Methods and instrumentation

Carbidopa and levodopa were dissolved in HCl 1.00 M to obtain 100 g L⁻¹ stock solutions. Stock solutions of a brand drug and the generic drug containing 5.00 g L⁻¹ of levodopa were prepared by stirring for 10 min one tablet (200 mg LD, 50 mg CD) in 40.0 mL of 1.00 M HCl. The working solutions were prepared within 24 h from tablet dissolution. The samples were heated by using thermomixer to control time and temperature of the reaction. All the solutions in different solvents (acetonitrile, methanol, ethanol, water, dimethyl sulfoxide, and EtOH:H₂O mixtures) contained 50.0 mg L⁻¹ (0.221 mM) of CD and 10.0 mM vanillin. The same CD and vanillin concentrations were used for EtOH:H₂O (1:1) solutions containing HCl 5 mM, 50 mM, or 500 mM. In all these cases the absorbance spectra were acquired after 4 h at 70 °C. The condition of CD 50.0 mg L⁻¹ in EtOH/H₂O 1:1 with HCl 0.500 M at 70 °C was applied also to different vanillin concentrations (0.00, 1.25, 2.50, 5.00, 10.0, 20.0 mM) for 4 h: vanillin 10.0 mM at 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C for 4 h; vanillin 10.0 mM at 70 °C for 15, 30, 60, 120, 240 min. For calibration curves, each concentration of CD, LD + CD 4:1, or pharmaceutical formulations was in HCl 1.00 M, and added then to vanillin 20.0 mM in ethanol, obtaining the final sample solutions in EtOH:H₂O 1:1, HCl 0.500 mM and vanillin 10.0 mM. The limit of detection (LOD) and the limit of quantification (LOQ) were calculated based on the standard deviation (SD) of the mean of the blank values, as 3 × SD/m and 10 × SD/m, respectively, where m indicates the slope of the calibration curve. The assay reproducibility is reported as (mean) coefficient of variation (CV_av). Absorbance spectra were acquired in disposable polystyrene 96-well microtest plates (Sarstedt, Milan, Italy) by using iMark™ microplate visible absorbance reader with optical filters (Bio- Rad, Milan, Italy), as well as in 1.0 cm cell by using a UV–Visible Spectrophotometer Evolution™ 201/220 from Thermo Scientific™ (Rodano, Milan, Italy).