Evaluation of in Silico Multifeature Libraries for Providing Evidence for the Presence of Small Molecules in Synthetic Blinded Samples

The current gold standard for unambiguous molecular identification in metabolomics analysis is comparing two or more orthogonal properties from the analysis of authentic reference materials (standards) to experimental data acquired in the same laboratory with the same analytical methods. This represents a significant limitation for comprehensive chemical identification of small molecules in complex samples. The process is time consuming and costly, and the majority of molecules are not yet represented by standards. Thus, there is a need to assemble evidence for the presence of small molecules in complex samples through the use of libraries containing calculated chemical properties. To address this need, we developed a Multi-Attribute Matching Engine (MAME) and a library derived in part from our in silico chemical library engine (ISiCLE). Here, we describe an initial evaluation of these methods in a blinded analysis of synthetic chemical mixtures as part of the U.S. Environmental Protection Agency's (EPA) Non-Targeted Analysis Collaborative Trial (ENTACT, Phase 1). For molecules in all mixtures, the initial blinded false negative rate (FNR), false discovery rate (FDR), and accuracy were 57%, 77%, and 91%, respectively. For high evidence scores, the FDR was 35%. After unblinding of the sample compositions, we optimized the scoring parameters to better exploit the available evidence and increased the accuracy for molecules suspected as present. The final FNR, FDR, and accuracy were 67%, 53%, and 96%, respectively. For high evidence scores, the FDR was 10%. This study demonstrates that multiattribute matching methods in conjunction with in silico libraries may one day enable reduced reliance on experimentally derived libraries for building evidence for the presence of molecules in complex samples.