CS273657B1 - Two-component agent for magnesium photometric determination - Google Patents

Abstract

The agent consists of two solutions: metallochrome indicator of 1-azo-2-hydroxy-3-(2,4-dimethyl-carboxy-anilido)- naphthalene-1'-(2-hydroxybenzene) in the specific mixture of two organic solvents of N,N-dimethylformamide and dimethyl sulfoxide, and tetraborate buffer containing acyclic alcohol with the total number of 1 to 3 carbon atoms. Both the components are contained in the agent in the volume ratios of 1 : 10 to 1 : 50. The two-component agent is characterized by high stability of the metallochrome indicator in the solution and it is especially useful for the analysis of magnesium in body fluids. The very analytic agent for determination of magnesium is prepared by mixing both the components in the above-mentioned ratios before carrying out the analysis.

Description

(57) The reagent consists of two solutions, namely a solution of 1-azo-2-hydroxy-3- (2,4-dimethylcarboxyanilido) -naphthalene-1 '- (2-hydroxybenzene) metalochrome indicator in a specific mixture of two organic solvents Ν, Ν-dimethylformamide and dimethyl sulfoxide and from a tetraborate buffer solution containing an acyclic alcohol having a total of 1 to 3 carbon atoms.

Both components are contained in the reagent in volume ratios of 1:10 to 1:50. The two-component reagent is characterized by high stability of the metalochromic indicator in solution and is particularly suitable for the analysis of magnesium in body fluids. The actual magnesium analytical reagent is prepared by mixing both components in the above proportions before performing the analysis.

Italy (11) (13) Bl (51) Int. Cl. ⁵ G 01 N 21/27 G 01 N 21/78

CS 273657 Bl

The present invention provides a two-component reagent for the photometric determination of magnesium.

It is known that magnesium is an essential activator of a number of enzymatic processes, in particular by transphosphorylation, and is an essential element even in oxidative phosphorylations. It plays an important role in the metabolism of ribonucleic acids, cholesterol, lipids, activation of cholesterol esterases and various lipases, and has a proactive effect in the synthesis of protein metabolism. The effect of magnesium on the central nervous pyntama is also significant; magnesium deficiency causes increased irritation to tetany. Reduced serum magnesium has been found in nervous, vascular and cardiac disorders, severe diarrhea, cirrhosis of the liver, and some of the dioxia. Increased magnesium content, on the other hand, is found mainly in renal insufficiency, diabetic coma and acidosis.

It follows that the determination of magnesium, particularly in the serum, is an important indicator of a number of serious disorders. Therefore, serum magnesium assays are included in a variety of biochemical examinations performed by the Clinical Biochemists Department.

A number of analytical methods have been designed to determine the magnesium content, especially in the aura of the urine, but recently the instrumental methods based on the atomic absorption spectrophotometer have clearly prevailed !! and photometric methods to measure the coloring of colored magnesium complexes with suitable analytical reagents such as metallo-chromic indicators. Photometric methods with respect to available instrumentation of most biochemical laboratories so far prevail.

In recent years, mainly 1-azo-2-hydroxy-3- (2,4-dimethylcarboxyanilido) -naphthalene-1 '- (2-hydroxybenzene), also called the commercial names Magon or Xylidile Blue II, has been used for the photometric determination of magnesium. A detailed study of the optical properties of Magon has been studied, for example, by Svoboda and Chromý (Anal. Chim. Acta 54, 121-131, 1971) and its application for photometric determination of magnesium in biological fluids has been described by Chromý et al. (Bioch. Med. 7, 208-217, 1973). In Magnesium photometric determination of Magon, the coloring of the relatively weak complex O composition MgR, where R is Magon, is measured with a stability constant K = 6.1. 10 and an absorption molar coefficient of 4.3 .10 1. mol. cm. Work with at least a triple excess of reagent and measure at 505 nm.

The complex should be measured in an environment containing 60 to 40 parts by volume of water and 40 to 60 parts by volume of alcohol. Otherwise the sensitivity of the assay decreases and both the reagent and the complex are eliminated from the reaction mixture. Higher amounts of alcohol, on the other hand, are unfavorable as they cause the precipitation of proteins from the sample when analyzing biological fluids. For this reason, a solution of Magon in alcohol is usually prepared and mixed with an equal amount of an aqueous buffer solution prior to analysis. Relatively very specific and precise adherence to optimal reaction conditions places high demands on the composition, stability and purity of the analytical reagent.

A disadvantage of the Magon metalochromic agent is its low solubility in water and polar solvents. Therefore, Magon solutions are usually prepared by dissolving in boiling ethanol or by dissolving in dimethylformamide and not diluting with ethanol. These solutions have also been found not to be stable and suitable for analytical purposes. Magon is gradually eliminated from these solutions as insoluble matter, its effective concentration in the reagent decreases and both the slope of the calibration line and the linear range of the calibration function change. Magon precipitated spontaneously from ethanol solution so no longer acts as a metalochromic indicator during aging, it is probably an oxidized form, which is not suitable for photometric purposes. These disadvantages, the low solubility of Magon, the changes it undergoes in ethanol solution, have an adverse effect on the photometric determination of magnesium, since the sensitivity of the reaction and the course of the calibration function change.

