JPH11242034A - Method for measuring iodine, reaction instrument for pretreating specimen, and airtight tool of reaction instrument for pretreating specimen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring iodine that can pretreat a specimen safely and can obtain a measurement value with improved reproducibility, and an airtight tool for a reaction instrument for pretreating a specimen that can also easily treat a number of specimens and is suited for measuring iodine. SOLUTION: A fixing supplementary material 4 is used as a component, where it can apply pressure to supports for fixing for making airtight two upper and lower supports 2 and 3 that can be fixed and the reaction region of a reaction instrument that sandwich and fix a reaction instrument 5 for pretreating a specimen for measuring iodine with a plurality of reaction regions that can heat and digest a plurality of specimens with an oxidizer and spacers 6 and 7 for covering the reaction instrument 5 for pretreating a specimen for measuring iodine and a plurality of reaction regions of the reaction instrument.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring iodine,
The present invention relates to a sample pretreatment reaction device and an airtight device for the sample pretreatment reaction device.

[0002]

2. Description of the Related Art Iodine is an indispensable trace element used as a material for thyroid hormone which promotes metabolism of a living body.
The iodine content in the body is 15-20 mg for healthy adults. Although the adult human thyroid weighs about 20 g, the thyroid selectively concentrates blood iodine and produces thyroid hormone.
The daily intake of iodine required by the human body is 100-1
It is 50 μg. This iodine deficiency causes goiter if left as it is, further causing hypothyroidism. In particular, in newborns, it leads to decreased intelligence, stunted growth, neurological symptoms, and the like. It is also said that iodine deficiency in pregnant women causes stillbirth and increased neonatal mortality.

[0003] There are approximately 1.6 billion people living in rural areas at risk for iodine deficiency, concentrated in developing countries. This is because in mountainous areas and floods or rainfall areas inland, the iodine runoff from the soil also causes the plants that grow there to be inadequate in the iodine content, resulting in iodine uptake by people and animals living there. It is said to be short due to iodine deficiency. Preventive measures include iodine supplementation,
For example, a relatively simple method such as ingestion of an iodized salt or the like is considered to be effective. In fact, in the United States and European countries, measures in iodine deficient areas have been effective by this method. Under these circumstances, the eradication of iodine deficiency was adopted at the 1990 United Nations Children's World Summit on the United Nations, with the goal of elimination of iodine deficiency being adopted. The International Organization for Countermeasures for Iodine Deficiency (ICCIDD), UNICEF and the World Health Organization (W
It is mentioned as one of the important issues of HO) and as a national measure of many countries. To that end, while accurately grasping the actual situation of iodine deficiency, diagnosing and treating it,
It is important to monitor this situation regularly. Diagnosis of iodine deficiency is made possible by measuring urinary iodine because urinary excretion and intake of iodine correlate well. The normal value of urine iodine which is an index in the diagnosis is 100 μg / L or more.

As a method for measuring iodine, EBSandell et al. Have reported a colorimetric method that utilizes the property of iodine ions as a catalyst to accelerate the redox reaction represented by the chemical reaction formula (I) ( Hereinafter, it is abbreviated as Sandell-Kolthoff reaction: EBSandell And Kolthoff, Mikrochemica Act
a, vol.1, P9-25 (1937)).

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[0005] That is, by adding arsenous acid reagent solution and ammonium cerium sulfate reagent solution as reagents to the iodine ion in the sample, iodine is reduced by the reaction of reducing yellow ammonium cerium sulfate (tetravalent ion) to form colorless trivalent cerium ion. It is a sensitive measurement method utilizing the fact that it acts as a catalyst as in the chemical reaction formulas (II) and (III).

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However, in the measurement of a sample such as urine, a large amount of interfering substances such as ascorbic acid and thiocyanate ions which affect the oxidation-reduction reaction are contained, and the sample cannot be directly applied to this reaction. In actual measurement, pretreatment such as removal of an interfering substance in a sample is required. As a pretreatment method, a method of incinerating and ashing a specimen (MCSa
nz et.al., Clinica Chemica Acta, 1,570-576,1956),
Wet ashing method in which oxidizing agents such as perchloric acid and chloric acid are added and heated to oxidize interference substances (Zak et.al, Anal.Chem., 24 (8), 1
345-1348,1952), dialysis and chromatography (WO96 / 2779)
Methods such as 4) for separating interfering substances have been proposed. Among these pretreatment methods, the wet incineration method of Zak et al. Is used as the simplest method. The mildest standard wet incineration method is to place a urine sample 250 in a glass test tube 10 cm long and 13 mm inside diameter in a local exhaust system.
μl, and 750 μl of a chloric acid reagent solution were added, and opened at 110 to 115 ° C. in a sand bath or an aluminum block.
After heating for 1 hour and cooling, 3.5 ml of sulfuric acid acidic arsenous acid TS
, And a reduction reaction was performed for 15 minutes, 350 μl of ammonium cerium sulfate reagent solution was added, and after a certain period of reaction,
A method for colorimetric quantification at 05 nm has been proposed and performed (Urine Iodine Measurement Method Manual, International Organization for Iodine Deficiency, 1993).

However, the container used in the pretreatment of urine samples using chloric acid described above is made of a material that can withstand heating even with a strong oxidizing agent such as chloric acid, and that the sandell following the pretreatment is used. -It is required that substances that interfere with the Kolthoff reaction do not elute. For this reason, in the pretreatment of a specimen using chloric acid, a container made of a polymer material has not been used. Further, the steam discharged in the pretreatment process is a steam containing chlorine or the like having an irritating odor, and it is necessary to perform the steam in a local exhaust facility having an exhaust device. Therefore, a method has been used in which a glass test tube resistant to heat and oxidant is used as a container in a local exhaust facility having an exhaust device, and the oxidant is heated in an open state due to a problem of strength. Further, in the method of heat-treating a specimen in an open glass container, in order to reduce a change in the amount of the liquid due to evaporation and scattering of the specimen and the test liquid, etc.
It was necessary to use long test tubes on a volume scale of ml or more.

Further, since a long test tube with a volume scale of 1 ml or more is used, a large processing space and a large heating device are required to process a large number of samples at the same time. Furthermore, in the pretreatment of a sample using chloric acid, a local exhaust facility is required to exhaust harmful chloric acid-derived vapor generated when the sample is heated together with chloric acid. In the measurement, the bleaching reaction (Sandell-Kolthoff reaction) after the addition of arsenous acid reagent solution and cerium ammonium sulfate reagent solution as reagents to iodine ions in the sample is a relatively quick and sharp reaction. Therefore, the measurement of the absorbance at a certain time after the start of the bleaching reaction requires strict measurement time. Therefore, there is a limit to the processing capacity of the sample in the measurement in which each cell is transferred to the cell for measuring the absorbance on a test tube scale.
There is a demand for a measurement method capable of processing a large number of samples. There is an Autoanalyzer manufactured by Technicon Instruments Inc. as an automatic device using the Sandell-Kolthoff reaction for processing a large number of sample urine, but it cannot be said that the device has easy operation and portability.

Recently, Pino et al. Have proposed a pretreatment method using ammonium persulfate as an oxidizing agent without using chloric acid (Cl
inical Chemistry, 42 (2), 239-243, 1996) has been reported as a safe method. However, this pretreatment method is also a method of heating using an open glass test tube, and has the same problem as the method using chloric acid, and it has been difficult to treat multiple samples. In this pretreatment method,
The safety of gas generated during heating has not been confirmed. Further, in the method for measuring iodine, toxic substances such as arsenic, cerium, and sulfuric acid and toxic substances are used as reagents. Therefore, when a large number of samples are measured, a large amount of environmentally problematic toxic waste is produced. In fact, according to the urine iodine measurement method manual, 4.85 ml of an arsenic-containing waste liquid is generated per sample.

