Хіміко-токсикологічне дослідження атомоксетину

Abstract

Дисертаційна робота присвячена розробці алгоритму проведення токсикологічного дослідження біологічного матеріалу та біологічних рідин на присутність атомоксетину, придатного для використання в судово-токсикологічній експертизі та клінічній токсикології. Визначено ступінь ізолювання атомоксетину з біологічного матеріалу загальноприйнятими методами. Розроблено метод ізолювання атомоксетину хлороформом з подальшою екстракційною очисткою у системі н-гексан – ацетонітрил. Встановлено оптимальні умови виділення атомоксетину з крові та сечі методом рідинно-рідинної екстракції. Розроблено методики ідентифікації та кількісного визначення атомоксетину в біологічних об’єктах методами ВЕРХ-УФД, ГХ-МС та ГХ-ПІД, УФ-спектрофотометрії та екстракційної спектрофотометрії у видимій області спектра з азобарвниками метиловим оранжевим та похідним теофілідину. Вперше розроблено умови виявлення атомоксетину та його метаболітів в сечі методом ТШХ. Проведено ідентифікацію метаболітів атомоксетину, виділених з сечі, методом мас-спектрометрії.

Диссертационная работа посвящена разработке алгоритма проведения токсикологического исследования биологического материала и биологических жидкостей на присутствие атомоксетина, пригодного для использования в судебно-токсикологической экспертизе и клинической токсикологии. Определена степень изолирования атомоксетина из биологического материала общепринятыми методами. Разработан метод изолирования атомоксетина хлороформом с последующей экстракционной очисткой в системе н-гексан – ацетонитрил. Установлены оптимальные условия выделения атомоксетина из крови и мочи методом жидкостно-жидкостной экстракции. Разработаны методики идентификации и количественного определения атомоксетина в биологических объектах методами ВЭЖХ-УФД, ГХ-МС и ГХ-ПИД, УФ-спектрофотометрии и экстракционной спектрофотометрии в видимой области спектра с азокрасителями метиловым оранжевым и производным теофилидина. Впервые разработаны условия обнаружения атомоксетина и продуктов его биотрансформации в моче методом ТСХ. Проведена идентификация метаболитов атомоксетина, выделенных из мочи, методом масс-спектрометрии.

The dissertation deals with the problems of creating an algorithm for conducting toxicological research on the content of atomoxetine in biological material and biological fluids, which will be suitable for use in forensic toxicological examination and clinical toxicology. The problem of prevalence of attention deficit hyperactivity disorder, which is treated with Atomoxetine, is considered. The mechanism of the drug action, the pharmacological properties, side effects, manifestations of the toxic action and causes of poisoning are given. The parameters of the chromatographic mobility of Atomoxetine and some of its pharmacological analogs (Melipramine, Doxepin, Fluoxetine, Fluvoxamine, Amitriptyline and Venlafaxine) have been determined in thin layers of the sorbent using mobile phases recommended by TIAFT for the general TLC-screening, as well as some other ones that are widely used in the practice of chemical and toxicological studies of basic medicinal substances. The chromatographic mobility has been determined on five types of chromatographic plates. The possibility of Atomoxetine detection in the urine by the products of its metabolic transformations using thin layer chromatography has been studied. The chromatographic mobility of two Atomoxetine metabolites in TLC screening systems, as well as staining of the main and minor metabolites of the antidepressant with a number of chromogenic reagents have been determined. The degree of isolation of atomoxetine from biological material by conventional methods has been determined. Taking into account the lipophilic properties of atomoxetine, a method for its isolation by chloroform was developed, with subsequent extraction purification in the n-hexane-acetonitrile system. The quantitative determination of the drug in extracts from the blood was performed by UV spectrophotometry, HPLC-UVD and GC-FID, in extracts from the urine – by the UV spectrophotometric method. Under the conditions proposed the degree of Atomoxetine isolation from the blood and urine was 35.8% and 69.3%, respectively. The study of the conditions of Atomoxetine extracting from aqueous solutions by organic solvents depending on the pH medium. Chloroform in the alkaline medium (pH 12), in the presence of sodium sulfate as a salting-out agent, has been shown to be the optimal extragent for the extraction of Atomoxetine. The lowest values of the degree of 23 Atomoxetine extraction were at pH 1 – 2 when using 1,2-dichloroethane, benzene and diethyl ether (the degree of extraction for the latter was equal to 0.2 – 1.6 %) as extragents. Methods for the identification and quantitative determination of atomoxetine in biological objects by methods of HPLC-UVD, GC-MS and GC-FID, UV spectrophotometry and extraction spectrophotometry in the visible spectrum with azole dyes and orange theodolite are developed. The simultaneous use of three mobile phases: methanol – 25 % solution of ammonium hydroxide (100:1.5), cyclohexane – toluene – diethylamine (75:15:10), toluene – acetone – ethanol – 25 % ammonium hydroxide solution (45:45:7.5:2.5) can be considered the most appropriate to separate Atomoxetine from some of its analogs studied. Based on the studies conducted on the development of the procedure for Atomoxetine visualization in the TLC screening the use of the following sequence of chromogenic reagents has been proposed: UV-light, ninhydrin solution, Dragendorff‘s reagent, Fröhde reagent, Marquis reagent, modified Mandelin reagent. Conditions for detection and identification of biotransformation products of this antidepressant in the human urine by TLC and mass spectrometry were established. Procedure of identification and the quantitative determination of Atomoxetine in the biological extracts using HPLC with multiwave spectrophotometric detection, UV spectrophotometry, extraction spectrophotometry in the visible region of the spectrum by the reaction with acid azo dyes, gas chromatography with flame ionization and mass spectrometric detection were developed. The highest selectivity of the UV-spectrophotometric method for determining Atomoxetine in the biological material in relation to matrix components was provided by chloroform isolation, the lowest one – by A. O. Vasilyeva method. The storage of Atomoxetine in the biological material during its putrefactive decomposition has been studied depending on the shelf life. It has been shown that 15 % of Atomoxetine can be isolated from a putrefactive biological object in 4 months. The possibility of detecting Atomoxetine in the urineby its biotransformation products was shown. The chromatographic mobility of the metabolites of Atomoxetine in screening TLC systems recommended by TIAFT have been determined. Based on the studies conducted the scheme for directed chemical and toxicological examination of the biological material and biological fluids on the presence of Atomoxetine has been developed.