High-performance liquid chromatographic assay of the diadenosine polyphosphates in human platelets

Diadenosine pentaphosphate (Ap5A) and diadenosine hexaphosphate (Ap6A) were recently identified in human platelets and were shown to be important modulators of cardiovascular function. Here we describe an HPLC assay for quantitating Ap3A, Ap4A, Ap5A, and Ap6A contents in human platelets simultaneously. Di(1,N6-ethenoadenosine) hexaphosphate was used as internal standard. The extraction procedure consists of (a) deproteinization, (b) selective concentration of the diadenosine polyphosphates with a boronate affinity chromatography, and (c) desalting prior to the HPLC analysis. The assay was validated by PSD-MALDI-mass spectrometry and by addition of authentic diadenosine polyphosphate to platelet samples. The assay was carried out by an ion-pair reversed-phase perfusion chromatography. In platelets from human blood the following amounts of diadenosine polyphosphates were determined: Ap3A, 192.5 +/- 151.0 nM; Ap4A, 223.8 +/- 172.3 nM; Ap5A, 100.2 +/- 81.1 nM; Ap6A, 32.0 +/- 19.6 nM (mean +/- SD, n = 105). The described assay can be used with less than 20 ml blood and allows quantitation of the diadenosine polyphosphates in the picomole range.