Electrochemical oxidation and protein adduct formation of aniline: a liquid chromatography/mass spectrometry study

Abstract

Historically, skin sensitization tests are typically based on in vivo animal tests. However, for substances used in cosmetic products, these tests have to be replaced according to the European Commission regulation no. 1223/2009. Modification of skin proteins by electrophilic chemicals is a key process associated with the induction of skin sensitization. The present study investigates the capabilities of a purely instrumental setup to determine the potential of commonly used non-electrophilic chemicals to cause skin sensitization by the generation of electrophilic species from the parent compound. In this work, the electrophiles were generated by the electrochemical oxidation of aniline, a basic industrial chemical which may also be released from azo dyes in cosmetics. The compound is a known sensitizer and was oxidized in an electrochemical thin-layer cell which was coupled online to electrospray ionization-mass spectrometry. The electrochemical oxidation was performed on a boron-doped diamond working electrode, which is able to generate hydroxyl radicals in aqueous solutions at high potentials. Without any pretreatment, the oxidation products were identified by electrospray ionization/time-of-flight mass spectrometry (ESI-ToF-MS) using their exact masses. A mass voltammogram was generated by plotting the obtained mass spectra against the applied potential. Oligomerization states with up to six monomeric units in different redox states of aniline were observed using this setup. This approach was extended to generate adducts between the oxidation products of aniline and the tripeptide glutathione. Two adducts were identified with this trapping experiment. Protein modification was carried out subsequently: Aniline was oxidized at a constant potential and was allowed to react with β-lactoglobulin A (β-LGA) or human serum albumin (HSA), respectively. The generated adducts were analyzed by liquid chromatography coupled to ESI-ToF-MS. For both β-LGA and HSA, aniline adducts were successfully generated and identified.

A Derivative Method with Free Radical Oxidation to Predict Resveratrol Metabolites by Tandem Mass Spectrometry

“…Using this type of instrument, tetrazepam metabolism has been investigated by comparing in vivo and in vitro methods [18, 19]. Similar EC/LC–MS/MS methods have been developed for many applications including protein adduct formation by thimerosal with human serum albumin and β-lactoglobulin A [20], protein adduct formation of aniline [21], nucleotide oxidative products generation and identification [22], and protein/peptide disulfide bond conformation by tracking electrolytic cleavage [23]. …”

Adduct formation of electrochemically generated reactive intermediates with biomolecules

“…The potential was ramped from 0 to 2.5 V versus Pd/H2. At around 750 mV, signals for GSH conjugates (monomer +O and dimer) appear with increasing signal intensities [51]. example, 92% of all proteins in yeast contain at least one cysteine moiety [59].…”

“…Next to pharmaceuticals and pesticides, skin sensitizers were studied in detail [48][49][50][51][52]. The skin-sensitizing process is assumed to be caused by the covalent binding of allergens or their metabolites to proteins present in the skin.…”

“…The results were in good accordance with conventional animal studies and underline the observation that isoeugenol is a more potent skin sensitizer. Melles et al also presented protein-binding studies of aniline, which is another known skin sensitizer and suspected to be released from the azo dye Red 2G [51]. Reactive quinone imine species of aniline and an aniline dimer were electrochemically generated and allowed to react with GSH.…”

Hyphenation of Electrochemistry with Mass Spectrometry for Bioanalytical Studies

“…Karst's group was concerned with metabolic pathways of skin sensitizers that can be released from cosmetic products such as hair dyes. Both aniline (phenylamine) [24] and p-phenylenediamine (1,4-diaminobenzene) [25,26] were oxidized electrochemically and trapped with various nucleophiles such as peptides or proteins. Tong et al [27] reported a novel approach for indirect quantification of parent drug and its unstable metabolites by complete online coulometric conversion.…”