Especially in the determination of serum magnesium in patients with severe renal disorders, when the magnesium content in serum increases rapidly, the analytical concentration of Magon in the reagent is not sufficient and there is an increased risk of incorrect analysis, diagnosis and treatment process progress. In many cases it is necessary to dilute the serum and further reduce the accuracy of the assay. Therefore, it is desirable

CS 273657 B1 eliminate these disadvantages and prepare such an analytical reagent for the photometric determination of magnesium that does not have the above drawbacks.

The two-component reagent for photometric determination of magnesium based on 1-azo-2-hydroxy-3- (2,4-dimethylcarboxyanilido) -naphthalene-1 '- (2-hydroxybenzene) and tetraborate buffer eliminates the disadvantages. It consists of one volume part of a solution containing 1 to 10 mmol / l of 1-azo-2-hydroxy-3- (2,4-dimethylcarboxyanilido) -naphthalene-1 (2-hydroxybenzene), 5 to 10 mmol / l. 7 mol / l of Ν, Ν-dimethylformamide and 4 to 9 mol / l of dimethylsulfoxide and 10 to 50 parts by volume of an aqueous-alcoholic solution containing 0,005 to 0,1 mol / l of alkali metal tetraborate and 4 to 15 mol / l of acyclic alcohol of 1 to 3 carbon atoms.

The Magon solution according to the invention is characterized by a very good stability, the Magon is not eliminated from the solution and its composition does not change in the long term. The combination of two specific organic solvents makes it possible to substantially increase the Magon content of the solution and to prevent its irreversible changes. The combination of an aqueous buffer solution with an aliphatic alcohol, which is usually an essential part of the reaction mixture in the Magon magnesium determination, stabilizes the buffer solution in the long term and protects it from bacterial contamination. For the actual photometric determination of magnesium, the reaction mixture is prepared by mixing the Magon solution with a buffer solution, for example 1 part of the Magon solution and 25 parts of the buffer solution according to the invention, which guarantee optimal composition of the reaction mixture for the photometric determination of magnesium. The mixture contains a sufficient excess of Magon sufficient to analyze biological fluids containing a pathologically elevated magnesium content. For example, sera of dialyzed patients containing up to 2 mmol of magnesium per liter of serum can be analyzed directly, without dilution.

The two-component reagent according to the invention can be industrially produced since both components are stable and guarantee long-term accurate and correct photometric determination of magnesium with the sensitivity and precision required for biochemical analyzes and in a concentration range suitable for analysis of common biological fluids, particularly serum and plasma. necessary biological dilutions prior to analysis. 5:

Example 1

(a) Prepare a solution containing 1,44 g (3,5 mmol) of 1-azo-hydroxy-3- (2,4-dimethylcarboxyanilido) -naphthalene-1 (2-hydroxybenzene) (hereinafter Magon), 450 ml ( 5.85 moles of Ν, Ν-dimethylformamide and 450 ml (6.34 moles) of dimethylsulfoxide.

b) Prepare 25 L of a buffer solution containing 97.6 g (0.25 mol) of sodium tetraborate decahydrate in 10.2 L (0.57 mol) of distilled water and 12.2 L (214.6 mol) of ethanol. The solution was stirred and adjusted to pH 10.9 by successive addition of sodium hydroxide (about 20 g of sodium hydroxide needed) and made up to 25 L with water.

For photometric determination, a reaction mixture containing one volume of solution a) and 25 volume of solution b) is prepared.

Example 2

a) Prepare a solution of 0.82 g (1.9 mmol) of Magon in a mixture of 365 g (5 moles) of N, N-dimethylforma.

mide and 702 g (8.98 mol) of dimethyl sulfoxide. \

b) Prepare 1000 ml of a buffer containing 0.005 moles of potassium tetraborate and 480 g (14.98 moles) of methanol and make up to 1000 ml with water. Its pH is adjusted to + 0.2.

Before use, mix two volumes of solution a) with 25 volumes of solution b),

M *

CS 273657 Bl

Example 3

a) Prepare. A solution containing 4.1 g (9.9 mmol) of Mngon, 511 g (6.99 moles) of Ν, Ν-dimethylformamide and 313 g (4 moles) of dimethylsulfoxide.

b) Prepare 1000 ml of a buffer containing 0.1 mol of potassium tetraborate and 241 g (4.0 mol) of 1-propanol, make up to 1000 ml with water and adjust the pH as in Example 1b).

Prior to the photometric determination of magnesium, mix 1 volume of solution a) with 50 volume of solution b).

Claims (5)

SUBJECT OF THE INVENTION Two-component reagent for photometric determination of magnesium based on l-azo- 2-hydroxy- 3- (2, 4-dimethylcarboxyanilido) -naphthalene-1 '- (2-hydroxybenzene) and tetraborate buffer, characterized in that it consists of one volume of solution containing 1 to 10 mmol / l of 1-azo-2-hydroxy-3- (2 4-dimethylcarboxyanilido) -naphthalene-1 '- (2-hydroxybenzene), 5 to 7 mol / l of Ν, Ν-dimethylformamide and 4 to 9 mol / l of dimethylsulfoxide and 10 to 50 parts by volume of an aqueous-alcoholic solution containing 0.005 to 0.1 mol / l of alkali metal tetraborate and 4 to 15 mol / l of acyclic alcohol having a total of 1 to 3 carbon atoms.