Isolation and quantification of fluoroacetate in rat tissues, following dosing of Z-Phe-Ala-CH 2-F, a peptidyl fluoromethyl ketone protease inhibitor

Abstract

Peptidyl fluoromethyl ketones (PFMK) are irreversible inhibitors of cathepsin B, a cysteine proteinase thought to be involved in the degradation of cartilage. It has been speculated that PFMK inhibitors may metabolize in rodents to form fluoroacetate (FAC), an extremely toxic poison. 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 [ 18O] 2-fluoroacetate ( 18O-FAC) as an internal standard. FAC and 18O-FAC were further isolated from matrix components using ion-exchange, solid-phase extraction. The pentafluorobenzyl esters of FAC and 18O-FAC were formed to facilitate the chromatographic separation. Two-dimensional gas chromatography coupled with selected-ion-monitoring detection provided the final measurement. The assay 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 S/ N ratio of 40:1. Linearity was established over two orders of magnitude, from 2–500 ng ml −1, with 5 μl injected on-column. The method was used to demonstrate that FAC was formed in rats following dosing with Z-Phe-Ala-CH 2-F, a PFMK cathepsin enzyme inhibitor.