Setting up the chromatographic analysis of anthelmintics using the “Crossed D-Optimal” experimental design methodology

Abstract

An “experimental design” methodology was applied to optimize the chromatographic separation and quantitative determination of six anthelmintics levamisole (LEV), thiabendazole (THI), mebendazole (MEB), albendazole (ALB), praziquantel (PRA) and niclosamide (NICL) by HPLC. Based on the “Crossed D-Optimal” criterion, the participation of three mixed solvents in the mobile phase (methanol, water and acetonitrile) was investigated, since binary systems presented low chromatographic resolution. Furthermore, this approach provides an opportunity to use a crossover design to study the effects of other chromatographic parameters, such as pH, which is considered important for achieving optimal results. Sample analysis was carried out on an HS C₁₈ column (250 × 4.6 mm) 5 μm at 40 °C and the detection was performed with both a UV-diode array detector and an ELSD (Evaporative Light Scattering Detector). The mobile phase consists of an aqueous ammonium acetate buffer (pH 5.5; 0.05 M), acetonitrile and methanol (40 : 37 : 23, v/v/v) with 1 mL min⁻¹ flow rate. The system was found to produce sharp and well resolved peaks for all analytes with their retention times ranging from 3.5 to 13.0 min. Linear regression analysis for the calibration curves showed a good relationship with regression coefficients higher than 0.999 for the UV detector and 0.997 for the ELSD respectively. The proposed method was validated and proven to be accurate, precise, reproducible and suitable for routine analyses.