Stacked Injections of Biphasic Extractions for Improved Metabolomic Coverage and Sample Throughput

Omics technologies attempt to provide comprehensive coverage of their target analytes. Comprehensive coverage of metabolites, the aim of nontargeted metabolomics applications, is hindered by the extreme diversity in physiochemical properties of the metabolome. One approach to deal with this challenge is the use of biphasic extractions. These methods generate two largely complementary extracts from a single sample, with an organic lipid-rich fraction and an aqueous fraction containing largely primary and secondary metabolites. To improve metabolite coverage, these two fractions are then independently analyzed resulting in a doubling of the experimental time. In this manuscript, we describe a novel injection approach, stacked injections of a biphasic extraction (SIBE), which enables simultaneous analysis of the two fractions. We demonstrate that SIBE offers nearly 3-fold more total peak area than a monophasic extract without dramatically increasing instrumentation time required for the analysis. The analytical variance is very slightly increased; however, significant improvements in retention time stability are obtained with SIBE vs monophasic injections. Collectively, these data indicate that SIBE is a viable injection approach whenever comprehensive metabolomic coverage is desired.