Broken into pieces: The challenges of determining sulfonated azo dyes in biological reactor effluents using LC-ESI-MS/MS analysis

Most studies on the biodegradation of textile azo dyes use color as parameter for measuring the efficiency of degradation. Although widely employed, spectrophotometric methods are susceptible to the interference of metabolites or degradation products from the biological treatment. We propose a method for determination of a model sulfonated azo dye (Direct Black 22, DB22) in wastewater using solid-phase extraction (SPE) and liquid chromatography - electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). MS analysis in negative electrospray ionization mode showed DB22 as the most abundant precursor ion, corresponding to [M-3Na + H]2-, which yields two radical anions of m/z 370.1 and m/z 645 after MS/MS fragmentation by collision-induced dissociation (CID). Calibration curve presented adequate linearity and precision in the range of 120-1500 ng mL-1, and recovery and detection limit were appropriate to the typically employed working concentrations. Nevertheless, we observed that standard heating of DB22 under alkaline conditions to simulate the production of wastewater during dye-baths resulted in loss of MS/MS signal, without affecting color. Further analysis showed that DB22 undergoes hydrolysis and does not remain unaltered in solution. Alternative methods of hydrolysis evaluated resulted in no MS/MS signal as well. SPE-LC-ESI-MS/MS analysis evidenced the structural change of DB22 in aqueous solution while the dyeing-capacity was preserved. This technique has also the potential of being tailored to consider the detection of the hydrolyzed fragments of azo dyes in wastewater for appropriate quantification, but it was not the scope of the current step of this research. Color remains as a more reliable parameter for monitoring azo compounds which are unstable in aqueous solution, while a more robust and holistic method needs to be developed for the speciation of the DB22 products of thermal hydrolysis.