PH Control in the fluorometric assay for selenium with 2,3-diaminonaphthalene

Area of Science:

• Analytical Chemistry
• Biochemistry

Background:

• Fluorometric assays for selenium in biological materials using 2,3-diaminonaphthalene (DAN) have shown inconsistencies.
• These issues are primarily linked to inadequate pH control during sample digestion and complex formation.

Purpose of the Study:

• To investigate and resolve reproducibility and duplication inconsistencies in the fluorometric assay of selenium.
• To establish an optimized method for accurate selenium determination in biological samples.

Main Methods:

• Utilized methyl orange as an internal indicator to control pH, establishing an initial pH of ~3 and a final pH of 1.8 after DAN reagent addition.
• Employed a single-tube digestion and extraction procedure with automatic sampling for fluorescence intensity measurement.
• Developed a method capable of analyzing 150 samples simultaneously.

Main Results:

• Optimized pH control at 1.8 maximized the fluorescence response of the DAN-selenol complex.
• Selenium standards yielded highly reproducible results (r = 0.9995, CV 2.3-3.3%).
• Analysis of Standard Reference Liver (1159 ± 34.7 ng/g) and recoveries from animal blood/liver (96-104%) demonstrated method accuracy and reliability.

Conclusions:

• Accurate pH control is critical for reproducible fluorometric selenium determination using DAN.
• The developed method offers a simple, high-throughput, and accurate approach for selenium analysis in biological matrices.
• This optimized assay significantly improves the reliability of selenium quantification in biological samples.