Determination of quifenadine by HPLC method in blood
Keywords:quifenadine hydrochloride (phencarol), extraction with chloroform, purification of extracts from impurities by TLC and extraction methods, identification and quantitative determination by HPLC, blood
Topicality. Quifenadine hydrochloride (phencarol) – quinuclidinyl-3-diphenyl carbinol hydrochloride – first generation H1-histamine receptor blocker. The drug reduces the content of histamine in tissues due to the activation of the enzyme diamine oxidase, which breaks down up to 30 % of tissue histamine. Quifenadine hydrochloride is superior to diphenhydramine in duration of antihistamine action. Unlike diphenhydramine and diprazine, quifenadine does not inhibit the CNS, is characterized by weak sedative properties. Quifenadine hydrochloride can be used in the development of tolerance to other sedative antihistamines. Quifenadine hydrochloride is used to treat anaphylactic shock, urticaria, hay fever, Quincke’s edema, dermatoses, allergic rhinitis, food and drug allergies. In case of overdose of quifenadine hydrochloride causes dryness of the mucous membranes, headache, vomiting, stomach pain and dyspepsia. At high doses, it can affect the cardiovascular system, gastrointestinal tract, liver and kidneys. Detection and quantification of quifenadine hydrochloride in pharmaceuticals and biological matrices during treatment are based on the choice of highly sensitive and selective research methods, which is an urgent task for monitoring the effectiveness of treatment of the population with antihistamines and diagnosis of drug intoxication.
Aim. To develop an algorithm for directed analysis of quifenadine in biological extracts from blood using a unified method of HPLC research.
Materials and methods. The extraction of quifenadine was carried out with chloroform at pH 9.0. The extracts were purified from impurities by a combination of TLC and extraction with hexane. TLC purification and identification of quifenadine were carried out under optimal conditions: system of organic solvents – chloroform-n-butanol-25 % solution of ammonium hydroxide (70:40:5) and chromatographic plates – Sorbfil PTLC-AF-A. For the detection of quifenadine, the most sensitive location reagents were used – UV light (λ = 254 nm) and reagent Dragendorff in the modification of Mounier. Chromatographic analysis has been carried out on a microcolumn liquid chromatograph “Milichrome A-02” (EkoNova, Closed Joint-Stock Company, Novosibirsk, Russia) using standardized HPLC conditions: reversed-phase variant with the use of metal column with non-polar absorbent Prontosil 120-5C 18 AQ, 5 μm; mobile phase in the mode of linear gradient – from eluent А (5 % acetonitrile and 95 % buffer solution – 0.2 М solution of lithium perchlorate in 0.005 М solution acid perchloric) to eluent B (100 % acetonitrile) as during 40 min. Regeneration of column has been conducted during 2 min with mixture of solvents; the flow rate of the mobile phase has been formed 100 μl/min, injection volume – 4 μl. Multichannel detection of the substance was carried out using a two-beam multi-wave UV spectrophotometer at 8 wavelengths of 210, 220, 230, 240, 250, 260, 280, and 300 nm; the optimal value of column temperature – 37-40 °С and pressure of pump – 2.8-3.2 MPa.
Results and discussion. Isolation of quifenadine from blood has been carried out according to the developed method, including extraction with chloroform at pH 9.0; extraction purification of extracts with hexane from impurities; TLC purification and identification of quifenadine. By using a unified HPLC method, quifenadine was identified by retention parameters and spectral ratios. For quantitative determination, a calibration graph or a straight line equation corresponding to this graph were used. The obtained results indicated the reliability and reproducibility of the method. It was found that the relative uncertainty of the average result in the analysis of quifenadine in blood was ε = ± 6.65 %, the relative standard deviation of the average result was RSDx = 2.25%.Conclusions. Quifenadine was extracted with chloroform at pH 9,0 from blood. Purification of extracts from co-extractive compounds was performed by combining TLC and extraction with hexane. It was established that when isolating quifenadine from blood according to the developed methods it is possible to allocate 32.5-37.6 % of substance (ε = ± 6.65 %, RSDx = 2.25 %). The method of TLC purification and identification of quifenadine in biogenic extracts was tested under optimal conditions: system of organic solvents – chloroform-n-butanol-25 % solution of ammonium hydroxide (70:40:5), location reagents – UV light, reagent Dragendorff in the modification of Mounier, Rf quifenadine = 0.25-0.30 (Sorbfil PTLC-AF-A). The unified HPLC method for identification and quantification of quifenadine has been tested in biogenic extracts from blood according to the developed algorithm of directed analysis. It was found that quifenadine can be identified by retention time – 20.27 ± 0.03 min; retention volume 2026.9 ± 0.34 μl; spectral ratios – 0.634; 0.255; 0.041; 0.022; 0.027; 0.001; 0.001. Equation was used to determine the quifenadine content S = 0.42·10-3 С + 0.94·10-3 the correlation coefficient was equal to 0.9985.Chromatographic techniques can be recommended for implementation in practice of the Bureau of Forensic Medical Examination, poison control centers, clinical laboratories regarding the study of medicinal substances in biological objects.
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