Anna P. Chernova

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.

Introduction: The study was aimed at a comparative analysis of the anti-inflammatory and analgesic activities of diterpene alkaloid songorine obtained from Aconitum barbatum and its cell culture. Materials and Methods: Experiments were performed on 60 male mice of the CBA line. The object of the study was the diterpene alkaloid of songorine (25 µg/kg) isolated from Aconitum barbatum, as well as obtained from the callus culture of cells. Comparative study of analgesic and anti-inflammatory activities of the alkaloid obtained from different sources was carried out under conditions of the formalin test and abdominal constriction test. Diclofenac sodium (10 mg/kg) and paracetamol (85 mg/kg) were used as comparison drugs. Control animals received distilled water. All substances were administered to mice per os for 5 days, the last time – 1 hour before the onset of the damaging effect. Results and Discussion: Under the formalin test conditions, the analgesic activity of songorine from both sources was manifested in the second phase of pain response. The anti-inflammatory activity in this model of both “standard” and ”culture” songorine was comparable to that of diclofenac sodium. Probably, pharmacological activity of alkaloid in the formalin test is provided by suppression of proinflammatory mediators and TRPA1-receptors. In the abdominal constriction test, all investigated substances showed a pronounced analgesic effect maximum under the action of songorine in vitro. In addition, songorine from both sources, in contrast to the reference drug, provided a reliable phlogolytic effect. Probably, the pharmacological activity of songorine in this model is provided by its suppressive action against endogenous mediators of pain and inflammation and of TRPV1-receptors. Conclusion: Comparative analysis of the analgesic and anti-inflammatory effects of songorine (25 µg/kg) from Aconitum barbatum after a 5-dose regimen demonstrated comparability with “culture” songorine (25 µg/kg).

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).

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.

ЦЕЛЬ Работы — изучение устойчивости 2,6-ди(пропан-2-ил)фенола в биологическом материале. В качестве методов анализа применены ГХ-МС (колонка DB-5MS EVIDEX (25 м × 0,2 мм); неподвижная жидкая фаза 5%-фенил-95%-диметилполисилоксан), ТСХ (пластины «Сорбфил», подвижная фаза гексан-диэтиловый эфир (9:1) и спектрофотометрия (растворяющая среда — 95% этанол). 2,6-ди(пропан-2-ил)фенол выделяли из биоматрицы (ткани печени) путем настаивания со смесью этилацетат-ацетон (7:3). Аналит очищали, комбинируя экстракцию (система вода-этилацетат) и полупрепаративную хроматографию в колонке силикагеля L 40/100 мкм, элюент — гексан-ацетон (7:3). Установлено, что при –22 °C, 0 °C, 12 °C, 20 °C и 30 °C. 2,6-ди(пропан-2-ил)фенол может присутствовать в ткани печени соответственно в течение 119, 98, 70, 56 и 42 суток. Изучена возможность математического описания динамики разложения аналита в биоматериале (ткани печени) при рассматриваемых температурах на основе уравнения гиперболы. Экспериментально рассчитанные коэффициенты в уравнения гиперболы (k_ср.) для температур –22 °C, 0 °C, 12 °C, 20 °C и 30 °C оказались равными соответственно 1823, 1130, 697, 510 и 255. Выявлена зависимость k_ср. от температуры сохранения t^о. Для описание зависимости предложено уравнение: k_ср.=30,61∙(50–t^о)–402,39. Показано, что это уравнение может быть основой для прогнозирования особенностей устойчивости 2,6-ди(пропан-2-ил)фенола в биоматериале (ткани печени) в диапазоне температур от –22 °C до 30 °C.

