RU2495425C1 - Method of extracting azathioprine from biological fluids - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: analysed sample is treated with a protein-depositing reagent which is acetone; the extract is separated from the precipitate by filtering; acetone from the filtrate is evaporated in an air current at room temperature; the aqueous residue is diluted with water; the formed solution is treated with ammonium sulphate, extracted with 0.9 mol/dm³ of dimethyl phthalate solution in a 1,4-dioxane-chloroform mixture, taken in volume ratio of 1:1, with ratio of the aqueous phase to the organic phase of 5:1 by volume; the organic extractant is separated, and the amount of the analysed compound transferred to the extract is determined from the optical density of the extract which is diluted tenfold with 1,4-dioxane while measuring at wavelength of 279 nm.

EFFECT: high degree of extraction of azathioprine from biological fluids.

2 ex, 3 tbl

Description

The invention relates to biology, toxicological and analytical chemistry, and in particular to methods for the extraction of azathioprine from biological fluids, and can be used in the practice of chemical toxicological and clinical laboratories to extract azathioprine from biological fluids with a view to its subsequent determination. The method belongs to the mass.

A known method of extracting derivatives of purine (caffeine) from a biological fluid (albumin solution), consisting in the fact that the analyzed sample is treated with a solution of pepsin in a solution of hydrochloric acid, the reaction of the resulting mixture is adjusted to pH 4, incubated at 37 ° C for 1 hour extracted three times with chloroform at a ratio of the volumes of aqueous and organic phases 1: 1, the extracts are combined, the residue is dissolved in phosphate buffer with a pH of 7.4, the resulting solution is filtered and the amount of the derivative extracted is determined purine by the value of the optical density of the filtrate, measured at a wavelength of 273 nm (Strelova O.Yu., Chuvina N.A. Isolation of caffeine from blood using enzymatic hydrolysis using an example of a model substance // Forensic medical examination. - 2008. - T. 51, No. 4. - S.28-31).

The method is labor intensive and has a relatively low degree of extraction.

A known method of extracting purine derivatives from biofluids (blood), which consists in the fact that the analyzed sample is treated with ammonium sulfate, kept for 30 minutes with stirring, the solution is centrifuged at 3000 rpm, the centrifugate is separated, its reaction is adjusted to pH 4.0- 5.0, extracted three times with chloroform at a ratio of aqueous and organic phases at each extraction stage of 1: 1, the extracts are combined, the extractant is evaporated, the residue is dissolved in phosphate buffer with a pH of 7.4, the resulting solution is filtered and the quantity determined the substance of the extracted substance by the optical density measured at a wavelength of 273 nm (Strelova O.Yu., Chuvina N.A. Evaluation of the effectiveness of methods of isolating toxic substances from the blood // Forensic medical examination. - 2008. - V. 51, No. 4. - S.22-24).

The method is characterized by an insufficiently high degree of extraction.

The closest is the method of extraction of purine derivatives (which includes azathioprine) from blood plasma, which consists in the fact that the analyzed sample is treated with a precipitating protein reagent, which is used as perchloric acid, the extraction is separated from the precipitate by centrifugation, the centrifugate is treated with sodium hydroxide solution, the excess alkali is neutralized with a solution of hydrochloric acid, the resulting solution is extracted with a mixture of organic solvents of chloroform-propanol-2, taken in a volume ratio 1: 1, the organic extract is separated by centrifugation, the extractant is evaporated to a dry residue, the residue is dissolved in the mobile phase and the amount of the analyte transferred to the extract is determined by HPLC by the area of the chromatographic peak (Dobrocky P., Bennet PN, Natarianny LJ Rapid method for the routine determination of caffeini and its metabolites by high-performance liquid chromatography // J. Chromatogr. Biomed. AppL - 1994. - Vol.652, No. 1. - P.104-108).

The method is characterized by an insufficiently high degree of extraction. The technical result of the present invention is to increase the degree of extraction.

