Determination of epoxides by high-performance liquid chromatography following derivatization with N,N-diethyldithiocarbamate.

A reversed-phase, high-performance liquid chromatography (RP-HPLC) method that allows quantitation of low levels of epoxides has been described. The method involved derivatization of epoxides using 100- to 1,000-fold excess N,N-diethyldithiocarbamate (DTC) at 60 degrees C for 20 min at neutral pH. The unreacted DTC was then decomposed to CS(2) and diethyl amine by acidification of the reaction mixture to pH 2 using orthophosphoric acid. The first two steps could be performed in the same reaction vessel by sequential addition of reagents. In the final step, an aliquot (20 microL) of the derivatized sample was analyzed for the presence of stable esters of DTC by RP-HPLC using a Supelcosil LC-18-S (150 x 4.6-mm) column and a mobile phase consisting of 40% (v/v) acetonitrile in water at a flow of 1 mL min(-1). Using UV detection at 278 nm, the epoxides gave linear responses in the concentration range of 0.25 to 50 microM. The method is robust, and as low as 5 pmol of the analyte could be successfully detected and quantified with recoveries of > or =94%. Following a minimal pretreatment such as ultrafiltration (molecular weight cutoff 5,000 Da), the method is suitable for analysis of epoxides in complex physiological fluids (e.g., fetal bovine serum). The method has been rigorously evaluated and adapted in our laboratory for routine analysis and determination of stability of epoxides of 1,3-butadiene and other alkenes added to cell cultures.