Stable Isotope Phosphate Labelling of Diverse Metabolites is Enabled by a Family of 18O-Phosphoramidites**

Stable isotope labelling is state-of-the-art in quantitative mass spectrometry, yet often accessing the required standards is cumbersome and very expensive. Here, a unifying synthetic concept for O-18-labelled phosphates is presented, based on a family of modified O-18(2)-phosphoramidite reagents. This toolbox offers access to major classes of biologically highly relevant phosphorylated metabolites as their isotopologues including nucleotides, inositol phosphates, -pyrophosphates, and inorganic polyphosphates. O-18-enrichment ratios >95 % and good yields are obtained consistently in gram-scale reactions, while enabling late-stage labelling. We demonstrate the utility of the O-18-labelled inositol phosphates and pyrophosphates by assignment of these metabolites from different biological matrices. We demonstrate that phosphate neutral loss is negligible in an analytical setup employing capillary electrophoresis electrospray ionisation triple quadrupole mass spectrometry.

Automated summary

A novel synthetic method for O-18-labelled phosphates is introduced to facilitate access to required standards for stable isotope labelling in quantitative mass spectrometry. The study demonstrates consistent >95% O-18 enrichment ratios and good yields in gram-scale reactions, enabling late-stage labelling and analysis of metabolites in biological samples. The utility of O-18-labelled inositol phosphates and pyrophosphates is shown through assignment of these metabolites from different biological matrices.