Capillary Zone Electrophoresis for the Analysis of Phenolic Preservatives in Plasma and Insulin Formulations Using Salting‐Out Assisted Liquid–Liquid Extraction

Phenolic preservatives are critical excipients in biopharmaceutical formulations such as insulin and its analogs. The determination of these analytes in insulin formulations and human plasma is crucial for various applications. This study reports the first use of a method combining salting-out assisted liquid-liquid extraction (SALLE) with capillary zone electrophoresis (CZE). The CZE-based separation of phenolic preservatives remains particularly challenging, as the alkaline conditions required for adequate resolution lead to elevated electric current and accelerated capillary deterioration. Analytical parameters, including the nature of the background electrolyte (BGE) and ionic strength, were optimized to ensure reliable separation of phenol, m-cresol, and the internal standard. SALLE was performed using acetonitrile (ACN) as the extraction solvent and ammonium acetate as the salting-out agent. CZE separation was completed in less than 6 min using a carbonate buffer at pH 10.3. The SALLE procedure yielded good recoveries (96%-102%) and repeatability (relative standard deviation [RSD] for concentration determination < 5.5%). The SALLE-CZE-UV methodology showed excellent linearity (r² > 0.994) and repeatability (RSD for corrected migration times: 0.4% for phenol and 0.5% for m-cresol; RSD for the ratio of corrected peak areas [analyte/internal standard]: 2.6% for phenol and 1.8% for m-cresol). Moreover, appropriate limits of detection (LOD) and quantification (LOQ) were obtained for insulin formulations in line with regulatory requirements. In addition, suitable LOD and LOQ (0.014; 0.047 g.L^-1 for phenol and 0.007; 0.022 g.L^-1 for m-cresol) were obtained for spiked human plasma.