[0010] In many cases, iodine is required to be measured in a region remote from a city. Therefore, a measuring method which can be carried out simply, quickly and safely, and a measuring instrument and a device having excellent portability are strongly desired. ing. Recently, Tsuda et al. (K. Tsuda et. Al., Clinical Chemistry, 41 (4), 581-5
85 (1995)), and Poluzzi et al.
Measurement method using LC (V. Poluzzi et al., J. of Anal. Ana)
l. Atomic Spectrometry, 11 (9), 731-734 (1996)) has also been reported and proposed. However, the introduction of these expensive and less portable devices is unacceptable.

[0011]

SUMMARY OF THE INVENTION According to the first aspect of the present invention, in pretreatment, evaporation of harmful steam and a change in the amount of a reaction solution are suppressed without requiring a special facility such as a local exhaust facility. An object of the present invention is to provide a method for measuring iodine, which can safely perform a pretreatment of a sample and obtain a measured value with good reproducibility. The second aspect of the present invention provides a method for measuring iodine capable of performing safe and highly reliable pretreatment of a sample because there is no concern about breakage of the container during the heat treatment. According to a third aspect of the present invention, in the pre-processing,
By suppressing the evaporation of harmful vapors and the change in the volume of the reaction solution without requiring special facilities such as local exhaust facilities,
An object of the present invention is to provide a method for measuring iodine which can perform safe and highly reliable pretreatment of a sample and can obtain a measured value with good reproducibility since there is no fear of damage to the container.

The invention of claim 4 provides a method for measuring iodine, which can facilitate pretreatment of a large number of samples quickly and easily and is suitable for the measurement of a large number of samples, in addition to the invention of the second or third invention. is there. The invention according to claim 5 is, in addition to the invention according to claims 2 to 4, in addition to the fact that there is no risk of damage to the container during the heat treatment, and that there is no elution of substances that affect the measured value. An object of the present invention is to provide a method for measuring iodine, which enables safe and reliable pretreatment of a sample. According to the invention of claim 6, in addition to the inventions of claims 2 to 5, inexpensive and highly reliable reactors are easily available, and there is no need to worry about damage to the container during the heat treatment, and the measurement can be performed safely. It is an object of the present invention to provide a method for measuring iodine, which allows a sample to be pre-treated without elution of a substance that affects the value.

According to the present invention, a large number of specimens can be pre-processed safely without fear of damage to the container during the heat treatment, and without elution of substances that affect the measured values. An object of the present invention is to provide a sample pretreatment reaction instrument suitable for measuring iodine. The invention according to claim 8 does not require a special facility such as a local exhaust facility in the pretreatment of the sample for iodine measurement, is not limited to a place where the sample is processed, and reduces the amount of evaporation of harmful vapor and the amount of harmful waste generated. An object of the present invention is to provide an airtight tool of a sample pretreatment reaction instrument suitable for iodine measurement, which can suppress the number of samples and can easily process a large number of samples.

[0014]

Means for Solving the Problems (1) The present invention provides a sample pretreatment step in which a sample is digested by heating with an oxidizing agent,
In a method for reacting an arsenous acid reagent solution and a cerium ammonium sulphate reagent solution and measuring or measuring the absorbance in the reaction solution, in which the iodine concentration in the sample is determined or detected, the sample pretreatment step is heat-treated in an airtight condition. And a method for measuring iodine, which is performed by cooling. (2) The present invention provides a method for preparing a sample, which comprises a pretreatment step of the sample in which the sample is heated and digested with an oxidizing agent, and a reaction measurement step of reacting the arsenous acid reagent solution and the ammonium cerium sulfate reagent solution and measuring the absorbance in the reaction solution. In a method for quantifying or detecting iodine concentration, the present invention relates to a method for measuring iodine, wherein a pretreatment step of a sample is performed using a reaction device made of a heat-resistant organic material. (3) The present invention provides a method for preparing a sample, comprising: a pretreatment step of a sample in which a sample is heated and digested with an oxidizing agent; and a reaction measuring step of reacting an arsenous acid reagent solution and an ammonium cerium sulfate reagent solution and measuring the absorbance in the reaction solution. In the method for quantifying or detecting the iodine concentration, the pretreatment step of the sample is performed by using a reaction device made of a heat-resistant organic material, and further, performing a heat treatment under airtight conditions and cooling. The measurement method.

(4) The present invention relates to the method for measuring iodine according to the above (2) or (3), wherein the reaction device made of a heat-resistant organic material has a plurality of reaction regions. (5) The invention is described in any one of the above (2) to (4), wherein the heat-resistant organic material is a polypropylene resin, a polycarbonate resin, a polysulfone resin, a polyethersulfone resin, a Teflon resin or a polymethylpentene resin. And a method for measuring iodine. (6) The present invention relates to the method for measuring iodine according to any one of (2) to (5), wherein the reaction device is a microtiter plate.

(7) The present invention comprises at least one heat-resistant organic material selected from the group consisting of a polypropylene resin, a polycarbonate resin, a polysulfone resin, a polyethersulfone resin and a polymethylpentene resin,
The present invention relates to a reaction device for sample pretreatment for iodine measurement, comprising a plurality of reaction regions capable of subjecting a plurality of samples to a heat digestion treatment together with an oxidizing agent. (8) The present invention provides a sample pretreatment reaction device for measuring iodine according to the above (7) and two upper and lower fixing members which can vertically interpose and fix a spacer covering a plurality of reaction regions of the reaction device. The present invention relates to an airtight device for a sample pretreatment reaction device, comprising a fixing auxiliary material capable of applying pressure to the fixing support so that the reaction region of the reaction support and the reaction region of the reaction device can be airtightly sealed by the spacer.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION In the method for measuring iodine of the present invention, a reaction apparatus used in a sample pretreatment step will be described. This reaction device must be able to withstand the process of heating and digesting the sample together with the oxidizing agent. In addition, the reaction device is capable of removing harmful vapors such as chlorine gas derived from chloric acid generated by heating in the pretreatment step of the sample heated together with the oxidizing agent. From the viewpoint of suppressing generation and minimizing reproducibility (variation) between measured values due to a change in liquid volume, it is necessary to be able to withstand airtight conditions.

The above-mentioned reaction apparatus preferably has a plurality of reaction regions. As such a reaction device, a container such as a test tube or a centrifugal test tube is used as a reaction region, and a plurality of the reaction regions are arranged and connected. Further, a well-shaped or column-shaped dent is dug in one plate. With this as the reaction area,
There is one in which a plurality of reaction regions are arranged. The shape of each reaction region may be U-shaped or flat-bottomed like a test tube, or V-shaped like a centrifugal test tube.

The capacity of each reaction zone is 200 to 10
It is preferably 00 μl. More preferably, 30
0-500 μl, more preferably 300-350 μl
l. If the volume of each reaction area exceeds 1000 μl, waste space tends to increase with the increase in the size of the container, which tends to make it difficult to perform a multi-sample processing operation.
When the volume is less than 1 liter, the miniaturization of the container size requires the minute dispensing operation of the sample and reagent and the necessity of precise operation, thereby increasing the risk of lowering dispensing accuracy and malfunctioning in multi-sample processing. Tend to.

It is preferable that the opening of each reaction region has a shape and a structure that can cover the reaction region and ensure airtightness by a spacer for airtightness. For example, the spacer may have a lid shape, a sheet shape, or a plate shape. When using a lid-shaped spacer,
If the surface shape or structure of the opening of each reaction area is complementary to the uneven lid, or if a flat sheet or plate-like spacer is used, the area around the opening of each reaction area It is preferable to make the surface shape flat.
Further, it is more preferable to increase the area where the spacer and the opening of each reaction region are in contact with each other, since airtightness can be ensured.

