Measurement of Endogenous Fatty Acid Esters of Hydroxy Fatty Acid (FAHFA) and Alkyl-diacylglycerol (TG(O)) from Human Breast Milk

Fatty acid esters of hydroxy fatty acids (FAHFAs) and alkyl-diacylglycerols (TG-(O)-s) are endogenous lipids that exert protective effects against diabetes, inflammation, and the development of childhood obesity. They have been identified as minor lipids in human breast milk, and an accurate characterization of FAHFAs and TG-(O)-s could further our understanding of changes in human milk lipid profiles across different phenotypes. However, due to differences in their polarities and chemical properties, current studies on FAHFAs and TG-(O)-s tend to be conducted independently. The study of these compounds is further complicated by a shortage of validated methods for the isolation of FAHFAs and TG-(O)-s from complex, lipid-rich biological matrices, such as human breast milk. Additionally, cohort studies on human tissues, including breast milk, are often confronted with limited sample availability. In this study, we describe the systematic validation of a workflow to reliably extract, isolate, and characterize both FAHFAs and TG-(O)-s from 1.6 mL of human breast milk. We have applied this method to the measurement of 16 and 19 endogenous FAHFA and TG-(O) species originating from two cohort studies, respectively. Our validated workflow also includes the application of liquid chromatography-multistage mass spectrometry approaches to identify FAHFA and TG-(O) molecular species as well as FAHFA regioisomers. This method has the potential to be adapted for the isolation and characterization of other minor lipids with different polarities from limited sample volumes of complex biological matrices.