Development of a qualitative spot assay for zinc detection in fortified cereal grain matrices

Zinc is an essential micronutrient included in cereal grain fortification programs in over 39 developing countries, yet no simple qualitative field test exists to verify its presence. We investigated the reaction between zinc(II) and chromogenic solutions containing xylenol orange (XO), dithizone (DTZ), or 2-(5-bromo-2-pyridylazo)-5-[(N-propyl-N-(3-sulfopropyl)amino])phenol (5-Br-PAPS) for visual or spectrophotometric evaluation of zinc fortification in wheat flour, rice, and maize flour. 5-Br-PAPS was eliminated due to its inability to differentiate between zinc and iron. XO formed a 1:1 Zn:XO complex, producing a purple–pink color with a peak absorption at 575 nm. While iron interfered with the XO-Zn reaction, this effect was reduced at pH 6.5, improving visual contrast. A 0.0223 mM XO solution was optimized for spot testing in fortified cereals and grains. XO successfully identified zinc in fortified white flour and rice but was less effective in whole wheat and maize flour. DTZ, while less water-soluble and sensitive to heat and light, also produced a clear pink complex with zinc and was particularly effective in darker or coarser matrices where XO was less reliable. These findings support the potential use of XO and DTZ as qualitative tools for field-level zinc fortification monitoring.