Bayfield R F, Romalis L F
Anal Biochem. 1985 Feb 1;144(2):569-76. doi: 10.1016/0003-2697(85)90155-1.
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 less than 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%.
研究了用2,3-二氨基萘（DAN）对生物材料中硒进行荧光测定时，重现性和重复性方面存在的不一致性。这些不一致性归因于样品酸消解后、在形成piazselenol络合物之前反应混合物中pH控制不当。通过使用甲基橙作为内部指示剂来确定初始pH约为3，并在0.1N HCl中加入DAN试剂后，最终pH为1.8，克服了这些问题。在此pH下形成的DAN-硒醇络合物在反应混合物的环己烷提取物中产生最大荧光响应。使用单个试管进行消解和提取程序，然后对环己烷提取物进行自动采样并打印荧光强度，提供了一种能够同时处理150个试管的简单分析方法。按照所述方法分析，硒标准品给出始终可接受的结果（r = 0.9995，P小于0.001，CV 2.3 - 3.3）；标准参考肝脏给出的值为1159±34.7 ng/g，与公布值1100±100 ng/g相比具有优势；动物血液和肝脏中硒的回收率在96%至104%之间。