The material of the above-mentioned reaction apparatus is preferably made of a heat-resistant organic material. Such a heat-resistant organic material has excellent heat resistance and chemical resistance to an oxidizing agent,
Further, it is preferable that the material has high physical strength and is easy to mold. Further, it is preferable that a substance that interferes with the reaction following the pretreatment by heating with the oxidizing agent does not elute from the container. Examples of such a material include polypropylene, polycarbonate resin, polysulfone resin, polyethersulfone resin, Teflon resin, and polymethylpentene resin.

A specific example of the reaction apparatus is a microtiter plate. In general, a microtiter plate has wells (reaction areas) having a diameter of about 8 mm and a depth of about 8 mm in a single plate, 9 rows in 8 columns and 12 rows.
One example is an integrated type in which six pieces are arranged. However, the microtiter plate that can be used in the method for measuring iodine of the present invention is not limited to the above, the number of wells, the diameter, the depth, and the like.For example, the diameter is reduced or the depth is increased, In addition, 96 wells are arranged in 8 rows or 1 row.
It is also possible to use a microtiter plate of a division type in which an arbitrary number of combinations can be divided in units of two rows. In the present invention, the reaction device is not limited to the above-mentioned microtiter plate. For example, a reaction region of a test tube for centrifugation, which is made of polypropylene and has a diameter of about 6 mm and a depth of about 20 mm, has a linear shape of 8 mm.
Eight linked tubes for PCR (Polymerase Chain Reaction) (imported by Assist Co., Ltd.) and the like can also be used. The number of reaction regions of the plurality of reaction devices made of these heat-resistant organic materials is not limited, and can be appropriately selected according to the number of specimens to be processed, the processing frequency, and the like.

In the method for measuring iodine according to the present invention, in the pretreatment step of a sample in which the sample is heated and digested together with an oxidizing agent, it is preferable that the reaction region of the reaction device is made airtight by a spacer. The spacer must be able to withstand sample pretreatment. The spacer has no gas permeability and is resistant to oxidizing agents.
It is preferable to use a material that also has heat resistance that does not cause deformation.
For example, polypropylene resin, polycarbonate resin,
Examples include polysulfone resin, polyethersulfone resin, Teflon resin, polymethylpentene resin, silicone rubber, butyl rubber, chlorobutyl rubber, and the like. As the spacer, a sheet-like or plate-like material having elasticity is preferable. Examples of the material of the sheet or plate spacer include silicone rubber, butyl rubber, chlorobutyl rubber, and the like. Further, as a film for preventing the deterioration of the cover, it is more preferable to coat a heat-resistant film such as a polypropylene film, a polyvinylidene chloride film or a fluoroethylene film. In addition, the surface of the opening of each reaction region and the spacer may be flattened to each other, or may have a complementary shape with irregularities, so as to ensure adhesion and airtightness.
It can be structured or processed. When the number of samples to be processed is small, it is preferable to use a lid shape from the viewpoint of economy.However, when processing multiple samples, the shape of the cover should be Is preferred.

The above-mentioned hermetic tools for the pretreatment reaction device are as follows:
Since the reactor and the spacer are accommodated and processed in a constant temperature bath or equivalent constant temperature heater, it is preferably made of a heat resistant material which does not deform at the processing temperature. Preferred examples of the heat-resistant material of the fixing support and the fixing auxiliary material include metal and heat-resistant hard resin.
More preferably, from the viewpoint of efficient heating in the pretreatment step of the specimen and cooling to room temperature after the heat treatment, aluminum, copper, a metal or carbon material such as stainless steel having good thermal conductivity is mentioned. . After heating, if the vapor of the processing liquid condenses on the spacers, the amount of liquid changes and the reproducibility of the measurement is impaired.Therefore, during cooling, the lower support plate of the fixing support is higher than the upper support plate in contact with the spacer. So that the lower support plate is forcibly brought into contact with water or a heat sink, or the upper support plate is made thicker than the lower support plate, or the upper support plate is made to have a higher thermal conductivity than the lower support plate. Low material, for example, a lower support plate aluminum, a material with high thermal conductivity such as copper, it is preferable to use a material with low thermal conductivity as compared to the lower support plate such as stainless steel for the upper support plate, It is more preferable to combine them.

FIGS. 1 to 3 show an example of an airtight tool, but the present invention is not limited to this. FIG. 1 is a diagram showing a front view (in use) of an example of an airtight device. FIG. 2 is a plan view (when used). FIG. 3 is a sectional view taken along the line aa 'of FIG.
It is a figure which shows a sectional view (however, the state which removed the snap screw as one fixing auxiliary material). The hermetic tool comprises an upper support plate 2, a lower support plate 3, and a fixing auxiliary member 4 such as a snap screw made of aluminum or the like attached to the lower support plate 3. A reaction device 5 such as a microtiter plate is sandwiched between two spacers 6 and 7 in a storage space formed by the upper support plate 2 and the lower support plate 3 and stored with the well opening upward. I have. Furthermore, the reaction equipment 5
The reaction region is tightly closed by a spacer 6 in a tightly closed state by tightening the lower support plate 3 and the upper support plate 2 from above and below by a fixing auxiliary material 4 attached to the lower support plate 3. In FIG. 3, the spacer 7 between the reaction device 5 and the lower support plate 3 may not be provided, and the spacer 6 between the reaction device 5 and the upper support plate 2 may be replaced with the reaction device 5.
It is necessary that the reaction region be thick enough to ensure airtightness.

The method for measuring iodine of the present invention will be described below. The method for measuring iodine of the present invention comprises a sample pretreatment step of heating and digesting a sample together with an oxidizing agent, and then a reaction measurement step of reacting an arsenous acid reagent solution and an ammonium cerium sulfate reagent solution and measuring the absorbance in the reaction solution. In the method of quantifying or detecting the iodine concentration in the sample, it is preferable that the pretreatment step of the sample is performed by heat treatment under airtight conditions and cooling. In the method for measuring iodine of the present invention, the pretreatment of the sample is performed, for example, in each reaction region of the reaction device, by a fixed amount of an iodine standard solution or a fixed amount of a sample such as urine and a fixed amount of an oxidizing agent such as a chloric acid reagent. The amount is added to each. Further, after the addition of the oxidizing agent, each reaction region is brought into an air-tight state using a spacer or the above-mentioned air-tight tool.
The airtight reactor is heat-treated at a constant temperature for a certain period of time using a thermostat or the like. After this heat treatment, the reactor is returned to room temperature. This pretreatment removes interference substances during measurement in the sample and oxidizes iodine ions and iodine to iodate ions.

In the method for measuring iodine of the present invention, examples of the specimen include urine of humans and animals. A known concentration of iodine standard solution or the like may be used. The specimen is not limited to these as long as it is a specimen that can be made into a solution or an aqueous solution that seems to have iodine. In the method for measuring iodine of the present invention, the liquid volume of the sample is preferably in the range of 2 μl to 100 μl, more preferably 3 μl to 80 μl.
1 and more preferably 5 μl to 50 μl. 2 μl
If less than 100 μl, the sensitivity of the measurement is poor, the dispensing error is large, and the reproducibility of the measurement tends to be poor. This tends to impair the rapidity and ability to process multiple samples.

