*2.3. High-Performance Liquid Chromatography (HPLC)*

Concentrations of ZON were also determined by HPLC instrumental analysis on the basis of peak areas in the chromatograms at the corresponding retention time (6.12 min) with excellent linear calibration characteristics in three parallel measurements. Peak areas determined at 236 nm for ZON concentrations between 10 and 2000 ng/mL were applied in linear regression, where regression coefficient of the concentration dependence was 0.999 in all measurements. Calibration curves with standard solutions were also investigated in MeOH:water = 7:3 and MeOH:phosphate buffer saline (PBS) = 1:1, where slope coefficients of linear regression were 26.90 ± 0.06 and 25.92 ± 0.06 for MeOH:water and MeOH:PBS, respectively. Peak purity was assessed by ratios of signal intensities (peak areas) recorded at 236 and 274 nm. These values for standard solutions were 2.15. Relative SDs established for different concentration levels for three parallel injections ranged between 0.65% and 1.76%. The LOD of the method was determined to be 10 ng/mL for ZON. A chromatogram and linear calibration (average of peak areas from three parallel measurements and their SDs) are presented in Figure 4.
