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 °C for 20 min at neutral pH. The unreacted DTC was then decomposed to CS2 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 μL) of the derivatized sample was analyzed for the presence of stable esters of DTC by RP-HPLC using a Supelcosil LC-18-S (150 × 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 μM. The method is robust, and as low as 5 pmol of the analyte could be successfully detected and quantified with recoveries of ≥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.
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Acknowledgements
This publication was made possible by National Institutes of Health (NIH) grants P20 RR 16456 (from the BRIN program of the National Center for Research Resources) and ES 10018 (from the ARCH program of the National Institute of Environmental Health Sciences), and NSF grant HRD 0450375 to RU. We thank Drs. Diedra Atkins-Ball and K. Sathishkumar for critical reading of the manuscript. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH or NSF. The assistance of Mr. Joseph Allison during the initial stages of setting up of RP-HPLC analysis is acknowledged.
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Dupard-Julien, C.L., Kandlakunta, B. & Uppu, R.M. Determination of epoxides by high-performance liquid chromatography following derivatization with N,N-diethyldithiocarbamate. Anal Bioanal Chem 387, 1027–1032 (2007). https://doi.org/10.1007/s00216-006-1003-3
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DOI: https://doi.org/10.1007/s00216-006-1003-3