JPH05322842A - Highly selective measurement method of chloro ion within body fluid specimen by ion electrode method - Google Patents

Abstract

PURPOSE:To measure Cl<-> within a body fluid specimen highly selectively, easily, and readily by adding a buffer liquid for dilution and Ag<+> for generating precipitate to a body fluid specimen and then measuring excessive Cl<-> or Ag<+> with a solid film type halogen electrode or a silver electrode which are provided. CONSTITUTION:After Good's buffer or Tris' buffer near pH 7 and Ag<+> for generating precipitate are added into a body fluid specimen, excessive Cl<-> or Ag<+> is measured by a solid model halogen electrode or silver electrode which is installed in the same system as measurement of Na<+> and K<+> according to a sodium electrode and a potassium electrode. In this case, the amount of added Ag<+> is reduced as compared with Cl<-> within the specimen, thus measuring Cl<-> which did not settle as AgCl or measuring excessive Ag<+> according to the silver electrode or the solid model halogen electrode to obtain Cl<->. A high selectivity can be obtained by this method since the silver electrode does not respond to various kinds of negative ions at all.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a plasma by the ion electrode method, serum, chloride ion in a body fluid sample such as urine (Cl ^-)
Relates to a highly selective measurement method of.

[0002]

2. Description of the Related Art Chlorine (Cl
^The solid film type ion electrode and the liquid film type ion electrode have been used for the measurement of-). The sensitive film of the solid film type ion electrode is formed by molding silver halide (AgX) alone or a mixed powder with silver sulfide, and the sensitive film of the liquid film type ion electrode is mainly made of polyvinyl chloride (PVC) or the like. It is a polymer in which a quaternary ammonium ion-exchange group is dissolved and fixed together with a plasticizer.

[0003] Solid film ion electrodes, thiocyanic ions (SCN ^-), a bromine ion (Br ^-), iodide ion (I ^-) is particularly poor selectivity for such, when these ions are present even a little, Cl ^- Measurement The value error is extremely large. In the case of a liquid membrane type ion electrode, Br ⁻ , I ^−, etc. are Cl
^- Although influence on the measured value is smaller than the solid film type, acetate ion (CH ₃ COO ^-), perchlorate ion (ClO ₄
^-), nitrate ion (NO ₃ ^-) is a relatively large impact like.

These interfering ions are usually less problematic because they are present in trace amounts in normal plasma and serum. However, when an analgesic or anesthetic containing bromide is taken or inhaled, the amount of Br in blood cannot be ignored.
^- will be there, Cl ^- measurement error becomes very large. In addition, sulfate ions (SO ₄ ²⁻ ), NO ₃ ⁻ and other ions may be present in the blood of critically ill persons such as cancer patients in several tens of times the normal amount, in which case the measurement error of Cl ⁻ also increases. ..

On the other hand, the problem becomes more complicated when a urine sample is used instead of blood. That is, since the urine component does not have a constant composition like the blood component, there are considerable amounts of interfering ions,
The reliability of the measured value of Cl ⁻ by the ion electrode method is remarkably low. What has been described above is Cl in biological samples.
^This is the main problem when measuring-by the ion electrode method. In addition to these problems, there are also problems that the liquid membrane type ion electrode has a short life, inferior stability of the liquid-liquid potential, and the selectivity decreases as the membrane deteriorates.

[0006] Cl ^- analysis of, was old is performed by argentometry. That is, a solution containing Cl ⁻ contains silver nitrate (A
gNO ₃ ) solution is added dropwise to form a precipitate of silver chloride (AgCl), and excess silver ion (Ag ⁺ ) is added after the equivalence point.
It is a method of determining the end point by detecting with a method using an indicator such as the Mohr method, the Forhard method, or the Fayans method. As a method for determining the end point, there is a potentiometric titration method using a silver indicator electrode in addition to the method using the indicator. This is a method of detecting excess Ag ⁺ with a silver electrode.

