Grant-funded Project Nr. 395/2004/B-CH/FaF
Final Report

Project title:HPLC method development for new drug candidate from pyridoxal isonycotinoyl hydrazone group and its application to biological samples analysis
Research leader:Mgr. Petra Kovaříková, 2001
Co-researcher: Doc.RNDr. Jiří Klimeš, CSc.; Renata Krutilová; Veronika Polová; RNDr. Milan Mokrý, CSc.
Period of project:2004-2004
Overall grant:173 000 CZK

Project Results

In this study, precise chromatographic methods appropriate for the determination of PIH, SIH and o-108 in the presence of their synthetic precursors (degradation products) were developed. These methods were validated with respect to accuracy, precision and linearity. Moreover these chromatographic conditions were employed to evaluate the influence of selected external conditions on the stability of these drugs. In was found that the chelators were sensitive to hydrolysis in aqueous media of different pH. The slowest degradation was determined at neutral pH, however especially in the case of PIH the important degradation was observed at this condition already at the laboratory temperature. Both acid and alkaline pH led to the acceleration of hydrolysis. The elevated temperature accelerated degradation as well. In comparison with PIH, SIH was more stable at the pH 7. Nevertheless, at low pH (2,5) the hydrolysis of SIH proceeded faster than the case of PIH. All chelators under the study were stable in organic solvents (methanol, acetonitrile). PIH was stable under both UV light and heat.

In order to develop the appropriate samples preparation techniques, different procedures (SPE, precipitation and L-L extraction) were evaluated. The absolute recoveries of all chelators were determined employing SPE columns with different type of sorbents (C18, C8, C4,CN, Phenyl) and several organic solvents as the eluents. Protein precipitation technique employing the organic solvents (methanol, acetinitril) with or without the addition of small amount of acid or base was evaluated as well. The highest recoveries of all chelators were reached by addition of small amount of HClO4 nearly before acetonitrile. In comparison with other sample treatment techniques employing L-L extraction  low recoveries of analytes were obtained.
Considering both the recovery of chelators and the general applicability of each technique for isolation of drugs from biomaterial, the most suitable procedure was chosen for each chelator under the study. In the case of PIH and o-108, SPE employing C18 and C8 columns, respectively, were chosen as the most suitable procedures. Since SPE didn’t lead to acceptable recovery of SIH, precipitation was chosen as an appropriate sample treatment technique.

The analytical methodology developed for the determination of SIH in the plasma samples was validated. Furthermore, it was employed in the preliminary in vivo experiment-determination of the concentrations of SIH in plasma after i.v. administration to rabbits. This experiment reveled for the first time the plasmatic concentrations which can be expected after administration of SIH to a living organism.