RU2265847C2 - Method for photometric detection of urinary iodine - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: the present innovation deals with laboratory techniques of investigation and includes centrifuging and evaporating a sample, transferring iodine into iodate-ions followed by treatment with a color reagent and measuring optic density of the solution obtained. Before centrifuging organic compounds in urinary sample should be transferred into residue, centrifugate should be supplemented with chloroform; afterwards the inferior chloroformic layer should be treated with chlorine water, aqueous phase should be separated to remove chlorine residues. Then one should add 1 n potassium iodide solution and 0.1 n sulfuric acid solution followed by photometering the solution at wave length being 400 nm. Organic compounds in a sample should be precipitated due to adding barium hydroxide and zinc sulfate.

EFFECT: higher sensitivity and accuracy of detection.

1 cl, 2 tbl

Description

The invention relates to biochemistry, in particular to methods for determining the microconcentration of iodine in urine, and can be used in biochemical, analytical and other laboratories for the diagnosis of iodine deficiency diseases by determining the excretion of inorganic iodine in urine.

Known methods for the determination of iodides in water by the titrimetric method [1] and with an ion-selective electrode [2]. However, the sensitivity of these methods is low (0.2-1.0 mg / dm ³ ), and they cannot be used to determine iodine in the urine.

The closest to the proposed method in technical essence and the achieved result is a catalytic method for the determination of iodides, based on measuring the reaction rate between cerium (IV) and arsenic (III) in an acidic solution. The method was developed by E. Sandle and J. Kolthoff [3, 4]. The solution containing cerium (IV) ion is yellow. During the reaction of cerium reduction during oxidation with arsenic in the presence of iodine, the solution becomes discolored. The rate of attenuation of the yellow color of the solution in the reaction is directly proportional to the iodine content in the sample. The sensitivity of the method is 0.04 μg / ml iodine.

Based on this method, a number of photometric methods have been developed for determining iodine in urine and blood [5, 6], however, the disadvantages of various variations of the method include the fact that the reaction between cerium (IV), arsenic (III) and iodine proceeds quite quickly, the solution from yellow it instantly becomes transparent, and it is impossible to photometer it at any wavelengths either on a spectrophotometer or on a photoelectric calorimeter. The authors of the developments suggest measuring optical densities of solutions and constructing a calibration graph for the dependence of optical density on the concentration of iodine in solution for calculations. However, in the original source [3], the authors construct a calibration graph of the dependence of the bleaching time of the solution on the iodine concentration in the solution, which is more true and reliable.

Thus, the disadvantages of the catalytic method include:

- the rapid disappearance of the color of the reaction, which does not allow to accurately measure the optical density of the solution;

- the use of expensive and scarce (cerium sulfate) reagents;

- osmium, rhenium [4] can interfere with the determination, which can lead to large errors.

When developing the method, the task was to increase the accuracy and sensitivity of the determination of iodine in urine.

To solve this problem, in the proposed method, which includes preparing the sample by centrifuging and evaporating it, converting iodine into iodate ions, followed by treatment with a color reagent and measuring the optical density of the resulting solution, the organic compounds are precipitated before centrifugation; chloroform is added to the centrifugate, after which the lower layer is treated with chlorine water, 1N is added. potassium iodide solution; photometer the colored solution at a wavelength of 400 nm. Barium hydroxide and zinc sulfate are used to precipitate organic compounds.

Distinctive features of the method is:

- the conversion of organic compounds into sediment, as a result of which their interfering effect is eliminated and the solution decolours;

- extraction of molecular iodine from the centrifugate with chloroform, followed by treatment of the bottom layer of the centrifugate with chlorine water to transfer iodine to iodate ions;

- processing the resulting solution 1 N. potassium iodide solution;

- photometry of the colored solution on a photoelectrocalorimeter at a wavelength of 400 nm;

- organic compounds are precipitated by adding barium hydroxide and zinc sulfate to the sample.

The sensitivity of the method increases to 0.025 μg / cm ³ due to the transfer of iodate ions into the triiodate complex with potassium iodide and photometry of the yellow-colored solutions. The accuracy of determination is improved by obtaining a stable color of the solutions, which lasts for a long time (6 hours), as well as eliminating the interfering effect of organic substances.