The technical result is achieved by the fact that the analyzed sample is treated with a protein-precipitating reagent, which uses acetone, the extraction is separated from the precipitate by filtration, the acetone from the filtrate is evaporated in an air stream at room temperature, the aqueous residue is diluted with water, the resulting solution is treated with ammonium sulfate, extracted 0.9 mol / dm ³ with a solution of dimethyl phthalate in a mixture of 1,4-dioxane-chloroform taken in a volume ratio of 1: 1 with a ratio of aqueous and organic phases of 5: 1 by volume, organically the th extractant is separated, and the amount of the analyzed compound transferred to the extract is determined by the optical density of the extract diluted 10 times with 1,4-dioxane, taking measurements at a wavelength of 279 nm.

The method is as follows: the blood plasma containing azathioprine is treated with a protein-precipitating reagent, which uses acetone, the extraction is separated from the precipitate by filtration, the acetone from the filtrate is evaporated in a stream of air at room temperature, the aqueous residue is diluted with water, the resulting solution is treated with sulfate ammonia, extracted with 0.9 mol / dm ³ solution of dimethyl phthalate in a mixture of 1,4-dioxane, chloroform, taken in a volume ratio of 1: 1 with a ratio of the aqueous and organic phases is 5: 1 to obe y, the organic extractant is separated and passed into the extract amount of the analyte is determined by the magnitude of the optical density of the extract diluted 10 times with 1,4-dioxane, taking measurements at a wavelength of 279 nm.

The method is illustrated by the following examples.

Example 1

Recovery of azathioprine from blood plasma

In 5 g of human blood plasma, 1 mg of azathioprine is added in the form of 0.5 cm ^{3 of a} 0.1% solution, the biological fluid is thoroughly mixed with the substance. The resulting mixture is treated with 10 cm ³ protein precipitating reagent, which is used as acetone, and left for 5 minutes with stirring. After the specified time, the liquid extraction is separated from the precipitate by filtration through a paper filter. The filter and filter cake are washed with 5 cm ^{3 of} acetone. Portions of the filtrate are combined in an evaporation cup and acetone is evaporated in a stream of air at room temperature. 5 cm of water was added to the aqueous residue of the filtrate, ammonium sulfate was added to the resulting solution to obtain a saturated aqueous salt solution, which was extracted with 2 cm ^{3 of a} 0.9 mol / dm ³ solution of dimethyl phthalate in a mixture of 1,4-dioxane-chloroform taken in volume 1: 1 ratio, on a vibratory mixer for 8 minutes, after which the solution is left for 3-5 minutes to exfoliate the system, the organic extract separates 0.2 cm of the extract and is introduced into a 10 cm volumetric flask, adjusted to the mark with 1,4-dioxane and measure the optical density of the resulting solution on spectrophotometer SF-2000 at a wavelength of 279 nm in cuvettes with a working layer thickness of 10 mm The measurements are carried out against the background of the solution obtained in the control experiment. The amount of azathioprine, transferred to the extractant layer, is determined by the optical density value using the equation of the calibration graph. The degree of extraction of azathioprine is calculated as the ratio of the amount of substance transferred to the extractant layer, as a percentage of its amount introduced into the biological fluid.

Building a calibration graph

In a series of volumetric flasks with a capacity of 10 ml add 0.1, 0.2, 0.4, 1.0, 1.6, 2.0 cm ^{3 of a} 0.01% solution of azathioprine in chloroform, 0.2 cm ³ 0, 9 mol / dm ^{3 of a} solution of dimethyl phthalate in a mixture of 1,4-dioxane-chloroform, taken in a volume ratio of 1: 1, and adjusted with 1,4-dioxane to the mark. The optical density of the resulting solutions is measured on an SF-2000 spectrophotometer at 279 nm in cuvettes with a working layer thickness of 10 mm. The measurements are carried out against the background of the solution obtained in the control experiment.

According to the results of measuring the optical density of a series of solutions with known concentrations of azathioprine, a graph of the dependence of optical density on the concentration of the analyte is constructed. The graph is linear in the concentration range 1.0 - 20.0 μg / cm ³ . The least squares method calculates the equation of the calibration graph, which in this case has the form:

A = 0.06285-C + 0.00124,

where A is the optical density; C is the concentration of the analyte (μg in 1 cm ³ photometric solution).

The results of the extraction of azathioprine from blood plasma are presented in table 1.

