The plasma concentrations of all components were determined using LC–MS/MS. The instruments consisted of an Agilent 1290 UPLC system (Agilent Technologies, Santa Clara, CA, USA) with a 6500QTRAP triple quadrupole mass spectrometer fitted with a TurboIonSpray electrospray ionization interface (AB Sciex, Framingham, MA, USA). A CAPCELL CORE AQ column (150 × 2.1 mm i.d., 2.7 μm particle size; Shiseido, Tokyo, Japan) was used for all components. The mobile phase consisted of 10 mM ammonium acetate (solution A) and acetonitrile (solution B) at a flow rate of 0.3 mL/min.
For quantification of 6,7-dimethylesculetin, geniposide and genipin, 50 µL of plasma was mixed with 50 µL of water and acetonitrile (4:1, v/v), 10 µL of internal standard solution (100 ng/mL niflumic acid), and 300 µL of acetonitrile. The mixture was centrifuged at 1800×g at 4 °C for 15 min. The supernatant was collected in a test tube at 40 °C in a dry bath under a stream of nitrogen gas. The residue was resolved with 100 μL of water and acetonitrile (9:1, v/v). An aliquot (20 µL) of each sample was injected onto the analytical column.
For quantification of 6,7-dimethylesculetin, geniposide and genipin, 50 µL of plasma was mixed with 50 µL of water and acetonitrile (4:1, v/v), 10 µL of internal standard solution (100 ng/mL niflumic acid), and 300 µL of acetonitrile. The mixture was centrifuged at 1800×g at 4 °C for 15 min. The supernatant was collected in a test tube at 40 °C in a dry bath under a stream of nitrogen gas. The residue was resolved with 100 μL of water and acetonitrile (9:1, v/v). An aliquot (20 µL) of each sample was injected onto the analytical column.