Basically the same as these silver titrations, but there is a coulometric titration method utilizing Faraday's law as a quantitative method. In this method, potassium nitrate or the like is used as an electrolytic solution, and silver is used as a working electrode, and a constant current is applied to the Ag electrode.
^{+ Is} generated and reacted with Cl ⁻ in the sample to generate Ag.
According to Faraday's law, the amount of Cl ⁻ is determined by the product of the time of the start and end of a constant current flowing between the working electrode and the counter electrode, which is the method of determining the precipitation of Cl and the end point with a silver electrode.

In addition to these, methods for measuring Cl ⁻ include ion chromatography, enzymatic analysis, and colorimetric method using mercury. Among the above methods,
Ion electrode method, coulometric titration method, ion chromatography, and enzymatic analysis method are mainly used as the analysis method of Cl ⁻ in blood. The advantages and disadvantages of these are summarized below.

The ion electrode method is the simplest as described above, but has a problem in selectivity. In the coulometric titration method, B
Although r ⁻ , I ⁻ , SCN ^{− and the} like interfere with Cl ^{− in} an equivalent amount, these interfering ions are not so much in normal blood, and therefore this method is currently used for measuring Cl ⁻ in blood. Although it is used as a standard, it is inferior in terms of simplicity as in the ion electrode method because the device becomes large in scale. Moreover, since sodium ion (Na ⁺ ) and potassium ion (K ⁺ ) cannot be measured by the coulometric titration method, these ions must be measured by another method that is different from Cl ⁻ in principle. , Has serious drawbacks from the point of view of speed.

Ion chromatography is the method with the least error, but it is the worst in terms of simplicity.
Moreover, this method is completely unsuitable for the analysis of many samples. The enzymatic analysis method utilizes the fact that the activity of α-amylase depends on Cl ⁻ and has high selectivity, but it is complicated in that an enzymatic reaction must be used. As described above, it has not been reported yet that a simple and highly selective method capable of simultaneously measuring Cl ⁻ and Na ⁺ and K ⁺ was achieved by the conventional technique.

[0011]

DISCLOSURE OF THE INVENTION The present invention provides a method for highly selective, simple and quick measurement of Cl ⁻ in a body fluid sample by using an ion selective electrode together with Na ⁺ and K ⁺ in the same measurement system. It is intended to be provided.

[0012]

Therefore, a method of highly selective measurement of Cl ^{− in} a body fluid sample according to the present invention is such that after adding a buffer for dilution and Ag ⁺ for producing a precipitate to a body fluid sample, a sodium electrode and It is characterized in that excess Cl ⁻ or Ag ⁺ is measured by a solid film type halogen electrode or silver electrode installed in the same system as the measurement of Na ⁺ and K ⁺ by a potassium electrode.

Since the chloric electrode according to the prior art is poor in selectivity, the method of the present invention succeeded in significantly increasing the selectivity by adding Ag ⁺ to the sample solution. That is, Ag ⁺ added to the sample solution has a smaller solubility product than Cl ⁻ and has a solubility product of Br ⁻ , I ⁻ , SCN ⁻ , and ClO _{4 in the} sample solution.
^- a 1: generates one insoluble precipitate, the remainder of Ag ⁺ is Cl ^- to form a precipitate. As a result, the influence of these interfering ions on the Cl ⁻ measurement is limited to the amount corresponding to the activity thereof, and there is no possibility of exerting the influence of many times the activity as in the conventional chlor electrode.

As described above, similar methods have been conventionally used in potentiometric titration and coulometric titration. However, these methods are completely different from the present invention because it is not necessary to consider how the reagents used and the precipitate affect the ion electrode. That is,
In an attempt to implement the principles of the present invention, the AgCl produced
And added Ag ⁺ , NO ₃ ⁻ (when AgNO ₃ is used as Ag ⁺ for precipitation generation), and further, the aggregated protein may affect the sodium electrode, the potassium electrode, the chloro electrode, and the silver electrode. All of them have to be solved, and such a study has never been attempted.

According to the present invention, the produced AgCl is not aggregated by the protein contained in the serum in the serum sample, and glycerol or polyethylene glycol is the same as the protein in the protein-free sample and standard solution such as urine. Good buffer solution (HEPES, HEPPS) around pH 7 that does not react with Ag ⁺
. O optimum) or tris (hydroxymethyl) aminomethane buffer (hereinafter, abbreviated as Tris buffer) if diluted about 100-fold _{^{using, Ag +, NO 3 -,}} AgCl
It was found that these do not affect the sodium electrode and the potassium electrode.

