Many small molecules require derivatization to increase their volatility and to be amenable to gas chromatographic (GC) separation. Derivatization is usually time-consuming, and typical batch-wise procedures increase sample variability. Sequential automation of derivatization via robotic liquid handling enables the overlapping of sample preparation and analysis, maximizing time efficiency and minimizing variability. Herein, a protocol for the fully automated, two-stage derivatization of human blood-based samples in line with GC-[Orbitrap] mass spectrometry (MS)-based metabolomics is described. The protocol delivers a sample-to-sample runtime of 31 min, being suitable for better throughput routine metabolomic analysis. Key features • Direct and rapid methoximation on vial followed by silylation of metabolites in various blood matrices. • Measure ~40 samples per 24 h, identifying > 70 metabolites. • Quantitative reproducibility of routinely measured metabolites with coefficients of variation (CVs) < 30%. • Requires a Thermo ScientificTM TriPlusTM RSH (or comparable) autosampler equipped with incubator/agitator, cooled drawer, and automatic tool change (ATC) station equipped with liquid handling tools. Graphical overview Workflow for profiling metabolites in human blood using automated derivatization.

CTC Analytics AG Zwingen Switzerland

EIRENE CZ Brno Czech Republic

RECETOX Faculty of Science Masaryk University Kotlarska 2 Brno Czech Republic

Thermo Fisher Scientific Milan Italy

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