Vladimir Kambulatovich Shormanov

Objective. To investigate the features of the chemical and toxicological determination of 4-methoxyhydroxybenzene (4-MOHOB). Material and methods. The methods of the study were TLC, reversed-phase column chromatography of ordinary pressure, GC-MS and HPLC. 4-MOHOB was isolated from liver tissue and blood by infusion with a mixture of ethyl «acetate—acetone» (7:3). Results. The expediency of eluting the analyte from the Silasorb C18 column (150×10 mm) with the mobile phase of «acetonitrile—water» (4:6) was shown, which in combination with extraction formed the basis for the developed version of purification of 4-MOHOB extracted from biomatrices. The advantages of the «hexane—ether» (6:4) mobile phase in the identification of 4-MOHOB by TLC (Merck plates) were proven. One of the variants of 4-MOGOB identification is based on its extinction properties (UV range; medium — 95% ethanol). GC-MS (mobile phase — helium, stationary phase «5% phenyl—95% methylpolysiloxane» in a column measuring 25 m × 0.2 mm) was used as the main method of confirmatory identification of 4-MOGOB. The mass spectrum of this compound included signals of the following ions (m/z): 38, 53, 65, 81, 109, 124. A 250×4.6 mm Discovery C 18 column and an eluent of «acetonitrile—acid-water solution with a pH of 5.5» (50:50) were proposed for determining 4-MOGOB by HPLC. Conclusion. The compliance of the developed methods with the key validation parameters was confirmed. The detection limits of 4-MOHOB in 100 g of organ (liver) and blood are 0.08 and 0.06 mg, respectively, the limits of quantitative determination are 0.14 and 0.10 mg.

Objective. To investigate the features of 1.4-dihydropyridine derivatives’ determination in biological material in chemical and toxicological studies. Material and methods. Liquid-liquid extraction, semi-preparative column chromatography, thin-layer chromatography (TLC) and gas chromatography with mass spectrometric detection (GC-MS) were used as analysis methods. Results. It has been established that extraction of 1.4-dihydropyridine derivatives from biomaterial is most appropriate to be carried out by consecutive (by 30 min) infusion with two portions of acetone-acetonitrile (7.5:2.5) mixture at a mass ratio of the extracting liquid and bioactive matrix equal 2:1. The optimum regimen of 1.4-dihydropyridine derivatives chromatography in C-18 «Silasorb» sorbent column using acetonitrile-aqua (7:3) eluant was determined, that was realized in combined purification including the extraction with ethyl acetate from aqua-acetonitrile medium and column chromatography of analytes. Butanol-acetone (5:5) eluant may be used to identify analytes by TLC method («Sorbfil» plates). Additionally, the identification of 1.4-dihydropyridine derivatives can be performed using GC-MS (HP-5 ms Ultra inert (30 000×0.25 mm) column, immobilized phase is 5% phenyl-95% dimethylpolysiloxane, mobile phase is helium by characteristic retention time and ion sets in the analytes’ mass-spectra. The same method was used for quantitative determination of studied compounds. Methods for the determination of amlodipine, nifedipine, nimodipine and felodipine were developed by GC-MS method in biomatrix model (liver tissue), that showed suitability to validation requirements by a number of key criteria. The maximum detectable and quantitatively determinable content levels of studied substances in biomatrix did not exceed 0.04—0.14 and 0.08—0.24 mg/g, respectively.

2-Amino-4,6-dinitrophenol (2-A-4,6-DNP), or picramic acid, is widely used as an intermediate in organic synthesis, a dye in hair coloring products, an agricultural fungicide, and an explosive. Similar to other related compounds (2,4,6-trinitrophenol (2,4,6-TNF) and N-methyl-N,2,4,6-tetranitroaniline), of which it is a metabolite, 2-A-4,6-DNP is toxic to humans and animals. However, certain aspects of its detection in toxicological screening remain poorly understood. In this study, a mixture of acetone and acetonitrile in equal amounts was identified as the most effective extractant for 2-A-4,6-DNP. The application of gas chromatography–mass spectrometry (GC–MS) for detecting 2-A-4,6-DNP in the biological material extracts was analyzed and justified. The proposed method, based on the indirect determination of 2-A-4,6-DNP through O-acetyl derivative quantification, was employed to assess its stability in the biolo- gical material. 2-A-4,6-DNP was found to be detectable in the biological medium within 25 (storage temperature 18–22 °C) to 45 (storage temperature 1–3 °C) days after the contamination.