The solid film type halogen electrode and silver electrode are N
O ₃ ^- and best use of that insensitive to, Cl by Ag ^{+ -} was devised to be used for the quantification ^- the precipitation reaction Cl. From the above, it was confirmed that it is possible to measure Na ⁺ , K ⁺ , and Cl ^{− in} the same system. In particular,
It was a key point of the method of the present invention that Ag ⁺ was confirmed to have no effect on the sodium electrode (glass electrode) and the potassium electrode (valinomycin liquid film electrode).

The method of the present invention includes two methods.
In the first method, the amount of Ag ⁺ added was set to be smaller than the amount of Cl ^{− in} the sample, and Cl that did not precipitate as AgCl was used.
^- a method for the residual amount measured by the solid-state membrane chlorine electrode.

In this method, the solid film type chloro electrode is different from the liquid film type chlor electrode in that the added AgNO ₃ NO ₃
^- it is insensitive ^{to, Br -, I -, SCN} - , etc. Although reaches several hundred times error solid membrane chlorine electrode when the ^{present, Br -, I -, SCN} - precipitation becomes silver It is no longer sensitive, and unlike the liquid film type, the solid film type chloric electrode responds almost only to these three ions, and if these ions are eliminated, it has three excellent properties. It is a method that skillfully used. In this method, a bromine electrode or an iodine electrode can be used instead of the chlorine electrode. Both of these electrodes are sufficiently sensitive to Cl ⁻ , and since Br ⁻ , I ⁻ , and SCN ⁻ have a much smaller solubility product than Cl ^−, they are preferentially converted to silver salts by precipitation and removed. Because it does not.

The second method is a method in which excess Ag ⁺ is added and excess Ag ⁺ is measured by a silver electrode or a solid film type halogen electrode to obtain Cl ⁻ . This method is a very highly selective method because the silver electrode is completely insensitive to various anions. As the solid film, AgCl, Ag
It is possible to use a single powder of Br, AgI alone or a mixed powder of one of these and Ag ₂ S formed into a disc shape by pressing, or a mixture of the powder with a support such as silicone or PVC.

[0020] When using a chlorine electrode in the first method, Cl of normal samples without addition of Ag ^{+ -}
It is also conceivable that the method of the present invention is applied only to abnormal samples. Now, when a chlor electrode is used as the detection means, the amount of Ag ⁺ to be added must be equal to or more than the maximum amount of interfering ions expected to be contained in the body fluid sample. This maximum amount is 30 mmol for serum
/ L is sufficient, but in the case of urine, 50 mmol / l is necessary. The remaining Ag ⁺ not consumed by the interfering ions reacts with Cl ⁻ and precipitates, and the remaining Cl ⁻ not converted to AgCl is measured by the chlor electrode.

The addition of the amount of Ag ⁺ added to this measured value is calculated as the amount of Cl ^{− in} the sample. According to this method, the calculated value increases by the total amount of interfering ions, which naturally causes an error, but the value is far smaller than the error in the measured value by the conventional chlor electrode. A solid film type chloro electrode is used as the chloro electrode, but it is convenient to use Br ⁻ , I ⁻ , SCN.
^-, ClO ₄ ^- for extremely high selectivity with respect to other anions except, this method is very effective. Furthermore, the maximum amount of Ag ⁺ added is 30 mmol even when using serum.
Since / l is sufficient, the amount of precipitate formed is small, and there is little interference.

The second method is to use a silver electrode as the detecting means. In this case, the amount of Ag ⁺ to be added must exceed the total amount of Cl ⁻ and interfering ions expected to be contained in the sample. The remaining Ag ⁺ not consumed by the reaction with Cl ⁻ and interfering ions in the sample is measured by the silver electrode. There is a silver sulfide film as a silver electrode, and this electrode has no interfering ions other than Hg ²⁺ and S ²⁻ , and is therefore superior in selectivity to the first method using the chlor electrode.

