Highly sensitive, selective and rapid LC-MS method for simultaneous quantification of diadenosine polyphosphates in human plasma

Background: Diadenosine polyphosphates (ApnAs) are endogenous mediators involved in large number of physiologic and pathophysiologic processes. The quantification of diadenosine polyphosphates in plasma and biological matrices is still challenging. Therefore, there is an urgent need for a simple and reliable quantification method suitable for clinical studies. The classical quantification of diadenosine polyphosphates is based on chromatographic separation and UV adsorption of the resulting fractions. These procedures are associated with low selectivity due to co-eluting plasma components. Therefore, we developed and validated a highly sensitive, selective and rapid LC-ESI-MS method for simultaneous quantification of ApnAs (with n=3-6) in human plasma within this study. The identities of the endogenous ApnAs (with n=3-6) were revealed by comparison of ESI-MS/MS fragment spectra of isolated endogenous compounds with those of authentic ApnAs.

Methods: Diadenosine polyphosphates were extracted from 100μl human plasma using weak anion-exchange extraction cartridges. The separation of ApnAs was achieved using capillary C18 columns. ESI-HCT mass spectrometer (Bruker Daltonik, Germany) operated in negative ion mode was used for detection and quantification of ApnAs.

Results: A calibration curve was established for diadenosine polyphosphate free plasma in the concentration range 1.9-125nM (r(2)>0.998) for all analytes. The intra- and inter-day accuracies were in the range of 91.4% and 110.9%. The intra- and inter-day precisions were determines as 0.1% and 11.4%, respectively. The mean plasma concentrations of ApnAs were quantified as 31.9±5.9nM for Ap3A, 40.4±6.6nM for Ap4A, 10.7±1.5nM for Ap5A and 10.0±18.9nM for Ap6A.

Discussion: The developed and validated ESI MS-based method for quantification of diadenosine polyphosphates in human plasma was successfully evaluated within the study. Conclusion Since the quantification is based on a volume of 100μl plasma, this method is highly applicable for clinical applications aiming at the validation of the impact of highly physiological and pathophysiological active diadenosine polyphosphates.