Neurotransmitters levels in ears from IMQ-induced mice or IMQ-induced mice treated with Scopolamine detected by LC/MS

Ears of 6 mice without any treatment, 6 IMQ-induced mice, and 6 IMQ-induced mice with Scopolamine treatment were subjected to detect neurotransmitters levels according to the LC/MS method. After being thawed and smashed, an amount of 50 mg of ears was mixed with 500 µL of 70% methanol/water. The sample was vortexed for 3 min under the condition of 2500 r/min and centrifuged at 12000 r/min for 10 min at 4°C. Take 300 μL of supernatant into a new centrifuge tube and place the supernatant in -20°C refrigerator for 30 min, Then the supernatant was centrifuged again at 12000 r/min for 10 min at 4°C. After centrifugation, transfer 200 μL of supernatant for further LC-MS analysis.

The sample extracts were analyzed using an LC-ESI-MS/MS system (UPLC, ExionLC AD; MS, QTRAP®n6500+System). The analytical conditions were as follows, HPLC: column, Waters ACQUITY UPLC HSS T3 C18 (100 mm×2.1 mm i.d., 1.8µm). The analytical conditions included a solvent system, water with 0.1% formic acid (A), and acetonitrile with 0.1% acetic acid (B). The gradient started at 5% B (0 min), increased to 95% B (0-8 min), 95% B (8-9.5 min), and finally ramped back to 5% B (9.6-12 min). The flow rate was 0.35 ml/min, the temperature was 40 °C, and the injection volume was 2 μl.

AB 6500+QTRAP® LC-MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in both positive and negative ion modes and controlled by Analyst 1.6 software (AB Sciex). The ESI source operation parameters were as follows: ion source, turbo spray; source temperature 550°C; ion spray voltage (IS) 5500 V (Positive), -4500 V (Negative); curtain gas (CUR) was set at 35.0 psi; DP and CE for individual MRM transitions was done with further DP and CE optimization. A specific set of MRM transitions was monitored for each period according to the neurotransmitters eluted within this period.

The mass spectrometry data were processed using Analyst 1.6.3 and MultiQuant 3.0.3.