[Development of selective method for identifying mebeverine in hair]. / Razrabotka selektivnoi metodiki opredeleniya mebeverina v volosakh.

OBJECTIVE: To develop the method for identification of mebeverine and its metabolites in biological materials (urine and hair) for solution of the problem of possible cross-reaction with psychoactive substances during preliminary studies by immunochromatographic method. MATERIAL AND METHODS: The study was carried out using «Mebeverine¼ tablets (NAO «Severnaya Zvezda¼, Russia), enzymes, namely trypsin, chymotrypsin and hyaluronidase, papain. The obtained extracts were analyzed using HELC-MS/MS on a modular liquid Nexera XR chromatograph with a tandem LCMS-8050 (Shimadzu) mass spectrometer. Guinea pigs weighing about 500 gr were used in modelling the long-term use of mebeverine. The drug was administered per os, once to 5 guinea pigs at a dosage of 200 mg calculated as an animal weighing 500 gr and daily urine was collected. The wool was collected 30 days after regular administration. RESULTS AND CONCLUSION: The main mebeverine's metabolites have been determined and native mebeverine and its marker metabolites (mebeverine and veratric acids) have been found in urine while taking this medicine in therapeutic doses for medical purposes. The application of enzymatic hydrolysis of hair method allowed to establish that hair tissue accumulates only a native molecule of mebeverine, therefore, only by results of hair analysis it is possible to conclude, if there was pharmacological use of this medicine or psychoactive substances from the group of phenylalkilamines. The effectiveness of enzymatic hydrolysis by hyaluronidase to isolate mebeverine from hair has been shown.

[Development of selective method for mebeverine detection in blood]. / Razrabotka selektivnoi metodiki opredeleniya mebeverina v krovi.

OBJECTIVE: To develop the method of biological fluids' sample processing and mebeverine detection to exclude false results' receiving when diagnosing drug intoxication. MATERIAL AND METHODS: The study was carried out using «Mebeverine¼ (NJCO «North star¼, Russia) medicine and hydrolysis by enzymes, namely papain, chymotrypsin, trypsin, chymopsin and hyaluronidase, was applied for sample processing. The extractions were analyzed by methods of HPLC-MS/MS on Nexera XR modular liquid chromatograph with LCMS-8050 (Shimadzu) tandem mass spectrometer and GC-MS on gas chromatograph connected with QP-2020 (Shimadzu, Japanese) mono quadrupole mass spectrometer. RESULTS AND CONCLUSION: It has been revealed that using selective method of sample processing, which consists of aqueous phase extraction at pH=2-4 and enzymatic hydrolysis by papain and hyaluronidase during isolation from blood, is required to detect mebeverine in biological liquids. It has been proven that the native mebeverine is hydrolyzed to veratric (3.4-dimethoxybenzoic) acid and mebeverine alcohol at alkalotic pH value of medium. It has been shown that mebeverine extraction is necessary to study using HPLC-MS/MS, which will allow to avoid the native mebeverine degradation in chromatograph injector as with GC-MS method analysis.

[Ways to solve the problem of cross-reactions during immunochromatographic examination of biological objects]. / Puti resheniya problemy perekrestnykh reaktsii pri provedenii immunokhromatograficheskogo issledovaniya biologicheskikh ob"ektov.

The purpose of this review is to study the causes of cross-reactions of a number of drugs (mebeverine, phenibut, tropicamide, ramipril, metoprolol, phenylephrine, sertraline, chloropyramine and diphenhydramine) during the preliminary stage of laboratory diagnostics by immunochromatographic method and to propose a possible algorithm for solving this problem. Conducting a hair study in order to identify the fact of the use of psychoactive substances will increase the reliability of analytical diagnostics and reduce the likelihood of false positive results of the analysis. The use of a validated method of enzymatic hydrolysis of hair will eliminate unreliable results of the analysis due to the detection of the native molecule of the toxicant, increase the efficiency and accuracy of the diagnostic procedure.

[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.

[Comparative characteristics of enzymatic hydrolysis methods for isolating toxic substances from blood and hair]. / Sravnitel'naya kharakteristika metodik fermentativnogo gidroliza dlya izolirovaniya toksichnykh veshchestv iz tsel'noi krovi i volos.

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).

[The development and validation of the methods for enzymatic hydrolysis for the extraction of toxic compounds from the uncoloured hairs]. / Razrabotka i validatsiia metodiki fermentativnogo gidroliza dlia izolirovaniia toksicheskikh veshchestv iz neokrashennykh volos.

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.

[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 development of the method for enzymatic hydrolysis for the extraction of toxic substances from the hair samples]. / Razrabotka metodiki fermentativnogo gidroliza dlia izolirovaniia toksichnykh veshchestv iz obraztsov volos.

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.