The amount of the oxidizing agent added is 2 times the amount of the sample.
It is preferably 10 to 10 times. If the amount of the oxidizing agent to be added is less than twice the amount of the sample, the recovery of iodine tends to decrease due to the decrease in the final concentration. Exceeding the number of times causes not only waste of the reagent but also an increase in the discharge of hazardous wastes that pose environmental problems. In particular, when processing a large number of specimens, it becomes a problem that cannot be ignored. Further, in this range, the maximum liquid volume obtained by combining the sample amount and the oxidizing agent addition amount according to the container volume is preferably 70% or less of the container volume, and more preferably 20 to 50% of the volume.
If the maximum liquid amount with respect to the capacity of the container exceeds 70%, stirring may become difficult, or the processing liquid may not be collected due to the adhesion of the processing liquid to the spacer.

Examples of the oxidizing agent include chloric acid and ammonium persulfate. When chloric acid is used, the final concentration in the pretreatment step is preferably 10 to 30 (W / V)%, and if it is less than 10 (W / V)%, the removal of interfering substances in the sample becomes insufficient, As a result, the recovery rate of iodine is lowered, and the dispersion of the recovery is increased depending on the sample, so that good reproducibility of measured values tends to be not obtained. If the final concentration of chloric acid exceeds 30 (W / V)%, not only will the chloric acid reagent be wasted, but also a large amount of harmful waste will be produced, and if it exceeds 40%, self-decomposition will occur. (Chloric acid dictionary).
When using ammonium persulfate, a final concentration of 15
~ 30 (W / V)%, preferably 15 (W / V)%.
If it is less than V)%, the recovery of iodine from the iodine in the sample becomes insufficient, the dispersion of the recovery increases, and good reproducibility of measured values tends not to be obtained. In addition, 30 (W
/ V)%, not only wasteful but also excessively high concentration of ammonium persulfate is produced by the subsequent reaction by adding arsenous acid test solution and ammonium cerium sulfate test solution (Sandell-Kolt).
hoff reaction) tends to inhibit the reaction,
Use at high concentrations is not preferred.

In the pretreatment of the sample, the reaction temperature at the time of digestion by heating with an oxidizing agent is preferably from 50 to 140 ° C., and from 80 to 1
The temperature is more preferably 20 ° C, particularly preferably 10 ° C.
0-110 ° C. If the temperature is lower than 50 ° C., the recovery of iodine in the sample becomes insufficient, and the dispersion of the recovery becomes large,
There is a tendency that good reproducibility of measured values cannot be obtained. 140
There is no problem with the recovery of iodine in the sample even if the temperature exceeds ℃, but if the heat resistance limit of the reaction equipment made of heat-resistant organic material is exceeded, the vaporization of harmful vapor and the There is a risk that scattering cannot be prevented. The reaction time for heat digestion with an oxidizing agent is preferably 30 to 120 minutes. More preferably, it is 45 to 90 minutes. 30
If the time is less than one minute, the recovery of iodine from the iodine in the sample becomes insufficient, the dispersion of the recovery becomes large, and good reproducibility of measured values tends not to be obtained. Even if the time exceeds 120 minutes, the dispersion of the collection and the reproducibility of the measured value do not change, but the rapid measurement and the processing ability of a large number of samples are impaired. Within the above range, the concentration, temperature, and time at which the organic matter and the like in the sample can be sufficiently oxidized and iodine can be recovered can be appropriately selected.

As described above, the pretreatment of the sample suppresses the generation of harmful vapors such as chlorine gas derived from chloric acid generated by heating, and also reduces the inter-measurement value due to the change in the liquid volume due to the evaporation of the reaction solution. From the viewpoint of minimizing the reproducibility (variation) of, the heating reaction is preferably performed under airtight conditions. In the pretreatment, it is more preferable that the airtight condition is such that the change in the liquid amount before and after the pretreatment is suppressed within a range of 2% or less. In order to achieve airtight conditions, it is preferable to apply the spacer described above. By setting the airtight condition in this way, the pretreatment of the sample can be performed more safely. When the air-tight state is insufficient, it may be performed in a local exhaust facility. Further, it is more preferable to use the above-mentioned air-tightness tool of the reaction device in order to more reliably ensure the air-tight state by the spacer and simultaneously process a plurality of samples. By using the airtight tool of the reaction apparatus, it is possible to process multiple samples simultaneously more safely than using only the spacer without requiring special facilities such as a local exhaust facility.

After the pre-treatment of the sample, the reaction temperature of the pre-treatment is actively reduced by cooling or the like in view of the safety of the pre-treatment to avoid harmful vapors such as chlorine gas and to shorten the cooling time. Is more preferably returned to room temperature or lower. From the viewpoint of safety, it is necessary not to open the spacer or the airtight device of the reaction apparatus until the reaction temperature of the pretreatment returns to room temperature. By performing this cooling process, emission of the harmful gas hardly occurs even when the airtight state is released.

As a process following the sample pretreatment process,
First, arsenous acid reagent solution was added to the pretreated treatment solution,
Further, a yellow ammonium cerium sulfate reagent solution is added, and a reaction (Sandell-Kolthoff reaction) in which the discoloration occurs is performed. For example, after returning the pretreatment solution to room temperature, a certain amount of this solution is transferred to a polystyrene microtiter plate, and a certain amount of arsenous acid reagent solution is added. This allows
The iodate ion is reduced to iodine ion. Then, a reduction reaction (fading reaction) of ammonium cerium sulfate with arsenous acid using iodine as a catalyst is started by adding a yellow ammonium cerium sulfate solution of ammonium cerium sulfate. In the present invention, the final measurement of the absorbance is a change in the bleaching reaction caused by the addition of the cerium ammonium sulfate reagent solution, which is measured as a change in absorbance or transmittance. The concentration of iodine in the sample is quantified or detected from the relationship between the measured value of the absorbance or transmittance and the amount of iodine. Each test solution can be prepared and used as follows, but is not limited to this composition.

The preparation of the arsenous acid test solution is performed, for example, by using arsenous acid 10
g was dissolved in 250 ml of purified water together with 7 g of sodium hydroxide, and the mixture was acidified by adding 32 ml of concentrated sulfuric acid, 25 g of sodium chloride was added, and purified water was added to make 1 liter. Store at room temperature protected from light. Preparation of cerium ammonium sulphate TS
g of ammonium cerium sulfate in 3.5N sulfuric acid to make 1 liter. Store at room temperature protected from light.

In the present invention, unless a transparent device (especially a transparent microplate) is used as a reaction device used in the pretreatment step, a transparent device capable of measuring an absorbance change before or after the addition of the arsenous acid test solution is used. It is necessary to transfer a certain amount of the processing solution to a measuring instrument (for example, a microplate). The transparent measuring instrument capable of measuring the change in absorbance, particularly the microplate, may be a plate made of polystyrene or the like capable of measuring the absorbance, but the volume of the well is preferably 300 to 500 μl. More preferably, it is 300 to 400 μl, further preferably, 300 to 350 μl. When the volume exceeds 500 μl, the container size becomes large and wasteful space is required, so that processing of multiple samples tends to be difficult. When the volume is less than 300 μl, the container size becomes small and the sample and reagent are dispensed. A minute operation such as an operation or the like is required to be minute, and the risk of erroneous operation in multi-sample processing tends to increase as the dispensing accuracy decreases.

In the present invention, the absorbance is measured by measuring the change in the fading reaction of the added ammonium cerium sulfate test solution as the change in absorbance or transmittance. The change in absorbance or transmittance is measured after the start of the fading reaction of the added ammonium cerium sulfate reagent solution. The change in the absorbance or the transmittance is measured by an end point measurement method (end point) in which the change in the bleaching reaction is measured for a certain time after the start of the reaction. It is preferable to measure the absorbance or the transmittance after the start of the reaction while the reaction is in progress, and measure the difference (reaction rate) by a reaction rate measurement method (rate assay). When the measurement is performed three or more times during the progress of the reaction, the reaction rate can be determined by a calculation method such as the least square method.

