Quantification of PA Concentrations by LC-MS/MS

The determination of PA concentrations after incubation with microsomes or supersomes was performed by LC-MS/MS as described earlier by Kaltner et al. (2019) and Enge et al. (2021) [16 (link),57 (link)]. Briefly, a 50 × 2.1 mm Kinetex 2.6 μm Core-Shell EVO C18 100 Å column (Phenomenex, Aschaffenburg, Germany) protected by a SecurityGuard ULTRA EVO C18 2.1 mm guard column (Phenomenex, Aschaffenburg, Germany) was used for chromatographic separation on a Shimadzu Prominence HPLC device (LC-20AB, SIL-20AC HT, CTO-20AC, CBM-20A, Shimadzu, Duisburg, Germany). Column oven temperature was maintained at 30 °C, the flow rate was consistently hold at 0.4 mL/min and the injection volume was 10 µL. The HPLC system was coupled to an API4000 triple quadrupole MS (Sciex, Darmstadt, Germany) which was operated in positive electro spray ionisation (ESI) mode with the following parameters: ionisation voltage: 2500 V; nebuliser gas: 50 psi; heating gas: 50 psi; curtain gas: 30 psi; temperature: 600 °C; collision gas: level 7. The selected PA analytes were determined in multiple reaction monitoring (MRM) mode and quantified by external calibration standards ranging from 10 to 125 nmol/mL in DMEM. The PA content was always normalized to the content at t = 0 h.