Development and Validation of an Isocratic HPLC Method for the Simultaneous Determination of Avobenzone and...

Avonenzone (AVO) and tris-biphenyl triazine (TBPT) are ultraviolet (UV) filters commonly used in sunscreen formulations. Their combination enables broad-spectrum photoprotection and improved photostability. Despite their widespread and emerging use, the simultaneous quantitative analysis of these highly hydrophobic compounds remains analytically challenging, particularly in complex formulation matrices. In this study, an isocratic HPLC method was developed and validated for the simultaneous determination of AVO and TBPT. Chromatographic separation was achieved using a reversed-phase RP-18 column (125 Å, 3.9 mm × 300 mm, 10 μm) with a mobile phase composed of acetonitrile, isopropyl alcohol, and 2% aqueous phosphoric acid (42:42:16, v/v/v). The flow rate was set to 1.0 mL/min, with an injection volume of 20 μL and a column temperature of 30 °C. Detection was performed at 358 nm for AVO and 310 nm for TBPT. Method validation was conducted in accordance with ICH Q2-(R1), including evaluation of specificity, linearity, limits of detection and quantification, accuracy, precision, and robustness. Method robustness was systematically assessed using a Box-Behnken Design (BBD) as a Quality-by-Design tool to identify critical chromatographic parameters and define a reliable analytical domain. The method demonstrated excellent linearity over the 0.5-32 μg mL^-1 range, high accuracy, acceptable precision, and adequate sensitivity for both analytes. Specificity was confirmed using a representative laboratory-prepared sunscreen formulation, demonstrating the method's ability to handle complex matrices without interference from formulation excipients. The BBD analysis confirmed the robustness and reproducibility of the method under small deliberate variations of critical parameters. Overall, the proposed method provides a simple, reliable, and reproducible isocratic analytical approach for the simultaneous quantification of AVO and TBPT. Its applicability to complex sunscreen matrices and its emphasis on robustness rather than ultrafast separation make it particularly suitable for routine quality control, formulation development, and stability studies, supporting current and future sunscreen research in an evolving regulatory landscape.