That is, NO ₃ ⁻ , SO ₄ 2−, P
Anions such as O ₄ ³⁻ , CH ₃ COO ⁻ , and HCOO ⁻ are
The first method has a slight influence on the measurement of Cl ⁻ , but since these anions do not react with Ag ⁺ and the silver electrode is not sensitive, no error occurs.

As a modification of the second method, there is a method of using a solid film type electrode instead of the silver electrode. Any of a silver chloride film, a silver bromide film, a silver iodide film and a silver thiocyanide film can be used. The reason is that these membrane electrodes are all A
This is because it responds to g ⁺ , and Br ⁻ , I ⁻ , SCN ^−, etc. existing in the sample in advance react with Ag ⁺ and precipitate as a silver salt, and in the sample almost pure Ag ⁺ is present. Since it remains, any solid film type electrode can be used.

In the method for selectively measuring Cl ⁻ according to the present invention as described above, initially, AgNO ₃ to be added should be added.
It was expected that the above would significantly interfere with the intended measurement, and that the produced AgCl colloid and aggregated protein would contaminate and destabilize the electrode. In fact, Ag
When the NO ₃ 0.1 M solution was measured by the direct method ion electrode method in which the sample was measured without diluting it, the serum sample was measured, and Na ⁺ in the serum was high. Further, it was found that the memory effect remained remarkably, and the memory effect did not disappear even if the standard aqueous solution was subsequently measured.

Therefore, in the method of the present invention, the serum was diluted about 100 times with the buffer solution. In that case, as shown in Table 1, no effect of Ag ⁺ and AgCl was observed on the sodium electrode and the potassium electrode. The memory effect is that Ag ⁺ remaining on the electrode surface or the channel reacts with Cl ^{− in} serum to form a precipitate, which is attached to the glass surface of sodium and Na ⁺ in the electric double layer Probably the cause.

[0027] As a countermeasure, to dilute the sample, Cl ^-
A method of reducing all the concentrations and AgNO ₃ concentration (so-called dilution method ion electrode method) is effective, but it may still cause contamination of the film surface in the long term. As a countermeasure, it was found that it is effective to regularly wash with a thiosulfate solution in order to remove the attached AgCl.
As shown in Table 2, it was found that the memory effect disappeared by measuring the sodium thiosulfate solution even in the direct method. However, sodium thiosulfate dissolves the surface of the solid-state membrane electrode, and thus has a drawback of causing potential shift. Similar effect is diluted ammonia water,
It is also provided by ammonia thiosulfate.

[0028] Next, it was found that the addition of glycerol is effective for the phenomenon of condensation of AgCl colloids, which has become large due to condensation, on the electrode surface and the channel. No aggregation of colloids was observed no matter how high the concentration of AgCl was produced by glycerol. The reason is that glycerol is Ag
It is considered to cover the surface of the nucleus of Cl precipitation. It was confirmed that glycerol did not affect any of the sodium electrode, potassium electrode, chlor electrode, and silver electrode. In the prior art, starch and polyvinyl alcohol have been found by coulometric titration as substances exhibiting the action of preventing the colloidal aggregation of AgCl.

A similar action to glycerol was seen with polyethylene glycol. That is, even in a high-concentration AgCl solution, colloid aggregation does not occur, and AgCl
Cl was dispersed as fine particles. In addition to this, as a unique action observed in polyethylene glycol, it can be said that the action of adhering an aggregated protein containing AgCl to the electrode surface and eliminating the abnormality of the electrode potential caused thereby is extremely strong. Now, 20 ml of the diluted solution is added to 1 ml of serum, and 1 M AgNO ₃ is added thereto to form a thick white colloid. Even if the solid film type chloro electrode is immersed in this and then washed with water, the potential of the chlor electrode is not restored. This phenomenon is considered to be because the ion dissociation equilibrium of the solid film is lost due to the adsorption of the AgCl colloid on the electrode surface.

Then, this chloro electrode was washed with a 2% aqueous solution of polyethylene glycol (using a molecular weight of 600),
Immediately after washing with water, the electrode potential immediately returned to the original zero potential. Glycerol has no such effect. It is considered that this is because polyethylene glycol has a function of peeling the adsorbed colloid. Further, as shown in Table 2, when 0.1 MAgNO ₃ is measured with a sodium electrode, a potassium electrode, and a chloric electrode and then serum is measured, an abnormal value appears. However, by washing the electrode with a polyethylene glycol aqueous solution,
The effect of 1 MAgNO ₃ could be completely eliminated.
As described above, polyethylene glycol has two functions, that is, it functions to prevent colloidal aggregation like glycerol, and functions to remove the adverse effect of AgCl on the electrode like sodium thiosulfate.

