Authentic standards and matrix-based single analytical platform for targeted metabolomic analysis of 1,044 metabolites

Abstract

Liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-MS/MS) has become the de facto standard platform for metabolite identification and quantification in metabolomics. For successful metabolomic assessment studies, it is essential to detect, identify, and quantify a wide range of metabolites with high sensitivity and reproducibility. Furthermore, although recent advances in targeted metabolomics have enabled the profiling of hundreds of known metabolites, metabolite coverage is currently limited due to the restricted availability of authentic standards. Here, we present an authentic standard and matrix-based analytical method capable of simultaneously detecting and identifying over 1,000 metabolites from various human matrices in a single analytical run. To expand metabolite coverage beyond commercially available standards, 302 MRM transitions were obtained directly from biological matrices based on characteristic MS/MS fragmentation patterns. This approach enabled the inclusion of diverse metabolite classes, including sphingolipids, glycerophospholipids, acylcarnitines, N-acyl taurines, and acyl CoAs. The present authentic standard- and matrix-based method offers broad metabolite coverage, high sensitivity, and excellent chromatographic behavior, demonstrating significant potential for advancing large-scale targeted metabolomics.