Design of experiment guided development of an UHPLC method for phytoplankton pigments analysis

Phytoplankton pigment analysis by high-performance liquid chromatography (HPLC) is widely used to characterize community composition, support satellite algorithm validation, and advance aquatic biogeochemical research. However, conventional HPLC methods often rely on toxic solvents, require relatively long analysis times, and may have difficulty resolving structurally similar pigments such as mono- and divinyl chlorophylls and xanthophyll isomers. In this study, we developed and validated a novel ultra-high-performance liquid chromatography (UHPLC) method optimized for the separation of ecologically significant phytoplankton pigments. The method used a core–shell C8 column (100 × 4.6 mm, 2.6 μm) maintained at 60 °C with a linear gradient of ethanol and solvent B (56 mM tetrabutylammonium hydroxide in water adjusted to pH 7) over 22.5 min, followed by a 3-min hold at 100% ethanol, reducing solvent toxicity while maintaining chromatographic performance. Method development was guided by a Design of Experiments (DoE) approach to optimize the resolution of challenging pigment pairs, such as divinyl/monovinyl chlorophyll a and lutein/zeaxanthin. The final method achieved identification of 33 pigments and showed excellent reproducibility, with retention time variation below 0.6% and a peak area variation below 12%. Validation was performed using pigment standards, laboratory cultures, and natural samples from the northern Adriatic Sea, and by comparison an established validated method, demonstrating robust performance across a wide range of concentrations and matrix complexities. Overall, the study provided a rapid, reproducible, and environmentally safer alternative UHPLC method for phytoplankton pigment analysis in marine research and operational monitoring programs.