Prebiotic chemistry analysis with UHPLCOrbitrap MS: Optimizing protocols to detect ribose, nucleobases, nucleosides, nucleotides, and short RNA oligomers.

Experimental prebiotic chemistry requires analytical methods that can sensitively detect and confidently distinguish RNA-related compounds within chemically complex reaction mixtures. Here, we develop and optimize two UHPLCOrbitrap MS workflows. The first workflow enables simultaneous high-confidence detection and structural certainty of 19 RNA-related building blocks relevant to origins-of-life studies, and the second complementary workflow enables the detection of short AMP oligomers. We systematically evaluate column chemistry, elution gradient, solvent composition, ionization mode, flow rate, column temperature, and collision energy to establish a robust analytical framework for prebiotic reaction products. Under the first workflow, analytical response varied across RNA-related compound classes: cyclic nucleotides produced the strongest signals, nucleosides showed in-source fragmentation to their corresponding nucleobases, and signal decreased with increasing phosphorylation state as highly phosphorylated analytes exhibit stronger interactions with metal components in the LC-MS system. Complementary conditions enabled reproducible detection of AMP dimers, trimers, and tetramers, although long-term monitoring showed substantial degradation at 4 °C, with stability decreasing with oligomer length and half-lives on the order of one month within mixed samples. By overcoming long-standing limitations in molecular-level structural confirmation, this workflow expands the analytical capabilities available for studying RNA-related chemistry in origins-of-life research.