An optimization protocol of the volatile organic compounds analysis in earwax samples for untargeted volatilomics.

Recent studies have highlighted the potential of earwax or cerumen, a non-conventional biomatrix, in volatilomics research as a valuable matrix for disease biomarker discovery. Despite that, there are still gaps in using non-conventional biomatrices in metabolomics research. In this sense, this study aimed to elucidate the main analytical factors involved in the extraction and analysis of volatile organic compounds (VOCs) in cerumen by headspace/gas chromatography-mass spectrometry (HS/GC-MS) using Design of Experiments (DoE) approaches. Furthermore, we present a repeatability study for the proposed method as a quality control process for cerumenomic assays. By applying factorial designs, it was possible to determine that the sample mass, splitless injector sampling time, headspace extraction time, headspace extraction temperature, injection volume, and vial volume were significant factors for the cerumen VOC analysis by HS/GC-MS. Throughout univariate and multivariate statistical approaches, we demonstrate that different analytical conditions lead to distinct chemical profiling of a sample. The most suitable analytical condition was determined after the optimization steps, and the proposed method's repeatability was evaluated by the metabolites coefficient variation (CV) calculation. Seventy-one earwax VOCs reached a CV considered adequate for untargeted metabolomics studies via GC-MS. In summary, this study describes a protocol for analysis optimization of a non-conventional biomatrix and also reports a quality control process in untargeted volatilomics assays using earwax. Our findings shed light on the potential of using earwax in volatolomic studies and establish analytical criteria to ensure quality in cerumenomic assays.