[Determining the time of intoxication due to non-drug use of tropicamide]. / Opredelenie srokov davnosti intoksikatsii pri nemedikamentoznom primenenii tropikamida.

The study objective is to develop approaches to the retrospective assay of tropicamide in biological fluids and hair. The study was performed using the substance of tropicamide. Sample preparation included hydrolysis with the following enzymes: papain, chymotrypsin, trypsin, chymopsin, and hyaluronidase. Extracts were analyzed using a gas chromatograph with mass selective detection Technologies (USA) 7890 A/5977 MSD. When modeling long-term use of tropicamide, male rats of white and brown natural color, about 6 months old and weighing 200-250 g, were used. Animals were injected with a tropicamide solution in the tail vein for 28 days at a dose of 40 mg/kg of body weight. After 28 days of administration of the tropicamide solution, daily urine and blood were collected, and hair was cut from the back and sides of the animal's body. After another 28 days, hair samples were taken again. Within the first 6 hours after the last tropicamide dose, its blood concentration reached the maximum (191.6 µg/ml) and within 4 days decreased by 10 times; in the urine, within the first 24 hours, tropicamide level decreased from 627.7 to 489.9 µg/ml, then for 2-3 days it remained approximately at the same level. From day 4, the tropicamide concentration significantly decreased, and on days 11-12, it was not detected in the urine. After 4 weeks, the tropicamide content in the hair was at the level of quantification (1.25-2.20 ng/mg) and could be detected only by sample preparation by enzymatic hydrolysis with papain. Thus, the developed and validated methods for the enzymatic hydrolysis of biological fluids and hair allowed retrospective studies of biological fluids and hair with high reliability.

[The chemical toxicological determination of isoniazid and metoclopramide in the objects used for animals poisoning and in biological fluids]. / Khimiko-toksikologicheskoe opredelenie izoniazida i metoklopramida v ob''ektakh, ispol'zuemykh dlia otravleniia zhivotnykh, i v biologicheskikh zhidkostiakh.

The number of mass casualty incidents among the dogs has significantly increased during a few recent years in the cities of this country; they are most frequently attributed to the activities of the so-called dog hunters. The dog hunters make use of a variety of chemical compounds for poisoning the dogs. Most of them are the multi-component substances, with their chemical composition being highly variable and continuously modified. The objective of the present study was to develop the method for the isolation and identification of isoniazid and metoclopramide introduced into the baits that are distributed by the dog hunters for the poisoning of the animals. The proposed method was tested with the use of biological fluids obtained from the laboratory animals. The effectiveness of isolation of the compounds of interest from these materials with the use of the liquid-liquid and fractional freezing extraction techniques was evaluated. In addition, the method for solid phase extraction with the use of the 'Oasis HLB' cartridges was developed.

[The use of enzymatic hydrolysis for isolation of barbituric acid derivatives from blood (as exemplified by phenobarbital and barbamyl)].

Modern isolation techniques by direct extraction with organic solvents or after protein precipitation by various sedimenting or salting-out agents are characterized by low efficiency and do not permit to liberate derivatives of barbituric acid from their complexes with blood proteins. The use of enzymatic hydrolysis makes it possible to break bonds between barbiturates and protein and thereby improve the efficiency of isolation. We performed enzymatic hydrolysis of the model phenobarbital-blood and barbamyl-blood complexes with the use of trypsin, pepsin, chymotrypsin, and papain. The degree of phenobarbital extraction with trypsin and barbamyl was estimated at 62.1 +/- 1.2% and 75.1 +/- 1.6% respectively; in other words, it was 32.7 +/- 1.0% and 51.1 +/- 1.0% higher than that achieved by traditional methods. Certain validation characteristics of the new method are presented.

[Isolation of blood caffeine as a model substance by enzymatic hydrolysis].

Organic solvent extraction of toxic compounds from blood either directly or following precipitation of serum proteins yields only free fractions. It is proposed to dissociate toxin-protein complexes in blood samples by enzymatic hydrolysis. Model caffeine complexes in an albumin solution were treated by tripsin, chemotripsin, and papain. The best results were obtained using chemotripsin and papain with which 70.4 +/- 1.6% and 62.2 +/- 1.2% of the caffeine was isolated from blood compared with 45.6% by direct extraction. Validation characteristics of the proposed method are specified.

[Assessment for efficiency of isolation of toxic substances from blood].

The authors gave assessment for efficiency of existing methods of isolation of toxic substances from blood with model substance. Some validation characteristics of the method of direct extraction from blood with organic solvent were determined.