5 shows the results.

Comparative Example 5 [Preparation of Ascorbic Acid Solution] Ascorbic acid was dissolved to a concentration of 0.4% and Triton X-100 to a concentration of 0.7% in a 0.4 M glycine buffer, and the pH was adjusted to 3.0 with hydrochloric acid. After preparation
It was stored for 3 months in a 25 ° C. incubator. [Measurement Method of Serum Iron] Serum iron was measured according to the method of Example 3 for the same serum as in Example 5 using the above ascorbic acid solution immediately after preparation and after storage for 3 months.
The results are shown in Table 5. Comparative Example 6 Using Fe C-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.), serum iron was measured in the same manner as in Example 5 in the same manner as in Comparative Example 3. The results are shown in Table 5.

[0030]

[Table 5]

As is clear from the results in Table 5, in Comparative Example 5 in which sodium pyrosulfite was not added, when ascorbic acid stored for 3 months was used, the measured values generally tended to be higher. However, in Example 5 to which sodium pyrosulfite was added, there was no difference between the measured values immediately after preparation and the values measured using ascorbic acid after storage at 25 ° C. for 3 months. Also, comparing the method of the present invention (Example 5) with the case of using the conventional reagent (Comparative Example 6), the ascorbic acid solution prepared by the method of the present invention shows that
Even when the solution after storage for 3 months was used, there was no effect on the measured values, indicating that the solution was sufficiently usable as a reagent for measuring serum iron.

[0032]

The present invention provides a method for effectively stabilizing a solution containing ascorbic acid and / or a salt thereof. According to the method of the present invention, the conventional method is unstable and can be stabilized. It is difficult to stably store a solution containing ascorbic acid or / and its salt for a long period of time, and the stabilization method is to use ascorbic acid or / and / or its salt as a trivalent iron reducing agent. The present invention has a remarkable effect in that it is sufficiently applicable to stabilization of a reducing agent solution in a measurement reagent or a UIBC measurement reagent.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁷ , DB name) A61K 31/375 A61K 9/08 A61K 47/02 G01N 33/90 CA (STN)

Claims (10)

(57) [Claims] 1. A method for stabilizing a solution containing ascorbic acid or / and a salt thereof, wherein pyrosulfite is coexistent with the solution. 2. The pyrosulfite is an alkali metal, ammonium, or alkaline earth metal salt of pyrosulfite.
A method according to claim 1. 3. The method according to claim 1, wherein the pyrosulfite is sodium or potassium pyrosulfite. 4. A solution containing ascorbic acid or / and a salt thereof, wherein a solution containing an aminoketone derivative represented by the following general formula [I] is provided, wherein a pyrosulfite is coexisted in the solution. Except for the case.) (Wherein, R ₁ represents a hydrogen atom or a C ₁ ~ ₄ alkyl group, R ₂ represents a phenyl group substituted by C ₁ ~ ₁₀ alkyl group in the para position, or R ₁ and R ₂ are C together _It may form a ₃ to ₅ alkylene group, and X represents a monovalent anion or one equivalent of a polyvalent anion.). 5. The pyrosulfite is an alkali metal, ammonium or alkaline earth metal salt of pyrosulfite.
The solution according to claim 4. 6. The solution according to claim 4, wherein the pyrosulfite is sodium or potassium pyrosulfite. 7. A method for measuring iron or unsaturated iron binding ability using ascorbic acid or / and a salt thereof as a reducing agent for trivalent iron, wherein a solution containing ascorbic acid or / and a salt thereof contains pyrosulfite. A method for measuring the binding ability of iron or unsaturated iron in a test sample, wherein a solution prepared by coexisting is used as a reducing agent solution. 8. The method according to claim 7, wherein the solution containing ascorbic acid and / or a salt thereof is co-existed with pyrosulfite at a concentration of 0.01 to 0.5 w / v%. 9. The pyrosulfite salt is an alkali metal, ammonium or alkaline earth metal salt of pyrosulfite.
The measurement method according to claim 7. 10. The method according to claim 7, wherein the pyrosulfite is sodium pyrosulfite or potassium pyrosulfite.