Mamina О.О., Kabachny V.I., Tomarovska T.O.

The research of prazosin in the biological objects by thin layer chromatography

Ukrainian National University of Pharmacy, t. Kharkov

The method of thin layer chromatography (TLC) is the most common method of analysis of drugs in biological objects, due to its high sensitivity, wide opportunities in determining the structure of various chemical compounds, high speed process chromatography [1].

Prazosin hydrochloride - 1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-(2-furoyl) piperazine hydrochloride, α₁-blocker is applied in medical practice in the treatment of hypertension and hypertrophy of the prostate. The medicinal preparation is characterized by toxicity in overdose, self-medication and can be causing of the intoxication of organism [1].

The aim is choice of the optimal conditions of preliminary and confirmatory stages of TLC-analysis of extracts from biological objects to prazosin, which includes methods of identification, screening and purification from impurities.

The investigations were conducted by the method [2]: into the chromatographic chamber (volume 500 cm³) was introduced solvent system for chromatography (50 ml), carefully closed the chamber followed by saturation of solvent vapors at least 30-60 minutes.

Chromatographic plates were cut into strips of a width according to the size of the chamber. At the starting line chromatographic plate at a distance of 1-2 cm from the edge to the point applied using calibrated capillary methanol solutions of extract after cleaning by hexane and drying. As witness applied 0,01% methanol solution of prazosin. The diameter of the spots was no more than 0,5 cm.

Chromatographic plate was placed in the chromatographic chamber. The length of the path of the front of the mobile phase was 7 cm. Chromatography ended when the solvent reached the finish line. Chromatographic plate was dried at room temperature, then identification was carried out using the most sensitive reagents for visualization of prazosin spots and definition of values Rf.

As a result of TLC studies, the optimal conditions for the previous stage of directional analysis of prazosin were established and included the identification and purification of drug in the presence of biogenic impurities.

For chromatography were used systems of mobile solvents - chloroform-methanol (90:10); toluene - acetone – ethanol - 25% ammonia solution (45:45:7,5: 2,5); ethyl acetate – hexane - methanol (80:10:10) and chromatographic plates company "Merck" (Germany) (type of sorbent - silicagel 60 F₂₅₄, granulation - 10-12 μm, the type of base - glass, plates size – 10x20 cm) (Rf _prazosin = 0,53–0,56). Impurities were placed on the starting line or at the finish line that did not prevent identification of prazosin.

For identification of prazosin previously was used UV light (λ = 254 nm), resulting in the chromatographic plates were observed purple spots of prazosin (sensitivity of analysis was 0,5 μg of substance in the sample). For confirmation plates were processed Dragendorff's reagent by Mounier and observed the orange spots of prazosin (sensitivity of analysis was 1,0 μg of substance in the sample) [3].

At the stage of confirmation of the results for chromatography were used systems of mobile solvents - ethyl acetate – methanol - 25% ammonia solution (85: 10:5), methanol, methanol - n-butanol (60:40) and chromatographic plates Sorbfil PSTH-AF-A (type of sorbent - silicagel STH-1A, granulation - 5-17 μm, layer thickness - 110 μm, bonding agent – silicasol, type base - aluminum foil, plates size - 10x10 cm) (Rf _prazosin = 0,76–0,78).

After TLC-identification of prazosin supporting research was performed by highly sensitive and selective methods: high performance liquid chromatography and ultraviolet spectrophotometry [1,4].

For screening studies in the first stage of the non-directional analysis were used chromatographic plates Sorbfil PSTH-AF-A and common solvent system recommended for TLC - screening of organic compounds [5]: chloroform - dioxane – acetone - 25% ammonia solution (47,5:45:5:2,5), ethyl acetate – methanol - 25% ammonia solution (85:10:2,5) (Rf _prazosin = 0,66-0,69) and toluene - acetone - ethanol-25% ammonia solution (45:45:7,5:2,5); chloroform - n-butanol - 25% ammonia solution (70:40:5) (Rf _prazosin = 0,71–0,77).

If positive results of research the second stage of the non-directional analysis was conducted in the specific systems of solvents that allow to effectively divide the compounds included in a chromatographic zone. A negative result in the first stage testified to the lack of prazosin in extracts from biological material.

Conclusions:

As a result of TLC studies of prazosin in extracts of biological material were found optimal conditions for preliminary and confirmatory stages of chromatography: mobile solvent systems, chromatographic plates, sensitive reagents for visualization, which can be recommended for using in the forensic medical expertise, toxicology and drug treatment centers, clinical laboratories for the study drugs in biological objects.

References:

1. Clarke, E. J. C. Isolation and Identification of Drugs in Pharmaceuticals, Body Fluids and Postmortem Material / E.J.C.Clarke .– London: The Pharm. Press, 2011. – 2463 p.

2. Kovalska, О. V. Identification of doxazosin by method thin layer chromatography in the presence of other antihypertensive drugs: Inform. letter № 120 / O. V. Kovalska, P. O. Byezugly, O.O. Mamina. - K., 2011. - Issue 5. - 4 p.

3. Kovalskaya, Ye. V. Analysis of alfuzosin hydrochloride by method thin layer chromatography / Ye. V. Kovalskaya, Ye. Ya. Levitin, Ye. A. Mamina // Pharmacy Kazakhstan. - 2014. - № 9. - P. 58-60.

4. Kovalska, О. V. Chromatographic investigation of antihyperthensis preparations / O. V. Kovalska, P. O. Byezugly, O.O. Mamina // Мaterials of the XI International Congress of Young Medical Scientists.– 13-14th May 2011. – Poznan, Poland – P.45.

5. The use of TLC- method for chemical-toxicological analysis of terazosin / О.О. Mamina, O.V. Kovalska, Z.I. Kovalenko, I.А. Zhuravel // News of science and education.- 2014, Science and education LTD, England – V.19 , № 19 – Р. 5–9.