2.3.1. Doxorubicin Quantification by HPLC-Fluorescence Detection

D content was determined by high-performance liquid chromatography (HPLC) analysis using fluorescence detection. The analytical quantification of D in the samples was performed using a Hitachi^® LaChrom Elite HPLC system (Hitachi High Technologies America, Inc., San Jose, CA, USA)equipped with an isocratic pump and an L-2485 fluorescence detector at 475 and 580 nm of excitation and emission wavelengths, respectively, with data acquisition and processing being performed using a EZChromElite Data System Manager software (3.1.3. version, Hitachi High Technologies America, Inc., San Jose, CA, USA). Chromatographic separations were carried out using a Zorbax SB^® C18 reverse phase column (4.6 × 150 mm, 3.5 µm particle size, Agilent, CA, USA). Analysis was performed with methanol and 0.1% of ammonia solution adjusted to pH 3.0 with formic acid (70:30, v/v) as the mobile phase at a flow rate of 1 mL/min in the isocratic mode. All solutions were filtered through 0.45 µm cellulose filters (Millipore^®, Barcelone, Spain) and then quantified by HPLC. For analysis, 20 µL of each sample were injected, and the run time was set at 6 min, with the retention time being observed at 3.5 min. D concentrations in the samples were quantified by relating the analytical peak area to the regression line of the calibration curve. For the calibration curve, a stock solution of 10 mg/mL D in distilled water was prepared. Subsequently, a six-point calibration curve was prepared over a range of 1.2 to 6.0 mg/mL by diluting several milliliters of stock solution with mobile phase. These solutions were prepared in triplicate. The calibration curve was constructed by plotting the mean peak area of D vs. D concentration.