Agilent 6530 lc qtof ms system

For CYP metabolite analysis of amlodipine and lovastatin, an Agilent 1260 binary pump HPLC system with an Agilent 6530 LC-QTOF MS system (Agilent Technologies) was used. The chromatographic separation was performed using a Thermo Hypersil GOLD column (2.1 mm × 150 mm, 5 µm; Thermo Fisher Scientific Inc., Waltham, MA, USA), with the column temperature maintained at 40 °C. The HPLC mobile phases consisted of solvent A, 0.1% formic acid in DW and solvent B, 90% acetonitrile in solvent A. A gradient elution was used with an initial concentration of 10% of solvent B and a flow rate of 0.3 mL·min⁻¹ for amlodipine. The solvent B composition was changed as follows: 0–20 min, 10%–70% (gradually increased); 20–21 min, 70%–10%; and 21–30 min, 10% (re-equilibrium). For lovastatin, the gradient elution was: initial, 30% B; 0–15 min, 30%–80%; 15–18 min, 80%; 18–18.1 min, 80%–30%; and 18.1–25 min, 30%. The injection volume was 5 µL for both amlodipine and lovastatin. A mass detection scan was performed in the positive ion mode.

The metabolite analysis was performed using a C18 column (2.1 × 100 mm, 2.1 μm) with an Agilent 6530 LC‐qTOF‐MS system (Agilent Technologies, Santa Clara, CA, USA) in both negative and positive ionization modes. The mobile phase included solvent A (0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid) with gradient elution (0–1 min, 90% B, 1–10 min 90%–10% B, 10–14 min 10% B, 14–15 min 10%–90% B, and 15–25 min 90% B). The flow rate was 0.3 mL/min. The injection volume was 10 μL. The analyses were performed at the electrospray ionization source with the following parameters: Capillary voltage of 4000 V and capillary temperature of 300°C. The auto‐tandem MS/MS spectra of the metabolites were recorded between 100 and 1700 m/z above the 200‐count threshold. To identify the peaks in the resulting data matrix, MS/MS spectra obtained by applying different collision energies (10, 20, and 40 eV) to the quality control samples (created by pooling the samples) were scanned against libraries.