2.6.3. Parameters of LC-MS
Samples were analyzed on an ExionLC instrument (AB Sciex, Stockholm, Sweden) with a 6500+ QTRAP triple-quadrupole mass spectrometer (AB Sciex, Stockholm, Sweden), equipped with an APCI ion source, operated in the negative-ion mode. Analytes were separated on a Waters Acquity UPLC BEH C18 column (1.7 um, 2.1 mm × 150 mm) (Birketoften 13, 3500 Værløse, Denmark) using a mobile phase consisting of MilliQ water (filtered through an LC-Pak Polisher (Merck, Molsheim, France) (A) and acetonitrile (B) delivered in a gradient: 0.5% solvent B 3 min, at 3 min, 2.5% solvent B ramping linearly to 17% solvent B at 6 min, then to 45% B at 10 min, and 55% B at 13 min, at a flow rate of 400 μL/minute. The column oven was set at 40 °C, the curtain gas and the ion source 50 psi, and the collision gas was set to medium. The ion source was 500 °C, nebulizer current −3 uA and the injection volume 12 μL. The autosampler was kept at 4 °C. Samples from the same individual were run in the same batch. After every five injections, a solvent blank was run (75% MeOH) and after every 15 injections, two solvent blanks were run. Multiple-reaction monitoring (MRM) was used to detect analytes. Two MRM transitions were used (Q1/Q3) per analyte, one serving as a method of quantitation and the other acting as a qualifier. Data were acquired using the Analyst 1.7 software and were processed with the MultiQuant 3.0.3. software (AB Sciex, Stockholm, Sweden).