Qtrap 5500 tandem mass spectrometer

The quantification of sphingolipids in plasma and colon, small intestine, and liver tissue was performed by liquid chromatography tandem mass spectrometry (LC–MS/MS) as described previously [53 (link)]. In brief, the analytes were extracted by liquid–liquid extraction with methanol/chloroform/hydrochloric acid (15:83:2, v/v/v). The organic phase was split, evaporated to dryness, and reconstituted in methanol/formic acid (95:5, v/v) for sphingoid base measurements and tetrahydrofuran/0.2% formic acid/10 mM ammonium formate (9:1, v/v) for ceramide measurement. For both analytical runs, chromatographic separation was performed using an Aglient 1290 Infinity UHPLC system (Aglient, Waldbronn, Germany) coupled to a QTRAP 5500 tandem mass spectrometer (Sciex, Darmstadt, Germany). The analysis was conducted in Multiple Reaction Monitoring (MRM) mode. Data were acquired with Analyst Software V 1.6.3, and quantification was performed with MultiQuant Software 3.0.3 (both Sciex, Darmstadt, Germany). Reference substances for the preparation of calibration standards and quality control samples as well as internal standards were obtained from Avanti Polar Lipids (Albaster, AL, USA).

Zebrafish were treated with 10 μM of olmesartan medoxomil (Sigma Aldrich cat# 144689-63-4, the pro-drug form of olmesartan) for 14 days. The drug was freshly dissolved in the system water and administered daily. On day 14, adult zebrafish were dissected to collect the body and the brain which were then pooled to obtain approximately 125 mg per sample (n = 10 males, 10 females). Brain samples were prepared by addition of PBS at a 1:5 ratio and homogenizing (Bertin Precellys 24). Homogenates were mixed with acetonitrile and methanol (1:1 v/v) containing 0.05 µg/mL niflumic acid as an internal standard before filtering with Captiva ND plates (0.2 um) into water and analyzed for the active olmesartan (Sigma Aldrich cat# 144689-24-7) with a QTRAP 5500 tandem mass spectrometer (Sciex) coupled with a Nexera X2 series UHPLC (Shimadzu).

Chromatography was performed on an Agilent 1260 LC system (Agilent Technologies, Mississauga, Canada). The compounds were eluted from a Kinetex C18 HPLC column (100 × 3 mm, 2.6 μm particle size from Phenomenex, Torrance, CA) in an isocratic gradient consisting in 40% (A) ultrapure water containing 0.01% formic acid and 60% (B) methanol 0.01% formic acid as mobile phases. The flow rate and column temperature were set at 0.5 mL/min and 40°C, respectively. The LC system was coupled to an SCIEX QTRAP 5500 tandem mass spectrometer (SCIEX, Concord, Canada) fitted with electrospray ionization (ESI) source.
The operating parameters were ionspray voltage 5000 V, curtain gas 10 (arbitrary units), nebulizer gas 40 (arbitrary units), auxiliary gas 45 (arbitrary units) and probe temperature 600°C. The compounds were monitored in positive ion mode using multiple-reaction monitoring (MRM); the transitions, declustering potentials, collision energies and collision cell exit potentials are summarized in Table 1. For rivaroxaban two MRM transitions were used, one quantifier and one qualifier.
The software packages Analyst 1.6.2, PeakView 2.2 and MultiQuant 3.0 (SCIEX, Concord, Canada) were used, respectively, for mass spectral data acquisition and quantitation.