3.5. Equipments and Chromatographic Conditions

The liquid chromatography–mass spectrometry system is composed of a Shimadzu CBM-20A HPLC pump controller (Shimadzu Corporation, Columbia, MD, USA), two Shimadzu LC-20AD pumps, a CTC HTS PAL auto-sampler (LEAP Technologies, Carrboro, NC, USA) and a Triple time-of-flight (TripleTOF^®) 5600 mass spectrometer (Sciex, Foster City, CA, USA) with an electrospray ionization (ESI) source. The chromatographic separation was achieved on a Phenomenex Kinetex XB-C18 column (2.1 × 100 mm for metabolite identification and 2.1 × 50 mm for method qualification), and the column temperature was set to 50 °C. The mobile phase consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile, with a binary flow rate of 0.4 mL/min and an injection volume of 10 μL. The LC gradients for SCH 58261 quantification and metabolite identification are summarized in Table 9.

Mobile phase condition for LC gradients. (a) LC gradient for quantification, (b) LC gradient for metabolite identification.

The TripleTOF^® 5600 mass spectrometer was equipped with a TurboIonSpray^® ion source Electrospray ionization (Sciex, Redwood City, CA, USA) was used for sample introduction and ionization in the positive ion mode. High-purity nitrogen gas was used for the nebulizer/Duospray^™ and curtain gases. Source-dependent parameters optimized were as follows: GS1 and GS2—both 50 psi; ion spray voltage—5500 V; temperature—500 °C, with a curtain gas flow of 30 L/min. The compound-dependent parameters such as the declustering potential (DP) and collision energy (CE) were optimized during tuning as 160 V and 35 V for SCH 58261, and 125 V and 30 V for verapamil, respectively. Based on the full scan and MS/MS spectrum, the transitions of precursors to the product ions were as follows: m/z 346.1 → 105.1 for SCH 58261 and m/z 455.2 → 165.2 for Verapamil. Data acquisition and analysis were performed using the Analyst^® TF 1.6 software (Sciex, Foster City, CA, USA).