Analysis of S-35 labeled WR-2721 and its metabolites in biological fluids

Studies with WR-2721 and related compounds have been hindered by the lack of a suitable assay for the drug and its major metabolites. We have developed a chromatographic method which requires no derivatization for the separation and detection of WR-2721, the free thiol, its symmetrical disulfide and other mixed disulfides. Our procedure involves ion-pairing for separation of ionizable compounds by causing polar molecules to become more lipophilic and hence separable using reverse phase HPLC. Detection is based upon liquid scintillation counting of S-35 incorporated during the synthesis of the parent compound. This method requires no pre-column preparation of samples and, by detecting the S-35 label, eliminates the chance that a coeluting species could interfere with detection, as might occur with post-column derivatization. Chromatography was done using a 10 micron C₈RP column and 35% MeOH/65% 0.0113M NaH₂PO₄, 0.005 M hexanesulfonate, pH 5.9, flowing at 1 ml/min. Half-minute fractions were collected into scintillation vials for counting. Retention volumes for the various compounds were: column breakthrough (3.5 ml), WR-2721 (4.5 ml), WR-1065 (9 ml), and WR-33278 (24 ml). This analytical technique employing radiotracers can be used to study radioprotective mechanisms by time dependent measurements of the tissue distribution and chemical form of labeled drug. Such chemical information can then be correlated with biological measures of radiation protection.