Determination of clemastine by the HPLC method in the blood

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, Rf 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, Rf 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.


INTRODUCTION
Сlemastine fumarate (tavegil)-1-methyl-2[2-α-methylp-chlorobenzhydryloxy)-ethyl]-pyrrolidine fumarate is the first generation H 1 -histamine receptor blocker. Сlemastine fumarate selectively inhibits histamine H 1 receptors and reduces capillary permeability. The drug has a pronounced anti-allergic and antipruritic effect, which reaches its maximum in 5-7 hours and lasts for 10-12 hours. Clemastine prevents the development of vasodilation and the smooth muscle contraction induced by histamine. The drug reduces the permeability of blood vessels, capillaries, inhibits exudation and formation of edema, reduces itching, and has the m-anticholinergic effect. Сlemastine fumarate has an isignificant anticholinergic activity, causes sedation [1,2]. The drug is used to treat pruritus in psoriasis [3], multiple sclerosis [4], and optic neuritis [5].
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 mani-fests itself in impaired consciousness with the development of generalized anticholinergic convulsive syndrome [6,7].
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.
According to the toxicological studies, one of the leading places among drug poisonings is occupied by intoxication with antihistamines due to the multi-vector pharmacological effects, the uncontrolled use of many combined drugs for the treatment of seasonal diseases. According to the literature, systematic studies of most antihistamines of the first generation are absent, the methods of their isolation, identification, quantification in biological objects have been insufficiently studied. All these determines the relevance of such studies using modern highly sensitive and selective methods of analysis [6,7].
The development of the analytical service in the directions of monitoring the treatment effectiveness of the population with antihistamines and diagnosis of intoxications with the use of these drugs is based on the creation ISSN 2311-715X (Print) ISSN 2519-8750 (Online) of databases of effective, economical and express methods of the drug analysis in biological objects using a unified HPLC method [8,9].
The most common highly sensitive and selective chromatographic methods of chemical and toxicological analysis of the first-generation antihistamines are HPLC and TLC. These methods are widely used for the separation of drug mixtures, detection of impurities and purification from them.
The literature presents HPLC methods for the study of сlemastine using different conditions (variants for detecting the test substance, the use of isocratic and gradient modes of elution, the use of different compositions of mobile phases, sorbents, buffer solutions).
According to the European Pharmacopoeia, the HPLC analysis of the methanol solution of clemastine was performed using the reversed-phase chromatography with an UV-detector. Detection of substances was carried out at a wavelength of 220 nm; the column (0.1 m × 4.6 mm) with octadecyl silica gel С18, 5 μm. The mobile phase was orthophosphoric acid -acetone -1 % solution of ammonium dihydrophosphate (0.1 : 45 : 55). The flow rate of the eluent in the column was 1.0 ml/min [13].
For HPLC separation of antihistamines, the use of different conditions is recommended: the сolumn: C 18 endcapped LiChrospher 100 RP-18, (125 × 4.0 mm, 5 μm) with the pre-column LiChrocart; the mobile phase: аddition of 146 μl of triethylamine and about 750 μl of phosphoric acid to 530 ml of water. The pH was adjusted to 3.3 using 10 % potassium hydroxide solution, then 470 ml acetonitrile was added. The flow rate was 0.6 ml/min; detection -UV diode-array; the retention time of сlemastine -14 min [9].
The disadvantages of the above HPLC methods are the use of isocratic elution regime [9,13], which limits the possibility of all sample components leaving the column in the form of narrow zones and effective separation of drug mixtures. Detection at the same wavelength [13] reduces the reliability of the results obtained in the identification and study of mixtures with other drugs since it allows using only the retention parameters without the use of spectral ratios.
Modern HPLC methods for the analysis of сlemastine indicate the absence of systematic studies, which does not allow the selection of the optimal conditions for the analysis of the drug in biological objects and pharmaceuticals.
The aim of the study is to develop an algorithm for directed analysis of clemastine in biological extracts from the blood using a unified method of the HPLC research.
To achieve these aims, the following tasks should be solved: 1. to carry out extraction of сlemastine according to the method developed for isolation of organic substances of the basic character from biological objects; 2. to select the optimal conditions for TLC purification and identification of сlemastine in biogenic extracts (thin layers of sorbents, organic solvent systems, substance detection agent); 3. to approve a unified HPLC method for identification and quantification of сlemastine in biogenic extracts from the blood according to the algorithm of directed analysis developed in biological extracts using a unified HPLC method.

