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Pulatova Lola Tairhanovna, doctor of Technical Sciences, professor of the Department "Classification and Certification of Goods" of the Higher Military Customs Institute of the Republic of Uzbekistan
E-mail: lorena 97@mail.ru Iskandarov Alisher Iskandarovich, doctor of Medical Sciences, professor Head of the Main Bureau of Forensic Expertise, Ministry of Health of the Republic of Uzbekistan Aripova Tamarkhan Uktamovna, director of the Republican Scientific Center of Immunology, Ministry of Health of the Republic of Uzbekistan, Academician of Sciences of the Republic of Uzbekistan, doctor of medical sciences, professor Polyarush Svetlana Vitalovna, candidate of Biological Sciences Senior Researcher of the Scientific Center of Immunology Ministry
of Health of the Republic of Uzbekistan Sirov Vladimir Nikolayevich, Head of the Department of "Pharmacology and Toxicology" of the Institute of Chemistry of Plant Substances, Academician of Sciences of the Republic of Uzbekistan
DEVELOPMENT OF IMPROVED METHODS OF THIN-LAYER CHROMATOGRAPHY AND UV-SPECTROPHOTOMETRY AT EXPERTISE OF PAROXETIN
Abstract: The practical application of the developed methods of thin-layer chromatography (TLC) and UV spectrophotometry with respect to paroxetine has been studied. The application of the obtained results to the practice of customs, forensic forensic medical examinations, as well as narcological and toxicological laboratories is considered. The validation of the methods was carried out by comparing the results with the traditional methods of analysis of solutions. High consistency between the values obtained in the analysis of substances allows us to declare the suitability of new techniques for routine analysis.
Keywords: thin-layer chromatography, antidepressants, depression, drug falsification, customs examination, sensitivity of the method, express analysis, paroxetin.
Psychopharmacology and psychopharmacotherapy of depressive states are dynamically developing areas, and antidepressants are drugs that are second only to all psychotropic drugs after benzodiazepines. According to the World Health Organization, such a high rating of these psychotropic drugs is due to the fact that about 5% of the world's population suffers from depression, often ending with suicide.
The main effect of antidepressants is that they block the disintegration of monoamines (serotonin, norepinephrine, dopamine, phenylethylamine) under the action of mono-amine oxidases or block the reverse neuronal capture of monoamines. In accordance with modern ideas, one of the leading mechanisms for the development of depression is
the lack of monoamines in the synaptic cleft - in particular serotonin and dopamine. With the help of antidepressants, the concentration of these mediators in the synaptic cleft increases, which leads to an increase in their action. Antidepres-sants are potent drugs, always requiring individual selection of a particular drug and dose. In this regard, when choosing a medicine for the treatment of depression, it is necessary to take into account the pharmacological and toxicological properties of certain preparations, as well as the symptomatic structure of the disease and the severity of the depressive state.
In a number of works the authors testify that antidepressants are used not only in psychiatric practice, but also in the treatment of certain neurovegetative and somatic diseases,
which can be considered as "disguised" forms of depression. Drugs like fluoxetine may increase the likelihood of suicide in the first months of therapy, especially in children and adolescents [1-5]. Self-administration of antidepressants without doctor's appointment is not recommended, because can be induced by hypomania, mania, psychoses both in patients with bipolar affective disorder, and in patients without it. Practice has shown that cases of inversion of affect (development of hypomania or mania) have been observed against the background of taking antidepressants of all groups [6-8]. With each form of pharmacological activity of antidepressants, certain clinical effects, including side effects, are associated.
Currently, globally, antidepressants are considered to be one of the most profitable drugs after antibiotics, anesthetics and antipyretic drugs. When studying the distribution of the main active substances in antidepressants, it was found that in 2012-2015 in the pharmaceutical market of the Republic of Uzbekistan, 70% of medicines were manufactured using 17 medicines related to antidepressants.
One such medication is paroxetine ((3S-trans) -3-(1.3-benzodioxol-5) -methyl) -4- (4-fluorophenyl) -piperi-dine), widely used in the treatment of depression. The mechanism of its effect is based on the inhibition of repeated seizure of serotonin by neurons. On the pharmaceutical market of the Republic of Uzbekistan, paroxetine preparations under the name Rexetin (Hungary), Palikson (Slovenia), Nondepress (England) are sold. The growing demand for these drugs increases the risk of falsified forms of them, which causes significant economic damage to the republic, as well as violating rights to intellectual property and trademarks. As practice shows, most falsified medicines are detected in the process of monitoring their quality for compliance with the requirements of regulatory documents for the indicator of "authenticity" [9].
