This paper presents the development and optimization of a liquid chromatographic method for the determination of fluconazole and its impurities by experimental design methodology. Four experimental design types were applied: two-level full factorial design, central composite design, Box-Behnken design, and three-level full factorial design. The advantages and drawbacks of each design are described and detailed statistical evaluation of mathematical models was performed. The central composite design and three-level full factorial design created significantly better models comparing to the other methods. As the central composite design requires a smaller number of experiments, its models were used for theoretical examination of experimental space. Multiobjective optimization aiming to achieve maximal separation of all investigated substances and minimal analysis duration was performed by a grid point search. The defined optimal separation was achieved on a C₁₈ (125 mm × 4 mm, 5 µm particle size) column with a mobile phase consisting of acetonitrile and water (5 mM ammonium formate) (15:85, v/v); a column temperature of 25°C; a flow rate of 1.2 mL min⁻¹; and a detection wavelength of 260 nm.

This paper presents the chemometrically assisted optimization and validation of the RP-HPLC method intended for the quantitative analysis of itraconazole and its impurities in pharmaceutical dosage forms. To reach the desired chromatographic resolution with a limited number of experiments in a minimum amount of time, Box- -Behnken design was used to simultaneously optimize some important chromatographic parameters, such as the acetonitrile content in the mobile phase, pH of the aqueous phase and the column temperature. Separation between itraconazole and impurity F was identified as critical and selected as a response during the optimization. The set optimal mobile phase composition was acetonitrile/ water pH 2.5 adjusted with o-phosphoric acid (50:50, V/V). Separations were performed on a Zorbax Eclipse XDB-C18, 4.6 × 150 mm, 5 μm particle size column with the flow rate 1 mL min^-1, column temperature set at 30 °C and UV detection at 256 nm. The established method was then subjected to method validation and the required validation parameters were tested. For the robustness evaluation, fractional factorial 2^4-1 design was utilized and factors that might significantly affect the system performance were defined. As other validation parameters were also found to be suitable, the possibility to apply the proposed method for the determination of itraconazole, its impurities B and F in any laboratory under different circumstances has been proven.

In this paper chemometrically-assisted optimization and validation of hydrophilic interaction liquid chromatographic (HILIC) method for the quantitative analysis of amlodipine besylate (AB) and bisoprolol fumarate (BF) in pharmaceuticals are presented. In this study, the development of HILIC method for AB and BF separation was performed by a systematic approach which included Design of Experiments (DoE) methodology. First, in method optimization, a central composite design was applied. Influence of the acetonitrile content in the mobile phase, pH of the aqueous phase, and concentration of ammonium acetate on selected chromatographic responses was investigated. Optimal chromatographic conditions in terms of adequate separation and minimal analysis duration were set by graphical optimization overlapping the contour plots as: acetonitrile–water phase (10 mM ammonium acetate, pH 4.0 adjusted with glacial acetic acid) (92:8, v/v), column Luna HILIC 200 A, 150 mm × 4.6 mm, 5 µm particle size, column temperature 30°C, flow rate 1 mL min⁻¹, and UV detection at 230 nm. After this step, method robustness was tested using a 2^5-1 fractional factorial design. Statistical and graphical evaluation of results confirmed robustness of the method and then the method was fully validated. Finally, the proposed method was successfully applied for AB and BF determination in pharmaceuticals.

Using theoretical models, retention mechanisms of amlodipine besilate and its impurities D, E, and F were tested, as well as bisoprolol fumarate and its impurities A, C, K, and L. Appropriate theoretical retention models for tested compounds were used, which enabled the determination of retention mechanisms responsible for the separation of tested compounds on three different HILIC column: silica, amino, and diol. In order to describe the effect of ratio of aqueous and organic portion of the mobile phase on the retention behavior of tested compounds, as well as the consideration of shares of partition and adsorption processes in HILIC retention mechanism in all three columns, partitioning and adsorption retention theoretical models were calculated. For this purpose, the water content was varied in the range from 4% to 29%, corresponding to the HILIC region of mobile phase composition. During the experiment, values of other factors of mobile phase were maintained at a constant level. Regression coefficients of partition and adsorption retention models and the corresponding determination coefficients were determined (R²). Partitioning models for all tested compounds on amino column had higher R² values, while adsorption models for all compounds tested on silica and diol column had higher R² value.