Ion-pair liquid chromatography-electrospray mass spectrometry for the analysis of cyclic nucleotides

An LC-MS method has been developed combining ion-pair chromatography with an electrospray interface linking microbore and capillary HPLC to mass spectrometry. Separation of cyclic nucleotides on C18 reversed-phase columns, using tetrabutylammonium bromide as an ion pairing agent was evaluated with different mobile phase compositions. It was found that low ion-pairing agent concentration (50-500 microM) used in combination with low flow-rates (5-10 microl min(-1)) allowed the system to operate for up to several days without observing a reduced signal caused by source pollution. The loss of sensitivity expected in ion-pair chromatography could be remedied by using a 2-propanol coaxial sheath flow. Optimal conditions for negative ion electrospray resulted in a linear detection response in the femtomole to picomole range. Using biological samples this method was evaluated and compared with a classical ion-suppression RP-HPLC method using UV detection.