2.12. Quantification of pitavastatin in plasma samples by LC–MS/MS

Before quantification, the plasma samples were thawed at room temperature and treated with liquid–liquid extraction. One hundred microliter of each plasma sample was added with 10 μL methanol, 10 μL of internal standard solution (IS, 100 ng/mL mebendazole in acetonitrile) and 800 μL of ethyl acetate. The mixture was mixed thoroughly and centrifuged at 14,000×g for 10 min. An aliquot of 600 μL upper organic layer was transferred to a new 1.5 mL tube afterwards, and dried with a gentle flow of hot nitrogen. The residue was then reconstituted with 100 μL methanol/water (1:1, v/v) and centrifuged at 14,000×g for 10 min. Finally 80 μL of supernatant was used for LC–MS/MS analysis.

The LC–MS/MS system consisted of an Agilent 1290 HPLC and a 6460 triple quadrupole mass spectrometer coupled with an Agilent Jet Stream ESI ion source (Agilent Technologies, Santa Clara, CA, USA). Chromatography separation was performed on a Zorbax Eclipse Plus C18 column (50 mm × 2.1 mm, 1.8 μm, Agilent Technologies), using a mobile phase system of water (A) and methanol (B) both containing 0.1% formic acid (v/v). The flow rate was 0.2 mL/min and the injection volume was 2 μL. The gradient elution program was used as follows: 0–0.5 min, 45% B; 0.5–3 min, 45%–95% B; 3–3.5 min, 95% B; 3.5–3.7 min, 95%–45% B; 3.7–5.5 min, 45% B. Pitavastatin and IS were both monitored in positive ESI mode, with the ion transition of 422.1 → 22.1I and 296.1 → 264.1, respectively.