Application of Thin Layer Chromatography in Analysis of Secnidazole, Ornidazole, Tinidazole and Nimorazole

Oksana V. Shovkova et al /J. Pharm. Sci. & Res. Vol. 10(12), 2018, 3411-3416 Application of thin layer chromatography in analysis of secnidazole, ornidazole, tinidazole and nimorazole Oksana V. Shovkova, Lina Yu. Klimenko*, Zoia V. Shovkova, Vera A. Ulanova, Oleg S. Shpychak National University of Pharmacy, Kharkiv, Ukraine 53, Pushkinska str., Kharkiv, 61002, Ukraine Abstract Secnidazole, ornidazole, tinidazole and nimorazole are the medicines of the group of 5-nitroimidazole derivatives and joint taking these medicines with alcohol leads to the strong intoxication syndrome; therefore they are potential objects of chemical toxicological investigations. The method of thin layer chromatography is widely used in the process of forensic toxicological examinations for screening and confirming investigations. The aim of work is integrated study of visualization conditions of sechnidazole, ornidazole, tinidazole and nimorazole on TLC-plates using standard and particular coloured reagents, and also chromatographic behaviour of the substances using standard mobile phases. To fix the results of visualization of the substances to be analysed some developing modes of TLC-plates with two types of substrate (plastic and glass) and with/without luminophor have been chosen – immediately after processing the substances with a reagent and after drying the plates at the ambient temperature; after heating the plates for 15 minutes at 110°C; in UV-light at two wavelengths – 254 nm and 365 nm – before and after heating. The Rf values of secnidazole, ornidazole, tinidazole and nimorazole in commonly used in forensic toxicological analysis solvents systems using different types of TLC-plates have been set. The individual mobile phases, which ensure optimal separation of the medicines on TLC-plates, have been chosen. Keywords: secnidazole, ornidazole, tinidazole, nimorazole, thin layer chromatography, colour tests INTRODUCTION The rats’ blood and urine were extracted using acetonitrile The method of thin layer chromatography (TLC) is widely with «salting-out» by ammonium sulphate [3, 4]. Obtained organ- used in the process of forensic toxicological examinations for ic extracts were evaporated and dry residues were dissolved in screening and confirming investigations – with the purpose of methanol (blank-samples) or reference solutions 1 of the sub- analytes detection and identification respectively [1, 2]. The main stances to be analysed (model samples). All experiments with rats focus is the chromatographic behaviour of the substances using and biological liquids were carried out at Pharmacology Depart- standard mobile phases (or solvents systems), as well as the con- ment of National University of Pharmacy. ditions of analytes spots visualization using standard coloured The colour reagents were prepared according to [1]. reagents. The data about Rf values in the solvents systems general- Weighing was carried out using digital analytical balance ly accepted in forensic toxicological analysis and spots coloura- АN100 (AXIS, Ukraine) with d = 0.0001 g. tion when developing with generally accepted reagents for medic- Glassware satisfied ISO 648:2008 «Laboratory glassware inal substances, which are potential toxic agents, are published in – Single-volume pipettes», ISO 1042:1998 «Laboratory glassware the well-known guidance «Clarke’s...» [1], and this information is – One-mark volumetric flasks» and calibrated according to ISO regularly updated and supplemented based on the results of re- 4787:2010 «Laboratory glassware – Volumetric instruments – searches monitoring. It should be noted that in the latest edition Methods for testing of capacity and for use» and «Guidelines for [1] there is only fragmentary information about chromatographic calibration in analytical chemistry. Part 2. Multispecies calibra- behaviour of the most popular 5-nitroimidazole derivative metro- tion» [5] was used throughout this study. nidazole and the results of its visualization on the chromatograph- The chromatographic plates Sorbfil® PTLC-IIH-UV (sili- ic plates using two coloured reagents – methanolic potassium ca gel STC-1HP, PETP, luminophor, silica sol, 8 ÷ 12 μm frac- hydroxide solution and acidified potassium permanganate solution, 100 μm layer thickness) were purchased from IMID LLC tion. Data about other 5-nitroimidazole derivatives such as sech- (Russia). The chromatographic plates Merck® TLC SILICA GEL nidazole, ornidazole, tinidazole and nimorazole are completely 60 (silica gel 60, glass, gypsum, 9,5 ÷ 11,5 μm fraction, 140 ÷ 160 absent. μm layer thickness) were purchased from Merck Group (Germa- The purpose of our work is integrated study of visualiza- ny). tion conditions of sechnidazole, ornidazole, tinidazole and