VMC was quantitatively determined by HPLC analysis, and chromatographic separation was performed on column Zorbax Eclipse® Plus C18, 4.6 × 150 mm, 5 μm equipped with a pre-column, and temperature-controlled at 22 °C. VMC aqueous solution was injected through a manual injector (volume 20 µL, Agilent 1260 Infinity Manual Injector). The pump (1260 Infinity Quaternary Pump VL) provided a constant and continuous flow at 1.0 mL/min. The mobile phase was a mixture of NH4H2PO4 (0.05M, pH 4.0 adjusted with H3PO4) and CH3CN at a ratio of 92:8, filtered through a cellulose filter membrane (pore size 0.22 µm) and sonicated for 5 min.
The detector (Agilent 1260 Series UV-visible detector) was set at 220 nm, referring to previous work in the literature [31 ]. VMC was determined from a standard calibration curve prepared starting from a stock solution containing 2 mg/mL VMC in mobile phase (Figure 2 ); the stock solution was diluted in a volumetric flask with mobile phase to obtain solutions of 1.95, 3.91, 7.81, 12.50, and 15.63 μg/mL of VMC. Each standard solution was tested in triplicate at 217 nm, and each point of the calibration curve is the average of the three analyses. Equation y = 0.1138x − 0.1319, R2 0.992.
The detector (Agilent 1260 Series UV-visible detector) was set at 220 nm, referring to previous work in the literature [31 ]. VMC was determined from a standard calibration curve prepared starting from a stock solution containing 2 mg/mL VMC in mobile phase (