We have proposed a method of TLC-UV spectrophotometry for the detection and quantification of ketotifen from the urine.. . The technique consists in the preliminary separation of ketotifen related endogenous substances present in urine by TLC. Further, the adsorption zone of ketotifen eluted and conduct a quantitative measurement of the drug by spectrophotometry in the UV region of the spectrum. The proposed method allows to determine 4.1±2,19 mkg/ml of ketotifen, which is 81.88% with a relative standard deviation RSD 3,18%.
Key words: ketotifen, thin-layer chromatography, chloroform, the urine, chemical and toxicological analysis.
Chemical and toxicological analysis (HTA) of medicines that are of interest in terms of the potential hazard to human acute poisoning and criminal acts, rather laborious process, where the objects of study appear biological objects (biofluid, secrets, organs, etc.) . Preparation of samples are very important in analytical toxicology, including selection (isolation), purification and concentration of toxic compounds, proper use of the capabilities of different methods of analysis, their rational combination and the ability to interpret the results. This analysis is very important quality measurements; a set of properties that lead to getting results with the required accuracy characteristics, in the required form and in a timely manner.
Ketotifen in the incidence of drug complications steadily occupies one of the first places in connection with wide application in medical and non-medical practice, the ability to cause poisoning, including fatal, accompanied by the negative reactions of the organism from simple allergies to anaphylactic shock. Adverse effects of the toxic nature tend to develop on the background of elevated concentrations of the drug in blood when used at doses higher than the recommended by accidental contact with the bloodstream, the rapid introduction of the drug, and when administered to patients with slow metabolism type.
The aim of the research is to develop methods of quantitative determination of ketotifen by TLC- UV spectrophotometry.
Materials and methods
We used drug substance of ketotifen (British Pharmacopoeia, 2009.-V.I & II); solvents and reagents of the category —reagent grade” and —analytical grade”. Thin-layer chromatographywas used to remove impurities: chromatographic chamber size 235x170x120, chromatographic plate “Sorbfil PTLC-UV” (—Sorbfil”, Russia); UV chromatoscope, microcapillaries of 2 ul (Russia). Quantitative determination was performed on an SF-2000 (OKB, Russia), in cells with a layer thickness of 10 mm.
Results and discussion
We have proposed a method of TLC-UV spectrophotometry for the detection and quantification of ketotifen from the urine. The technique consists in the preliminary separation of ketotifen related endogenous substances present in urine by TLC. Further, the adsorption zone of ketotifen eluted and conduct a quantitative measurement of the drug by spectrophotometry in the UV region of the spectrum.
The identification and quantification of drugs in extracts of bioliquids important condition is the choice of pH. pKa of ketotifen is 8.58, based on which it follows that it is optimal for separation of accompanying substances is slightly alkaline and alkaline environment. To 25 ml of urine was added an alcoholic solution containing 5 mg of ketotifen. The resulting model was left for 24 hours in the refrigerator at a temperature of 40C. To the mixture was added 25% ammonium hydroxide solution to pH 10-11 and extracted with chloroform three times the volume of 10 ml. After each extraction the organic solvent layer was separated from the aqueous phase. The resulting organic extract was combined for further research.
Detection and separation of ketotifen in extracts of urine was performed in the optimum TLC solvent system of chloroform-25% ammonium hydroxide solution with conc. (50:0.5). Then, in order to clean contaminants from endogenous elution test sample to the chromatographic plate. For this purpose, the adsorption zone ketotifen eluted with 0.1 M hydrochloric acid solution.
The absorption spectrum of the resulting solution was measured in the 200-400 nm using a spectrophotometer cuvettes working layer with a thickness of 10 mm with respect to the reference solution (extract of a control experiment without adding ketotifen). The spectrum is characterized by extracting the maximum absorption at a wavelength of 300 ± 2 nm, which corresponds to the literature data. In extracts from a control sample of urine absorption was observed. The proposed method allows to determine 4.1±2,19 mkg/ml of ketotifen, which is 81.88% with a relative standard deviation RSD 3,18%.
Thus, the developed methodology is correct and reproducible, making it suitable for chemical and toxicological analysis of ketotifen.
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