Chemical Exposomics in Human Plasma by Lipid Removal and Large Volume Injection Gas Chromatography High-Resolution Mass Spectrometry

For comprehensive chemical exposomics in blood, analytical workflows are evolving through advances in sample preparation and instrumental methods. We hypothesized that gas chromatography high-resolution mass spectrometry (GC-HRMS) workflows could be enhanced by minimizing lipid co-extractives, thereby enabling larger injection volumes and lower matrix interference for improved sensitivity and molecular discovery. A simple protocol was developed for small plasma volumes (100-200 µL) by using isohexane (H) to extract supernatants of acetonitrile-plasma (A-P). The HA-P method was quantitative for a wide range of hydrophobic multiclass target analytes (i.e. log Kow >3.0), and the extracts were free of major lipids, thereby enabling robust large-volume injections (LVI; 25 µL) in long sequences to a GC-Orbitrap HRMS. Without lipid removal, LVI was counter-productive because method sensitivity suffered from the abundant matrix signal, resulting in low ion injection times to the Orbitrap. The median method quantification limit was 0.08 ng/mL (range 0.005–4.83 ng/mL), and good accuracy was shown for a certified reference serum. Applying the method to plasma from a Swedish cohort (n=32; 100 µL), 51 of 103 target analytes were detected. Simultaneous nontarget analysis resulted in 112 structural annotations (12.8% annotation rate), and Level 1 identification was achieved for 7 of 8 substances in follow-up confirmations. The HA-P method is potentially scalable for application in cohort studies and is also compatible with many liquid-chromatography-based exposomics workflows.