Gradient, reversed-phase HPLC method for simultaneous determination of chemical and enantiomeric impurities of dexibuprofen in a single run

Mhammad, Ali and Dombi, Gergely and Dobó, Máté and Simon, Balázs and Szabó, Zoltán-István and Tóth, Gergő (2025) Gradient, reversed-phase HPLC method for simultaneous determination of chemical and enantiomeric impurities of dexibuprofen in a single run. Journal of Chromatography A, 1758. No.-466210. ISSN 00219673

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Abstract

In this work, a gradient reversed-phase HPLC method is presented for the simultaneous determination of four potential chemical impurities in dexibuprofen, as well as its chiral isomer, (R)-ibuprofen. Among nine polysaccharide-based chiral columns tested, enantioselectivity was observed only with the column containing cellulose tris(4-methylbenzoate) under polar organic mode conditions. To enhance enantioresolution, a reversed-phase mode was employed by adding water to the mobile phase. Examination of the water content's impact on retention time revealed a typical reversed-phase retention behavior in alcohol–water mixtures, whereas acetonitrile–water mixtures displayed HILIC-like retention characteristics. A full factorial design optimization strategy was applied to identify the ideal conditions for determining organic and enantiomeric impurities in dexibuprofen. Under the optimized conditions—Lux Cellulose-3 stationary phase using a methanol:water:acetic acid 70:30:0.1 (v/v/v) mobile phase, at a flow rate of 0.5 mL/min and temperature of 35 °C—all target compounds were effectively separated with clear baseline resolution. The initial analysis required 60 min, primarily due to the prolonged retention of highly lipophilic ester impurities. To reduce the analysis time, a gradient elution strategy was developed, successfully cutting the runtime by half. Furthermore, the stability and robustness of the gradient system were confirmed by consistent performance over one hundred consecutive injections. Following regulatory validation guidelines, the developed HPLC method was effectively applied for the quality assessment of commercially available pharmaceutical formulations.

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