Enhanced Environmental Hydroxylamine Detection: A Chromatographic Adaptation to the Indooxine Approach

Hydroxylamine (NH₂OH) plays an important role as an intermediate species in many nitrogen transformation reactions. Since it readily reacts with O₂, NH₂OH is often present in submicromolar concentrations in environmental systems. Existing NH₂OH quantification protocols are limited by sensitivity, cost, or both; consequently, the extent and role of NH₂OH in many biological and abiotic processes are poorly understood. In the work presented here, a colorimetric method is improved and adapted for use in high-performance liquid chromatography (HPLC). The method entails the reaction of NH₂OH with a reagent, 8-hydroxyquinoline, to form a larger compound, indooxine, that absorbs strongly in the visible range at 705 nm and at 261 nm in the UV window. Using a HPLC-UV system detecting at the 261 nm peak provided a significantly enhanced NH₂OH detection limit and improved measurement reliability. This chromatographic method was tested for sensitivity and performance in wastewater and river water matrices, in addition to pure water cases, achieving reliable results in each solution. The protocol achieved accurate measurements of NH₂OH concentrations as low as 0.05 μM, vastly improving previous detection limits. Sampling procedures that ensure the preservation of NH₂OH were also established; samples acidified below a pH of 2.0 remained stable under room temperature conditions for up to two months. Given that HPLC-UV systems are relatively common in environmental laboratories, the adapted method reported here is expected to significantly improve the accessibility of accurate, highly sensitive NH₂OH detection.