Example 2

Urinary azathioprine recovery

In 5 g of human urine, 1 mg of azathioprine is added in the form of 0.5 cm ^{3 of a} 0.1% solution, the biological fluid is thoroughly mixed with the substance. The resulting mixture is treated with 10 cm of protein precipitating reagent, which is used as acetone, and left for 5 minutes with stirring. After the specified time, the liquid extraction is separated from the precipitate by filtration through a paper filter. The filter and filter cake are washed with 5 cm ^{3 of} acetone. Portions of the filtrate are combined in an evaporation cup and acetone is evaporated in a stream of air at room temperature. 5 cm ^{3 of} water are added to the aqueous residue of the filtrate, ammonium sulfate is introduced into the resulting solution to obtain a saturated aqueous salt solution, which is extracted with 2 cm ^{3 of a} 0.9 mol / dm ³ solution of dimethyl phthalate in a mixture of 1,4-dioxane-chloroform taken in a volume ratio of 1: 1, on a vibratory mixer for 8 minutes, after which the solution is left for 3-5 minutes to exfoliate the system, the organic extract is separated, 0.2 cm ^{3 of the} extract is introduced into a volumetric flask with a capacity of 10 cm ³ , adjusted to label 1, 4-dioxane and measure the optical density of the resulting solution and on an SF-2000 spectrophotometer at a wavelength of 279 nm in cuvettes with a working layer thickness of 10 mm. Measurements are carried out against the background of the solution laid in the control experiment. The amount of azathioprine, transferred to the extractant layer, is determined by the optical density value using the equation of the calibration graph. The degree of extraction of azathioprine is calculated as the ratio of the amount of substance transferred to the extractant layer, as a percentage of its amount introduced into the biological fluid.

The scheme for constructing a calibration graph for determining azathioprine and the equation of this graph are presented above in Example 1.

The results of the extraction of azathioprine from urine are presented in table 2.

The proposed method in comparison with the prototype by 7-8% increases the degree of extraction of azathioprine from biological fluids.

Comparative characteristics of the proposed and known methods are presented in table 3.

Table 1 The results of the extraction of azathioprine from blood plasma (n = 5; P = 0.95) Azathioprine added to blood plasma, mg Found Metrological characteristics mg The degree of extraction of R,% 1.00 0.9810 98.10

=97,33 $$\overline{x}$$

= 97.33 1.00 0.9621 96.21 S = 1.06 1.00 0.9812 98.12 $$S_{\overline{x}}$$

= 0.47 1.00 0.9808 98.08 $$\Delta \overline{x}$$

= 1.32 1.00 0.9613 96.13 ε = 1.35

table 2 The results of the extraction of azathioprine from urine (n = 5; P = 0.95) Vnesono azathioprine in urine, mg Found Metrological characteristics mg The degree of extraction of R,% 1.00 0.9792 97.92

=98,11 $$\overline{x}$$

= 98.11 1.00 0.9811 98.11 S = 0.66 1.00 0.9849 98,99 $$S_{\overline{x}}$$

= 0.29 1.00 0.9833 98.33 $$\Delta \overline{x}$$

= 0.82 1.00 0.9768 97.18 ε = 0.83

Table 3 Comparative characteristics of the proposed and known methods (n = 5; P = 0.95) Compared Indicators The proposed method Known method 1. The degree of extraction from blood plasma 97.33% 90.26% 2. The degree of extraction from urine 98.11% 90.43%

Claims (1)

The method of extracting azathioprine from biological fluids, namely, that the analyzed sample is treated with a precipitating protein reagent, the extraction is separated from the precipitated precipitate, extracted with a mixture of organic solvents and the amount of the analyte transferred to the extract is determined, characterized in that acetone is used as the precipitating protein reagent after separating the extract from the precipitate, acetone is evaporated in a stream of air at room temperature, the aqueous residue is diluted with water, processing It is extracted with ammonium sulfate. extracted with 0.9 mol / dm ³ with a solution of dimethyl phthalate in a mixture of 1,4-dioxane-chloroform taken in a volume ratio of 1: 1 at a ratio of aqueous and organic phases of 5: 1 by volume, and the amount of the analyzed compound transferred to the extract is determined by the optical the density of the extract, diluted 10 times with 1.4-dioxane, taking measurements at a wavelength of 279 nm.