A combined two-step high-performance liquid chromatographic (HPLC) method was developed for the analysis of endogenous levels of cyclic adenosine diphosphoribose (cADPR) in cell extracts. The detection sensitivity for cADPR was about 10 pmol. Linearity of the HPLC detection system was demonstrated in the range of 10 pmol up to 2 nmol. The method was validated in terms of within-day and between-day reproducibility of retention times and peak areas of standard nucleotides. The method was applied to the analysis of endogenous cADPR in human T cell lines. Sequential separation of perchloric acid extracts from cells on strong anion-exchange and reversed-phase ion-pair HPLC resulted in a single symmetrical peak co-eluting with standard cADPR. The identity of this endogenous material was further confirmed by its ability to be converted to ADPR upon heating the cell samples at 80 degrees C for 2 h. Recoveries of the combined perchloric acid extraction-HPLC analysis procedures were 48.3 +/- 10.2%. The determined intracellular concentrations of cADPR in quiescent Jurkat and HPB.ALL human T cells were 198 +/- 41 and 28 +/- 9 pmol/10(8) cells, respectively. In conclusion, a non-radioactive HPLC method presenting a specificity and sensitivity suitable for precise quantification of cADPR in cell extracts was developed.