In the measurement of these absorbances, the measurement wavelength is selected from the range of 400 to 450 nm.
It is preferable to measure the absorbance at a wavelength. further,
The main wavelength is set to 400 to 4 in order to reduce fluctuations in measured values due to contamination or scratches between wells of the microtiter plate.
One wavelength selected from a range of 50 nm and a sub wavelength of 450
It is preferable to measure as a difference in absorbance of two different wavelengths selected from the range of -700 nm. Furthermore, after starting the fading reaction by adding the ammonium cerium sulfate reagent solution, when measuring the progress degree of the fading reaction as a change in absorbance or transmittance, the measurement time is preferably from 30 seconds to 60 minutes after the fading reaction starts. Preferably 5 to 3
It is necessary to perform at least one measurement during 0 minutes. Also, in order to reduce the dispersion of the measurement,
It is also preferable to perform the measurement more than once.

In the present invention, the apparatus for measuring the absorbance is not limited as long as it can measure the absorbance at 400 to 450 nm, but it is widely used in ELISA and the like, is excellent in portability, and can be continuously measured automatically. A general-purpose microtiter plate reader capable of processing a sample can be used as a preferable measuring device. Actually, the concentration of iodine in the sample is measured in advance or when measuring the sample under exactly the same conditions using an iodine standard solution of a known concentration instead of the sample, so that the absorbance or transmittance and the iodine concentration can be measured. A calibration curve for the relationship is created, and the amount of iodine in the sample is quantified or detected based on the calibration curve. The standard concentration of iodine can be appropriately selected depending on the measurement range. However, in the case where urine is used as a sample, for example, a concentration of 100 μg / L for determining a normal value is sandwiched. It is necessary to select a plurality of densities of at least two points.

[0039]

The present invention will be described below with reference to examples. Example 1 (Preparation of Reagent) 1) Chloric acid reagent solution (20 (W / V)%) 500 g of potassium chlorate was added to 900 ml of water and dissolved by heating. 400 ml of 62 (W / V)% perchloric acid was added little by little (about 20 ml / min) to the resulting solution while stirring. The resulting aqueous solution was ice-cooled, and allowed to stand in a freezer (−20 ° C.) overnight, and then filtered under reduced pressure to collect a filtrate.
This was stored in the refrigerator until use. 2) Arsenous acid reagent solution 20 g of arsenous acid was added to 500 ml of water, and 14 g of sodium hydroxide was added and dissolved. Further, 64 ml of concentrated sulfuric acid was added, 50 g of sodium chloride was added, and then water was added to make a final volume of 2 L. Stored at room temperature protected from light until use.

3) Ammonium cerium sulfate reagent solution 24.0 g of ammonium cerium sulfate dihydrate
It was dissolved in 5N sulfuric acid to make the final liquid volume 1 liter. Stored at room temperature away from light. 4) Iodine standard stock solution 10 μg / ml 168 mg of potassium iodate (100 mg of iodine) was dissolved in water to make a final solution volume of 100 ml (1 mg / m of iodine).
l). 1 ml of this solution was taken up to 100 ml to prepare a standard stock solution of 10 μg / ml. Stored at room temperature until use.

1. Sample Pretreatment 8 wells using a 96-well microtiter plate made of polypropylene (manufactured by Corning Coaster Japan) (hereinafter abbreviated as Plate A) to which 96 reaction regions (that is, wells) were connected as a reaction device for sample pretreatment. 0, 25, 50, 75, in 16 wells in rows 1 and 2
40 μl of a 100, 200, 300, or 400 ng / ml iodine standard solution was added to each well at two concentrations per concentration. In addition, 40 μl of a urine specimen of 20 children in an iodine-deficient area and 40 μl of an iodine-added urine prepared by adding 100 μl of an aqueous potassium iodate solution containing 50 ng in terms of iodine to 1 ml of the urine specimen.
1 was placed in each well. Then, 100 μl of a chloric acid reagent was added to each well of the plate A.

Further, in order to make each well of the plate airtight, the airtight tool shown in FIGS. 1 to 3 was used as a gastight tool for a 96-well microtiter plate. Specifically, as the upper support plate 2, (vertical 2
As a lower support plate 3 made of aluminum having a size of 00 mm × width 108 mm × height 30 mm, (length 200 mm ×
An aluminum support having a size of 108 mm (width) x 15 mm (height) is used. A snap screw made of aluminum is used as an auxiliary fixing member 4, and an elastic covering-like elastic body coated with a polyvinylidene chloride film is used as a spacer 6. Silicon rubber plate (length 113 mm x width 81 mm x height 3 mm), elastic silicone rubber plate (length 1) covered with a polyvinylidene chloride film as spacer 7
08 mm x width 75 mm x height 3 mm). The size of the storage space formed when the upper support plate 2 and the lower support plate 3 are combined is 108 mm in length × 90 mm in width × 18 mm in height, and the upper support plate is formed so that the storage space is formed. Board 2
The upper side of the lower support plate 3 and the upper side of the lower support plate 3 are respectively provided with recesses (upper support plate: 108 mm long × 90 mm wide × 16 mm high) Lower support plate: 108 mm long × 90 mm wide × 2 m high
m).

The plate A (reactor 5) was sandwiched between two elastic members of the spacers 6 and 7, and the plate A was stored with the well opening facing upward. Further, by fastening the lower support plate 3 and the upper support plate 2 from above and below by the fixing auxiliary material 4 attached to the lower support plate 3, each well of the plate A (reactor 5) increases the elasticity of the upper portion. The spacer 6 has a close contact and is in an airtight state. This was placed in a thermostat and heated at 105 ° C. for 1 hour. After heating, the airtight tool for the microtiter plate was taken out from the thermostat, placed on a heat sink, allowed to cool to room temperature, and then cooled to plate A (reaction). The device 5) was taken out.

2. Reagent reaction (Sandell-Kolthoff reaction)
Measurement of Absorbance After confirming that plate A after the pretreatment step of the sample returned to room temperature, 40 μl of the reaction solution of this plate was added to a new polystyrene 96-well microtiter plate (Nalgen Nunc International Co., Ltd.) using a multipipettor. (Hereinafter abbreviated as plate B). Then, 150 μl of the arsenite solution was added to all wells of plate B. Immediately (within 1 minute), add 40 μl of ammonium sulphate ammonium sulphate reagent solution to all the wells using a 12-well multipipettor, and after 20 minutes, mount the plate B on a microtiter plate reader (manufactured by Tosoh Corporation), 40
The absorbance at 5 nm was measured.

A calibration curve having the concentration on the horizontal axis and the logarithm of the absorbance on the vertical axis showed a straight line having a correlation coefficient of 0.99 or more. This suggests that no interfering substance that would affect the measured value was eluted from the pretreatment container. FIG. 4 is a plot showing the results of measurement of each human urine sample and the urine sample to which iodine was added in an iodine-deficient area, that is, the results of an addition recovery test. All the samples showed a concentration of 100 ng / ml or less, which was regarded as a normal value. Further, since the iodine added in the iodine-added urine is quantitatively recovered, it indicates that it is possible to measure a urine sample of an iodine-deficient subject, and indicates the reliability of the measurement method of the present invention. Things.

