Integrated biological chemical approach for the identification of polyaromatic mutagens in surface waters

摘要

Surface waters are essential for human life, to supply of drinking water and as an important resource for agricultural, industrial and recreational activities. However, tonnes of pollutants enter these surface waters every year. Amongst the substances discharged into the environment, a large number are known to be mutagenic. Effect-directed analysis (EDA) is a tool to identify chemicals responsible for the observed toxic effects. It is based on a combination of chemical and biological analysis. The chemical analysis enable the separation and isolation of toxicants from complex samples, while the biological analysis enables the “tracking” of the toxicants during the separation of the chemicals, which allows decreasing the complexity of the sample in focusing only on chemicals causing the biological effects. Among planar aromatic compounds such as azaarenes, polyaromatic hydrocarbons (PAHs), keto-, nitro-keto-, hydroxyl-, amino- and nitro-PAHs, as well as quinones and hydroxy-quinones there are many known environmental mutagens often present in surface waters in complex mixtures and at low concentrations. Planar aromatic compounds may be sampled by selective adsorption to blue rayon (BR). The first part of the thesis work aimed to provide a specific fractionation method for these compounds applicable in effect-directed analysis. This procedure relies on three fractionation steps: (i) solid phase extraction using mixed-mode cation- and anion-exchange sorbents, (ii) reversed-phase HPLC polymeric C18 column and (iii) reversed-phase HPLC phenyl-hexyl column. Based on 47 analytical standards and a BR extract tested with the Ames fluctuation test, the ability of the method to recover and isolate mutagenicity is shown. Standard group recoveries ranked from 37% (quinones) to 85% (keto-polyaromatic hydrocarbons and amides) and these chemicals were separated satisfactorily, with little overlap between neighbouring fractions. The recovery of the BR mutagenicity was over 75 %. As the sample mutagenicity was mainly present in only 7 of 50 fractions, this three step fractionation method has been shown to be a reliable method to decrease sample complexity. The aim of the second part of the EDA study was to develop and apply an identification strategy to handle the data produced by liquid chromatography-high resolution mass spectrometry (LC-HRMS) and to identify chemicals that are responsible for the mutagenic effects in selected fractions. This was done in two steps: (i) Reference standards were analysed in order to understand their ionisation behaviour by electrospray (ESI) and atmospheric pressure chemical ionisation (APCI) and their MS/MS fragmentation behaviour with the aim of deriving rules that can be used for the unknown identification, and (ii) the method was then applied in the frame of a target-, suspect compounds- and non-target-screening for two mutagenic environmental fractions, N-2-8 and N-7-12 (target-and suspect-compounds-screening for this latter). The target screening based on the exact mass and retention time of the reference standards did not provide any hit and hence, no compounds were identified in the two fractions. The suspect-screening strategy based on the exact mass of the compounds enabled the identification of pigment yellow 1 in fraction N-7-12. Finally non-target screening was applied, which relied on (i) the determination of the empirical formula based on the exact mass of the parent ion, its isotopic pattern and its MS2 fragmentation, (ii) the selection of the candidates based on the comparison of MSn fragments of the unknown and compounds present in chemical database such as ChemSpider or PubChem, and (iii) the match of the physico-chemical properties of the unknown compound with candidates. This strategy enabled to identify and confirm the presence of benzyl(diphenyl) phosphine oxide in the fraction N-2-8 and to propose a list of 92 candidates, including 46 aromatic amines. The aim of the third part of this study was to apply two different approaches to further decrease the candidates list, containing 92 candidates for fraction N-2-8. This was done by applying (i) retention prediction in reversed phase liquid chromatography calculating the Chromatographic Hydrophobicity Index (CHI) using Linear Solvation-Energy Relationship (LSER), which is based on the Abraham equation and (ii) the mutagenicity prediction of aromatic amines on the basis of the stability of corresponding nitrenium ions as the ultimate electrophiles attacking the DNA. Applying the LSER model, the list of candidates was decreased to 22. The mutagenicity prediction method identified nine amino-candidates as mutagens, 24 amino-candidates for which the mutagenicity could not be predicted and thirteen as non-mutagens. In combining the results of these two methods, 22 compounds remained on the list. Hence, these two methods allowed to drastically decrease the number of candidates.