Preparation of styrene-maleic anhydride copolymer nanobrush chromatographic stationary phase and its application in phospholipid analysis of exosomes.

BACKGROUND: Phospholipids in serum exosomes are promising biomarkers for breast cancer diagnosis. However, the separation of phospholipids by liquid chromatography presents several challenges due to their inherent structural heterogeneity and the presence of isomeric forms. Traditional columns often struggle with the overlapping retention times of phospholipid species, which can result in poor resolution and difficulty in accurate quantification. Furthermore, the varying polarities and hydrophobicities of different phospholipid classes necessitate specialized stationary phases to achieve optimal separation. This study aimed to develop a novel chromatographic material for the comprehensive analysis of phospholipids in complex biological matrices.

RESULTS: In this study, a novel silica-based Sil-SMA-GMS copolymer brush stationary phase was fabricated for the first time via reversible addition-fragmentation chain transfer (RAFT) polymerization, using styrene-maleic anhydride (SMA) copolymer as the functional matrix and glycerol monostearate (GMS) as the derivatization reagent. Benefiting from the synergistic effects of hydrophobic moieties (benzene rings and long alkyl chains) and hydrophilic groups (carboxyl and hydroxyl groups) in the SMA copolymer, the prepared stationary phase exhibited a unique mixed hydrophilic interaction chromatography/reversed-phase liquid chromatography (HILIC/RPLC) retention mechanism. It demonstrated excellent separation selectivity and stability for both hydrophilic and hydrophobic small molecules, achieving a maximum column efficiency of 81,029 N/m. Notably, this stationary phase overcame the limitations of traditional single-mode chromatographic columns, enabling the simultaneous separation and analysis of phospholipids that vary in class, acyl chain length, and degree of unsaturation. Furthermore, by coupling the Sil-SMA-GMS column with mass spectrometry (MS), a non-targeted analytical method for serum exosomal phospholipids was developed. Applying this method to serum exosomes from breast cancer patients and healthy controls led to the identification of 9 major classes encompassing 200 phospholipid molecular subtypes, as well as the screening of phospholipid markers closely associated with breast cancer pathogenesis.

SIGNIFICANCE: This work presents a novel chromatographic material for the efficient separation of phospholipids in complex biological samples and establishes a technical foundation for the construction of multidimensional lipidomics platform. This advancement provides crucial support for the screening of cancer-related biomarkers, elucidating tumor metabolic mechanisms, and exploring targeted therapeutic targets.