Quality by Design framework in the development of a capillary electrophoresis method for the enantioseparation and impurity determination of cinacalcet hydrochloride

Cinacalcet hydrochloride (CIN) is the first agent of a new class of calcimimetic compounds, which act by decreasing the secretion of the parathyroid hormone by activating calcium receptors. CIN is the only approved drug by the US FDA in 2004 acting as regulator of ionized calcium and is marketed as single R-enantiomer, which is about 75 times more potent than its corresponding S-enantiomer. CIN is useful in the treatment of chronic kidney disease where hyperparathyroidism is a common complication of chronic pathologies. In this study, a capillary electrophoresis (CE) method for the simultaneous determination of the enantiomeric purity and of impurities of CIN has been developed following the Quality by Design principles. Comparing to HPLC, CE utilizes the wide spectrum of chiral selectors as buffer additives for chiral separation without the need for expensive columns. Moreover, CE mostly provides shorter analysis time and better resolution and offers the advantage of altering the enantiomer migration order based on the change of the type of chiral selector. The most commonly used chiral selectors are the cyclodextrins (CDs), due to their wide variety of cavity size, side chain, degree of substitution and charge. In the scouting phase of the development, preliminary experiments were carried out to select the separation operative mode and to identify a suitable chiral selector in order to reach an adequate selectivity. Among the tested CDs, (2-carboxyethyl)-β-CD and (2-hydroxypropyl)-γ-CD (HPγCD) showed chiral resolving capabilities at lower concentration values. The best results were obtained by using solvent modified capillary zone electrophoresis with the addition of HPγCD. Voltage, buffer pH, concentration of methanol as organic modifier and concentration of HPγCD were investigated as critical process parameters. Critical quality attributes were represented by the critical resolution value between the enantiomers and analysis time. A Box-Behnken Design for four factors allowed the contour plots to be drawn and the design space to be identified with a desired probability level π≥ 90%. The working conditions, with the limits of the design space, corresponded to the following: capillary length, 48.5 cm; temperature, 18 °C; voltage, 25 kV (23-28 kV); background electrolyte, 150 mM phosphate buffer pH 2.8 (2.7-3.0), 3.1 mM (2.2-4.0 mM) HPγCD; 2% (0.0-7.0 v/v%) methanol. Applying the selected conditions, the full separation of analytes was obtained in about 10 min. The method was validated and finally applied to a real sample of CIN tablets.