High-performance liquid chromatographic determination of diadenosine 5',5'"-p1,p4-tetraphosphate with precolumn fluorescence derivatization and its application to metabolism study in whole blood.
Diadenosine 5',5'"-p1,p4-tetraphosphate (Ap4A) was converted with chloroacetaldehyde to the fluorescent di-1,N6-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 microgram/ml in plasma when 10 microliters of the sample was applied to the column. The rate of degradation of Ap4A added to whole blood (5 micrograms/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.
Iwata
K
K
Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Haruki
S
S
Kimura
T
T
D000241:Adenosine / Q000031:analogs & derivatives; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D015226:Dinucleoside Phosphates / Q000097:blood*; D004285:Dogs; D005456:Fluorescent Dyes; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D051381:Rats; D013045:Species Specificity; D013050:Spectrometry, Fluorescence
D015226:Dinucleoside Phosphates; D005456:Fluorescent Dyes; C007640:1,N(6)-ethenoadenosine; C020733:diadenosine tetraphosphate; D000241:Adenosine