Investigation of the mebhydroline by chromatographic methods
Keywords:mebhydroline napadisylate (diazolin), identification, quantitation, HPLC (high performance liquid chromatography), TLC (thin layer chromatography)
Topicality. Mebhydroline napadisylate (diazolin) – 3-methyl-9-benzyl-1,2,3,4-tetrahydrocarbolyn naphthalene-1,5- disulfonate – is a first-generation antihistamine, used for the prevention and treatment of seasonal and allergic rhinitis, food and drug allergies, dermatosis. With an overdose, mebhydroline affects the gastrointestinal tract, liver, central nervous system. According to toxicological studies, one of the leading places among medical poisoning is occupied by intoxication with antihistamines, due to multivectoral pharmacological effects, uncontrolled use as part of many combined medications for the treatment of seasonal diseases. The development of highly sensitive and selective methods for the study of mebhydroline suitable for analysis in biological objects is an actual problem.
Aim. To develop an algorithm for the directed analysis of mebhydroline in biological extracts using unified methods of research by HPLC and TLC methods.
Materials and methods. TLC studies were performed using 16 mobile solvent systems and three types of chromatographic plates. To identify the mebhydroline we applied universal visualization. HPLC studies were performed on a microcolumn liquid chromatograph “Milichrom A-02” in a reversed-phase variant using a metal column with a non-polar sorbent Prontosil 120-5C 18 AQ. Elution was performed in a linear gradient mode, the elution rate was 100 μl/min; column temperature – 40 ºС; pump pressure – 2.8-3.2 MPa; sample volume for input – 4 µl. Multichannel detection of the substance was performed using a two-beam multi-wavelength UV spectrophotometer at 8 wavelengths 210, 220, 230, 240, 250, 260, 280 and 300 nm.
Results and discussion. TLC conditions for purification of mebhydroline from biogenic impurities and its identification have been established. Based on the results of the studies, the behavior of mebhydroline by the HPLC method was studied. Identification was carried out using retention parameters, spectral relationships, and quantitative determination using the absolute calibration method. The method is validated by parameters – range of linearity, limit of detection and quantitative determination, accuracy and precision.
Conclusions. The chromatographic behavior of mebhydroline by the TLC method was studied. The most suitable conditions (system of organic solvents – chromatographic plates) for identification and purification of mebhydroline from biogenic impurities were established: а) chloroform – acetone (80 : 20), Sorbfil PTLC-P-V-UV (Rf mebhydroline = 0.52 ± 0.03), Merck glass plates (Rf mebhydroline = 0.59 ± 0.03); b) methanol, Sorbfil PTLC-AF-A (Rf mebhydroline = 0.63 ± 0.03), Sorbfil PTLC-P-V-UV (Rf mebhydroline = 0.50 ± 0.03); c) benzene-ethanol-25 % ammonium hydroxide solution (80 : 20 : 1), glass plates produced by Merck (Rf mebhydroline = 0.63 ± 0.03). The identification parameters of mebhydroline under unified conditions of the HPLC method were established – the retention time was 21.17 ± 0.02 min; retention volume 2117.21 ± 0.34 μl; spectral ratio: 1.151; 0.717; 0.141; 0.089; 0.147; 0.024; 0.047. A technique has been developed for the quantitative determination of mebhydroline by HPLC method. It was established that the linearity range of the calibration curve is 50.0-200.0 µg/ml, which corresponds to the content of mebhydroline in the sample from 200.0 ng to 800.0 ng, respectively. The content of mebhydroline was determined by the equation S = 0.90 · 10-4 С + 1.05 · 10-3; correlation coefficient 0.9990. An algorithm for the directed analysis of biological extracts to mebhydroline using a combination of HPLC and TLC methods has been developed. 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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