Determination of selenium in human blood by high-performance liquid chromatography with fluorescence detection

Selenium was determined in human whole blood or red cells by high-performance liquid chromatography (HPLC) using a new internal standard. The method provides a 0.15-ng detection limit, a between-day standard deviation of 1% at the 20-ng level, and a 3% within-day standard deviation at the 1-ng level. Samples were wet-ashed, and a selenium-diaminonaphthalene derivative was formed, followed by addition of tetraphenylnaphthacene internal standard, reverse-phase HPLC separation (10 min/run), and fluorescence detection. The detection limit was reduced by discrimination of fluorescence excitation of the selenium complex from the background using long-wavelength excitation (480 nm), removal of stray light in the excitation beam, and other optimizations described. Representative aliquots of frozen and thawed whole blood samples were obtained by using a nitric acid predigestion at ambient temperature. Procedures to validate the method included standard addition and neutron activation analysis.