pH control in the fluorometric assay for selenium with 2,3-diaminonaphthalene

Inconsistencies in reproducibility and duplication in the fluorometric assay of selenium in biological materials with 2,3-diaminonaphthalene (DAN) were investigated. These were attributed to inadequate pH control in the reaction mixture following acid digestion of samples and preceding formation of the piazselenol complex. The problems were overcome by using methyl orange as an internal indicator to establish an initial pH of approximately 3 and, after addition of the DAN reagent in 0.1 n HCl, a final pH of 1.8. Formation of the DAN-selenol complex at this pH gave maximum fluorescence response measured in cyclohexane extracts of the reaction mixture. Use of a single tube for the digestion and extraction procedures, followed by automatic sampling of the cyclohexane extract and printout of fluorescence intensities, provided a simple method of analysis capable of handling 150 tubes simultaneously. Analyzed by the method described, selenium standards gave consistently acceptable results (r = 0.9995, P < 0.001, CV 2.3–3.3); Standard Reference Liver gave values of 1159 ± 34.7 ng/g, which compared favorably with the published value of 1100 ± 100 ng/g; and recoveries of selenium from animal blood and liver varied from 96 to 104%.