The method is as follows.

To 2 cm ^{3 of} urine in vitro add 2 cm ^{3 of a} 0.3% solution of barium hydroxide and 5 cm ^{3 of a} 5% solution of zinc sulfate. The contents of the tube are thoroughly shaken and centrifuged for 4 minutes. The centrifuge is poured into a separatory funnel.

The sediment in a test tube is washed with 3 cm ^{3 of} distilled water and centrifuged again for 2 minutes. The centrifuge is poured into the same separatory funnel, 8 cm ^{3 of} chloroform is added and shaken. The lower chloroform layer is transferred to another separatory funnel, add 1 cm ^{3 of} chlorine water and 7 cm ^{3 of} distilled water, shake well.

The aqueous phase is transferred to cups and placed in a sand bath for 10 minutes to remove residual chlorine.

The cooled solution is transferred into a volumetric flask of 25 cm ³ , poured 3 cm ³ 1 N. a solution of potassium iodide and 4 cm ³ 0.1 N. sulfuric acid solution, adjusted to the mark with water. The yellow colored solution is photometered on a photoelectrocalorimeter at a wavelength of 400 nm in 10 mm cells in relation to a blank sample (distilled water).

The iodine content in the urine is found according to the calibration schedule, the construction of which is carried out similarly to samples from a standard solution of potassium iodide with a concentration of 0.25 μg / cm ³ (see table 1).

Table 1
Standard scale No. standard KI standard solution with an I ₂ content _of 0.25 μg / cm ³ Distilled water, cm ³ Sulfuric acid 0.1 n. rr, cm ³ The content of I ₂ , mcg 0 0 21.0 4 0 1 0.1 20.9 4 0,025 2 0.2 20.8 4 0.05 3 0.4 20.6 4 0.10 4 0.6 20,4 4 0.15 5 0.8 20,2 4 0.20 6 1,0 20,0 4 0.25

Table 2 presents the calibration dependence of the optical density on the concentration of iodine in solution.

table 2
Calibration graph №№ The concentration of iodine in solution, mcg The value of optical density (D) 1 0,025 0,03 2 0.05 0.06 3 0.10 0.125 4 0.15 0.175 5 0.20 0.24 6 0.25 0.30

The scale is stable for 6 hours, the values of the optical density of the calibration scale, measured after 6 hours, were as follows: 0,028; 0.063; 0.12; 0.18; 0.24; 0.305.

The proposed method is characterized by high sensitivity, accuracy and reproducibility.

Used sources

1. Unified methods of water research. - M., 1977.

2. Guidelines 4.1.0-97 FU Medbioextrem. Determination of iodine with an ion-selective electrode "Eco-J".

3. Sandell E.B., Kolthoff I.M. Chronometric catalytic Method for the Determination of micro Quantities of Jodine // J. Am. Chem. Soc., 56, 1426 (1954).

4. Yatsimirsky K. B. Kinetic methods of analysis. - And: "Chemistry", 1967. - p.168-169.

5. Selyatitskaya V. G., Palchikova N. A., Galkin P. S. The experience of determining iodine in urine by the kinetic cerium-arsenate method // f. Clinical laboratory diagnostics. - No. 5. - 1996, - p. 22.

6. Stepanov G.S. Determination of iodine bound to blood proteins. // w. Laboratory work. - No. 7, 1965. - P.594.

Claims (2)

1. The method of photometric determination of iodine in urine, including centrifugation and evaporation of the sample, the conversion of iodine into iodate ions, followed by treatment with a color reagent and measuring the optical density of the resulting solution, characterized in that the organic compounds are precipitated before centrifugation, chloroform is added to the centrifugate, after whereby the lower chloroform layer is treated with chlorine water, the aqueous phase is separated, the remaining chlorine is removed from it, 1N is added. potassium iodide solution and 0.1 n. sulfuric acid solution, photometrically colored solution at a wavelength of 400 nm. 2. The method according to claim 1, characterized in that the organic compounds are precipitated by adding barium hydroxide and zinc sulfate to the sample.