Tsq system

After reconstitution in 60% acetonitrile spiked with internal standards, samples were analysed using HPLC and triple-quadruple mass spectrometry and tandem mass spectrometry. Specifically, the system consisted of a TSQ system (Thermo Fisher Scientific) in line with an electrospray source and a Vanquish (Thermo Fisher Scientific) UHPLC consisting of a binary pump, degasser and auto-sampler outfitted with a XBridge Amide column (Waters; dimensions, 3.0 mm × 50 mm and a particle size of 3.5 μm). The mobile phase A contained 95% (v/v) water, 5% (v/v) acetonitrile, 10 mM ammonium hydroxide, 10 mM ammonium acetate, pH 9.0; B was 100% acetonitrile. The gradient was as follows: 0 min, 15% A; 3 min, 45% A; 10 min, 60% A; 10.1–11 min, 75% A; 11.1 min, 15% A; 11.1–15 min, 15% A with a flow rate of 150 μl min⁻¹. In positive/negative polarity switching mode, the capillary of the electrospray ionization source was set to 300 °C, with sheath gas at 35 arbitrary units, auxiliary gas at 5 arbitrary units and the spray voltage at 3.5 kV. The selective reaction of the protonated precursor ion and the related product ions were monitored. The transitions are listed as follows: dopamine (+) 154→137, dopamine_D4(+) 158→141. The peak area was integrated. Data acquisition and analysis were carried out using Xcalibur v.4.1 and Tracefinder v.4.1, respectively (both from Thermo Fisher Scientific).