Triple tof 6500

LC-MS/MS was performed by Triple TOF 6500 (AB Sciex LLC, Framingham, MA, USA) to determine whether CY-09 crosses the BBB and enters the brains of the mice. In this experiment, metabolites were extracted from brain tissues using a 300 μL methanol acetonitrile mixture (methanol: acetonitrile = 2:1), as previously described [39 (link)]. After vortexing for 1 min, sonicating for 10 min at 0 °C, and centrifuging for 15 min at 13,000× g, the samples were held at −20 °C until further detection. The conditions for the chromatographic separation of metabolites were as follows—flow rate: 0.3 mL/min; injection volume: 6 μL; mobile phase: phase A, water (containing 0.1% formic acid); phase B, acetonitrile (containing 0.1% formic acid). The ion source parameters of mass spectrometry were as follows—curtain gas: 35 psi; ion spray voltage: −4500 V; source temperature: 550 °C; ion source gas1: 55psi; ion source gas2: 55psi. MultiQuant 3.02 (AB Sciex LLC, Framingham, MA, USA) and ProteoWizard 1.3.5.0 (ProteoWizard, Palo Alto, CA, USA) were used to process the data.

Multiple reaction monitoring (MRM) assays were developed to quantitatively validate target proteins. Samples were digested as described, and data were normalized by the addition of 50 fmol of β‐galactosidase. MRM analysis was conducted using a QTRAP 6500 mass spectrometer (SCIEX, Framingham, MA) equipped with an LC–20AD nano‐HPLC system (Shimadzu, Kyoto, Japan). The mobile phase comprised solvent A (0.1% formic acid aqueous solution) and solvent B (98% acetonitrile plus 0.1% formic acid). Peptides were separated on a C18 column (15 cm long, 75 μm diameter, 3.6 μm beads) at a flow rate of 300 nL/min, following a gradient elution of 5% to 30% solvent B for 38 minutes, 30% to 80% solvent B for 4 minutes, and maintained at 80% for 8 minutes.
For the QTRAP 6500 mass spectrometer, a spray voltage of 2400 V, 23 psi of nebulized gas, and a dwell time of 10 milliseconds were used. Multiple MRM transitions were monitored in Q1 and Q3 quadrupoles using unit resolution to maximize specificity. A spectral library of MS/MS data was generated on an TripleTOF6500 (AB SCIEX, Foster City, CA) and searched using Mascot version 2.3 (Matrix Science, UK) against a Homo sapiens database (20 307 entries). MRM data were analyzed with Skyline software.