RU2701524C1 - Method for quantitative determination of disulphiram in biological media - Google Patents

Abstract

FIELD: measurement technology.

SUBSTANCE: invention relates to a method for quantitative determination of disulphiram in biological media, involving extraction of the substance to be determined with ethyl acetate from biological tissue with subsequent determination thereof by high-performance liquid chromatography-mass spectrometry with a gradient elution mode using 0.1 % solution of formic or acetic acid in water as a mobile phase and 0.1 % solution of formic or acetic acid in methanol or acetonitrile.

EFFECT: method enables quantitative determination of disulphuram in various biological tissues.

1 cl, 1 dwg, 9 tbl, 9 ex

Description

The invention relates to medicine, namely to pathophysiology, pharmacology and toxicology, and can be used to determine disulfiram in biological media, including in the tissues of muscles and heart of mammals.

New drugs containing disulfiram are currently being developed. One of the urgent tasks in the development of a new drug is the study of its pharmacokinetics in order to assess the absorption, distribution, metabolism and excretion in animals and humans. To conduct pharmacokinetics studies of new drugs, it is necessary to develop bioanalytical methods for the quantitative determination of the active substance in biological media. In particular, for topical drugs administered intramuscularly or intramyocardially (into the heart muscle), it is necessary to develop a method for the quantitative determination of the active substance in such biological media as muscle and heart tissues.

For the quantitative determination of the active substance in biological media, chromatographic methods are used, which have specificity, high sensitivity, accuracy and reproducibility. One of the most common methods for studying the pharmacokinetics of a drug is high performance liquid chromatography combined with tandem mass spectrometric detection (HPLC-MS / MS), which has several advantages over other methods, namely: high sensitivity, which allows the determination of microquantities substances, and high speed analysis.

Currently, only methods for the quantitative determination of disulfiram in the blood and urine of mammals are known. So, to determine disulfiram in human plasma, solid-phase extraction is used at the sample preparation stage, and for analysis, the HPLC-MS / MS method is used, in which a mixture of water and methyl alcohol (22:78) containing 0.1% formic acid is used as an eluent, isocratic elution. Detection was carried out in MRM mode with a transition registration of 296.95 / 115.94 (Zhang L., Jiang Y., Jing G., Tang Y., Chen X., Yang D., Zhang Y., Tang X. A novel UPLC-ESI-MS / MS method for the quantitation of disulfiram, its role in stabilized plasma and its application. J. Chromatogr. B. Analyt. Technol. Biomed. Life Set 2013, v. 937, p. 54-59). In the study (V. O. Doroschuk, AB Grogul, YS Mandzyuk, OG Makukha, NO Grytsyk. Cloud point extraction of disulfiram for its HPLC-MS / MS determination in synthetic urine. Chem. Papers 2016, v. 70 No. 10, p. 1316-1321) describes a method for the quantitative analysis of disulfiram in artificially prepared urine. The method consists in liquid-liquid micellar extraction of a urine sample, followed by analysis of the extract by HPLC-MS / MS. Chromatographic separation was carried out using a column with a C18 reversed-phase sorbent, 0.25% aqueous acetic acid solution (eluent A) served as a mobile phase and acetonitrile (eluent B), elution was performed in a gradient mode with an increase in the proportion of eluent B from 10% to 100%. Detection was carried out in the MRM mode with the registration of the transition 297.3 / 116.0. However, to determine the disulfiram in the tissues of the muscle and heart, this method is not applicable due to the significant differences between the tissues of the muscle and heart as a biological matrix from blood plasma and urine.

The technical result of the invention is the development of a new highly effective method for the quantitative determination of disulfiram in various biological tissues, including muscle and heart tissues of mammals. The method has the required accuracy and precision within the cycle for the quantitative determination of disulfiram in samples of homogenates of the heart tissues of mammals, and also has the required accuracy and repeatability of the quantitative determination of disulfiram in the homogenate of tissues of the muscle and heart of mammals. The present invention allows to obtain the necessary and reliable information about the method for quantitative determination of the substance disulfiram in biological tissues, including tissues of the muscles and hearts of mammals.

The problem is solved by the method of quantitative determination of disulfiram in biological media, including in the tissues of the muscles and heart of mammals, including extraction of the analyte with ethyl acetate from a sample representing tissue homogenate in physiological saline in a ratio of 20:80 (organ mass, g / volume of saline , ml), followed by its determination by high performance liquid chromatography with mass spectrometric detection with a gradient elution mode using ETS components of the mobile phase 0.1% aqueous solution of formic acid or acetic acid and 0.1% solution of formic acid or acetic acid in methanol or acetonitrile.

