Method of preparing reagent for determining sugars in presence of reducing substances

FIELD: chemistry.

SUBSTANCE: method involves dissolving 855 mg of a crystalline hydrate of copper chloride (CuCl2·2H2O) in 100 ml of distilled water (concentration of Cu2+ ions in the prepared solution is 50 mmol/l) and adding 1 ml of the prepared solution to 100 ml of a standard reagent used in glucose oxidase test. The ascorbic acid oxidant used is copper chloride solution in end concentration in the glucose oxidase reagent of 500 mcmol/l.

EFFECT: method enables correct determination of glucose content.

1 tbl, 1 ex

 

The invention relates to the field of analytical chemistry for the determination of sugars in the presence of high concentrations of reducing substances that create barriers for correct determination of sugar content methods, based on the reducing properties of sugars.

To achieve the correct measurement of the content of sugars in the presence of reducing substances last pre-oxidised copper salts (II) (table 1). The invention can be used to determine the sugar content in biological fluids (blood, urine, spinal fluid and other), in fruits, vegetables, fruit juices, dairy products in the presence of reducing substances.

Most methods for the determination of sugars based on the reducing properties due to the aldehyde group - iodometric method, Bertrand and ferricyanide method (1). On the reducing properties of glucose based specific enzymatic glucose oxydase method for the determination of its content in blood and other biological fluids (2). These methods do not allow the correct measure of glucose in the presence of significant amounts of reducing substances (>100 µmol/l). In particular, the use of glucose oxydase method for the determination of glucose in blood (3, 4, urine (5) or in the cerebrospinal fluid (6) in the presence of reducing substances leads to underestimated values of glucose, because compounds having reducing properties, reduce the intensity of the color developing due to the recovery of the resulting colored complex. There is therefore a need to develop a reagent for the determination of sugars, which would correctly measure the content of sugars in the presence of significant amounts of reducing substances.

Famous Regent-based enzyme ascorbicacid, specifically oxidizing ascorbic acid and used for the determination of glucose in blood in the presence of significant quantities of ascorbic acid (3). The reagent is added to glucoseoxidase reagent at a final concentration of askorbatoksidazy ME/L. However, due to the high cost of ascorbicacid order of magnitude greater than the cost of glucoseoxidase, this reagent does not seem economically feasible. In addition, ascorbicacid cannot be used in non-enzymatic methods for the determination of sugars based on the regenerative properties of the latter.

Closest to the proposed reagent is the reagent on the basis of a acetate mercury (II). Adding this reagent at a final concentration of 3 mmol/l for blood samples the urine allows accurate determination of glucose in samples of glucose oxydase method (4). The main disadvantage of this reagent is too high oxidative ability of the cation Hg2+(redox potential +0,789), in excess of the oxidizing power of the oxidizing agents used in iodometric and ferricyanides methods, method Bertrand. In particular, the redox potential of the iodine is +0,535 and ferricyanide +0,36. Therefore mercury compounds (II) oxidizes not only reducing agents like ascorbic acid, but also sugar, thus reducing the development of color painting and leading to low values defined sugars. In addition, mercury compounds are toxic, and pose a danger to personnel and pollute the environment.

The aim of the invention is to provide a reagent for the determination of sugars in the presence of reducing substances, devoid of these shortcomings.

This goal is achieved using as reagent salts of copper (II), for example chlorine copper (CuCl2), at a final concentration of 500 μmol/L. the Proposed reagent allows to correctly measure the sugar content in different samples due to the oxidation of the reducing substances ions Cu2+. The redox potential of the ions si2+(+0,19) is relatively small, and therefore the ions si2+not oxidize sugar at neutral and acid value of the reaction medium. Compounds of copper (II) budget and znachitelnaya toxic, than mercury compounds (II).

The proposed reagent used for the determination of sugars in the presence of reducing substances in the following way. The reagent is prepared by dissolving 855 mg of hydrated chloride of copper (CuCl2·2H2O) in 100 ml distilled water (concentration of ions of Cu2+in the reagent 50 mmol/l). It contains sugar sample (fruit juices, extracts from fruits and vegetables, biological fluids) add the proposed reagent, keeping the volume ratio of reagent and sample 1:100 (final concentration of ions si2+in the analyzed sample of 500 µmol/l). When determining the content of sugars (glucose) in biological fluids to 100 ml of a standard reagent used in glucose oxydase method, add 1 ml of the proposed reagent. Next, perform a determination of sugar content in accordance with accepted methods.

Example 1.

Patient diabetes takes 2 g of ascorbic acid daily in the morning. The patient taken from a finger capillary blood glucose determination glucose oxydase method. For preparation of the claimed invention of the reagent in 100 ml distillirovannoi water dissolve 855 mg of hydrated chloride of copper (CuCl2·2H2O) in 100 ml distilled water (concentration of ions of Cu2+in the reagent 50 mmol/l). 1 ml of prepared is eagent added to 100 ml of a standard reagent, used in glucose oxydase method (concentration uCl2in a modified glucose oxydase the reagent is 500 µmol/l). Carry out the determination of glucose using the standard and the proposed reagents. The results are presented in the table.

Table
ReagentStandardOffer
The glucose content in the blood plasma (mol/l)5,86,5

The results show that when using a standard reagent amount determined glucose is 5.8 mmol/l and within the limits of normal values of blood glucose (3,5-6.1 mmol/l). True glucose (6.5 mmol/l), measured using the proposed reagent, 12% more and is beyond the limits of normal values, indicating that the patient has hyperglycemia, requiring correction of drugs.

Literature

1. Interstate standard. GOST 3628-78. Milk products. Methods for determination of sugar, http://www.gosthelp.ru/gost/gost32390.html.

2. Balakhovskiy I.S. // Lab research methods in the clinic. The Handbook. (Von is - as amended). - M.: Medicine, 1987. S.

3. Leary N.O., Pembroke A. and Duggan P.F. Improving Accuracy of Glucose Oxidase Procedure for Glucose Determinations on Discrete Analysers // Clinical Chemistry. 1992. Vol.38, No. 2, P.298-302.

4. White-Stevens, R.H. Interference by ascorbic acid in test systems involving buffer. I. Reversible indicators and the effects of copper, iron, and mercury // Clinical Chemistry. 1982. Vol.28, No. 4, P.678-788.

5. Nagel D, Seiler D, Hohenberger EF, Ziegler M. Investigations of ascorbic acid interference in urine test strips // Clinical Laboratory. 2006. Vol.52, No. 3-46. P.149-153.

6. Maguire G.A. and Price C.P. Evidence for interference by ascorbate in the measurement of cerebrospinal fluid glucose by a kinetic glucose oxidase buffer procedure // Clinical Chemistry. 1983. Vol.29. No. 10, P.1810-1812.

The way to create a reagent for the determination of glucose glucose oxydase method in the presence of ascorbic acid, which consists in the fact that 855 mg of hydrated chloride of copper (CuCl2·2H2O) dissolved in 100 ml distilled water (concentration of ions of Cu2+in a prepared solution of 50 mmol/l), add 1 ml of the prepared solution to 100 ml of a standard reagent used in glucose oxydase method, characterized in that as the oxidant ascorbic acid using a solution of chloride of copper in the final concentration of glucose oxydase the reagent 500 µmol/L.



 

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