Example 2 (Preparation of reagent) Prepared according to Example 1. 1. Specimen pretreatment 8 sample PC made of polypropylene as a reaction device for specimen pretreatment
Using five R tubes (manufactured by Assist Co., Ltd.), 0, 25, 50, 75, 10
0, 200, 300, 400 ng / ml iodine standard solution 10μ
l, human urine 5 specimens 10 μl and its iodine-added urine specimen 1
0 μl (adjusted by adding 10 μl of an aqueous solution of potassium iodate containing 50 ng in terms of iodine to 1 ml of each urine sample) was placed in a standard solution of one concentration or two tubes (reaction areas) per sample. Further, 100 μl of a chloric acid solution was added to each tube, and the tube was capped with an 8-tube polypropylene tube cap (manufactured by Assist Co., Ltd.) as a lid to make the tube airtight. Then, put this in a thermostat,
For 1 hour. Then, it was left to cool to room temperature.

2. Reagent reaction (Sandell-Kolthoff reaction)
Measurement of Absorbance 150 μl of the arsenite solution was added to the treatment solution after the above pretreatment, and 100 μl thereof was transferred to a new 96-well microtiter plate made of polystyrene using a multipipette. Furthermore, 40 μl of ammonium cerium sulfate solution was added with a multipipette, and after 30 minutes, a 96-well microtiter plate made of polystyrene (Nargen Nunc International Co., Ltd., polystyrene) was attached to a microtiter plate reader (Tosoh Corporation). The absorbance at a wavelength of 405 nm was measured.

Table 1 shows the measured average value (n = 2) of each urine sample and iodine-added urine sample and the results of the recovery rate by adding iodine. All samples in iodized urine were 9
A good recovery rate of 3 to 103% was shown. A polypropylene 8-tube PCR tube is used as a ligation reaction region,
It was shown that the interfering substance can be removed even if the pretreatment step is performed under the airtight condition of only covering with the cap.

[0049]

[Table 1]

Example 3 (Preparation of reagent) Prepared according to Example 1. 1. Sample Pretreatment Step As a sample pretreatment reaction instrument, a PCR polycarbonate 8-well (NucleoLink strip, manufactured by Nargennunc International Co., Ltd.) was used.
Made of ABS (acrylonitrile-
Butadiene-styrene) frame (Nalgen Nunc International Co., Ltd., catalog No. 2491)
82). 0, 5, 10, 25, 50, 10
0, 150, 200, 250, 300, 400, 600
ng / ml iodine standard was placed in two wells per concentration. In addition, 6 human urine samples and 10 μl of the iodine-added urine sample (50 ml in terms of iodine were added to 1 ml of each urine sample).
ng-containing aqueous solution of potassium iodate (10 μl) was added to each of the 6 wells (reaction area).
Put in. 100 μl of chloric acid solution in all these wells
l, and covered with a silicon rubber plate 5 in order to make it airtight, sandwiched from above and below by two stainless steel support plates (thickness 3 mm, width 100 mm, length 150 mm) as fixing supports, and The airtight state was achieved with the screw of the fixing auxiliary material. Thereafter, this was placed in a thermostat and heated at 105 ° C. for 60 minutes. Then, it was left to cool to room temperature.

2. Reagent reaction (Sandell-Kolthoff reaction)
Measurement of Absorbance The measurement was performed in the same manner as in Example 2. Table 2 shows the measured average value (n = 6) of each urine sample, iodine-added urine sample, and the result of the recovery rate by iodine addition. All samples in iodized urine showed a good recovery rate of 83 to 102%, and the interference was completely removed by the pretreatment. The container itself was colored yellow by the oxidizing agent, but the interference was eluted from the container. Did not appear, indicating that pretreatment was possible even with a polycarbonate container.

[0052]

[Table 2]

Example 4 (Preparation of Reagent) A pretreatment was prepared according to Example 1 except that the chloric acid reagent solution as an oxidizing agent was changed to the following ammonium persulfate. 1) Ammonium persulfate reagent solution 200 g of ammonium persulfate was dissolved in water to make the final liquid volume 1 liter. 1. Sample Pretreatment As a sample pretreatment reaction instrument, 0, 5, 10, 25, 50, 100, 150, 20 were added to wells of a 96-well microtiter plate made of polypropylene (plate A).
0, 250, 300, 400, and 600 ng / ml iodine standard solutions were placed in each of two wells at a concentration of 12 human urine specimens and 12 iodine-added urine specimens (1 ml of each urine specimen contained 50 ng of iodine-equivalent potassium iodate). Aqueous solution 10
10 μl each of which was adjusted by adding each to three wells. Further, 50 μl of ammonium persulfate reagent was added to all wells of the plate.

The plate A was made airtight in the same manner using the airtight tool of the reaction apparatus used in Example 1. afterwards,
Plate A was placed in a thermostat and heated at 105 ° C. for 1 hour. After that, take out the airtight tool for the microplate from the thermostat, put it on the heat sink, and let it cool down to room temperature.
Plate A was taken out.

2. Reagent reaction (Sandell-Kolthoff reaction)
Measurement of Absorbance The reaction was carried out in the same manner as in Example 2 except that the reaction time after the addition of the ammonium sulfate ammonium cerium sulfate reagent was 60 minutes. The reaction rate of the Sandell-Kolthoff reaction was slightly slower than that in the case of pretreatment with chloric acid, and required about twice as long as one hour. Tables 3 and 4 show the measured average value (n = 2) of each urine sample and iodine-added urine sample, and the results of the recovery rate by adding iodine. Good recovery rate of 87-116% for all samples in iodized urine, complete removal of interfering substances by pretreatment, and complete removal of interfering substances by pretreatment with ammonium persulfate showed that.

[0056]

[Table 3]

[0057]

[Table 4]

Comparative Example 1 Standard conventional method (manual for measuring iodine in urine, International Organization for Countermeasures for Iodine Deficiency, 1993) (Preparation of Reagents) Prepared according to 1. 1. Sample pretreatment Using 28 glass test tubes (inner diameter 13 mm, length 10 cm), 0, 25, 50, 75, 100, 200, 300, 4
250 μl each of a 00 ng / ml iodine standard solution and 20 human urine samples were placed in a test tube of one concentration or two test tubes per sample. Then, 750 μl of chloric acid TS was added to all test tubes. Insert the test tube into the hole of the aluminum block thermostat (Iwaki Glass Co., Ltd.), which was previously adjusted to 115 ° C, in the local exhaust system with the exhaust device.
Heated for hours. After that, remove the test tube from the thermostat,
Allowed to cool.

2. Reagent reaction (Sandell-Kolthoff reaction)
Measurement of Absorbance After confirming that the test tube after the pretreatment of the sample almost returned to room temperature, 3.5 ml of an arsenous acid solution was added to the test tube. Then, in the first test tube, ammonium cerium sulfate TS 3
50 μl was added and stirred quickly. To the remaining test tubes, ammonium cerium sulfate reagent was added every 20 seconds using a stopwatch, and the absorbance at a wavelength of 405 nm was measured exactly 20 minutes after the addition of the ammonium cerium sulfate reagent.

FIG. 5 shows a correlation diagram between the measured values of 20 urine samples in Comparative Example 1 and the measurement results of the samples of Comparative Example 1 measured according to Example 1. As a result, a good correlation was recognized.
However, the generation of steam having a pungent odor peculiar to the oxidizing agent was observed from the test tube due to the pretreatment heating, and about 4 ml of harmful waste liquid such as arsenic and the oxidizing agent was discharged per sample. Further, as a result of measuring the change in the liquid amount, about 0.5 g per line was evaporated by heating, and the error due to the change in the liquid amount was not negligible.