The most optimal, but not necessary modes of high performance liquid chromatography are:

eluent flow rate - 0.15 ml / min; sample volume - 10 μl; detector - tandem mass spectrometric; analysis time - 8 min; column thermostat temperature - 35 ° C.

The implementation of the proposed method is presented in the examples.

Example 1. The selection of the conditions of the chromatography-mass spectrometric analysis

The determination of the active substance by disulfiram in biological media was carried out by HPLC-MS / MS on a QTRAP 3200 mass spectrometer (AB Sciex, USA) in combination with an LC-20AD liquid chromatograph (Shimadzu, Japan).

Preparation of mobile phase A (PF A)

In a 2-liter volumetric flask, 2 ml of formic acid was placed, the volume of the solution was adjusted to the mark with purified water and mixed.

Preparation of mobile phase B (PF B)

In a 1 L volumetric flask, 1 ml of formic acid was placed, the volume of the solution was adjusted to the mark with methanol and mixed.

Preparation of 0.1 mg / ml disulfiram substance

About 1 g (accurately weighed) of disulfiram substance is placed in a 10 ml volumetric flask, 8 ml of acetone are added and stirred until complete dissolution, the solution volume is adjusted to the mark with the same solvent and mixed.

Preparation of initial calibration solutions of disulfiram substance

From a solution of the disulfiram substance with a concentration of 0.1 mg / ml, solutions of the disulfiram substance with concentrations of 250, 200, 100, 50, 25, 10, 5, and 2.5 μg / ml were prepared by successive dilution with acetone.

Selection of mass spectrometric conditions

A solution of the disulfiram substance with a concentration of 500 ng / ml was analyzed by direct injection mass spectrometry (without using a chromatograph) to obtain a complete mass spectrum.

When analyzing the solution in Q1 mode (simple mass scanning) in the range of 50-400 Da, a mass spectrum (presented as a separate document) with an intense ion with m / z 297.4 corresponding to the protonated disulfiram molecule is observed. When this ion is fragmented in the mode of low impact energy (CE = 10 V), fragmentation with m / z = 116.1 and 88.1 is observed.

In order to obtain the most intense fragment ions, the CE parameter (Collision Energy) was optimized, as a result of which the optimal value of this indicator was established.

Based on the conducted experiments, the quantitative determination of disulfiram in the samples was carried out using the MRM transition 297.4 → 116.1.

Preparation of calibration solutions of disulfiram in rat tissue muscle and heart homogenates

190 μl of single homogenate of rat muscle or heart tissue was placed in a 2 ml Eppendorf plastic microcentrifuge microtube, 10 μl of a calibration solution of disulfiram substance of various concentrations were added, mixed on a Microspin shaker (Biosan, Latvia) at 2000 rpm for 2 min . Then, 1 ml of ethyl acetate was added, thoroughly mixed on a shaker at 4000 rpm for 10 minutes. The resulting mixture was centrifuged at 11500 g for 5 minutes. 900 μl of the organic phase was taken and evaporated in vacuo to a dry residue. 1000 μl of methanol was added to the dry residue and stirred on a shaker at 2000 rpm for 10 minutes. The resulting solution was centrifuged at 11500 g for 5 minutes. The supernatant was used for chromatography-mass spectrometric analysis.

Preparation of rat muscle tissue or heart homogenates samples containing disulfiram

200 μl of rat muscle or heart tissue homogenate was placed in a 2 ml Eppendorf plastic microcentrifuge microtube, 1 ml of ethyl acetate was added, and thoroughly mixed on a shaker at 2000 rpm for 10 min. The resulting mixture was centrifuged at 11500 g for 5 minutes. 900 μl of the organic phase were taken and evaporated in vacuo to a dry residue. 1000 μl of methanol was added to the dry residue and mixed on a shaker at 2000 rpm for 10 minutes. The resulting solution was centrifuged at 11500 g for 5 minutes. The supernatant was used for chromatography-mass spectrometric analysis.

Chromatographic conditions

Eluent Flow Rate - 0.15 ml / min; Column Thermostat Temperature - 35 ± 1 ° C; Autosampler Thermostat Temperature - 10 ± 1 ° C; Sample Volume - 10 μl; Analysis time - 8 min; Disulfiram retention time - 5.8 ± 0.2 min.

