Xbridge c18 analytical column

TLC experiments were performed using 0.2-mm silica-coated aluminum plates, being a 95:5 dichloromethane/methanol mixture the mobile phase. The plates were observed under UV light.
Agilent 1100 HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with a Xbridge™ C18 analytical column (2.1 × 30 mm, 3.5 μM; Waters, Dublin, Ireland) was used in the chromatographic separation of LC-MS/MS analysis. An elution gradient of acetonitrile and water, with 0.1% of formic acid, was used as a mobile phase. MS spectra were taken at Applied Biosystems MDS Sciex API 200 triple quadrupole mass analyzer (Concord, Ontario, Canada) with an electrospray ionization (ESI) interface between HPLC and MS.

A Nexera UHPLC system (Shimadzu, Kyoto, Japan) was coupled to a tandem quadrupole mass spectrometer (LCMS-8040, Shimadzu). Electrospray ionization with MRM was operated in positive mode. The MS parameters were as follows: capillary voltage, 4.0 kV; interface temperature, 300 °C; desolvation line temperature, 250 °C; heat-block temperature, 400 °C; heating gas (air), 10 L/min; nebulizing gas (N₂), 3.0 L/min; and drying gas (N₂), 10 L/min. Chromatographic separation was achieved using a Waters Xbridge C18 analytical column (100 mm × 2.1 mm; 3.5 μm particle size, Milford, MA, USA). The column oven was kept at 40 °C. The mobile phase consisted of (A) deionized water and (B) methanol, both containing 0.1% formic acid. The gradient program for chromatographic separation was programmed as follows: 95% of mobile phase A for 0.5 min from the start, which was decreased to 5% (A) linearly over 2.5 min and kept for 3 min, followed by an increase to 95% (A) in 0.5 min, which was maintained for 3.5 min. The flow rate was 0.2 mL/min, and the injection volume was 5 µL. The MRM transitions and parameters were optimized carefully by the injection of individual standard solutions (0.1 µg/mL) on UHPLC without an analytical column under an isocratic flow of 1:1 (mobile phase A and B).

Concentrations of RIS and the active metabolite, PAL, in plasma and bone marrow were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis as previously reported [17 (link)]. Briefly, RIS and PAL were extracted from both plasma and bone marrow via protein precipitation with acetonitrile. Separation was accomplished using a Waters XBridge C18 analytical column (3.0 x 50 mm, 3.5 μm). Mobile phase consisted of 0.1% formic acid in purified water (A) and 0.1% formic acid in acetonitrile (B). The flow rate was 0.4 mL/min, and heated to 60°C. Gradient elution was employed, with initial conditions 95% A and 5% B. Solvent composition was held at the initial conditions for 1.0 minutes, and then was ramped over the following 1.5 minutes to 95% B. Composition was maintained at 95% B for 1 minute. RIS and PAL were detected via an Agilent (Waldbronn, Germany) 6460 triple quadrupole mass spectrometer operated in positive ion MRM mode. The following transitions were monitored: RIS (411.2→191.0) and PAL (427.2→207.0). Significant differences between groups were determined by two-tailed T test using GraphPad Prism.