Single File Injections (SFI) Enable Thousand-Sample-per-Day Analysis by Liquid Chromatography-Mass Spectrometry

Mass spectrometry-based untargeted analysis, or metabolomics, typically requires chromatography to separate charged ions from different compounds. However, analyzing a large number of samples using serial injections limits throughput and can introduce batch effects or retention time shifts during extended data acquisition. To address these limitations, we propose single file injections (SFI), a method for parallel data acquisition from multiple samples without instrument modifications. In SFI, pooled samples are initially injected to serve as reference peaks for subsequent analyses. Repeated injections of individual samples are then performed at fixed time intervals using isocratic elution. This approach significantly increases throughput, potentially allowing for the analysis of 1000 samples per day, with a marginal time cost per sample equivalent to the fixed injection interval. To ensure compatibility with conventional analysis techniques, an open source software is developed to analyze data from SFI, which can recover peaks back to their original samples and is available online (https://github.com/yufree/sfi).