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

Background: Diadenosine polyphosphates (Ap(n)As) 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 Ap(n)As (with n = 3-6) in human plasma within this study. The identities of the endogenous Ap(n)As (with n = 3-6) were revealed by comparison of ESI-MS/MS fragment spectra of isolated endogenous compounds with those of authentic Ap(n)As. Methods: Diadenosine polyphosphates were extracted from 100 mu l human plasma using weak anion-exchange extraction cartridges. The separation of Ap(n)As 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 Ap(n)As. Results: A calibration curve was established for diadenosine polyphosphate free plasma in the concentration range 1.9-125 nM (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 Ap(n)As were quantified as 31.9 +/- 5.9 nM for Ap(3)A, 40.4 +/- 6.6 nM for ANA, 10.7 +/- 1.5 nM for Ap(5)A and 10.0 +/- 18.9 nM for Ap(6)A. 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 100 mu l plasma, this method is highly applicable for clinical applications aiming at the validation of the impact of highly physiological and pathophysiological active diadenosine polyphosphates. (C) 2014 Elsevier B.V. All rights reserved.