Advancements in sample preparation and extraction techniques for metabolomic analysis of animal fluids

Abstract

Metabolomics has become an extremely valuable tool in investigating biochemical processes in animal disease diagnosis, nutrition, and health. The precision of metabolomic data, however, is heavily dependent on sample preparation and extraction, which are still the most error-susceptible and variable steps in the workflow. Animal biofluids such as milk, urine, plasma, and serum pose especially challenging problems due to their complex protein, salt, and lipid matrices that make it difficult to detect metabolites. This review gives a systematic and critical evaluation of traditional, modern, and mixed extraction techniques, including protein precipitation, liquid–liquid extraction, solid-phase extraction, and microextraction-based methods such as SPME, DLLME, and QuEChERS. Mini case studies demonstrate their actual use in animal metabolomics, and provide context-dependent advantages, limitations, and method selection strategies. We also address the expanding role of hybrid and automated systems, including robotic platforms and microfluidics, to enhance reproducibility and scalability for large-cohort research. Analytical platform integration with GC–MS, LC–MS, CE–MS, and NMR is addressed, centring on platform selection criteria and complementary multi-platform approaches. Besides extraction, we address major areas of quality control, standardization, and inter-laboratory reproducibility, and then move to advances in data pre-processing, normalization, and pathway analysis based on bioinformatics. Finally, we touch on translational applications in animal and human disease, and outline future directions that integrate automation, artificial intelligence, and green extraction chemistries. Through the integration of methodological detail with pragmatic case examples, this review at once fulfills state-of-the-art summarization and forward-looking vision and offers pragmatic guidance for optimizing metabolomic investigations of animal fluids.