Determination of clemastine by the HPLC method in the blood

Abstract

Topicality. Сlemastine fumarate (tavegil)-1-methyl-2 [2-α-methyl-p-chlorobenzhydryloxy)-ethyl]-pyrrolidine fumarate is the first generation H₁-histamine receptor blocker. Сlemastine fumarate selectively inhibits histamine H₁ receptors and reduces capillary permeability. The drug has a pronounced anti-allergic and antipruritic effect. Clemastine prevents the development of vasodilation and the smooth muscle contraction induced by histamine. Сlemastine fumarate has an isignificant anticholinergic activity, causes sedation. The drug is used to treat pruritus in psoriasis, multiple sclerosis and optic neuritis. Clemastine is characterized by the following side effects: increased fatigue, drowsiness, sedation, weakness, lethargy, impaired coordination of movements; nausea, vomiting, decreased blood pressure, palpitations, hemolytic anemia, skin rash, anaphylactic shock. In case of an overdose, the drug has a neurotoxic effect, which manifests itself in impaired consciousness with the development of generalized anticholinergic convulsive syndrome. The urgent task for monitoring the treatment effectiveness of the population with сlemastine fumarate and diagnosis of drug intoxication is the choice of highly sensitive and selective research methods of its analysis in pharmaceuticals and biological matrices during the treatment.

Aim. To develop an algorithm for directed analysis of clemastine in biological extracts from the blood using a unified method of the HPLC research.

Materials and methods. The extraction of clemastine was performed with chloroform at Ph 9.0. The extracts were purified from impurities by a combination of TLC and extraction with hexane. The TLC purification and identification of clemastine were carried out under optimal conditions: the system of organic solvents – methanol – 25 % solution of ammonium hydroxide (100 : 1.5) and chromatographic plates – Sorbfil PTLC-AF-A, R_{f сlemastine} = 0.60 ± 0.03. To detect clemastine, the most sensitive location reagents were used –UV light (λ  = 254 nm) and Dragendorff’s reagent modified by Mounier. The chromatographic analysis was performed on a “Milichrome A-02” microcolumn liquid chromatograph (EkoNova, Closed Joint-Stock Company, Russia) under standardized HPLC conditions: the reversed-phase variant using a metal column with a non-polar absorbent Prontosil 120-5C 18 AQ, 5 μm; the mobile phase in the linear gradient mode – from eluent А (5 % acetonitrile and 95 % buffer solution – 0.2 М solution of lithium perchlorate in 0.005 М solution of perchloric acid) to eluent B (100 % acetonitrile) for 40 min. Regeneration of the column was conducted for 2 min with the mixture of solvents; the flow rate of the mobile phase was 100 μl/min, the injection volume – 4 μl. The multichannel detection of the substance was performed 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 the column temperature – 37-40 °С and the pump pressure – 2.8-3.2 MPa.

Results and discussion. Isolation of clemastine from the blood was performed according to the method developed, including the extraction with chloroform at pH 9.0; the extraction purification of extracts with hexane from impurities; the TLC purification and identification of clemastine. Using the unified HPLC method clemastine was identified by retention parameters and spectral ratios. For the quantitative determination, a calibration graph or the straight line equation corresponding to this graph were used. The results obtained indicated the reliability and reproducibility of the method. It was found that the relative uncertainty of the average result in the analysis of clemastine in the blood was ε = ± 4.63 %, the relative standard deviation of the average result was RSDx = 1.67 %.

Conclusions. Clemastine was extracted with chloroform at pH 9.0 from the blood. Purification of extracts from co-extractive compounds was performed by combining TLC and extraction with hexane. It has been found that when isolating сlemastine from the blood according to the methods developed it is possible to determine 36.05-39.55 % of the substance (ε = ± 4.63 %, RSDx = 1.67 %). The method of TLC purification and identification of сlemastine in biogenic extracts was tested under the optimal conditions: the system of organic solvents – methanol – 25 % solution of ammonium hydroxide (100 : 1.5), the use of reagents – UV light, Dragendorff’s reagent modified by Mounier, R_{f сlemastine} = 0.60 ± 0.03 (Sorbfil PTLC-AF-A). The unified HPLC method for identification and quantification of сlemastine was tested in biogenic extracts from the blood according to the algorithm of the directed analysis developed. It has been found that сlemastine can be identified by the retention time – 25.997-26.011 min; the retention volume – 2599.7-2601.1 μl; spectral ratios – 0.741; 0.536; 0.096; 0.023; 0.027; 0.005; 0.003. The сlemastine content was determined by the equation S = 0.15 · 10^-3 С + 0.14 · 10^-3; the correlation coefficient was equal to 0.9998. Chromatographic methods 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.