In leading foreign pharmacopoeias, complex mobile phases or expensive reagents are used to analyze antidepres-sants. The analysis techniques described in foreign pharmacopoeias are more adapted for the use of TLC plates of foreign manufacture. Spectrophotometric methods allow solving a wide range of problems, namely, quantitative determination of substances in a wide range ofwavelengths (185-1100 nm), as well as quantitative analysis of multicomponent systems; determine the composition, stability constants and photometric characteristics of light-absorbing compounds. The application of molecular spectroscopy methods for the forensic chemical study of biological objects is based on the individuality of the absorption spectra of chemical compounds [10-11].
Spectrophotometry in the ultraviolet region is based on measuring the properties of not the whole molecule, but only of its part - the chromophore grouping. In this case, the chemical nature of the latter determines the wavelength at
which the maximum absorption takes place. In the process of measurement, the substance is not destroyed and it can be determined in parallel by another method. Spectrophotometry in the ultraviolet region makes it possible to carry out studies with fairly dilute solutions of the investigated substances (10-3-10-5 M), provided that the solvent is properly selected.
For this purpose, the solvents must have the ability to dissolve the substance under study, be resistant to the radiation of the used wavelength and optically stable, should not fluoresce, react with the cuvette material, and absorb light in the same spectral region as the solute. The usual practice of recording absorption spectra involves the use of solvents such as hexane, heptane, methanol, ethanol, chloroform, carbon tetrachloride, etc. [12]. The advantage of this method over others is its high sensitivity, simplicity, expressiveness of the analysis, the uniqueness of the equipment used. This is especially important from the point of view of carrying out analytical work in forensic institutions, where substances that are in extremely small quantities are analyzed. UV spectrometry methods are also of great importance in the detection of counterfeit medicines. Using a ready-made collection of spectra greatly simplifies the analysis.
Despite the development of high-performance analytical instruments, for the final determination of analytes in pharmaceutical products and biological samples, in sample preparation, it is necessary to extract, isolate, concentrate the interesting analytes from a complex matrix. Sampling and sample preparation take more than 80% of the time from the general analysis, because these stages are very important and determine success in the analysis of components in a complex matrix. The method chosen for sample preparation depends on the overall analysis strategy.
Taking into account the foregoing, in this paper the results of studies on the development of optimal conditions for determination of paroxetine using TLC and UV spectrophotometry are presented, followed by their introduction into practice of customs, forensic forensic medical examinations, and also narcological and toxicological laboratories.
Materials and methods
To develop the TLC method, the studies were carried out on various sorbents, which differed in character and grain size:
- finished plates "Silufol", made of a large porous sorbent LS5-40, fixed on aluminum foil, binder-starch. The sorbent is processed by a luminescent substance intended for viewing at a wavelength of 254 nm;
- finished "Sorbfil" plates, PTSH-V-UV grades, grain size 8-12 microns. The sorbent is processed by a luminescent substance intended for viewing at a wavelength of 254 nm. The plates have a layer thickness of 100 mkm, fixed with the help of silicazole on polyethylene terephthalate plates. All plates
before the assays were activated at 105 °C for 30 minutes and stored in a desiccator before research.
To develop the UV spectrophotometry method, the studies were performed on a UV-VIS-8453 spectrophotometer from Agilent Technologies within the wavelength range of 220-400 nm.
Separation of paroxetine from the tablet. One tablet is crushed in a ceramic container, placed in a glass and 20-30 ml of ethyl alcohol is added and dissolved in a magnetic mixer. The filter separates the solution from the insoluble part of the tablet and is transferred to a 50 ml flask for qualitative and quantitative analysis.