Objective. Study of 2.6-di(propan-2-yl)phenol (2.6-di(P-2-yl)F) distribution nature in warm-blooded in case of fatal poisoning due to intragastric administration of the substance. Material and methods. The warm-blooded model consisted of Wistar male rats aged about 4 months, administered 1.800 mg 2.6-di(P-2-yl)F per 1 kg of animal body weight (≈3 LD50). After the rats died, 2.6-di(P-2-yl)F was isolated from their corpses’ biomatrices by infusing an ethyl acetate-acetone mixture (7:3), purified by extraction (distribution in the systems trichloromethane-aqueous solution (pH 11.0—11.5) and aqueous solution (pH 2—4) — ethyl acetate), and normal pressure chromatography in a silica gel column L 40×100 µm (eluent hexane-acetone (7:3). The analyte was identified using gas chromatography with mass spectrometry (capillary (0.2 mm) column, stationary phase — polysiloxane with a ratio (in %) of methyl and phenyl substituents of 95:5), thin layer chromatography (Sorbfil plates, dynamic phase hexane-diethyl ether (9:1)), and electronic spectrophotometry (solvent — ethanol). The analyte content was quantified by spectrophotometry. Results. In the study, 2.6-di(P-2-yl)F mainly accumulated in the stomach contents (378.94±41.51) and tissues (175.20±17.12). Among other biomatrices obtained from poisoned rat corpses, a significant amount of the investigated toxic compound was found in the heart (142.79±13.08) and spleen (117.83±16.23), a bit smaller amounts — in the lungs (112.32±29.54) and small intestine (97.10±11.66). Conclusions. A scheme for determining 2.6-di(propan-2-yl)phenol in biomaterial has been developed and applied to study the analyte localization in the body of animals poisoned by it. The highest amount of the toxicant (mg/100 g) was found in the stomach contents (378.94±41.51), its tissues (175.20±17.12), heart (142.79±13.08) and spleen (117.83±16.23).

The aim of the work is to study the nature of the distribution of 2-A-4.6-DNP in the organisms of warm-blooded animals with intragastric administration of a toxicant. The study was carried out using the methods of TLC, UV-Visible spectroscopy, and GC-MS using derivatives of 2-A-4.6-DNP. Male Wistar rats at the age of 4 months were considered as a model of the body of a warm-blooded animal. An oily suspension of 2-A-4.6-DNF was administered intragastrically in an amount of three times the LD₅₀. Extraction of the target substance from the biomaterial was carried out by double infusion (30 minutes each) with a mixture of acetone-acetonitrile (1:1), the amount of the mixture exceeded the weight of the biomaterial by 2 times. Extractions were purified by TLC method using «Sorbfil» plates and acetone-chloroform (7: 3) mobile phase. Preliminary identification was carried out at the same time using a standard substance. Confirmatory identification was carried out by the absorption of dimethylformamide eluates in «SF-2000», as well as by the retention time and mass spectra of the major compound of the corresponding chromatographic peaks after GC-MS analysis. The quantitative content was determined spectrophotometrically, in DMF, by optical density at the analytical wavelength (490 nm). 2-Amino-4.6-dinitrophenol was found unchanged in the blood and in all the studied hollow and parenchymal organs of poisoned rats. The largest amount of 2-amino-4.6-dinitrophenol (mg/100 g) was found in the stomach walls (199.39±25.43) and stomach contents (143.14±22.63), a significant amount of the substance was found in the heart (33.49±3.66), skeletal muscles (30.70±2.64), as well as in the spleen (24.30±1.96).

Objective is to develop a method for determining 2.4-dimethylhydroxybenzene (2.4-DMHOB) in biological material. The analytical methods used in the experiments were extraction, column chromatography of normal pressure, TLC, GC-MS and HPLC. To extract the analyte from the bioactive matrix, maceration with the binary insulating agent acetone—ethyl acetate (3:7) was used, observing the 2:1 mass ratio of the «insulating agent—matrix». Optimal conditions of semi-preparative analyte chromatography were achieved in column (150×10 mm) of «Silasbor» S-18 sorbent in elution with a mixture of acetonitrile—water (7:3), which was used in the proposed cleaning scheme, combining extraction and reversed-phase column chromatography. The application of the mobile phase of tetrachloromethane-dioxane (9.5:0.5) has been substantiated for the selective determination of 2.4-DMHOB by TLC («Sorbfil» plates). The expediency of confirming identification of the analyte in the form of 2.4-dimethyltrymethylsilylphenol using GC-MS (DB-5MS EVIDEX column (25.000×0.2 mm), stationary phase (5%-phenyl)-methylpolysiloxane, carrier gas — helium) has been shown. The group of characteristic particles in the mass spectrum of trimethylsilyl analyte derivative was represented by 45; 59; 73; 82; 91; 105; 119; 135; 149; 163; 179; 194 m/z ions. HPLC (Discovery C18 250×4.6 mm column, eluting liquid — acetate buffer solution with pH 5.5 — acetonitrile, 50:50) was used to confirm the identification and quantification of 2.4-DMHOB. A method for determining 2.4-DMHOB by the HPLC method in biological material (liver tissue) is proposed, which corresponds to the criteria of linearity, selectivity, accuracy, precision and stability. The minimum detectable quantity of 2.4-DMHOB in the bioactive matrix is 0.5 µg/g, the minimum determined quantity is 1.2 ug/g.

