Mass spectrometry-based measurements of cyclic adenosine monophosphate in cells, simplified using reversed phase liquid chromatography with a polar characterized stationary phase

Abstract

3′, 5′ – Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that is involved in many cellular functions and biological processes. In several cell types, cholera toxin will increase the level of cAMP, which mediates toxic effects on cells. In this context, we have developed a fast and simple method based on extraction with 5% trichloroacetic acid (TCA) and quantitation with liquid chromatography-mass tandem spectrometry (LC-MS/MS) for measuring cAMP in cells. A main feature of the LC-MS method was employing a reversed phase C18 column (2.1 mm × 50 mm, 1.6 µm particles) compatible with a 100% aqueous mobile phase, providing retention of the highly polar analyte. Isocratic separations allowed for fast subsequent injections. Negative mode electrospray ionization detection was performed with a triple quadrupole (QqQ)MS. cAMP was extracted from cell samples (~106 cells per well) and spiked with a labelled internal standard, using 200 µL of 5% TCA. The extraction solvent was fully compatible for direct injection onto the reversed phase column. After 10 min incubation, the supernatant was removed, and 10 µL of the supernatant was directly analysed by LC-MS. The method was characterized by the simplicity of the extraction, and the speed (3 min retention time of cAMP), sensitivity (250 pg/mL detection limit), and selectivity (separation from interferences e.g. isomeric compounds) of the LC-MS method, and could be used for quantitation of cAMP in the range 1–500 ng/mL cell extract.