Determination of paroxetine by thin layer chromatography. The TLC method is one of the main methods of substance research and is used in preliminary screening studies of a sample of unknown nature and confirmatory (private) studies on a specific drug or substance that confirm or deny its presence in the test sample. One of the advantages of TLC, in comparison with other types of distribution chromatography, is the rapid separation of substances, the stability of the sorbent-carrier layer with respect to aggressive developers and heating, the ability to fabricate thin-layer plates with any layer thickness under laboratory conditions [13-15]. In the course of our studies, we found that the detection reagents, the specificity of the chromatographic method of detecting paroxetine with respect to other toxicologically important compounds, and the influence of coextractive substances on the detection of an antidepressant have not been practically
studied analytically. In this connection, the task was to study and select the optimal conditions for the detection of paroxetine in unknown samples.
A few drops of the alcohol solution were applied to the starting arrow of the chromatographic plate "Sorbophil", a few drops of the paroxetine starting solution were applied to the opposite side as a control substance and allowed to dry at room temperature. For the development of chromatograms, a number of organic solvents were used: ethanol: chloroform: benzene (2:1:2), taken in various combinations and ratios.
Chromatography was carried out according to the following procedure: A suitable solvent system was poured into the hermetically sealed chromatographic chamber (140 x 200 mm) 2-3 hours before the plate was established, in order to saturate the space of the chamber with the vapors of these solvents, so that the height of the liquid layer in the chamber was 1 cm. The chromatographic plate was marked with a start line, which was 1.5 cm above the lower edge of the plate. A sample of paroxetine solutions was applied using graded capillaries or a microsyringe MSh-10. After drying at room temperature, the sample plates were placed in a chromatographic chamber preliminarily saturated with solvent vapors for 15 minutes. When the front of the solvents reached 10 cm from the start line, the plate was removed from the chamber and dried at room temperature. To detect paroxetine, a Dragendorff-modified reagent was used [10, 16-17]. The results of the studies of paroxetine are given in (Table 1).
Table 1. - Opening reagents and their sensitivity in the analysis of paroxetine TLC
Name of reagents Colors of spots and their sensitivity (^g)
Bromine phenol blue Bluish (4.0)
Dragendorff's reagent, made on the basis of Mounier Yellow-brown (1.0)
Erdman's Reagent Violet (4.5)
Brand reagents Yellow-green (1.5)
Brand reagents followed by washing with clean water and with UV rays Brown-green (1.0)
Bushard'sReagent Yellow-white (2.5)
ReactiveFrede Blue (5.0)
Table 2 shows the results of the amount of Rf paroxetine conditions of TLC are individual for paroxetine, which is Rf= in various chromatographic plates. The developed analytical = 0.32.
Table 2.- The amounts of Rf paroxetine in various chromatographic plates
The quantity system Rf of paroxetine in various chromatographic plates The amounts of Rf paroxetine in various chromatographic plates
Ethanol: chloroform: benzene(2:1:2) 0.39-0.41 (CSC) 0.31-0.34 (Sorbphil)
As a result of the conducted experiments it was established that the best separation of paroxetine is achieved on the plates of "Sorbophil". On these plates, the spots turn out to be rounded with distinct edges, the color of the spots differs in good intensity. As the developers for the detection
of paroxetine localization zones in chromatographic plates, many chemical reagents and their mixtures were used in various sequences. Interactions of paroxetine with the following reagents were determined: Dragendorf modified by Mounier, Buke, Bouchard, Wagner, Lieberman, Mayer, Mark, Marme,
Mandelin sodium cobaltnitrate, pyridine thiocyanate, bis- studies presented in (Table 3). We have established reagents muth nitrate, alcohol solution of ammonium molybdate, that allow us to detect and differentiate paroxetine in the test ammonium molybdate sulfate, acid [16]. As a result of the samples.
Table 3.- Color reactions of the paroxetine localization zones by TLC
Reagent Substance (paroxetine) and staining
Bromophenol blue blue
Iodine vapor brown
Bushard's Reagent brown
Dragendorf modified by Mounier orange
UV light -
As a result of the studies (Table 3), reagents were established, which allow detecting and differentiating paroxetine.
Based on the data given in Table. 2 and 3, one can judge the specificity of the recommended chromatographic conditions.
