5500 qtrap triple quadrupole linear ion

For the inter-instrument cross-validation of the plasma quantification, targeted LC-MRM analyses were performed using a 5500 QTrap triple-quadrupole linear ion trap mass spectrometer equipped with a Turbo V Ion Source (AB SCIEX, Darmstadt, Germany) with polarity switching. The LC, ionization, detection, and MRM transition parameters for selected lipids were set as previously described^{22 (link),48 (link)} and are provided as a Source data file.

A total of 20 μL of the solution of extracted eCBs were injected and separated on a Phenomenex Luna 2.5 μm C18(2)-HST column, 100 × 2 mm, combined with a pre-column (C18, 4 × 2 mm; Phenomenex, Aschaffenburg, Germany), by increasing acetonitrile containing 0.1% formic acid over 2 min from 55 to 90%, and maintaining it at 90% for 5.5 min. The separated eCBs were flow-through analyzed using MRM on a 5500 QTrap triple-quadrupole linear ion trap mass spectrometer equipped with a Turbo V Ion Source (AB SCIEX, Darmstadt, Germany). Positive and negative ions were simultaneously analyzed using the ‘positive-negative-switching’ mode. The following MRM transitions were monitored for quantification of eCBs: AEA, m/z 348.3 to m/z 62.3; 2-AG, m/z 379.1 to m/z 287.2; AA, m/z 303.05 to m/z 259.1. Calibration solutions were prepared using commercially available standards of high purity, spiked with a mixture of deuterated eCBs and run in triplicates. Quantification of eCBs was carried out using Analyst 1.6.1 software (AB SCIEX, Darmstadt, Germany). The eCB concentrations were normalized to protein content (for tissues) measured by BCA and to serum volume.