LC-MS/MS analysis sequence data

Male C57BL/6J mice (6-8 weeks old, purchased from Nanjing Jisihuiyuan Biotechnology Co., Ltd.) were housed under constant temperature and humidity conditions (22 ± 1℃ , 55 ± 10% humidity, 12 h light/dark cycle), with free access to water. Animals were fasted for 12 h prior to dosing. Twenty-four mice were randomly divided into eight groups (n=3) and administered intraperitoneal (IP) injections of SPAM1 solution (100 μmol/kg) dissolved in physiological saline. Blood and brain tissue samples were collected at 10 min, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h post-administration. Blood collection: Approximately 200 μL whole blood was collected via the orbital venous plexus into EDTA-K2 anticoagulant tubes. After gentle mixing, plasma was separated by centrifugation (8000 rpm, 5 min, 4℃). Brain tissue collection: Following euthanasia, brain tissue was immediately removed, rinsed with physiological saline, blotted dry with filter paper, rapidly frozen in liquid nitrogen, and stored at -80℃.
Plasma Samples: 30 μL of plasma was vortex-mixed with 120 μL of acetonitrile solution containing the internal standard (IS; m/z 455.2/165.0) for 3 min, followed by centrifugation (12,000 rpm, 5 min, 4℃). Transfer 80 μL supernatant and dilute with 40 μL 50% acetonitrile aqueous solution. Brain Tissue Samples: the tissue was homogenized in 5 volumes of 30% acetonitrile in water using a frozen tissue grinder. Subsequently, 30 μL of the homogenate was processed in an identical manner to the plasma samples.
Quantification of SPAM1 was performed using an ExionLC-AD system coupled with an API 4000 mass spectrometer (SCIEX) operating in positive ion multiple reaction monitoring (MRM) mode. Chromatographic separation was achieved on an ACE Excel SC18 column (50 × 3.0 mm). The mobile phase consisted of (A) 0.1% formic acid in water and (B) acetonitrile, delivered at a flow rate of 0.3 mL/min. The gradient program was as follows: 0-0.2 min, 20% B; 1-2.2 min, 95% B; 2.21-3 min, 20% B. The mass spectrometric parameters were set as follows: monitor ion transitions for SPAM1 (366.1 → 214.8) and IS (455.2 → 165.0); ion spray voltage: 5500 V; temperature: 500℃.
Using Phoenix WinNonlin 8.1 software, non-compartmental models were employed to calculate pharmacokinetic parameters, including Cmax, Tmax, AUClast, AUCinfinite, AUC%Extrap, T1/2, CL, and Vss.