SU1663516A1 - Method for determination of sulphanilamide drugs in objects of biological origin - Google Patents

Abstract

The invention relates to medicine and can be used in clinical, chemical toxicological and forensic chemical studies. In order to improve the accuracy of research, sulfa drugs are isolated from objects of biological origin with an aqueous acid solution. The resulting acid extraction is subjected to sorption purification on microcolumns with polyamide sorbent from endogenous substances capable of forming azo dyes, and during the photometric study, a mixture obtained by mixing an aliquot part of the purified extract under study and all reagents is used as a comparison solution, the reaction mixture after adjusting its PH to a value in the range of 10.10 to 12.25 with 10% sodium hydroxide solution. 9 tab.

Description

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The invention relates to medicine and can be used in clinical, chemical toxicological and forensic chemical studies.

The aim of the invention is to improve the accuracy of the method for determining sulfanilamide preparations in objects of biological origin.

The method is carried out as follows.

40 ml of the analyzed acid, extracts from the tissue of the organ are introduced into a 50 ml volumetric flask, then 3.8 ml of 40% sodium hydroxide solution are introduced into the flask and the pH of the mixture is adjusted to the value indicated in Table 2 (column 3 ) with a solution of 10% sodium hydroxide, added dropwise, after which the liquid level in the flask was made up to the mark with distilled water.

5 ml of blood tested for the sulfanilamide preparation are added to

centrifuge tube and add 10 ml of distilled water there too. The mixture is stirred and after 5 minutes, 5 ml of 4N solution is added to the tube with stirring. perchloric acid, the contents of the tube centrifuged at 500D for 5 min. Centrifugal transfer to a 50 ml volumetric flask. The pellet in the centrifuge tube is treated twice more in the same way with a solution of 1N. perchloric acid in portions of 10 ml. To the centrifugates combined in a 50 ml volumetric flask, 3.8 ml of 40% sodium hydroxide solution are added and, after stirring, the pH of the solution is adjusted to the value indicated in Table 2 (column 3) with a solution of 10% sodium hydroxide, add it dropwise, after which the total volume of the solution in the flask is made up to the mark with distilled water.

5 ml of the studied urine are introduced into a 50 ml volumetric flask and the volume of liquid in

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the flask is adjusted to the mark with a buffer solution with an appropriate pH value (Table 2, column 3),

In the four prepared microcolumns with a polyamide sorbent, identical, precisely measured volumes of the bioinvolvement to be purified (but not more than indicated in Table 2, column 4), treated as described above, are introduced. The microcolumns are centrifuged for 3-5 minutes at a centrifugal acceleration of 300-5QOg, at which the flow rate of the centrifugate to the receiver is about 1 ml / min. After centrifuging, the receivers are freed from the centrifugate and proceed to washing the columns with buffer solutions with the corresponding pH value (Table 2, column 3), make them three times in microcolumns in the volumes indicated in Table 2 (column 5). After the necessary volumes of buffer solutions are added, the microcolumns are centrifuged in the mode described above. The centrifugates obtained in the receivers are discarded. After the microcolumns are washed, the elution of the sulfanilamide preparation under study is eluted. To this end, a buffer solution with a pH of 11 in the volumes indicated in Table 2 (column 7) is added to the microcolumns and centrifuged three times. The eluates obtained from the four columns are collected in a 50 ml volumetric flask. The total volume of liquid in the flask is made up to the mark with distilled water. After mixing, the solution is used to determine the content of sulfanilamide preparations,

For this purpose, 0.05-10.0 ml of the obtained solution is introduced into four 25 ml volumetric flasks, after which 1 ml of 4N each is added to the contents of the flasks. hydrochloric acid and in the first three flasks of 1 ml of 0.2% aqueous solution of sodium nitrite. After stirring for 15 s, 1 ml of 0.2% alcoholic solution of 3-a, y-dicarboxypropylrodanine is added thereto. After mixing, a 10% sodium hydroxide solution is added to the flasks in an amount sufficient to produce a clear pink-red color when mixing (the pH of the solutions should be in the range indicated in Table 2), a similar amount of a 10% aqueous caustic solution. The soda is added to the fourth flask and, after stirring, 1 ml of a 0.2% aqueous solution of sodium nitrite, 1 ml of a 0.2% alcoholic solution of 3-cc, and y-dicarboxypropylrodanine are added thereto. After stirring for 15-20 s

The volume of liquid in flasks is made up to the mark with distilled water.

