Hybridspe phospholipid ultra cartridge

The brains of mice injected intraperitoneally with A_2ARPAM-1, optoA_2ARPAM-1 or optoA_2ARPAM-2 at 21:00 were collected 30 min after treatment. Each brain was combined with 300 μL of acetonitrile with 1% formic acid (v/v) and vortexed for 3 min under normal light conditions to convert all brain-penetrating optoA_2ARPAM-1 or optoA_2ARPAM-2 to A_2ARPAM-1. The precipitated proteins were then removed by centrifugation (1000 × g for 5 min), and the supernatant was transferred to a HybridSPE-Phospholipid Ultra cartridge (Supelco) and eluted from the cartridge by applying a vacuum. The eluate was injected into a Waters ACQUITY UPLC-MS/MS system with an electrospray ionization interface and operated in the negative ion mode. An ACQUITY UPLC BEH C18 column (1.7 μm, 50 mm × 2.1 mm; Waters) with a graded acetonitrile/water mobile phase at a flow rate of 500 μL min⁻¹ was used for UPLC separation. A_2ARPAM-1 was detected by single ion (m/z 392) monitoring.

The brains of mice injected intraperitoneally with A_2ARPAM-1 at 21:00 were collected 1 h after treatment. Each brain was combined with 300 μL of acetonitrile with 1% formic acid (v/v) and vortexed for 3 min. Then, the precipitated proteins were removed by centrifugation (3000 rpm for 5 min), and the supernatant was transferred to a HybridSPE-Phospholipid Ultra cartridge (Supelco) and eluted from the cartridge by applying a vacuum. The eluate was injected into a Waters ACQUITY UPLC-MS/MS system with an electrospray ionization interface operating in negative ion mode. An ACQUITY UPLC BEH C18 column (1.7 μm, 50 mm × 2.1 mm; Waters) with a graded acetonitrile/water mobile phase at a flow rate of 500 μL/min was used for UPLC separation. A_2ARPAM-1 was detected by single-ion (m/z 392) monitoring.