Purification of paroxetine from impurities by the TLC method. Before recommending the TLC method for the purification of paroxetine from impurities, studies were conducted on the elution process, the eluents reacting to it, and the amount of elution. Chromatography by TLC is carried out simultaneously for additional purification and preliminary detection of paroxetine. To this end, the chloroform solution was evaporated to a volume of 0.5-1.0 ml. To the start line of the chromatographic plate, 3-5 drops were placed in the form of a spot (not more than 5 mm in diameter) of the concentrated solution. At a distance of 2 cm from the spot examined, an alcohol solution of paroxetine was applied as a "witness". The remaining test solution was applied in the form of a strip 3 cm wide and 0.3-0.5 cm wide, chromatographed in chloroform: acetone (9:1). After raising the phase to a height of 10 cm, the plate was removed, dried until the smell of solvents was removed, and treated with Buschard's reagent in that part of the plate corresponding to the solution under investigation and to the "witness". In the area of the location of the paroxetine spot, the "witness" and the test extraction, spots with a value of Rf 0.31-0.34 (Sorbphil) and 0.39-0.41
(Silica Gel KSK) were shown. In the absence of spots on the chromatographic plate, it is concluded that paroxetine is not found in the test samples.
The validation characteristics of the developed TLC method. The sensitivity of the developed chromatographic conditions of the analysis was then determined. The sensitivity of the developed procedure was determined as follows: samples of a solution with different paroxetine contents were applied to chromatographic plates and chromatographed under the above conditions.
In the course of the studies, the suitability of the TLC method for the detection of paroxetine in chloroform extracts obtained from the liver of a human corpse containing and containing this preparation (model mixtures and expert material) was tested. The obtained results showed that the extracts from the biological material that does not contain the studied preparations do not give spots on the chromatograms of the spots, Rf of which is close to Rf of the paroxetine stain, which indicates the absence of the influence of possible impurities, which turn into extracts from the biological material, on the detection of paroxetine. When studying model mixtures and expert material, they came to the conclusion that, based on the results presented in (Table 4), developed by the TLC detection method is suitable for forensic analysis.
Table 4.- Limits of paroxetine detection by TLC
Reagent Detection limits, ^g (in the sample applied to the plate)
Bromophenol blue 3.0
Iodine vapor 2.5
Bushard's Reagent 2.0
Dragendorf modified by Mounier 0.45
UV light -
The next stage of the research presented in the work was the development of optimal conditions for determining paroxetine by UV spectrophotometry. Ethanol, methanol, 0.1 M sodium hydroxide solution and 0.1 M hydrochloric acid solution
were used as solvents. Paroxetine was chosen as the model sample, the solution ofwhich was prepared for the determination of absorption spectra as follows: 1, 2, 3, 4, 5, 6, 7, 8, 9 ml of a 0.01% solution of paroxetine were introduced into a row of
tubes (in one of the above solvents). The volume of solutions in the tubes was adjusted to 10 ml with an appropriate solvent and mixed thoroughly, after which the optical density of these solutions was measured, as well as 0.01% solution of paroxetine. The wavelengths Xmax = 235, 264, 295 nm correspond to the presence of paroxetine in the composition of the test samples. A comparative solution was a solvent corresponding to that used in the preparation of paroxetine solutions. The above absorption maxima were not observed in the study of
The work also tested the possibility of the influence of extraneous coextractive substances of biological and plant objects (not containing these preparations) on the identification of paroxetine by UV spectrophotometry method, based on the detection of characteristic paroxetine peaks at the wavelengths indicated in (Table 5). To solve this problem, a control sample of the crushed liver was taken and processed as in the isolation of the studied preparations from biological and plant material.
pure solvents not containing paroxetine.
Table 5. - UV spectrophotometry characteristics of paroxetine
Name of the drug Maxima of the wavelength of \max "spice" and antidepressants, (nm)
Ethanol Methanol 0.1 М HCl
Paroxetine 235, 264, 271, 295* 235, 264, 271, 295* 233, 264, 270, 294*
Notes: 1. "-" - negative result; 2. "*" is the value of the wavelength recommended for measuring the optical density
ИЮПАК: (3S-frans)-3-[(l.3-Benzodioxol-5-yloxy)methyl]-4-(4-fluorophenyl)-piperidine; C19H20FNO3 329.4 г/моль
Figure 1. UV spectrum of paroxetine
Absorption maxima at these wavelengths were not observed in the control sample under the conditions described. The spectra obtained in this way are shown in (Figure 1).
The obtained results showed that the extracts from the biological material that do not contain the studied preparations do not have absorption maxima characteristic for par-oxetine, which gives grounds to judge the absence of foreign substances influence when detecting the characteristic peaks of the studied preparations by UV spectroscopy.