The determination of the optical density of the colored solutions of azorodanines is carried out using a KFK-2 photocolorimeter with a Yaeff light filter. 490 ± 10 nm in a cuvette with a working layer thickness of 50 mm. As a comparison solution, the solution obtained in the fourth volumetric flask is used.

The content of sulfa drugs in the samples is calculated according to the appropriate calibration graphs.

To build calibration graphs, 25 ml volumetric flasks are added.

by 0.01, 0.05, 0.1, 0.2.0.3, 0.4 ml of a standard solution of the drug in 1.0 n. hydrochloric acid with a concentration of 100 µg / ml, the amount of 1 n is missing up to 1 ml. hydrochloric acid and 1 ml of 0.1% aqueous

sodium nitrite solution. After 15s, 1 ml of a 0.1% alcoholic solution of 3-, y-dicarboxypropylrodanine is added to the flasks. After thorough mixing, the total volume of the liquid in the flasks is adjusted to

labels with a mixture of 0.05 M solution of sodium tetraborate and 0.1 n. sodium hydroxide solution (1: 2.95). After mixing the contents of the flasks, the optical density of the colored solutions of azanodanines was measured using a KFK-2 photocolorimeter in a cuvette with a working layer thickness of 50 mm at a light filter with a wavelength of Aeff 490 ± 10 nm.

Absorption is in all cases subject to the Bouguer-Lambert-Beer law at concentrations of 1-40 µg of the drug in 25 ml of the final volume. A mixture of the above reagents is used as a comparison solution.

The quantitative content of sulfanilamide preparations in the studied samples is calculated by the following formulas: when determining the content of sulfanilamide preparations in tissues of organs

515.625-C-Usob

where X is the content of sulfanilamide preparation (µg) in a sample of the tissue of the organ taken for the study;

0С-quantity (mcg) of sulfanilamide

drug found on the appropriate calibration schedule; Total - total volume (ml) of acid extraction, obtained from a sample of organ tissue;

5 VK - the volume of the test solution (m) deposited on one microcolumn;

Vc is the volume of the final solution (ml) taken to perform one analysis,

when determining the content of sulfa drugs in the blood and urine

x 125 s

VK Vc

where X is the content of sulfa drug in the blood or urine (µg / ml);

С - amount (mcg) of sulfanilamide preparation, found according to the corresponding calibration graph;

/ K - the volume of the test solution (ml) deposited on one microcolumn,

Vc is the volume of the final solution (ml) taken to perform one analysis.

Preparation of microcolumns for work.

Purification of the obtained extracts from tissues of organs, blood and urine samples prepared for analysis is carried out by the method of sorption on a polyamide sorbent (TU 6-09-1-822- 73).

The microcolumn is a 65-70 mm long tube with an internal diameter of about 13 mm, sealed at one end with a perforated partition. To fabricate microcolumns, a porous disk made of filter paper is placed on a perforated partition, after which the required amount (table 2, column 2) of dry sorbent is added to the column and another porous disk of filter paper is placed on top, then the layer of sorbent in the micro column is sealed with a piston from the same syringe. A clamping ring with an outer diameter of 13 mm and an inner diameter of 10 mm is placed over the porous disc of filter paper. The receiver is a glass tube 45–55 mm long with an inner diameter of about 13 mm.

The chromatographic microcolumn is connected to the receiver with a piece of adhesive tape measuring 15x30 mm.

