Optimized Lipid Extraction and Hybrid Annotation Pipeline for Individual Chitinous Mesozooplankton Using UPLC-HRMS

High-resolution lipidomics at the scale of individual mesozooplankton offers a powerful tool for understanding trophic interactions and carbon cycling in marine ecosystems, but chitinous exoskeletons present challenges for efficient lipid extraction. Here, we develop and validate an optimized Bligh and Dyer-based extraction protocol that incorporates in-line glass bead homogenization, yielding a 2.5-fold increase in lipid recovery. When combined with an increased injection volume, this protocol achieves a 4.4-fold gain in signal intensity. This workflow enables the robust detection of intact lipid species from single Calanus copepods, eliminating the need for additional homogenization equipment or extended extraction steps, thereby making it broadly accessible for analytical applications. To address the limitations of current annotation pipelines, we directly compared adduct-hierarchy (LOBSTAHS) and fragmentation-based (MS-DIAL) approaches, finding systematic biases that reshape lipidomic profiles depending on the computational strategy employed. Additionally, by integrating a wax ester-specific fragmentation library, we demonstrate improved annotation of marine-relevant lipid classes largely absent from conventional databases. Together, this extraction and hybrid annotation pipeline enables high-resolution, compound-specific lipidomics of individual mesozooplankton, capturing biological heterogeneity while remaining scalable to pooled samples. Our approach provides a critical methodological advance for tracing lipid metabolism across trophic levels and for quantifying the role of mesozooplankton lipids in marine biogeochemical cycles.