To validate the identification of paroxetine by UV spec-trophotometric method, validation of the developed method was carried out. To this end, standard solutions of paroxetine were prepared in those solvents in which they had the best solubility. Standard solutions of paroxetine were prepared in methanol (ethanol).
As is known, measurement of optical density with a relative error of 2-3% is possible in the optimal range of its values, namely from 0.2 to 1.0 [12]. Thus, suitable concentrations of the analytes were selected by dilution. Based on the measurement results, a plot of the optical density versus the solution
concentration was constructed and the regression equation and correlation coefficient were calculated. Specific ( E11°%n ) and molar absorption (s) paroxetine were also determined. Calculation of the value of the specific absorption index was carried out according to the formula:
E" =D (L1)
where D is the optical density of the solution, cond. units; C is the concentration of the test substance,%; I is the thickness of the absorbing layer, The molar absorption coefficient is interrelated with the specific index and is expressed by the formula:
.C x M
£ = -
10
(1.2)
where M is the molecular mass of the test substance, cond. units
We also studied the linear dynamic range of definitions and subordination to the combined Bouguer-Lambert-Beer law. The results of the developed spectrophotometric method and validation are given in (Table 6).
Table 6.- Parametersof UV-spectrophotometricparametersparoxetine
Options Results (n = 5)
HM 295
The range of subordination to the Lambert-Beer law, ^g/ml 5-60
The free term of the regression equation (a) 0.0069
Coefficient. regression (b) 0.0179
Coefficient. correlation (r2) 0.9998
E'% 1cm 181.90
A(l /mol/cm) 6005.62
PO, mcg/ml 0.7732
EXAMPLE, mcg/mL 2.3195
Relative art. deviation,% 1.4236
Notes: 1.-the specific absorption index for each model of the spectrophotometer must be specified; 2. y is the optical density of the standard solution, condition; x-concentration, pg/ml.
In addition to the calibration graphs, we recommended a formula for calculating the quantitative content of the anti-depressants and spices tested, isolated from various objects: ,, D -V, '1000
X =-nrJ--(1.3)
E,1% -100 -V,
lew 2
where X - the number of "spice" and antidepressants in mg (ml), isolated from 100 g of biological object; n is the weight of a biological object, g (ml);
El% - specific absorption index (for each model of the spectrophotometer it is necessary to specify); V1 - initial chloroform extraction, ml; V2 is the volume taken for quantitative determination from V1, ml.
Table 7. - The results of studying the accuracy of the method of UV spectrophotometric analysis of paroxetine (n = 6)
Solvent concentration, (^g/mT) Defined Metrological characteristics of the method
(^g/ml) %
0.5 0.491 98.2 = 5 T(95%.5) = 2.57 XXav= 99.8 S2= 0.8960 SS= 0.9466 S= 0.3864 AXa= 1.9931 £= 0.9951
0.5 0.501 100.2
0.5 0.505 101.0
0.5 0.501 100.2
0.5 0.497 99.4
0.5 0.499 99.8
The above results indicate that when calculating the statistical indicator ofthe results ofthe experiment, the error did not exceed 0.9951%. The received validation data prove that the metrologi-cal indices of the developed method of UV spectrophotometric analysis of paroxetine meet generally accepted requirements. Conclusions
Based on the results of the conducted studies, it is possible to draw the following conclusions:
1. The optimal conditions for extraction of paroxetine from aqueous solutions are determined, depending on the nature of the organic solvent, the duration of extraction. It has been established that the pH of the medium, as well as the nature of the organic solvent, affects the process of isolating paroxetine by liquid-liquid extraction.
2. A unified method for the detection of paroxetine in aqueous solutions by TLC with the use of optimal solvent systems with detection of the zones of its localization Bromphenol blue and iodine vapor was developed. The detection limit was 3 ^g and 2.5 ^g in the sample.
3. Methods for determining paroxetine by UV spectrophotometry have been developed. Molar and specific absorption rates are determined. Calibration charts have been constructed to determine the quantitative content of paroxetine in objects of different origins.
4. It was found that the solution of paroxetine in 95% ethyl alcohol with UV spectrophotometry has an index of high absorption of rays at a wavelength of 235, 264, 295 nm. The linearity range of the method in the sample was 5-60 ^g/ml, the sensitivity index was 0.77 ^g/ml.
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