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HPLC-MASS SPECTROMETRY AS METHOD FOR QUANTITATIVE DETERMINATION OF 2-ETHYL-6-METHYL-3-HYDROXYPYRIDINIUM N-ACETYL-6-AMINOHEXANOATE IN RAT BLOOD PLASMA

DOI: https://doi.org/10.29296/25877313-2021-10-06
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Issue: 
10
Year: 
2021

N.S. Popov Ph.D. (Pharm.), Head of the Research Laboratory, Tver State Medical University of the Ministry of Health of Russia (Tver, Russia) E.N. Egorova Dr.Sc. (Med.), Head of the Department of Biochemistry with Clinical Laboratory Diagnostics Course, Tver State Medical University of the Ministry of Health of Russia (Tver, Russia) M.B. Petrova Dr.Sc. (Biol.), Professor, Head of the Department of Biology, Tver State Medical University of the Ministry of Health of Russia (Tver, Russia) E.V. Andrianova Assistant, Department of Biochemistry with Clinical Laboratory Diagnostics Course, Tver State Medical University of the Ministry of Health of Russia (Tver, Russia) O.A. Shikunova Assistant, Department of Farmation, Tver State Medical University of the Ministry of Health of Russia (Tver, Russia) E-mail: education@tvgmu.ru

Relevance. Studies of the effectiveness use of 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-6-aminohexanoate (acexamate) as an ointment for the treatment of burn wounds in animals have shown the prospects of its use as a regenerant and reparant. One of the important stages of the pharmaco-logical study medicines for external application is the assessment of resorption. These studies require the implementation of precise and reproducible bioanalytical techniques. Aim: to design and validate a chromatography-mass spectrometric technique for the quantitative determination of 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-6-aminohexanoate in rat blood plasma for subsequent evaluation of the substance resorption from medicines for external application. Material and methods. The object of the study was 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-6-aminohexanoate, which was quantified in rat blood plasma with HPLC method using Agilent Technologies 1260 Infinity II and AB Sciex 3200MD mass spectrometer. Chromatograms of 2-ethyl-6-methyl-3-hydroxypyridine, acexamic acid and sulfacetamide (internal standard) were constructed in the MRM mode. Chromatographic separation of ana-lytes was carried out using a Phenomenex Synerdgi C18 column of 4 microns, 2×50 mm, elution was fulfilled in a gradient mode with a mixture of water and absolute methanol. Calibration standards, quality control samples and blank samples were analyzed. For the developed method, the analytical range, the detection limit, the lower limit of quantitative determination, linearity were determined. The method was validated by the following indicators: selectivity, matrix effect, degree of extraction, substance transfer, accuracy and precision, stability. The developed technique was used to evaluate the resorption of 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-6-aminohexanoate from ointment after a single application to the surface of burn defects of the rat skin. Results. 2 hours after applying the ointment, the concentrations of 2-ethyl-6-methyl-3-hydroxypyridine and acexamic acid in the rat blood plasma were 18.05±0.96 ng/ml and 21.62±1.12 ng/ml, respectively, in terms of 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-6-aminohexanoate. The obtained re-sults indicate a slight resorption of the test substance through the surface of the burn defect. Conclusion. The developed method for the quantitative determination of 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-6-aminohexa¬noate in rat blood plasma is selective, accurate, precise, linear and meets the requirements for the validation of bioanalytical methods in all parameters.
Keywords: 
HPLS-MS/MS
chromatography
mass-spectrometry
2-ethyl-6-methyl-3-hydroxypyridine
N-acetyl-6-aminohexanoic (acexamic) acid

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