After centrifugation, aliquots of the supernatant were analyzed both beforeand after dryingin an ovenat 60ÂCand comparedwith activity in the original homogenate to assess recovery and the presence of volatile activity, respectively. The remaining superna tant was concentrated by vacuum centrifugation and resuspended in ethanol. The resulting concentrate as well as [3H] MTBZ stan dards were applied to silica TLC plates and developed with chlo roform: methanol (96: 4). The locations of labeled compounds were determined by contact autoradiography against radiographic film at — 70ÂCwith the use of a fluorographic enhancer (EN3HANCE, Dupont, Boston, MA). The identified bands were then collected for liquid scintillation spectrometry. The TLC system clearly sep arated MTBZ from more polar labeled metabolites. The experi ments were repeated with a 200-g male Sprague-Dawley rat fol lowing administration of 370 MBqIkg (10 mCi/kg)[3H] MTBZ, in which the liver was also processed for isolation of large amounts of the radiolabeled metabolites. Together with [3H] MTBZ, they were used to develop a rapid column chromatographic procedure to isolate [†œC] MTBZfrom labeled metabolites in human plasma (see below).