Example 5 (Preparation of reagent) Prepared according to Example 1. 1. Sample Pretreatment As a reaction device for sample pretreatment, using 96-well polypropylene microtiter plates (manufactured by Corning Coaster Japan), 0, 25, 50, 75, 100, 200, 3
40 μl of an iodine standard solution of 00 or 400 ng / ml was added to each well at 2 wells per concentration, and 80 human urine samples were placed in other wells. Then, 100 μl of ammonium persulfate test solution was added to all wells of the plate, and the weight was measured. Using the airtight tool of the reaction apparatus used in Example 1, the microtiter plate was similarly made airtight. After that, the airtight tool equipped with the microtiter plate was placed in a thermostat and heated at 105 ° C. for 1 hour. Thereafter, the microtiter plate was taken out of the thermostat, placed on a heat sink, allowed to cool to room temperature, taken out of the microtiter plate, and weighed again.

[0062] 2. Reagent reaction (Sandell-Kolthoff reaction)
Measurement of Absorbance The measurement was performed in the same manner as in Example 2. As a result, the weight of the plate pretreated using the hermetic tool of the reaction apparatus for performing the hermetic condition was reduced by 0.3 g. The calibration curve of the relationship between the absorbance and the iodine concentration is shown in FIG.

Comparative Example 2 In the pretreatment reaction of Example 5, the same method and the same method as in Example 5 were used except that the reaction apparatus was not placed in an airtight device, but was placed in an open system, placed in a thermostat, and pretreated. Performed under conditions. As a result, the weight of the plate pretreated in the open system was about 9.2.
g, and the liquid volume in each well varied. In addition, generation of steam having a pungent odor peculiar to the oxidizing agent was observed by pretreatment heating. In terms of the amount of liquid evaporated per well (solution specific gravity 1), an average of 96 μl was evaporated. Crystals were precipitated from the two wells in the plate pretreated in the open system by evaporation of the solution.
This is due to the difference in the degree of evaporation in each well due to the temperature deviation of the plate as a result of performing the pretreatment in an open system, and the scattering of the solution when the oxygen bubbles generated by decomposition of ammonium persulfate burst. it was thought. FIG. 7 shows the relationship between the absorbance and the iodine concentration. As shown in the figure, a one-to-one relationship between the iodine concentration and the absorbance was not obtained, and the calibration was not possible.

Example 6 (Preparation of reagent) Prepared according to Example 1. 1. Sample pretreatment Four human urine samples and an iodine-added urine sample obtained by adding 50 ng / ml of iodine to the sample (50 ml of iodine conversion per 1 ml of each urine sample)
ng-containing aqueous potassium iodate solution (10 μl) was added to each of the samples, and the heating operation in the sample pretreatment step was air-tight. Example 2 except that the heating time was changed
And went exactly the same.

2. Reagent reaction (Sandell-Kolthoff reaction)
Measurement of Absorbance The measurement was performed in exactly the same manner as in Example 2. Table 5 shows the measured average value (n = 2) of each urine sample and iodine-added urine sample and the results of the recovery rate by iodine addition. In the iodine-added urine, all the samples showed a good recovery of 94 to 108%. The average (n = 2) of the measured values of each urine sample and the measured values of the iodine-added urine sample indicated that the pretreatment removed the interfering substance.

Comparative Example 3 (Preparation of reagent) Prepared according to Example 1. 1. Sample pretreatment Four human urine samples and an iodine-added urine sample obtained by adding 50 ng / ml of iodine to the sample (50 ml of iodine conversion per 1 ml of each urine sample)
ng-containing aqueous solution of potassium iodate (10 μl) was added to each of the samples, and the heating operation after airtightness in the pretreatment of the sample was omitted, except that the heating operation was omitted. 2. Reaction of test solution (Sandell-Kolthoff reaction) and measurement of absorbance The same procedure as in Example 2 was carried out. Table 5 shows the measured average value (n = 2) of each urine sample and iodine-added urine sample and the results of the recovery rate by iodine addition. That is, the recovery rate when chloric acid was added and the heating operation was not performed was compared with the recovery rate after heating at 105 ° C. for 30 minutes and the heating rate after heating for 1 hour. From Table 5, when the heating operation was not performed, the recovery rate of addition of iodine was only 58% on average, but it was 0.5% in Example 5.
The average recoveries after heating for 30 minutes and 1 hour were 97% and 101%, respectively.

[0067]

[Table 5]

Example 7 (Preparation of reagent) Prepared according to Example 1. 1. Sample Pretreatment As a sample pretreatment reaction instrument, 0, 5, 10, 25, 50, 100, 150, 20 were added to wells of a 96-well microtiter plate made of polypropylene (plate A).
10 μl of 0, 250, 300, 400, and 600 ng / ml iodine standard solutions were added to two wells per concentration. 10 μl each of 6 human urine specimens and 6 iodine-added urine specimens (adjusted by adding 10 μl of an aqueous potassium iodate solution containing 50 ng in terms of iodine to 1 ml of each urine specimen) in 4 wells, respectively. 100 μl of a chloric acid solution was added to all wells, and the temperature of the thermostat was set to 80 ° C.
C. and 120.degree. C., respectively, except that heating was performed for 30 minutes each. 2. Reaction of test solution (Sandell-Kolthoff reaction) and measurement of absorbance The reaction was performed in exactly the same manner as in Example 3. FIG. 8 shows the relationship between the reaction temperature of the pretreatment and the recovery of the measured value due to the addition of iodine.

Example 8 (Preparation of reagent) Prepared according to Example 1. 1. Specimen pretreatment The same procedure as in Example 7 was carried out except that the temperature of the thermostat was set to 50 ° C and heating was performed. 3. Reaction of test solution (Sandell-Kolthoff reaction) and measurement of absorbance The reaction was performed in exactly the same manner as in Example 3. 12 human urine samples and an iodine-added urine sample obtained by adding 50 ng / ml of iodine to the sample (adjusted by adding 10 μl of a potassium iodate aqueous solution containing 50 ng in terms of iodine to 1 ml of each urine sample)
The recovery rate of iodine when the reaction was performed at a pretreatment temperature of 50 ° C. for 30 minutes was examined. The reaction temperature (heating temperature) of the pretreatment was compared with the recovery of the measured value due to the addition of iodine. FIG. 8 shows the relationship between the iodine addition and recovery rates in Example 7 and Example 8.

From the results, it was found that the pretreatment temperature in Example 8 was 5
As for the recovery at 0 ° C., the average recovery was low, and the variation was large depending on the sample. This was presumed to depend on the urinary interfering substance content in the sample. On the other hand, at 80 ° C. or higher in Example 7, a recovery rate of 90% or higher was obtained. From these results, it is preferable that the pretreatment temperature for removing the interfering substance in urine be higher than 50 ° C and the pretreatment temperature be 80 ° C or higher and 120 ° C or lower.

Example 9 (Preparation of reagent) Example 1 was repeated except that the conditions for the concentration of chloric acid used in the pre-heating treatment were changed.
The same was done. That is, the chloric acid reagent solution (2
0 (W / V)%), add an equivalent amount of purified water and adjust the chloric acid concentration to 10
(W / V)%. 1. Sample pretreatment Five human urine samples and five iodine-added urine samples (each urine sample 1
The same procedure was followed as in Example 2 except that 10 μl of an aqueous solution of potassium iodate containing 50 ng in terms of iodine was added to each ml, and 10 (W / V)% chloric acid was used for the pretreatment. Was. 2. Reaction of test solution (Sandell-Kolthoff reaction) and measurement of absorbance The same procedure as in Example 2 was performed. Table 6 shows the results of addition and recovery rates of urine iodine in Examples 2 and 9. In the pretreatment with 10% chloric acid in Example 9, the recovery rate varies greatly depending on the sample, and the influence of the interfering substance in urine is presumed. Rate. That is, the concentration of chloric acid to be added for the pretreatment is preferably 20% or more than 10%.