Further, the polyethylene glycol aqueous solution itself has pH, Na, K, Cl, Ca, PO ₂ , PCO ₂
Since it has no effect on any of the ion electrodes and the glass electrodes, it can be used as a cleaning solution for these electrodes. In particular, as described above, the cleaning action of silver ions on the sodium electrode and the cleaning action of polyanions (anions composed of proteins, colloids, etc.) on the chlor electrode are excellent. This is due to the fact that polyethylene glycol has both hydrophilic and lipophilic properties, is a nonionic surfactant, and is related to the properties used as the internal liquid of electric field capacitors and lubricants. Thought to be a thing.

From the above-mentioned experiments, it was found that it is particularly excellent in the adsorption action of other ions to the glass electrolysis such as Na and pH, the adsorption action of polyanion, and the similar action to the solid film type chloric electrode. It was As for simple proteins and lipids, they naturally have an action as a surfactant. The same action as polyethylene glycol,
It also has a low molecular weight, water-soluble polypropylene glycol. The above action of polyethylene glycol enables it to be used as an electrode cleaning liquid for potentiometric and coulometric titration methods using a silver electrode or a chloro electrode.

The reaction rate of AgCl precipitate formation in the method of the present invention is extremely fast and reacts instantaneously, so that it can be sufficiently applied to a measurement system having a high detection rate such as the conventional ion electrode method. Hereinafter, the method of the present invention will be described in more detail with reference to Examples.

[0034]

EXAMPLES Example 1 50 ml of 10 mM Tris buffer (HNO ₃ was used instead of HCl to adjust the pH to 7.4 in the preparation) was placed in a beaker and stirred with a stirrer for 0.100.
Add 1.00 ml of MAgNO ₃ aqueous solution. In the beaker, place a reference electrode (double junction type, salt bridge solution is K
NO ₃ ) and a silver electrode (AgS solid film electrode) are immersed, then 0.5 ml of a 1% glycerol aqueous solution is added, and then the electrodes are offset. While stirring with a stirrer, 500 ml of serum containing Cl ⁻ was added. Similarly, the same operation was performed for the silver standard solution to prepare a calibration curve.

As shown in FIG. 1, the remaining amount of Ag ⁺ is 4 ×.
Below 10 ^-4 M, Ag ⁺ generated by AgCl dissolution
Therefore, the calibration curve becomes non-linear. That is, with respect to the total liquid volume of 50 ml, the maximum Cl ⁻ concentration in serum was 0.1.
Assuming a 16M of the sample, including Cl to be added ^- the maximum amount of the sample becomes 250 [mu] l. Therefore, the dilution ratio of the sample was 250 μl / 50 ml = 1/500, and simultaneous measurement of Na ⁺ and K ⁺ was impossible because the serum concentration of K ⁺ was low and the sensitivity was insufficient when diluted 500 times. Become.

Therefore, in order to make the dilution ratio 100 times or less, the addition amount of the 0.1MAgNO ³ solution needs to be 1 ml or more. Under these conditions, serum Na ⁺ , K ⁺
And Cl ⁻ can be measured, and the calibration curve becomes linear.

The coulometric titration Cl obtained by ^- concentration, two types of serum samples 97.7mmol / l and 86.3 mmol / l, Cl by the methods described above ^- was measured density, respectively 97.5mmol / L,
The result was 87.5 mmol / l, and good agreement was obtained.

Next, I ^- 2 mM, Br ^- 10 mM, ClO
₄ ^- When a solution containing 2mM was determined errors in the case of a sample, Cl ^{- -} 4 mM and SCN in terms of a concentration of 15
It was a positive error of mM. This is ClO ₄ ^- other than I ^-,
This is because Br ⁻ and SCN ⁻ consume Ag ⁺ , which should theoretically have a positive error of 14 mM, but the above values indicate that they are close to the theoretical values.

