Rapid quantification of 50 fatty acids in small amounts of biological samples for population molecular phenotyping

Efficient quantification of fatty-acid (FA) composition (fatty-acidome) in biological samples is crucial for understanding physiology and pathophysiology in large population cohorts. Here, we report a rapid GC-FID/MS method for simultaneous quantification of all FAs in numerous biological matrices. Within eight minutes, this method enabled simultaneous quantification of 50 FAs as fatty-acid methyl esters (FAMEs) in femtomole levels following the efficient transformation of FAs in all lipids including FFAs, cholesterol-esters, glycerides, phospholipids and sphingolipids. The method showed satisfactory inter-day and intra-day precision, stability and linearity (R² > 0.994) within a concentration range of 2–3 orders of magnitude. FAs were then quantified in typical multiple biological matrices including human biofluids (urine, plasma) and cells, animal intestinal content and tissue samples. We also established a quantitative structure-retention relationship (QSRR) for analytes to accurately predict their retention time and aid their reliable identification. We further developed a novel no-additive retention index (NARI) with endogenous FAMEs reducing inter-batch variations to 15 seconds; such NARI performed better than the alkanes-based classical RI, making meta-analysis possible for data obtained from different batches and platforms. Collectively, this provides an inexpensive high-throughput analytical system for quantitative phenotyping of all FAs in 8-minutes multiple biological matrices in large cohort studies of pathophysiological effects.