The HPLC system consisted of two LC

The HPLC system consisted of two LC-10AD pumps, a RF-550A fluorescence spectrometer, an FCV-2AH six-port switching valve, a CTO-10AC column oven, a DGU-3A degasser, a C-R4A integrator, and a SIL-10A autoinjector, all of which were controlled by a SCL-10A controller (all from Shimadzu, Kyoto, Japan). The precolumn was a BSAODS (5 m, 4.6 mm ID 3.5 cm; Tosoh, Tokyo, Japan), and the analytical column was a TSK-gel ODS (5 m, 4.6mm ID 15 cm; Tosoh) heated at 40°C. The sample was injected into the HPLC system and washed with 0.1% aqueous phosphoric acid for 5 min at a flow rate of 1.0 ml/min in the precolumn. After switching the valve, analyte was transferred onto the analytical column from the precolumn for back-flashing. Analysis was performed with a mixture of acetonitrile–5 mmol/liter sodium sulfate–tetrahydrofuran–acetic acid (750:1250:20:60, v/v) at a flow rate of 1.0 ml/min. Detection was performed using fluorescence employing a 330-nm wavelength for excitation and a 375-nm wavelength for emission. The actual tissue concentration was calculated based on the dilution ratio.