ni- The part of plates were previously processed with 0.1 morazole on TLC-plates using standard and particular coloured mole/L potassium hydroxide solution in methanol and then dried reagents, and also chromatographic behaviour of the substances at 110°С for 30 min. The part of plates were previously processed using standard mobile phases. with 0.1 mole/L sodium bromide solution [1]. To choose the developing colour reagents in 10 µL of ref- MATERIALS AND METHODS erence solutions 1, blank-samples and model samples were ap- Secnidazole, ornidazole, tinidazole and nimorazole were plied on the plates of both types, and then the reagents were of pharmacopoeial purity. Chloroform (≥99%, anhydrous, con- sprayed or poured onto the plates. The results were fixed visually tains 0.5 – 1.0% of ethanol as stabilizer), ethyl acetate (99.8%, at once and after drying the plate, then the plates were developed anhydrous), methanol (≥99.8%, puriss. p.a., ACS reagent), am- in UV-light with the wavelength of 254 nm and 365 nm. At the monium hydroxide solution (≥25% NH3 in H2O, puriss. p.a. plus) next stage the plates were heated for 15 min. at 110°C (the plates were purchased from Sigma-Aldrich Co. LLC (USA). All other were covered with glass plate), and then colours of spots were reagents were of analytical grade. fixed in visible and UV-light one more time. The reference solutions 1 (1000 μg/mL) were prepared by To determine sensitivity of the developing colour reagents dissolving 50.0 mg of the respective substance to be analysed the same experiments were carried out using 1 and 10 µL of refer- (secnidazole, ornidazole, tinidazole or nimorazole) in methanol ence solutions 2 of the substances. and the solutions were diluted to 50.0 mL with the same solvent. Chromatographing was carried out in cells with the vol- The reference solutions 2 (100 μg/mL) were prepared by diluting ume of 500 mL; 50 mL of the respective TLC-systems were 5.00 mL of the reference solutions 1 to 50.0 mL with methanol. placed into them. The cell was saturated for 30 min. In 10 µL of 3411 Oksana V. Shovkova et al /J. Pharm. Sci. & Res. Vol. 10(12), 2018, 3411-3416 reference solutions 1 of the substances to be researched were ap- 2) in UV-light at two wavelengths – 254 nm and 365 nm; plied on the start line in the distance of 1 cm from the plate edge. 3) after heating the plates for 15 minutes at 110°C (the The solvents path length was 8 cm. After reaching the finish line plates are covered with glass); by the mobile phase the plate was taken out from the cell, dried at 4) in UV-light at two wavelengths – 254 nm and 365 nm – the ambient temperature and developed with the respective rea- after heating. gents. The choice of developing modes is due to the following reasons: RESULTS AND DISCUSSION • there is no heating of plates after processing with the most Secnidazole, ornidazole, tinidazole and nimorazole are the of reagents according to the recommendations [1], but in medicines of the group of 5-nitroimidazole derivatives and widely the case of carrying out the researches at high environment used for treatment of infectious diseases [6 – 8]. Administration of temperatures we can see the results of substances visuali- 5-nitroimidazole derivatives is accompanied by a number of side zation, which are not typical for normal conditions; so in effects [9 – 11]. They block the enzymes of alcohol dehydrogen- our work we have recorded the results of substances col- ase and acetaldehyde dehydrogenase, therefore when joint taking ouration under normal conditions and after heating – for the medicines with alcohol it is observed the strong intoxication all investigated reagents; syndrome [12 – 14]. Therefore we can make the conclusion that • in some cases processing with a reagent may not lead to secnidazole, ornidazole, tinidazole and nimorazole are potential formation of coloured compounds, but at the same time objects of chemical toxicological investigations. changes the fluorescence (intensification, change the col- The structural formulae of secnidazole, ornidazole, ouration, quenching) of the substances to be analysed; tinidazole and nimorazole are shown on Figure 1. such signs can become an important differentiating fea- N ture; in addition, coloured reaction products may have a N characteristic fluorescence that can increase the reliability O O N CH of detection and identification of the substances to be ana- N CH N 3 lysed; N 3 O • in preliminary experiments it has been found that in the O OH case of covering the plates with glass when heating the re- OH sults of spots visualization become more stable, reliable and reproducible; • the type of substrate of the chromatographic plates can in- Cl fluence the results of the substances visualization, espe- cially when heating; therefore, it

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