5500 qtrap triple quadrupole linear ion trap mass spectrometer

Liquid chromatography-multiple reaction monitoring (LC-MRM) analyses were performed on a LC-MS/MS system consisting of a 5500 QTrap triple-quadrupole linear ion trap mass spectrometer equipped with a Turbo V Ion Source (AB SCIEX, Darmstadt, Germany), an Agilent 1200 series LC system (degasser, pump; Agilent, Waldbronn, Germany), and a CTC HTC PAL autosampler (CTC Analytics AG, Zwingen, Switzerland). Data acquisition and analysis were performed using Analyst software (version 1.6.1; AB SCIEX).

LC-MRM measurements were performed as described before^{8 (link)}. In detail, frozen lung lobes were homogenized in cold extraction tubes with ceramic beads containing spiking solution (deuterated endocannabinoids in acetonitrile), 0.1 M formic acid (buffer) and ethylacetate/hexane (9:1). After homogenization with Precellys 24 samples were centrifuged and kept at −20 °C for 10 min to freeze the aqueous phase. Then, the upper organic phase was recovered, evaporated under a gentle stream of nitrogen at 37 °C and reconstituted in a solution of water/acetonitrile (1:1). The aqueous phase was used for determination of protein concentration. For LC-MRM, samples were injected and separated on a Phenomenex Luna 2.5-µm C18(2) HST column combined with a SecurityGuard precolumn (Phenomenex Inc., USA). Separated endocannabinoids were flow-through analyzed by MRM with the 5500 QTrap triple-quadrupole linear ion trap mass spectrometer armed with a Turbo V Ion Source (AB Sciex Germany GmbH, Germany). For quantification of the endocannabinoids in lung tissues, triplicate calibration curves were used. Protein concentrations of lung samples were determined using the Pierce BCA Assay Kit (Thermo Fisher Scientific, USA) and the endocannabinoid concentrations were normalized to total protein.