No abstract

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.

A fluorometric, reversed-phase high-performance liquid chromatography (RP-HPLC) method that allows quantitation of low levels of alloxan has been described. The method involved derivatization of alloxan with 500-200,000-fold excess of 1, 2-phenylenediamine (PD) in 0.1 M acetate buffer, pH 4.5 for 15 min at room temperature. The fluorescent product alloxazine (excitation: 382 nm; emission: 435 nm) was then analyzed by RP-HPLC using an Eclipse XDB-C18 (4.6 x 150 mm) column and a mobile phase consisting of 0.1% trifluoroacetic acid in 15/85 (v/v) acetonitrile/water at a flow of 1 mL/min (injection volume: 20 microL). The method is robust, and as low as 0.1 pmol of the analyte could be successfully detected and quantified. Following a minimal pre-treatment such as ultrafiltration (molecular weight cut-off 5000 Da) or protein precipitation using perchloric acid, acetonitrile, or phosphotungstic acid, the method is suitable for analysis of alloxan in complex physiological fluids (e.g. fetal bovine serum) and tissue homogenates (e.g. heart and kidney). The method has been rigorously evaluated and adapted in the laboratory for routine analysis and determination of alloxan added to cell cultures.