Reversed-phase liquid chromatographic separation of 3',5'-cyclic ribonucleotides

A rapid, reversed-phase "high-performance" liquid-chromatographic separation of the five naturally occurring cyclic ribonucleotides is described. The separation, optimized for the measurement of these compounds in biological samples, is short (25 min), sensitive (50-100 pmol), and requires no sample pre-concentration steps. We also report an alternative isocratic elution mode, optimized for a rapid and selective analysis for adenosine 3',5'-cyclic phosphate. The identity of chromatographic peaks in biological extracts is confirmed by several methods: retention times, co-chromatography with the reference compounds, absorbance ratios, enzymatic peak-shift with cyclic nucleotide phosphodiesterase, and stopped-flow ultraviolet-scanning techniques.