Mass spectrometric conditions:

Polarity - positive; Ion source - turboelectrospray; Ion source voltage - 5500 V; Ion source temperature - 150 ° C; Gas curtain - nitrogen, 25 psi; Gas atomizer - air, 20 psi; Gas dryer - air, 20 psi; Scan type - MS / MS;

Mode: MRM

Quantitative determination of disulfiram in samples of tissue homogenates of the muscle and heart was performed using the MRM transition m / z 297.4 → 116.1, which corresponds to the most intense signal in the mass chromatogram.

In FIG. 1 shows the mass spectrum of disulfiram decomposition upon fragmentation of ion 297.4

Example 2 Evaluation of the lower limit of the quantitative determination of the substance disulfiram in the homogenate of rat muscle tissue using acetic acid as an eluent modifier

Preparation of mobile phase A (PF A)

2 ml of acetic acid was placed in a volumetric flask with a capacity of 2 L, the volume of the solution was adjusted to the mark with purified water and mixed. Preparation of mobile phase B (PF B)

1 ml of acetic acid was placed in a 1-liter volumetric flask, the volume of the solution was adjusted to the mark with methanol and stirred.

The chromatographic and detection conditions on the mass spectrometer coincide with the conditions given in example 1.

The minimum concentration of analyte in the homogenates of rat heart tissues was established, which can be quantified in a biological sample with the required precision. Acceptance criteria: the signal-to-noise ratio should be at least 10/1.

The results of the evaluation of the signal-to-noise ratio for the lower limit of the quantitative determination of disulfiram in the homogenate of rat muscle tissue are shown in Table 1.

The signal-to-noise ratio for the disulfiram peak with a concentration of 2.5 μg / ml is 156, therefore, a concentration of 2.5 μg / ml is the lower limit for the quantification of disulfiram substance in rat muscle tissue homogenate samples.

Example 3. Evaluation of the lower limit of the quantitative determination of the substance disulfiram in the homogenate of rat heart tissue.

Preparation of mobile phase A (PF A)

In a 2-liter volumetric flask, 2 ml of formic acid was placed, the volume of the solution was adjusted to the mark with purified water and mixed.

Preparation of mobile phase B (PF B)

In a 1 L volumetric flask, 1 ml of formic acid was placed, the solution volume was adjusted to the mark with acetonitrile and stirred.

The chromatographic and detection conditions on the mass spectrometer coincide with the conditions given in example 1.

The minimum concentration of analyte in the homogenates of rat heart tissues was established, which can be quantified in a biological sample with the required precision. Acceptance criteria: the signal-to-noise ratio should be at least 10/1.

The results of evaluating the signal-to-noise ratio for the lower limit of the quantitative determination of disulfiram in the homogenate of rat heart tissues are shown in Table 2.

As can be seen from table 2, the signal-to-noise ratio for the peak of disulfiram with a concentration of 2.5 μg / ml is 159: 1, which meets the criterion of the signal-to-noise ratio of at least 10/1.

Example 4. Evaluation of the linearity of the quantitative determination of the substance of disulfiram in homogenates of rat muscle tissue.

Preparation of mobile phase A (PF A)

2 ml of acetic acid was placed in a volumetric flask with a capacity of 2 L, the volume of the solution was adjusted to the mark with purified water and mixed.

Preparation of mobile phase B (PF B)

1 ml of acetic acid was placed in a 1-liter volumetric flask, the volume of the solution was adjusted to the mark with acetonitrile and stirred.

The chromatographic and detection conditions on the mass spectrometer coincide with the conditions given in example 2.

Linearity is the ability of a technique to give values directly proportional to the concentration (amount) of an analyte in a sample. To construct a calibration curve, model samples of rat muscle tissue homogenates containing and disulfiram were prepared and analyzed in concentrations: 2.5; 5, 10, 25, 50, 100, 200 and 250 μg / ml, table 3 presents the results.

The equation of the calibration curve has the form: y = 130142 × - 73894.

Acceptance criterion: the square of the correlation coefficient of the calibration curve must be at least 0,990. The results obtained satisfy this criterion; the value of the squared correlation coefficient is 0.9974.

Thus, the linearity of the bioanalytical method in the concentration range from 2.5 to 250 μg / ml was confirmed. The range under consideration is suitable for the quantitative determination of disulfiram in rat muscle tissue homogenate.

Example 5. Evaluation of the lower limit of the quantitative determination of the substance disulfiram in the homogenate of rat muscle tissue using formic acid as an eluent modifier

The lower limit of quantification is the minimum concentration of an analyte in a sample that can be quantified with the required accuracy and precision.

