Quantification of cyclic AMP and cyclic GMP levels in Krebs-Henseleit solution by LC-MS/MS: Application in washed platelet aggregation samples

•For the first time, nucleotide level is measured in washed platelet by LC-MS/MS.•This method shows high precision with the lowest LLoQ to date for cAMP and cGMP.•6-Nitrodopamine does not affect platelet nucleotide levels when stimulated. Cyclic Nucleotides are important in regulating platelet function. Increases in 3′5′-cyclic adenosine monophosphate (cAMP) and 3′5′-cyclic guanosine monophosphate (cGMP) inhibit platelet aggregation and are pharmacological targets for antiplatelet therapy. Here we report an improved method for determining cAMP and cGMP concentrations and, for the first time, in washed platelet supernatants by combining high-performance liquid chromatography and tandem mass spectrometry (LC-MS/MS). Characteristic peaks of the substrates, cGMP or cAMP and their internal standards were identified in negative-ion electrospray ionisation using multiple reaction monitoring. Compared with previously reported methods, the method presented here shows high precision with the lowest lower limit of quantification (LLoQ) to date (10 pg/mL). The effect of a novel catecholamine, 6-nitrodopamine, on cyclic nucleotide levels was quantified. Our results showed that this new method was fast, sensitive, and highly reproducible.