ISOLATION AND QUANTIFICATION OF FLUOROACETATE IN RAT-TISSUES, FOLLOWING DOSING OF Z-PHE-ALA-CH2-F, A PEPTIDYL FLUOROMETHYL KETONE PROTEASE INHIBITOR

Peptidyl fluoromethyl ketones (PFMK) are irreversible inhibitors of ca thepsin B, a cysteine proteinase thought to be involved in the degrada tion of cartilage. It has been speculated that PFMK inhibitors may met abolize in rodents to form fluoroacetate (FAC), an extremely toxic poi son. A highly selective and sensitive separation and detection scheme was developed to measure trace levels of FAC in rat tissues following PFMK dosing. The procedure consisted of extracting FAC from tissue and spiking the extract with [O-18](2)-fluoroacetate (O-18-FAC) as an int ernal standard. FAC and O-18-FAC were further isolated from matrix com ponents using ion-exchange, solid-phase extraction. The pentafluoroben zyl esters of FAC and O-18-FAC were formed to facilitate the chromatog raphic separation. Two-dimensional gas chromatography coupled with sel ected-ion-monitoring detection provided the final measurement. The ass ay had a limit of detection of 2 ng FAC per g tissue, and was capable of accurately quantitating as little as 10 ng FAC per g tissue with a SIN ratio of 40:1. Linearity was established over two orders of magnit ude, from 2-500 ng ml(-1), with 5 mu l injected on-column. The method was used to demonstrate that FAC was formed in rats following dosing w ith Z-Phe-Ala-CH2-F, a PFMK cathepsin enzyme inhibitor. (C) 1997 Elsev ier Science B.V.