Simplified fluorometric assay of total selenium in plasma and urine

We describe here a single-tube assay that may be applied to the whole range of selenium status in adult and pediatric patients, including depletion during parenteral or other nutrition. A specimen or aqueous standard, 100 microL, is digested with 0.5 mL of HNO3/HCIO4 (4/1 by vol, at 190 degrees C for 90 min), reduced with 0.5 mL of concentrated HCI (150 degrees C, 30 min), and complexed with 0.5 mL of 6.3 mmol/L 2,3-diaminonaphthalene (DAN) reagent in the presence of EDTA (60 degrees C, 30 min). The resulting fluorophore is extracted into cyclohexane and its fluorescence measured (excitation at 366 nm; emission at 544 nm). It is not necessary to control pH during the complexing step or to protect the DAN from light. The limit of detection of selenium is 10 micrograms/L (0.126 mumol/L); linearity of results extends to 2000 micrograms/L (25.3 mumol/L). Between-batch precision is 5%, analytical recovery 90%-96%. Performance is good as tested against Reference Materials and by participation in a National Quality Assurance Scheme.