Effects of titanium contamination caused by iron-free high-performance liquid chromatography systems on peak shape and retention of drugs with chelating properties.

Iron-free HPLC systems, better known as biocompatible systems, are generally regarded to be chemically more inert compared to conventional HPLC systems. In this work, we studied the chromatographic behavior of some classes of compounds of pharmaceutical interest, analyzed with iron-free systems. Issues typically associated with metal contamination, i.e. strong peak tailing, were observed when using an amide polar-embedded column. Effects of the contamination were visible when anhydrous methanol-acetonitrile was used, indicating that this solvent, albeit generally considered safe for conventional HPLC systems, induce corrosion of iron-free systems. The confirmation of titanium as main acting contaminant came from systematically studying the contribution of each wetted component of the HPLC system on peak shape of affected molecules. Quantification of titanium by ICP-MS analysis of effluents provided further evidence on the source of contamination. A mechanistic description of the complex interaction between titanium ions, organic molecules, and column stationary phase is proposed. In the perspective of developing methods that are fully portable between stainless steel and titanium systems, recommendations are given in terms of potentially sensitive molecules, suitable mobile phase conditions, and type of column to be used.

Analysis of Arabidopsis glutathione-transferases in yeast.

The genome of Arabidopsis thaliana encodes 54 functional glutathione transferases (GSTs), classified in seven clades. Although plant GSTs have been implicated in the detoxification of xenobiotics, such as herbicides, extensive redundancy within this large gene family impedes a functional analysis in planta. In this study, a GST-deficient yeast strain was established as a system for analyzing plant GSTs that allows screening for GST substrates and identifying substrate preferences within the plant GST family. To this end, five yeast genes encoding GSTs and GST-related proteins were simultaneously disrupted. The resulting yeast quintuple mutant showed a strongly reduced conjugation of the GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl). Consistently, the quintuple mutant was hypersensitive to CDNB, and this phenotype was complemented by the inducible expression of Arabidopsis GSTs. The conjugating activity of the plant GSTs was assessed by in vitro enzymatic assays and via analysis of exposed yeast cells. The formation of glutathione adducts with dinitrobenzene was unequivocally verified by stable isotope labeling and subsequent accurate ultrahigh-resolution mass spectrometry (ICR-FTMS). Analysis of Arabidopsis GSTs encompassing six clades and 42 members demonstrated functional expression in yeast by using CDNB and NBD-Cl as model substrates. Subsequently, the established yeast system was explored for its potential to screen the Arabidopsis GST family for conjugation of the fungicide anilazine. Thirty Arabidopsis GSTs were identified that conferred increased levels of glutathionylated anilazine. Efficient anilazine conjugation was observed in the presence of the phi, tau, and theta clade GSTs including AtGSTF2, AtGSTF4, AtGSTF6, AtGSTF8, AtGSTF10, and AtGSTT2, none of which had previously been known to contribute to fungicide detoxification. ICR-FTMS analysis of yeast extracts allowed the simultaneous detection and semiquantification of anilazine conjugates as well as catabolites.