Desenvolupament d'un mètode analític per la determinació de neurotòxics en sèrum de ratolí per HPLC-DAD

Abstract

3,3'-iminodipropionitrile (IDPN) is a neurotoxin that can metabolize in 3-Aminopropionitrile (BAPN) and β-alanine, among other metabolites. In this study we developed a chromatographic method for the determination of IDPN and two of its possible metabolites, BAPN and β-alanine in biological samples using a high-performance liquid chromatography (HPLC) system equipped with diode array detector (DAD). The developed method has been evaluated by analyzing spiked mouse serum samples. The aim of this study is to study the matrix effects and to calculate the recoveries of these compounds in this type of samples. Derivatization of the amino group is required to facilitate UV-Vis detection of the three compounds of interest. In this study, dansyl chloride was selected as the derivatization agent. The conditions of the derivatization reaction such as temperature, time, pH and concentration of the derivatitzing agent were studied and especially those required for obtaining IDPN derivatives. Finally, despite the extreme conditions tested, IDPN was not derivatized. Therefore, this compound was not determined and only BAPN and β-alanine were included in the method. The chromatographic separation and detection conditions by HPLC-DAD for these compounds were then studied. Once established the optimum conditions of derivatization and chromatographic separation, we calculated calibration curve for BAPN and β-alanine, setting the range of linearity and limits of detection and quantification of this method for the determination of these compounds. To study the recovery of compounds in mouse serum samples we tested different procedures of sample treatment after spiking the samples with BAPN and β-alanine. The results show a linearity interval of 0,67-2 ppm for β-alanine and a detection limit of 0,2 ppm despite we could not achieved enough sensitivity with this method to determine lower concentrations. For BAPN, the sensitivity is even lower than for β-alanine and the calibration curve has linearity interval of 0,61-2 ppm with a detection limit of 0,18 ppm. In order to determine the recoveries of these compounds from the serum samples, we tested several sample treatments to remove serum proteins: HClO4, acetone-methanol, centrifugal filters AMICON 3 KDa and combinations of centrifugal filters with the other two treatments. The results were not positive in any of the procedures tested to treat samples and we could not prevent chromatographic peaks of serum components did not interfere in the peaks of the compounds of interest. The matrix effect is strong in this case and therefore we could not calculate the recovery percentage of β-alanine and BAPN in serum samples