MATERIALS AND METHODS
Сlemastine fumarate (tavegil) was isolated from "Tavegil" tablets (10 pcs), 1 mg (Novartis AG, pharmaceutical company, Basel, Switzerland) as follows: the number of tablets containing 200 mg was transferred to a porcelain mortar and triturated to a homogeneous state. 100.0 ml of methanol was added to the mixture and mixed thoroughly. The resulting mixture was filtered through a paper filter in a porcelain cup and evaporated on a water bath at a temperature not higher than 40° C to remove the organic solvent; the residue was dried.
0.1000 g of the test substance was added to a 100.0 ml volumetric flask, dissolved in methanol, and the solution was diluted to the volume with the solvent (the standard solution with the concentration of 1000.0 μg/ml).
According to the algorithm for the study of biological objects for the presence of drugs, the following steps were performed: isolation of the substance from the biological object, purification from nutrients, identification and quantification. Purification of extracts from co-extractive compounds was performed by combining TLC and hexane extraction [16,17].
A model mixture of the blood with сlemastine fumarate was used for the research. To 10 ml of the appropriate biological fluid, 1000.0 μg of сlemastine fumarate using the methanol solution of the drug substance containing 1000.0 μg/ml, as well as control samples, were added. The samples were left for 24 h at room temperature. A day later, the studies were performed according to the extraction procedures developed.
Methods of isolation of сlemastine from the blood and extraction purification by hexane. To 10.0 ml of a model mixture of the blood with сlemastine fumarate, 5.0 ml of 10 % solution of trichloroacetic acid was added for breaking bonds with proteins, mixed and checked with an universal indicator of the mixture pH of 2.0-2.5, allowed to stand for 2 h with constant stirring at room temperature.
The mixture was centrifuged at 3000-5000 rpm for 10 min, the liquid over the precipitate was separated. Lipid impurities were extracted three times with hexane in portions of 5 ml. The hexane phases were not studied.
The aqueous layer was alkalinized with 0.1 M solution of sodium hydroxide to pH 9.0, and the сlemastine base was extracted twice with chloroform in portions of 10.0 ml followed by centrifugation at 3000-5000 rpm for 10 min to destruct water-chloroform emulsions. Chloroform extracts were combined and filtered through a paper filter ("red tape") with 1.0 g of anhydrous sodium sulphate, and TLC-purification of the extracts was performed.
Methods of TLC purification of biogenic extracts. The purified chloroform solutions were evaporated at room temperature to dryness, dissolved in 2.0-3.0 ml of methanol, then quantitatively transferred to a 5.0 ml volumetric flask, and diluted to the volume with methanol.
The TLC purification and identification of clemastine were performed under conditions: chromatographic plates -Sorbfil PTLC-AF-A, the system of organic solvents -methanol -25 % solution of ammonium hydroxide (100 : 1.5), R f сlemastine = 0.60 ± 0.03, impurities were located at the start line or at the finish line. The detection of сlemastine was performed under uniform conditions using the most sensitive developers: UV light (λ = 254 nm) -a purple color of spots, sensitivity in the sample -0.3-0.5 μg, Dragendorff's reagent modified by Mounier -an orange color of spots, sensitivity of the developer -1.0-3.0 μg of the substance in the sample [12].
The TLC purification of сlemastine in the extracts was performed according to the following method: 1.0 ml of the methanol solution of сlemastine after extraction purification was evaporated to 0.3-0.5 ml. At the starting line of the chromatographic plate at a distance of 1-2 cm from the edge to the point, 0.01 % methanol solution of сlemastine was applied using a calibrated capillary witness. At a distance of 1-2 cm from the witness, the extract from the control sample was applied, the diameter of the stain should not exceed 0.5 cm. The methanol solution of сlemastine after extraction purification was applied in the form of a strip of 1.0-1.5 cm long.
The chromatographic plate was placed in a chromatography chamber, which was a glass vessel with a ground lid with a volume of 500 cm 3 ; the system of solvents for chromatography (50 ml) was introduced into it. The chamber was carefully closed, and there was the solvent vapor saturation for at least 30-60 min. The length of the mobile phase front was 7 cm. The chromatography was completed when the solvent reached the finish line. The chromatographic plate was dried at room temperature, after that its part with spots of the witness and extracts from the control sample was developed using UV light and Dragendorff's reagent modified by Mounier.
At the level of the spot of the standard methanol, 0.01 % solution of сlemastine from the part of the plate that was not treated with the developer removed a layer of the sorbent with an area of 4-5 cm 2 , and was transferred to the filter. The substance was eluted three times with 5.0 ml of methanol, and the resulting solution was filtered through a filter ("red tape").
