Analytical quality by design development of an ecologically acceptable enantioselective HPLC method for timolol maleate enantiomeric purity testing on ovomucoid chiral stationary phase

Abstract

Official method in Ph. Eur. for evaluation of timolol enantiomeric purity is normal-phase high perfor-mance liquid chromatography (NP-HPLC) method. Compared to other HPLC modes, NP is depicted asquite expensive with high consumption of organic solvents which leads to chronic exposure of analyststo toxic and carcinogenic effects. In order to overcome above-mentioned drawbacks, the aim of thisstudy was to develop new method with better eco-friendly features. This was enabled by using proteintype Chiral Stationary Phase (CSP) in reversed-phase mode that required up to 10 % (v/v) of organicsolvent. Therefore, an enantioselective HPLC method was developed and validated for quantification of(S)-timolol and its chiral impurity, (R)-isomer. Optimized separation conditions on ovomucoid columnwere set using Analytical Quality by Design (AQbD) approach in method development. Optimizationstep was performed following the Box-Behnken experimental plan and the influence of three criticalmethod parameters (CMPs) towards enantioseparation of the above-mentioned peak pair was exam-ined. CMPs included variation of acetonitrile content in the mobile phase (5–10 %, v/v), pH value of theaqueous phase (6.0–7.0) and ammonium chloride concentration in the aqueous part of the mobile phase(10−30 mmol L−1). The most relevant critical method attributes (CMAs) in this case were the separa-tion criterion between studied critical pair and retention factor of the second eluting peak, (S)-timolol.Qualitative Design Space (DS) was defined by Monte Carlo simulations providing adequate assurance ofmethod’s qualitative robustness ( = 95 %). The selected working point situated in the middle of the DSwas characterized by following combination of CMPs: acetonitrile content in the mobile phase 7 % (v/v),pH value of the aqueous phase 6.8 and concentration of ammonium chloride in aqueous phase 14 mmolL–1. In the next step, the quantitative robustness was tested by Plackett-Burman experimental design. Thevalidation studies confirmed adequacy of the proposed method for its intended purpose. Finally, Ana-lytical Eco-Scale metric tool was applied to confirm that developed method represents excellent greenanalytical method compared to the official one.