To establish the lower limit of quantification, the signal-to-noise ratio was used. The signal values obtained for the control experiment (in the absence of the analyte) and for the sample with a low concentration of the analyte were compared. For the lower limit of quantification, the signal-to-noise ratio should be at least 10/1.

Preparation for analysis and the analysis itself was carried out as described in example 1. The results of evaluating the signal-to-noise ratio for the lower limit of the quantitative determination of disulfiram in the homogenate of rat muscle tissue are shown in Table 4.

The signal-to-noise ratio for the peak of disulfiram with a concentration of 2.5 μg / ml is 163, therefore, a concentration of 2.5 μg / ml is the lower limit for the quantification of the substance of disulfiram in samples of rat muscle tissue homogenate.

Example 6. Evaluation of the lower limit of the quantitative determination of the substance disulfiram in the homogenate of rat heart tissue using acetic acid as an eluent modifier

The minimum concentration of analyte in the homogenates of rat heart tissues was established, which can be quantified in a biological sample with the required precision. Acceptance criteria: the signal-to-noise ratio should be at least 10/1.

Preparation for analysis and the analysis itself was carried out as described in example 2. The results of evaluating the signal-to-noise ratio for the lower limit of the quantitative determination of disulfiram in the homogenate of rat heart tissues are shown in table 5.

As can be seen from table 2, the signal-to-noise ratio for the peak of disulfiram with a concentration of 2.5 μg / ml is 159: 1, which meets the criterion of the signal-to-noise ratio of at least 10/1.

Example 7. The linearity assessment of the quantitative determination of the substance disulfiram in homogenates of rat heart tissue

To construct a calibration curve, model samples of rat muscle tissue homogenates containing and disulfiram were prepared and analyzed in concentrations: 2.5; 5, 10, 25, 50, 100, 200 and 250 μg / ml, table 6 presents the results.

The equation of the calibration curve has the form: y = 236005 × - 272006.

Acceptance criterion: the square of the correlation coefficient of the calibration curve must be at least 0,990. The results obtained satisfy this criterion, the value of the squared correlation coefficient is 0.9966.

Thus, the linearity of the bioanalytical method in the concentration range from 2.5 to 250 μg / ml was confirmed. The considered range is suitable for the quantitative determination of disulfiram in the homogenate of rat heart tissue.

Example 8. Evaluation of the accuracy and precision of the quantitative determination of the substance disulfiram in homogenates of rat muscle tissue

To assess accuracy and precision, model samples of rat muscle tissue homogenates were prepared and analyzed, containing disulfiram at concentrations of 2.5, 10, 100, and 200 μg / ml, while calculating the quantitative content according to the calibration graph. Accuracy was calculated by the input-found method; in the case of precision assessment, the relative standard deviation (RSD) was calculated. The results of determining the correctness and repeatability are shown in table 7.

The obtained accuracy values lie in the range of 96.7-103.6%, which meets the requirements (85-115%), the values of the relative standard deviation also meet the requirements - not more than 15%.

Example 9. Evaluation of the accuracy and precision of the quantitative determination of the substance disulfiram in homogenates of rat heart tissue

To assess the accuracy and precision, model samples of rat heart tissue homogenates containing disulfiram in concentrations of 2.5, 10, 100 and 200 μg / ml were prepared and analyzed, calculating the quantitative content according to the calibration graph. Accuracy was calculated by the input-found method; in the case of precision assessment, the relative standard deviation (RSD) was calculated. The results of determining the correctness and repeatability are shown in table 8.

The calculated accuracy between cycles varies between 97.2-105.5%. The obtained values of the relative standard deviation correspond to the requirements of not more than 15%.

Thus, the developed technique has the required accuracy and precision within the cycle for the quantitative determination of disulfiram in rat heart tissue homogenates samples.

As a result of the experiments, it was proved that the developed bioanalytical method for the quantitative determination of disulfiram in the tissue tissue homogenate of rat muscle and heart possesses the required correctness and repeatability.

As can be seen from the above examples, the present invention allows to obtain the necessary and reliable information about the method for the quantitative determination of the substance disulfiram in biological media, including in the tissues of the muscle and heart of mammals.

Claims (1)

A method for the quantitative determination of disulfiram in biological media, including extraction of the analyte with ethyl acetate from biological tissue, followed by its determination by high-performance liquid chromatography-mass spectrometry with gradient elution using a 0.1% solution of formic or acetic acid in water as a mobile phase and 0.1% solution of formic or acetic acid in methanol or acetonitrile.

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