[0072]

[Table 6]

Example 10 (Preparation of reagent) Prepared according to Example 1. 1. Preparation of Sample Pretreatment Reaction Device (Plate A) As a sample pretreatment reaction device, four 96-well polypropylene microtiter plates (manufactured by Corning Coaster Japan) were used. 25, 50, 75, 100, 150, 20
10 μl of 0, 250, 300, 400, or 600 ng / ml iodine standard solution was added to each well (Plate A). 2. Pretreatment of the sample Before each well without adding the iodine standard solution,
Human urine 28 measured by the autoanalyzer method (Technicon Instruments) and the measured values are known.
Three samples were placed in each well. Further, 100 μl of a chloric acid solution was added to each well containing an iodine standard solution and a human urine sample.
Was added respectively. Performed in the same manner as in Example 1.

3. Reagent reaction (Sandell-Kolthoff reaction)
After measuring that the temperature of the plate A after the pretreatment of the sample was almost returned to room temperature, the arsenous acid solution 15 was added to all the wells of the plate B.
0 μl was added. 60 μl of the reaction solution from plate A was transferred to a new 96-well microtiter plate (Nargen Nunc International Co., Ltd., polystyrene) (plate B) using a multipipette.
Immediately (within 1 minute), add 40 μl of ammonium cerium sulfate reagent solution to all the wells with a 12-multiple pipettor, and after 30 minutes, mount the plate B on a microtiter plate reader (manufactured by Tosoh Corporation), and measure the absorbance at a wavelength of 405 nm. Was measured. Autoanalyzer method for 283 human urine samples (Technicon Instruments)
The correlation between the measurement results of Example 10 and Example 10 was examined, and the results are shown in FIG. The results showed good correlation with a correlation coefficient of about 0.92 and a slope of 1.03.

[0075]

According to the method for measuring iodine according to the first aspect of the present invention, in the pretreatment, evaporation of harmful vapor and a change in the amount of the reaction solution can be suppressed without requiring a special facility such as a local exhaust facility. In addition, the sample can be safely pre-processed and a measured value with good reproducibility can be obtained. According to the method for measuring iodine according to the second aspect, since there is no concern about breakage of the container during the heat treatment, a safe and highly reliable sample pretreatment can be performed. According to the method for measuring iodine according to the third aspect, in the pretreatment, the evaporation of harmful vapor and the change in the amount of the reaction solution are suppressed without requiring a special facility such as a local exhaust facility, and the container is damaged. Since there is no need to worry about such a problem, safe and reliable pretreatment of the sample can be performed, and a measured value with good reproducibility can be obtained.

According to the method for measuring iodine according to the fourth aspect, in addition to the invention according to the second or third aspect, pretreatment of a large number of samples can be quickly and easily performed, which is suitable for measurement of a large number of samples.
According to the method for measuring iodine according to claim 5, claims 2 to
In addition to the invention described in 4, the sample can be safely and highly reliably pre-treated because there is no risk of damage to the container during the heat treatment and there is no elution of substances that affect the measured value. . According to the method for measuring iodine according to the sixth aspect, in addition to the inventions according to the second to fifth aspects, an inexpensive and highly reliable reactor is easily available, and there is no worry about damage to the container during the heat treatment and the like. In addition, the sample can be pre-treated without elution of a substance that affects the measured value.

According to the reaction apparatus for sample pretreatment according to the seventh aspect, there is no need to worry about breakage of the container during the heat treatment, and safely, and without elution of substances that affect the measured value. Pre-processing of many samples is possible. According to the hermetic tool of the reaction device for sample pretreatment according to claim 8, in the pretreatment of the sample for iodine measurement, special equipment such as a local exhaust facility is not required, the place where the sample is processed is not limited, and the harmful vapor is not restricted. And the amount of hazardous waste generated can be suppressed, and the processing of a large number of samples can be facilitated.

[Brief description of the drawings]

FIG. 1 is a diagram showing a front view when used as an example of an airtight tool for a microtiter plate.

FIG. 2 is a plan view when used as an example of an airtight tool for a microtiter plate.

FIG. 3 is a sectional view taken along a line aa ′ of FIG. 2 (however, a state in which a snap screw as one fixing auxiliary member is removed).

FIG. 4 is a graph showing the results of an iodine addition and recovery test in Example 1.

FIG. 5 is a diagram showing a correlation between a measurement value of Comparative Example 1 of 20 urine samples and a measurement result of the same sample measured by the method of Example 1.

FIG. 6 is a diagram showing a calibration curve of a relationship between iodine concentration and absorbance in Example 5.

FIG. 7 is a diagram showing the relationship between iodine concentration and absorbance in Comparative Example 2.

FIG. 8 is a graph showing the relationship between the reaction temperature of pretreatment and the recovery rate of iodine addition in Examples 7 and 8.

FIG. 9: Autoanalyzer method for human urine specimen and Example 1
It is a figure which shows the correlation of the measured value of 0.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Fixing support 2 Upper support plate 3 Lower support plate 4 Fixation auxiliary material 5 Reaction device 6 Spacer 7 Spacer

Claims (8)

[Claims] 1. An iodine concentration in a specimen comprising a pretreatment step of a specimen in which the specimen is digested by heating with an oxidizing agent, followed by a reaction measuring step of reacting an arsenous acid reagent solution and an ammonium cerium sulfate reagent solution and measuring the absorbance in the reaction solution. In a method for quantifying or detecting, a pretreatment step of a sample is performed by heat-treating the sample under airtight conditions and cooling the sample, followed by cooling. 2. An iodine concentration in a specimen comprising a pretreatment step of the specimen in which the specimen is digested by heating with an oxidizing agent, and a reaction measuring step of reacting the arsenous acid reagent solution and the cerium ammonium sulfate reagent solution and measuring the absorbance in the reaction solution. A method for measuring or measuring iodine, wherein the step of pretreating a sample is performed using a reaction device made of a heat-resistant organic material. 3. An iodine concentration in a specimen, comprising a pretreatment step of the specimen in which the specimen is digested by heating with an oxidizing agent, and a reaction measuring step of reacting the arsenous acid reagent solution and the cerium ammonium sulfate reagent solution and measuring the absorbance in the reaction solution. In the method for quantifying or detecting the amount of iodine, the pretreatment step of the sample is performed by using a reaction device made of a heat-resistant organic material, and further, by performing a heat treatment under airtight conditions and cooling. Method. 4. The method for measuring iodine according to claim 2, wherein the reaction device made of a heat-resistant organic material has a plurality of reaction regions. 5. The method for measuring iodine according to claim 2, wherein the heat-resistant organic material is a polypropylene resin, a polycarbonate resin, a polysulfone resin, a polyethersulfone resin, a Teflon resin, or a polymethylpentene resin. . 6. The method for measuring iodine according to claim 2, wherein the reaction device is a microtiter plate. 7. A heat digestion treatment of a plurality of samples together with an oxidizing agent, comprising at least one heat-resistant organic material selected from the group consisting of a polypropylene resin, a polycarbonate resin, a polysulfone resin, a polyethersulfone resin and a polymethylpentene resin. A reaction device for sample pretreatment for iodine measurement, comprising a plurality of reaction regions capable of performing iodine measurement. 8. A sample pretreatment reaction device for iodine measurement according to claim 7, and a spacer for covering a plurality of reaction areas of the reaction device, which are vertically sandwiched and fixed to two upper and lower fixing supports. And an airtight device for a sample pretreatment reaction device, comprising a fixing auxiliary material capable of applying pressure to these fixing supports so that the reaction region of the reaction device can be airtightly sealed by the spacer.