The resulting solutions were evaporated at room temperature to dryness, dissolved in 2.0-3.0 ml of methanol, and then quantitatively transferred to a 5.0 ml volumetric flask, and diluted to the volume with methanol.
Methods of studying сlemastine by HPLC. The chromatographic analysis was performed on a "Milichrome A-02" microcolumn liquid chromatograph (EkoNova, Closed Joint-Stock Company, Russia) according to the unified HPLC method developed by Baram G.Y. [14]: 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.

RESULTS AND DISCUSSION
Identification of сlemastine was performed by retention parameters and spectral ratios. It was found that the absolute retention time of сlemastine was 25.997-26.011 min, and the absolute retention volume was 2599.7-2601.1 μl.
Peak symmetry coefficients and capacitance coefficients were determined to verify the choice of chromatography conditions. It was found that the values of the coefficients of symmetry of the peaks were equal to 0.92 ± 0.07 (less than 2.0-2.5), and the coefficients of capacity -16.34 ± 0.02 (more than 0.5-2.0) showed the suitability of the chromatographic system HPLC analysis [15].
The method of the сlemastine determination by HPLC was validated by the following parameters: the range of linearity, limits of the quantitative determination (LOQ), accuracy and precision in the areas of low, medium and high concentrations of the test substance [18]. To quantify сlemastine, the absolute calibration method was applied using the area of the peaks of the substances.
The concentration of сlemastine in the methanol solution (C, μg/ml) obtained after purification of biogenic extracts by TLC and extraction methods was calculated using a calibration graph or the equation of the line corresponding to the calibration graph.
The linearity of the calibration graph was observed in the concentration ranges of 5.0-100.0 μg/ml, which corresponded to the content of сlemastine in the sample (4 μl) from 20.0 ng to 400.0 ng. The lower limit of the сlemastine determination by HPLC was 5.0 μg/ml (20.0 ng in the sample).
When performing the HPLC analysis of сlemastine in model solutions using the method proposed the relative uncertainty of the average results did not exceed ± 1.90 %, indicating the suitability of HPLC conditions for analysis in biological objects.
The amount of сlemastine in the blood samples was calculated by the formula: where: С х, % -is the mass fraction of сlemastine in 10 ml of the blood, %; V 1 -is the volume of a volumetric flask with the methanol extract of the substance from the blood after extraction purification, 5 ml; V 2 -is the volume of the methanol extract of the substance from the blood taken for TLC purification, 1 ml; V 3 -is the volume of the volumetric flask with the extract after TLC purification using methanol, 5 ml; k -is the coefficient of recount equal to the ratio of molecular weights of salt/base; а -is the weight of the sample of сlemastine fumarate in 10.0 ml of the blood, 1000.0 μg.
The results of the study are shown in Tab. 1.
According to the results of research, 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 ( = ± 4.63 %, RSD = 1.67 %).
According to the results of HPLC and TLC studies, an algorithm for the directed blood analysis for сlemastine was developed (Tab. 2).
The algorithm developed as a result of the present research had a number of advantages. They were: • the use of a unified HPLC method made it possible to identify сlemastine by retention parameters and spectral ratios, which made the results accurate, reliable and reproducible; • the use of the linear gradient mode of сlemastine elution allowed to obtain symmetrical, sharp chromatographic peaks (the peak symmetry coefficient did not exceed 2-2,5) and perform their reliable processing. The results of the identification and quantification by HPLC were calculated using a "Multichrome" computer program (CJSC "Ampersend", Russia) included in the chromatograph; • a high degree of purification of extracts from impurities allowed to obtain reliable and reproducible results in accordance with metrological characteristics.  The mixture was centrifuged at 3000-5000 rpm for 10 min, and the liquid over the precipitate was separated. Lipid impurities were extracted three times with hexane in portions of 5 ml. The hexane phases were not studied Chloroform extraction of the сlemastine base The aqueous layer was alkalinized with 0.1 M solution of sodium hydroxide to pH 9.0, and the сlemastine base was extracted twice with chloroform in portions of 10.0 ml followed by centrifugation at 3000-5000 rpm for 10 min to destruct water-chloroform emulsions TLC-purification and the preliminary identification of сlemastine in extracts The range of linearity of the calibration graph was 5.0-100.0 μg/ml corresponding to the content of сlemastine in the sample from 20.0 ng to 400.0 ng, respectively. To determine the content of сlemastine, the equation S = 0.15 · 10 -3 С + 0.14 · 10 -3 was used, the correlation coefficient was 0.9998