Before operation, the microcolumn is centrifuged twice with 96% ethyl alcohol (3-5 ml portions), after which it is washed twice with a buffer solution with the required pH value (Table 2, column 3) with 3-5 ml portions, after Such training microcolumn is ready to work. For the analysis, it is desirable to prepare a new column. Used columns can be used up to three times, subjecting them to regeneration, for this the microcolumns are centrifuged three times in 3–5 ml portions of 96% ethanol, after which the microcolumns are centrifuged twice in 3–5 ml portions of the buffer solution required pH value (table 2, column 3). It is possible to store microcolumns in a dry form, preparing it before working according to the procedure described above.

PRI me R 1. Determination of sulfanilamide preparations in liver tissue.

Fresh liver of a human body that died as a result of an injury is finely ground and 100 g of each is placed in 250 ml centrifuge cups. The cups are added with 40 ml of a solution of the corresponding sulfanilamide preparation with a concentration of 100 µg / ml in a buffer solution with a pH of 7.4 and left for a day at room temperature, stirring occasionally. In parallel, blank tests are carried out. After 2 hours, 80 ml of 1N perchloric acid solution are added to the beakers. The contents of the glasses are thoroughly mixed and centrifuged at 500d at 0 for 5 min. The centrifugate is poured into a 1-liter beaker, and 80 ml of 1N are again added to the solid residue in the centrifugal beaker. perchloric acid, mix thoroughly with a glass rod 5 and centrifuge under the same conditions. The isolation operation is carried out 6 times. The total volume of the combined extracts is measured using a 1 L measuring cylinder. About 50 ml of the recovered extract is placed in a 0 centrifuge tube and centrifuged at 500d for 10 minutes. 40 ml are taken from the obtained protein-free centrifugate and then proceed as described above.

The results of determining the content of 5 sulfa drugs in the liver tissue (model mixture) are given in table 4. As can be seen from Table 4, the error of the method in determining the content of sulfanilamide preparations in the liver tissue is 0 3.83–9.56%. The poor results (73.13%; 71.04%) obtained for some drugs are not connected with the disadvantages of the proposed method of determination, but with the limited capabilities of the method for isolating sulfanilamide preparations with solutions of acids from biological tissues.

To compare the accuracy of the methods, the content of sulfonyllamine preparations was determined in control experiments (i.e., in extracts from liver tissue not treated with sulfonamides) using the method proposed and the method chosen as the prototype (Table 5). 5From table 5 it follows that the proposed

the method makes it possible to accurately determine the content of sulfa drugs in acid extraction from biological tissue. Using the method of direct determination

sulfonamide preparations in acidic stretching by the azo-dye formation reaction does not allow to obtain accurate data, which can lead to significant errors, especially in forensic chemical studies, since even in the absence of a sample of the studied preparations, the analysis data their presence in the analyzed sample. The proposed method completely eliminates these disadvantages.

PRI mme R 2. Determination of sulfa drugs in the blood.

In 100 ml volumetric flasks, 10 ml of a solution of the corresponding sulfanilamide preparation in a buffer solution of pH 7.4 with a concentration of 400 µg / ml is added, after which human citrate blood is added to the flasks to bring the liquid in the flasks to the mark. The contents of the flasks are thoroughly mixed and incubated for 24 hours. After the indicated period, samples are taken of 5 ml each and then proceed as described above.

The results of determination of the content of sulfonamide drugs in the blood are presented in Table 6.

The error in the determination of sulfa drugs in the blood is 0.69% -4.17%. .

To compare the accuracy of the proposed method and the method chosen as a prototype, sulfanilamide preparations were determined in uncured blood.

The results of the study are presented in table 7.

PRI me R 3. Determination of sulfanilamide drugs in the urine.

In 100 ml volumetric flasks, 10 ml of a solution of the corresponding sulfanilamide preparation in a buffer solution with a pH of 7.4 with a concentration of 400 µg / ml is added, after which fresh human urine is added to the flask to bring the liquid to the mark. The contents of the flasks are thoroughly mixed and incubated for 24 hours.

After the specified period, samples are taken of 5 ml and then proceed as described above.

The results of the determination of sulfa drugs in human urine are presented in Table 8.

As follows from the data presented in Table 8, the error in the determination method is 2.18-4.12%.