Example 2 This example is an illustration of a method using a solid film chloro electrode. 10 mM Tris-HNO ₃ buffer 50
Transfer ml to a beaker and add 0.5 ml of 1% polyethylene glycol aqueous solution. Then Cl ^- serum 50 containing the
Add 0 μl and stir with a stirrer. At this time, the chlor electrode shows a potential corresponding to the halogen concentration in serum. In this example, Br ^- 2 mM, I ^- 0.4 mM, SCN
^- since with sera containing 0.4 mM, chloro electrode strongly responsive to these interfering ions, actual Cl ^- 10
150 mM as Cl ⁻ despite being 5 mM
A potential corresponding to the degree was shown.

Next, 0.100 MAgNO ₃ aqueous solution 25
0 μl was added with stirring with a stirrer. as a result,
The potential of the chlor electrode showed a chlor concentration of 58 to 60 mM. However, the relationship between potential and concentration is 0.080-
A 0.120 M NaCl aqueous solution was prepared. From the above, 108 mM and 110 mM were obtained as the measured values of Cl ⁻ according to the present invention. This sample was originally a serum sample containing 105 mM Cl ⁻ (this 105 mM is a dilution method ISE
(Value obtained with the device), there is 100 mM Ag ⁺
Add half of the above, the rest will be 55 mM. 5
The reason why the concentration is 8 to 60 mM is that the total amount of Br ⁻ , I ⁻ , and SCN ⁻ is 2.8 mM.

As described above, although the conventional method causes a large error in the measurement of only the chloric electrode, by using the method of the present invention, 5 mM / 50 mM = 1/1.
0, that is, the disturbance is reduced to about 1/10. The method of Example 2 is particularly effective for the analysis of a body fluid such as a urine sample in which the range of contained Cl ⁻ concentration is extremely high. In other words, the added Ag ⁺ concentration is Cl ^- 50m
It suffices to add an amount corresponding to M. In the method of Example 1,
In practice it would be difficult to use as the added Ag ⁺ must be above the maximum Cl ^- concentration possible for urine.

[0042]

According to the method of the present invention, by adopting the dilution method with Good or Tris buffer, Na ⁺ ,
By the ion electrode method based on the same detection principle, K ⁺ and Cl ⁻ can be measured by the same measurement system, and the measurement speed and accuracy are comparable to those of the conventional ion electrode.

The selectivity of Cl ⁻ measurement is superior in the method using a silver electrode for detection as compared with the conventional chloric electrode, solid film type chloric electrode, etc., and Br ⁻ , I ⁻ , SCN.
^-, ClO ₄ ^- or the like 1: a only provides 1 of the effects, the accuracy of coulometric titration equivalent measurements are obtained. Further, in the method using the solid film type chloro electrode for detection, B
The selectivity of r ⁻ , I ⁻ , SCN ⁻ , ClO ₄ ^−, etc. is remarkably improved as compared with the conventional method using the solid film type chloro electrode, and far exceeds the liquid film type.

As described above, according to the method of the present invention, Cl ⁻ with high selectivity can be measured in a very short time together with Na ⁺ and K ⁺ , and, like the enzymatic analysis method, the enzyme can be used. Since it is not necessary to use an expensive reagent necessary for the color development system or its color development system, it largely contributes as a measurement method in the medical field where rapid and low cost is required. In addition, high selectivity can be obtained, and the method of the present invention can bring about great effects such as elimination of erroneous diagnosis of diseases and the need for double test of verification by other methods.

[Brief description of drawings]

FIG. 1 is a diagram showing a calibration curve for a silver standard solution.

Claims (2)

[Claims] 1. A solid film type halogen electrode installed in the same system as the measurement of sodium ion and potassium ion by a sodium electrode and a potassium electrode after adding a diluting buffer and a silver ion for producing a precipitate to a body fluid sample, or A method for highly selective measurement of chloride ions in a body fluid sample, which measures excess chloride ions or silver ions with a silver electrode. 2. The measuring method according to claim 1, wherein one or a mixture of two or more selected from the group consisting of glycerol, polyethylene glycol and polypropylene glycol is added to the sample solution.