A sensitive method for the separation and quantification of low-level adenine nucleotides using porous graphitic carbon-based liquid chromatography and tandem mass spectrometry

A liquid chromatography coupled to heated electrospray ionization/tandem mass spectrometry (LC-HESI-MS/MS) method was developed for the simultaneous quantitative analysis of low nanomolar level adenine nucleotides AMP, ADP, ATP, cyclic AMP (cAMP), and the nucleoside adenosine. For analyte retention and separation, reverse phase chromatography using porous graphitic carbon (PGC) was employed as it provided full resolution. The erratic chromatographic behaviour characteristic of PGC, including deterioration of analyte resolution and increased peak tailing (leading to decreased sensitivity), was mitigated by incorporating acidic equilibration within runs using a quaternary gradient. Analyte resolution and chromatographic sensitivity were still lost after a period of column inactivity; hence a pre-conditioning protocol was implemented between batches to regenerate the column. These column regeneration measures also allowed elution of AMP, ADP and ATP in the sequence of mono- to tri- nucleotides, differing from conventional reverse phase elution where analytes elute with decreasing polarity. This nucleotide elution sequence has the advantage of overcoming potential mis-annotation and inaccurate quantification of smaller nucleotides caused by in-source fragmentation of ATP. The method was validated in granulosa cell conditioned media, with the LLOQs ranging between 10-50 nM for most analytes. To verify the method using biological samples, nucleotide secretion was measured in granulosa cell conditioned media under various treatments known to alter their levels. Moreover, the method was applied to cumulus-oocyte complex cell lysates to examine its linearity in a complex matrix.