Reaction profiling by ultra high-pressure liquid chromatography/time-of-flight mass spectrometry in support of the synthesis of DNA-encoded libraries

Abstract An ultra high-pressure liquid chromatography/mass spectrometry (UHPLC/MS) separation and analysis method has been devised for open access analysis of synthetic reactions used in the production of DNA-encoded chemical libraries. The aqueous mobile phase is 100 mM hexafluoroisopropanol and 8.6 mM triethylamine; the organic mobile phase is methanol. The UHPLC separation uses a C 18 OST column (50 mm × 2.1 mm × 1.7 μm) at 60 °C, with a flow rate of 0.6 mL/min. Gradient concentration is from 10 to 40% B in 1.0 min, increasing to 95% B at 1.2 min. Cycle time was about 5 min. This method provides a detection limit of a 20-mer oligonucleotide by mass spectrometry of better than 1 pmol on-column. Linear UV response for 20-mer extends from 2 to 200 pmol/μL in concentration, same-day relative average deviations are less than 5% and bias (observed minus expected) is less than 10%. Deconvoluted mass spectra are generated for components in the predicted mass range using a maximum entropy algorithm. Mass accuracy of deconvoluted spectra is typically 20 ppm or better for isotopomers of oligonucleotides up to 7000 Da.