Особенности распределения эналаприла в организме теплокровных животных (крыс)

ЦЕЛЬ РАБОТЫ Изучение распределения 2,4,6-тринитрофенола (2,4,6-ТНФ) у теплокровных животных с использованием физико-химических методов анализа после внутрижелудочного введения токсиканта. В процессе исследования применялись методы тонкослойной хроматографии, спектрофотометрии и высокоэффективной жидкостной хроматографии. Как модель теплокровных организмов рассмотрены четырехмесячные крысы линии Wistar (мужские особи). Исследуемое вещество в количестве трехкратной LD₅₀ вводили внутрижелудочно в водно-суспензионном состоянии. 2,4,6-ТНФ изолировали из биоматриц подопытных животных смесью ацетон—ацетонитрил (1:1) в режиме двукратного (по 0,5 ч) настаивания, выдерживая массовое отношение изолирующего агента и биоматрицы 2:1. Очистка и предварительная идентификация аналита проводилась на пластинах Сорбфил (подвижная фаза — ацетон—хлороформ (7:3)), подтверждающая идентификация — по поглощению в среде диметилформамида и по времени удерживания в колонке (64×2 мм) сорбента Сепарон С-18 при элюировании смесью ацетонитрил—вода (2:8). Оценка количественного содержания 2,4,6-тринитрофенола проводилась по оптической плотности диметилформамидного раствора аналита при 379 нм. У подопытных животных аналит обнаруживался в неизменном виде в крови, паренхиматозных и полых органах, их содержимом и крови. Наибольшее содержание 2,4,6-ТНФ (мг/100 г) отмечено в содержимом желудка (149,88±22,70), ткани желудка (97,89±4,86), крови (15,91±0,90) и мышцах (10,87±1,91).

Explosives such as tetryl and picric acid, which were common in the past, now have lost their combat relevance. However, they are often used for peaceful purposes individually and in combination with other trinitroaromatic compounds (e.g., trinitrotoluene). As a result of their use, environmental pollution occurs, followed by intoxication of plants, animals, and people. Cases of explosive poisoning during their production are also described. The symptoms of poisoning include both of general disorders and specific phenomena such as skin staining, impaired physiological efficiency of NADPh-dependent enzymes, genotoxicity, and immunotoxicity. Previous scientific studies established a trend toward the development of chemical-analytical probes. Various options for the sensor surface of the device and methods for detecting compounds are considered. To determine the explosives, ion mobility spectrometry is widely used, which is very rare for the chemicaltoxicological analysis of other groups of compounds. Simultaneously, methods commonly used in the analysis of narcotic and psychotropic substances (gas chromatography/ combination of high-performance liquid chromatography and mass spectrometry methods) are also applicable to determine trinitroaromatic explosives. However, the presence of nitro groups in their structure complicates such an analysis. This problem can be resolved by injecting cold samples directly to the column. Despite the availability of various developed techniques and methods, the possibility of their application to study biological matrices remains insufficient. Therefore, further studies of the chemicaltoxicological nature should be conducted to establish the optimal conditions for extracting the substances in question, the parameters of instrumental analysis, and the possibility of storing samples and for solving other problems of forensic medical examination.

The aim of the study is to investigate the lercadipine distribution in warm-blooded animals (rats). The experimental study used rats of Wistar race. TLC, GC-MS and UV-spectrophotometry methods were used for physical and chemical analysis. Semilethal (890 mg/kg) dose of lercadipine, previously suspended in water, was injected into stomach of laboratory animals. Examined substance was isolated from the thick tissues and animals’ blood by acetone, cleaned with a change of solvent and macrocolumn chromatography using 30 µm «Silasorb S-18» sorbent and acetonitrile-water (8:2) polar eluent. The analyte was identified by chromatographic behavior in the thin sorbent layer, retention time and set of positive ions in its mass spectrum, as well as by UV-spectrum. The analyte was determined quantitatively in bioactive matrix using UV-spectrophotometry. The methods were validated by criteria of linearity, selectivity, accuracy, precision, detection limits and quantitative determination. The main content of lercanidipine (mg/100 g) was determined in the stomach content (198.183±29.541), the stomach (195.312±21.579), the small intestine (47.096±3.947), the spleen (38.952±3.532) and the liver (26.211±2.232).