Serum concentration analyses of atomoxetine
During the time course of the retrospective data collection, two
slightly different, cross-validated routine LC-MS/MS methods were
applied for atomoxetine analyses. For the most recent routine LC-MS/MS
method, serum samples were prepared by protein precipitation in 96-deep
well plates using a Microlab Star pipetting robot (Hamilton, Reno, NV,
USA) in a semi-automated sample preparation procedure. The LC system was
a Vanquish-UHPLC (Thermo Fisher Scientific, Waltham, MA, USA), and
chromatographic separation was performed by an XBridge BEH C18-column
(2.6 μm, 2.1x75 mm; Waters, Milford, MA, USA) using gradient elution at
35°C with a mix of ammonium acetate buffer (pH = 4.8) and acetonitrile
(20-52%). The retention time was 2.08 min for atomoxetine. Detection
used a QExactive Orbitrap mass spectrometer (Thermo Fisher Scientific,
Waltham, MA, USA), operated in positive ionization mode acquiring full
scan data at a resolution of 70,000 within the 100-1,500 Da scan range.
Atomoxetine was quantified in full scan acquisition mode, while accurate
data dependent MS2 analysis was simultaneously triggered to permit
confirmation of its identification. The lower limit of quantification
(LLOQ) was 5 nM. The precision and accuracy of atomoxetine
concentrations were <5%.