Reliable quantification of citrate isomers and isobars with direct-infusion tandem mass spectrometry

Direct-infusion tandem mass spectrometry (DI-MS/MS) is an excellent tool for large cohort high-throughput quantitative metabolomics, MS imaging and single cell studies but incapable of discriminating isomers/isobars with similar MS spectral features. With experimental and density-functional theory (DFT) approaches, here, we comprehensively investigated the fragmentation pathways and characteristics of differential ion-mobility spectrometry (DMS) for three citrate isomers (citrate, isocitrate, glucaro-1,4-lactone) and an isobar (quinate) co-existing in biological sample such as urine. Results showed that all these compounds gave better MS spectra in negative-ion mode than positive-ion one and had numerous fragment ions under collision-induced dissociation (CID) with sequential losses of H₂O and CO₂. All observed fragment ions were assignable by combining experimental with DFT calculation results. A DI-DMS-MS/MS method was then developed to simultaneously quantify these four isomers/isobars with m/z 191-87 (CoV, -5.5 V), 191-73 (CoV, -3.5 V), 191-85 (CoV, -29.5 V) and m/z 191-93 (CoV, -41.5 V) for citrate, isocitrate, glucaro-1,4-lactone and quinate, respectively. The low limit-of-quantification was below 5.5 nM whilst accuracy was above 94% for all above compounds. The urinary concentrations of them in human and C57BL/6 mouse samples were further quantified showing clear inter-individual and inter-species level differences with significantly higher levels of isocitrate, glucaro-1,4-lactone and quinate in human urine samples than mouse ones. This provides an approach to understand the detailed fragmentation pathways for organic isomers/isobars and a high-throughput MS strategy to quantify them in complex mixtures for metabolomics, lipidomics, foodomics and exposomics especially when chromatographic separations are not useable.