HPLC-MASS spectrometry as method for quantitative determination of 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-6-aminohexanoate in rat blood plasma

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 pharmacological 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 analytes was carried out using a Phenomenex Synerdgi C18 column of 4 microns, 2x50 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 results 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.

HPLS-MS/MS, chromatography, mass-spectrometry, 2-ethyl-6-methyl-3-hydroxypyridine, N-acetyl-6-aminohexanoic (acexamic) acid