A novel purity analysis method of bovine milk-derived exosomes by two-dimensional high-performance liquid chromatography

Exosomes have been implicated in many biological processes as intercellular communication carriers. Because exosomes are increasingly explored as natural vehicles for cell and gene therapies, and drug delivery applications, it is critical to have high-quality samples. Protein:particle ratio, ELISA, western blotting, polymerase chain reaction, and size-exclusion high-performance liquid chromatography are the main methods that have been extensively applied to analyze exosomes purity in recent years. However, there is currently no well-established method that is convenient for routine quality analysis of small-size impurities in exosomes samples. Here, a size-exclusion high-performance liquid chromatography (SE-HPLC), an ion-exchange high-performance liquid chromatography (IEX-HPLC), and a novel two-dimensional high-performance liquid chromatography (2D-HPLC) method were used to detect the purity of bovine milk-derived exosomes with different processes of exosome isolation in detail. The results showed that the 2D-HPLC method could enhance the accuracy of detecting exosomal purity with higher precision and accuracy of instrumental, reduced personal error and experimental cost, shortened analysis time greatly, and more automation. The 2D-HPLC method is rapid, exhibits high selectivity, and has good sensitivity, thus making it well-suited for application in the pharmaceutical and toxicological analysis of exosomes.