To compare the accuracy of the proposed method and the method chosen as a prototype, the content of sulfanilamide preparations in urine samples, in which there were no determinable preparations, was determined.

The results of the determination are presented in Table 9.

The data given in Tables 7 and 9 indicate that the use of the method chosen as a prototype does not accurately determine the content of sulfanilamide preparations in the blood and urine. Using the proposed method completely eliminates this disadvantage.

Results of quantitative determination of sulfa drugs in extracts from biological material

As follows from the data presented in Table 1, the absolute error of the method chosen as a prototype can reach 45%, while the absolute error of the proposed method does not exceed 11%.

Claims (1)

Invention Formula The method of determining sulfa drugs in objects of biological origin by removing the drug from a biological object with acid, purifying, obtaining a colored solution with azo dye and photometry, characterized in that, in order to improve the accuracy, the stretch is passed through a column with polyamide sorbent, sodium hydroxide solution is added to pH 10.1-12.85 and photometrically using, as a comparison solution, a mixture obtained by mixing an aliquot part of the investigated purified extract of all reagents. Table 1 Conditions for purification of biological extracts containing sulfa drugs on microcolumns with Spreadsheets The ranges of pH values within which the optical density of airborne is maximal and stable A drug PH range Table Determination of sulfanilamide preparations in liver tissue extracts Norsulfazole 318879.70 305976.48 298474.60 288272.05 295673.90 Streptocid 353388.33 325181,28 322080.50 341685.40 343285.80 X 75.35 C 2.90 Gx 1.30 IMS-A 4.79 M 75.351 4.79 And 84.26 G 3.29 Gx 1,47  4.08 A 4.84 M 84.26i 4.08 Table Determination of sulfanilamide preparations in control experiments by the proposed method 0.00 0.00 0.00 0.00 0.00 976.17 828.13 614.87 718.72 990.12 Table 6 Determination of sulfa drugs in human blood 4000 4000 4000 4000 4000 Norsulfazole 3671 3575 3671 3858 3763 4000 4000 4000 4000 4000 Streptocid 3858 3954 3763 3763 3954. 4000 4000 4000 4000 4000 Sulfalen 4000 3960 4000 3960 4000 Sulfamonomethoxin 4000 4000 4000 4000 4000 3933 3823 3600 3713 3823 4000 4000 4000 4000 4000 Etazol 3643 3820 3908 3643 3643 X 92.69 G 2.68 Nx 1,20 B ,, h 3.33 A 3.59 M 92.69t 3.33 X 96.46 G 2.39 Gx 1.07 Io 45 2.97 A 3.08 M 96,461 2.97 X 99.60 G 0,55 Gx 0,25 Ibffi 0,69 A 0,69 M 99.60i 0.69 X 94.46 G 3.16 Gx 1.41 I0 |, f 3.91 A 4.14 M 94.46 3.91 X 93.29 G 3.12 Gx 1.40 ToflV 3.89 A 4.17 M 93,29i 3,89 Table 7 Determination of sulfanilamide preparations in non-poisoned blood (in control experiments) Table Determination of sulfanilamide preparations in human urine 4000 4000 4000 4000 4000 Norsulfazole 3858 3763 3762 3671 3763 4000 4000 4000 4000 4000 Streptocid 3763 3671 3763 3954 3954 4000 4000 4000 4000 4000 Sulfalen 4000 4000 4000 3920 3980 Sulfamonomethoxin 4000 4000 4000 4000 4000 3600 3490 3600 3600 3713 eight X - 94.09 G - 1.65 Gx 0.74 1yt A 2.18 M 94.08 t2.05 X 95.53 G 3.17 Gx 1.42 W 3.94 A 4.12 M 95.53 3.94 X 99.50 G 1.33 Gx 0.77 Ip 4f 2.11 A 2.13 M “99.50 t2, 11 X 90.02 G 1.96 Gx 0.88 Chht "2.44 A 2.71 M 90.02 12.44 TYPE OF I Etazol Table 9 Determination of sulfanilamide preparations in non-poisoned urine (in the control experiment) Continued table. eight and:::::::::