High performance liquid chromatography system

The teneligliptin and teneligliptin sulfoxide concentrations were measured as described previously (Park et al., 2020 (link)). Briefly, the sample was injected into a high-performance liquid chromatography system (Shiseido Co., Ltd, Japan) coupled with an API 4000 mass spectrometer (Applied Biosystems-SCIEX, MA, United States) equipped with a Capcell Pak C18 column (2.0 mm × 150 mm, 5 μm, Tokyo, Japan) and a guard column. The isocratic mobile phase was a mixture of acetonitrile (100%) and methanol (50%; diluted in distilled water) (1:1; v:v). The flow rate of the mobile phase was 0.25 mL/min. The mass spectrometer was equipped with an electrospray ionization source and operated in positive ion mode with multiple reaction monitoring. The mass transition ion pairs of teneligliptin and teneligliptin-d8 were selected as m/z 427.2→ 243.1 and m/z 435.2→ 251.3, respectively. Standard working solutions of teneligliptin (1,000, 500, 100, 50, 10, 5, and 2 ng/mL) were prepared by diluting the stock solution with blank plasma. A linear calibration curve of standard teneligliptin was established (r² = 0.9996).

The atorvastatin and 2-hydroxy (2-OH) atorvastatin concentrations were measured as described in a previous study, with a slight modification [26 (link)]. Briefly, the sample was injected into a high-performance liquid chromatography system (Shiseido Co., Ltd., Tokyo, Japan) coupled with an API 4000 mass spectrometer (Applied Biosystems-SCIEX, Framingham, MA, USA) equipped with a Capcell Pak C₁₈ column (2.0 mm × 150 mm, 5 μm, Tokyo, Japan). The isocratic mobile phase was a mixture of acetonitrile (25%), methanol (40%), and 0.01% formic acid in water (35%). The flow rate of the mobile phase was 0.2 mL/min. The mass spectrometer was equipped with an electrospray ionization source and operated in negative ion mode with multiple reaction monitoring. The mass transition ion pairs of atorvastatin, 2-OH atorvastatin, and atorvastatin-d5 (internal standard) were selected as m/z 557.4 → 278.1, m/z 573.5 → 278.1, and m/z 573.5 → 278.1, respectively. Standard working solutions of atorvastatin and 2-OH atorvastatin (0.5, 1, 2, 5, 10, 20, 50, 100, and 200 ng/mL) were prepared by diluting the stock solution with blank plasma. Linear calibration curves of standard atorvastatin and 2-OH atorvastatin were established (r² = 0.999).