ЦЕЛЬ Работы — анализ литературы по применению, фармакологическим свойствам, токсичности и методам определения внутривенного анестетика пропофола. Рассмотрены области и формы использования пропофола, его фармакокинетика, особенности биотрансформации, протекающей более чем на 90% в печени, и побочные эффекты от применения вещества для анестезии. Приведены данные о синдроме инфузии пропофола (известен также как PrIS) и смертным случаям в результате передозировки пропофола вследствие ошибок медицинских работников, злоупотреблений, попыток самоубийства и убийства. Систематизированы способы идентификации и количественного определения пропофола на основе методов высокоэффективной жидкостной хроматографии (ВЭЖХ), хромато-масс-спектрометрии (ГХ-МС) и жидкостной хроматографии (ЖХ). Указаны особенности воспроизведения описанных способов, а также биологические материалы — объекты исследования, преимущественно кровь и плазма, а также моча, желчь, волосы и др. Обозначена актуальность широкого судебно-химического изучения пропофола, количество исследований которого в выбранном научном направлении пока сравнительно невелико.

The purpose of this work is to study the stability of 2-methoxy-4-(2-propenyl)hydroxybenzene [2-MO-4-(2-P)HOB] in biological material. For analysis, gas chromatography-mass spectrometry (GC-MS) was used: column DB-5MS EVIDEX (25 m × 0.2 mm) with a stationary phase of 5%-phenyl-95%-dimethylpolysiloxane; thin layer chromatography (TLC): Sorbfil plates, hexane-dioxane-propanol-2 mobile phase (40:5:1) and UV spectrophotometry (solvent - 95% ethanol). 2-MO-4-(2-P)HOB was isolated from the biomatrix (liver tissue) by infusion with a mixture of ethyl acetate-acetone (7:3). Purification of the analyte was carried out by combining extraction (water-ethyl acetate system) and semi-preparative column chromatography [sorbent - silica gel L 40/100 μm, eluent - hexane-dioxane (8.5:1.5)]. It was established that at -22 °C, 4 °C, 12 °C, 20 °C and 30 °C 2-MO-4-(2-P)HOB is stored in the liver tissue for 385, 357, 301, 245 and 217 days, respectively. We studied the possibility of mathematical description of the dynamics of analyte decomposition in a biomaterial (liver tissue) at the indicated temperatures using the hyperbolic equation. The coefficients in the hyperbola equation (k_av), calculated according to the results of the experiment, for temperatures of -22 °C, 4 °C, 12 °C, 20 °C and 30 °C amounted to 6223, 3036, 2387, 1903 and 932, respectively., which is described by the equation: kav=101.19∙(50-to)-1272.78. It was established that on the basis of this equation it is possible to predict the nature of the stability of 2-MO-4-(2-P)HOB in the biomaterial (liver tissue) at temperatures in the range from -22 °C to 30 °C.

ЦЕЛЬ РАБОТЫ Изучение особенностей определения и динамики разложения 2-метокси-4-(1-пропенил)гидроксибензола в биологическом материале. В качестве методов анализа применили экстракцию, полупрепаративную хроматографию, ТСХ, ВЭЖХ, ГХ-МС и УФ-спектрофотометрию. 2-Метокси-4-(1-пропенил)гидроксибензол извлекали из биоматериала двукратным (по 45 мин) настаиванием с этилацетатом при соотношении масс-изолирующего агента и биоматрицы 2:1. Очистку проводили экстракцией и хроматографией в полупрепаративной (190×10 мм) колонке силикагеля L 40/100 мкм с использованием элюента гексан-диоксан (7:3). Аналит определяли методами ТСХ (пластины «Сорбфил», подвижная фаза гексан-ацетон 9:1), ВЭЖХ [колонка Discovery C18 HPLC Column (250×4,6 мм), подвижная фаза — ацетонитрил-ацетатный буфер pH 5,5 (5:5)], ГХ-МС [колонка DB-5MS EVIDEX (25 м×0,2 мм) с неподвижной фазой — 5%-фенил-95%-диметилполисилоксан], УФ-спектрофотометрии (растворитель — 95% этанол). Предлагаемая методика определения 2-метокси-4-(1-пропенил)гидроксибензола в биоматериале (ткань печени) валидирована по показателям линейности, селективности, правильности и прецизионности. По результатам исследования установили, что с ростом температуры сохранения скорость разложения аналита возрастает. При температуре 0—2; 8—10 и 18—22 ^оС продолжительность сохранения 2-метокси-4-(1-пропенил)гидроксибензола составляет соответственно 480, 390 и 260 сут.

Применение классических методов для изолирования 2-метоксигидроксибензола (гваякола) из биологического материала