HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC DETERMINATION OF DIADENOSINE 5',5'''-P(1),P(4)-TETRAPHOSPHATE WITH PRECOLUMN FLUORESCENCE DERIVATIZATION AND ITS APPLICATION TO METABOLISM STUDY IN WHOLE-BLOOD

Diadenosine 5',5'''-p(1),p(4)-tetraphosphate (Ap4A) was converted with chloroacetaldehyde to the fluorescent di-1,N-6-ethenoadenosine derivative within 60 min at 80 degrees C. It was separated by reversed-phase HPLC and detected fluorimetrically (excitation and emission wavelengths of 275 and 410 nm, respectively). The detection limit of Ap4A was ca. 0.2 mu g/ml in plasma when 10 mu l of the sample was applied to the column. The rate of degradation of Ap4A added to whole blood (5 mu g/ml) was examined using this method. Half-lives (means +/- S.E., n = 3) were 0.88 +/- 0.30 min (in rat blood), 13.7 +/- 3.6 min (in dog blood) and 17.2 +/- 1.4 min (in human blood). A marked species difference in the degradation rate of Ap4A in blood was observed.