2.5. HPLC

DTX and DiI were quantified using a Waters Corporation (Milford, MA, USA) HPLC system consisting of a separations module (e2695), an ultraviolet (UV) detector (e2489), and a data station (Empower 3). DTX was separated on a Kromasil^® C18 column (5 μm, 4.6 × 250 mm; Akzo Nobel, Bohus, Sweden) using an isocratic mobile phase consisting of acetonitrile and water (55:45, v/v) at a flow rate of 1 mL/min at 25 °C. The eluate was monitored at a UV wavelength of 230 nm, and the injection volume was 50 μL. For DiI quantification, a fluorescence detector (W2475) was used. Chromatography was performed on a C18 column (5 μm, 4.6 × 150 mm; Shiseido, Tokyo, Japan) at a flow rate of 1.5 mL/min using a mobile phase consisting of 0.05 M dimethyl sulfate and methanol (2:98, v/v). The injection volume was 50 μL, and the excitation and emission wavelengths were 549 and 565 nm, respectively. The standard calibration curves of DTX and DiI were linear in the ranges of 0.5–100 μg/mL and 0.005–10 μg/mL, respectively, with coefficient of determination (r²) values of greater than 0.999. The analysis method offered a limit of detection of 0.1 μg/mL for DTX and 0.001 μg/mL for DiI at a signal-to-noise ratio of 3:1.