ABSTRACT
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.
Subject(s)
Chymotrypsin , Tropicamide , Animals , Hyaluronoglucosaminidase , Male , Papain , Rats , Reproducibility of Results , Retrospective Studies , TrypsinABSTRACT
The aim of study was to develop methods for the hydrolysis with hyaluronidases for the isolation of drugs (phenobarbital, diphenhydramine hydrochloride and phenibut) from biological objects (blood, hair) and to compare their effectivenes with previously developed methods of enzymatic hydrolysis. The studies were carried out using model «blood - model drug substance (MDS)¼, a natural and artificially colored wool (hair) of laboratory animals - guinea pigs of white, red and black natural colors, which were daily given a solution of MDS and then were subjected to cosmetic effects - coloring. The isolation of MDS from the model complex «blood - MDS¼ and from wool was carried out using the developed methods of hydrolysis with proteolytic enzymes (papain, chymopsin and chymotrypsin) and hyaluronidase. Phenobarbital and diphenhydramine from hydrolysates were extracted by liquid-liquid extraction method, phenibut - by direct freezing extraction with acetonitrile. The analysis of the extracts was carried out by gas chromatography with mass selective detection. The results showed that the developed methods of enzymatic hydrolysis can be recommended for isolating substances of various properties (acidic, amphoteric and alkaline) from blood, natural and artificially colored wool (hair).
Subject(s)
Chymotrypsin , Hair , Animals , Guinea Pigs , Hydrolysis , PapainABSTRACT
The objective of the present study was the development of the method for the extraction of toxic compounds from the uncoloured hairs with the use of enzymatic hydrolysis by proteolytic enzymes of the toxic compounds comprising the group of pharmaceutical substances present on the uncoloured hairs collected for the forensic medical expertise. The experiments were carried out using laboratory animals, viz. guinea pigs having hair of natural white and black colour and white rats that were given per os a phenobarbital solution and a dimedrol solution as well as daily intravenous injections of tropicamide hydrochloride administered every 28 days. The hair obtained from the experimental animals was subjected to acidic, alkaline, and enzymatic hydrolysis with chymotrypsin, trypsin, and papain. The analysis of the extracted materials was performed using the gas chromatographic technique with mass-selective detection. The results of the study give evidence that the completeness of the extraction of the toxic substances from animal hairs with the use of enzymatic hydrolysis is twice and thrice that of acidic and alkaline hydrolysis respectively. The enzymatic hydrolysis methods developed in the present study are equally efficient using the naturally coloured white and black hairs of the laboratory animals which allows to recommend them for the extraction of poisonous substances from such material. The methods were validated.
Subject(s)
Chymotrypsin/chemistry , Forensic Medicine/methods , Hair/chemistry , Hydrolysis , Papain/chemistry , Trypsin/chemistry , Animals , Guinea Pigs , Molecular Weight , RatsABSTRACT
The objective of the present study was to develop and validate the method for the extraction of toxic substances from the hair samples as exemplified by enzymatic hydrolysis of barbituric acid derivatives. The experiments were carried out with the use of laboratory animals (white female rats and albino guinea pigs) that had been daily given a phenobarbital solution per os during 4 months preceding the study. The hairs obtained from the experimental animals were subjected to acid hydrolysis with a 6 mole hydrochloric acid and enzymatic hydrolysis with the use of chymopsin, trypsin, chymotrypsin, and papain solutions. The analysis of the extracted materials was performed by means of gas chromatography with mass-selective detection. The application of the proposed method for enzymatic hydrolysis produced the better results than acid hydrolysis. This technique was validated. The results of the study made possible the comparative characteristic of the effectiveness of acid and enzymatic hydrolysis.
