Development and application of an LC–MS/MS method for measuring the effect of (partial) agonists on cAMP accumulation in vitro

Abstract

Cyclic-adenosine monophosphate (cAMP) plays an important role in cell signalling and is widely used as a marker for receptor activation and as a target for treating various diseases. In this paper we present the development and validation of a new method for the determination of cAMP and ATP (adenosine triphosphate) and other nucleotides in a biological system by combining zwitterionic hydrophilic interaction liquid chromatography (HILIC) and tandem mass spectrometry (MS/MS).The HILIC–MS/MS method was developed for the simultaneous quantitative analysis of cAMP and ATP, and was validated by assessment of linearity (over a range from 0.5 to 100nM for cAMP and 50nM to 50μM for ATP (r2>0.999)), resolution, limit of detection (0.5 and 50nM for cAMP and ATP, respectively) and reproducibility. Furthermore, the method was validated and applied in vitro to determine cAMP accumulation in biological samples. The effect of several dopamine D2 (partial) agonists and antagonists on cAMP accumulation was assessed by determination of the cAMP/ATP ratio in cells transfected with the human dopamine D2L receptor. Quinpirole, dopamine and ropinirole produced agonist effects on cAMP accumulation, with a potency of quinpirole>ropinirole>dopamine. Lisuride, terguride and bifeprunox were found to be partial agonists with efficacies of lisuride>terguride>bifeprunox. As expected, haloperidol, (−)-sulpiride and LY-741626 were antagonists.These results demonstrate that the present analytical method was robust, fast, sensitive, and selective. Moreover, it showed utility in determining cAMP/ATP in biological systems and the ability to study the effect of (partial) agonists and antagonists which makes it a useful tool for drug discovery.