DOI: 10.5281/zenodo.18246460
title: Dataset for the manuscript "Lipidomic analysis of single and combined effects of polyethylene microplastics and polychlorinated biphenyls on human hepatoma cells"

description: This dataset contains the untargeted lipidomics datasets generated from HepG2 human hepatoma cells exposed to polyethylene microplastics (PE-MPs), two polychlorinated biphenyl (PCB) congeners, and their combinations, as described in Menéndez-Pedriza et al. (2022).

Lipid extraction procedures and instrumental conditions for lipidomic profiling followed protocols previously optimized in our laboratory. In brief, each cell pellet was mixed with 1 mL of MTBE/MeOH (3:1, v/v). The extraction solvent was supplemented with 200 pmol of each recovery standard, including 1,2,3–17:0 triglycerides (TG), 17:1 lysophosphatidylethanolamines (lyso-PE), 17:1 lysophosphatidylglycerol (lyso-PG), and 16:0-d31–18:1 phosphatidylethanolamine (PE). Samples were vigorously vortexed and subsequently sonicated for 10 min. Afterwards, 500 µL of H2O/MeOH (3:1, v/v) were added and the mixture was vortexed again. Following centrifugation at 13,000 rpm for 10 min, the organic upper layer was transferred to fresh tubes and evaporated under a gentle nitrogen stream.

The dried extracts were reconstituted in 170 µL of acetonitrile and centrifuged at 13,000 rpm for 10 min to remove insoluble material. Aliquots of 130 µL of the clarified supernatants were transferred to conical UHPLC vials and stored at −25 °C until LC–MS analysis. To monitor analytical performance, quality control (QC) samples were prepared by pooling 20 µL from each extract. Prior to injection, ceramide (Cer) C12:0, glucosylceramide (GluCer) C12:0, and sphinganine (17:0) were added to the QC mixture at 100 pmol each and served as instrumental internal standards.

Chromatographic separations were performed by injecting 10 µL of each sample onto a Kinetex® C8 reversed-phase column (100 Å, 100 mm × 2.1 mm i.d., 1.7 µm particle size) coupled to a Waters Acquity UPLC system maintained at 30 °C. Mobile phase A consisted of water containing 1% of 1 M ammonium acetate and 0.1% acetic acid, whereas mobile phase B was composed of acetonitrile/isopropanol (7:3, v/v) with the same additives. Lipids were eluted at a flow rate of 400 µL min⁻¹ using a multistep linear gradient: starting at 45% A for 1 min, decreasing to 35% A over 3 min, followed by ramps to 11% A over 8 min and to 1% A over 3 min. The column was then flushed at 1% A for 3 min and re-equilibrated for 4 min, resulting in a total runtime of 22 min.

Mass spectra were acquired using a Waters LCT Premier orthogonal accelerated time-of-flight (oa-TOF) mass spectrometer operated in both positive and negative electrospray ionization modes. Capillary voltages were set to 3.0 kV and 2.5 kV for positive and negative ionization, respectively. In both modes, the desolvation temperature was maintained at 350 °C with a desolvation gas flow of 600 L h⁻¹. Full-scan data were collected over an m/z range of 50–1800, and spectra were averaged to generate one data point every 0.2 s. Continuous mass calibration was ensured through leucine enkephalin introduced via the LockSpray interface.

Instrument-specific RAW files were converted to the open CDF format using the Databridge utility within MassLynx 4.1 software (Waters, Milford, MA, USA).