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

Diadenosine pentaphosphate (Ap(5)A) and diadenosine hexaphosphate (Ap(6)A) were recently identified in human platelets and were shown to be important modulators of cardiovascular function. Here we describe an HPLC assay for q uantitating Ap(3)A, Ap(4)A, Ap(5)A, and Ap(6)A contents in human platelets simultaneously. Di(1,N-6-ethenoadenosine) hexaphosphate was used as interna l standard. The extraction procedure consists of (a) deproteinization, (b) selective concentration of the diadenosine polyphosphates with a boronate a ffinity chromatography, and (c) desalting prior to the HPLC analysis. The a ssay was validated by PSD-MALDI-mass spectrometry and by addition of authen tic diadenosine polyphosphate to platelet samples. The assay was carried ou t by an ion-pair reversed-phase perfusion chromatography. In platelets from human blood the following amounts of diadenosine polyphosphates were deter mined: Ap(3)A, 192.5 +/- 151.0 nM; Ap(4)A, 223.8 +/- 172.3 nM; Ap(5)A, 100. 2 +/- 81.1 nM; Ap(6)A, 32.0 +/- 19.6 nM (mean +/- SD, n = 105). The describ ed assay can be used with less than 20 mi blood and allows quantitation of the diadenosine polyphosphates in the picomole range. (C) 1999 Academic Pre ss.