Iodinated disinfection byproducts (I-DBPs) are highly toxic, but few precursors of I-DBPs have been investigated. Tyrosine-containing biomolecules are ubiquitous in surface water. Here we investigated the formation of I-DBPs from the chloramination of seven tyrosyl dipeptides (tyrosylglycine, tyrosylalanine, tyrosylvaline, tyrosylhistidine, tyrosylglutamine, tyrosylglutamic acid, and tyrosylphenylalanine) in the presence of potassium iodide. High resolution mass spectrometry and tandem mass spectrometry (MS/MS) analyses of the benchtop reaction solutions found that all seven precursors formed both I- and Cl-substituted tyrosyl dipeptide products. Iodine substitutions occurred on the 3- and 3,5-positions of the tyrosyl-phenol ring while chlorine substituted on the free amino group. To reach the needed sensitivity to detect iodinated tyrosyl dipeptides in authentic waters, we developed a high performance liquid chromatography (HPLC)-MS/MS method with multiple reaction monitoring mode and solid phase extraction. HPLC-MS/MS analysis of tap and corresponding raw water samples, collected from three cities, identified four iodinated peptides, 3-I-/3,5-di-I-Tyr-Ala and 3-I-/3,5-di-I-Tyr-Gly, in the tap waters but not in the raw waters. The corresponding precursors, Tyr-Ala and Tyr-Gly, were also detected in the same tap and raw water samples. This study demonstrates that iodinated dipeptides exist as DBPs in drinking water.

We report a new method of ascorbic acid assisted high performance liquid chromatography (HPLC) with high resolution tandem mass spectrometry (HRMS/MS) for the differentiation of isomeric N-chloro (N-Cl) from phenol ring C-chloro (C-Cl) peptides produced during chlorination of water. Using the specific reductive nature of ascorbic acid, we successfully identified the N-Cl isomers and C-Cl isomer, overcoming the difficulty that, due to lack of standards, these isomers cannot be separated by HPLC-HRMS. Using the new approach, we identified 36 new chlorinated products including mono-, di-, tri-, and tetra-Cl-tyrosyl dipeptides in the reaction mixture based on retention time, accurate mass, Cl/Cl isotopic pattern, and characteristic MS/MS fragments. The method was further applied to investigate competitive reactions when mixed tyrosyl dipeptides were chlorinated. Tyrosyl histidine was the most reactive tyrosyl dipeptide in the mixture. The chlorinated products formed are identical when the dipeptides are chlorinated separately or as a mixture. The formation conditions and stability of the chlorinated products were also examined. With increasing chlorine dose, the number of chlorine substituents on the tyrosyl dipeptides increased from products with one/two to three/four Cl atoms. Most of the chlorinated products are stable for up to 9 days. By chlorination of tyrosyl dipeptides spiked into raw water, we projected that chlorinated tyrosyl dipeptides can form during treatment of raw water containing tyrosyl dipeptides even at low μg/L levels. This new method can be utilized for the discovery of a wide range of chlorinated peptide DBPs and the study of their formation and occurrence in water.

Dipeptides are widely present in surface water and serve as precursors to form disinfection byproducts (DBPs) during disinfection (e.g., chloramination). Bromide (Br–) and iodide (I–) are common in many source waters, enhancing Br- and I-DBP formation. Recently Cl-, I-, and Cl-I-dipeptides were identified after chloramination of tyrosyl dipeptides in the presence of I– and were detected in authentic disinfected drinking water samples. However, the formation and occurrence of Br- and mixed halogen (Cl, Br, and/or I)-dipeptides in disinfected water have not been studied. Here we investigated the formation of halogenated dipeptides from three aromatic dipeptides, phenylalanylglycine (Phe-Gly), tyrosylalanine (Tyr-Ala), and tyrosylglycine (Tyr-Gly), under chloramination in the presence of Br– and I– at environmentally relevant levels ([Br–] and [I–], 0 and 0 μg L–1, 6 and 30 μg L–1, 30 and 30 μg L–1, 150 and 30 μg L–1, 300 and 30 μg L–1, and 900 and 30 μg L–1, respectively). For the first time, N-Br- and N,N-di-Br- as well as N-Br-N-Cl- and N-Br-3-I-tyrosyl dipeptides were identified using infusion electrospray quadrupole time-of-flight mass spectrometry. Tyrosyl dipeptides produced N-Cl-, 3-I-/3,5-di-I-, and N-Cl-3-I-tyrosyl dipeptides, while Phe-Gly formed only N-Cl-/N,N-di-Cl-Phe-Gly. To determine halogenated dipeptides in authentic water samples, we developed a new method of solid phase extraction and high-performance liquid chromatography with quadrupole ion trap mass spectrometry using reaction monitoring. 3,5-Di-I-Tyr-Ala and N-Br-Tyr-Ala were detected in treated water but not in the corresponding raw water, warranting further investigation into the occurrence of halogenated peptides in other drinking water systems.

Nitrogenous organics in urine can react with chlorine in swimming pools to form volatile and irritating N-Cl-amines. A urinary marker is desirable for the control of pool water quality. The widespread consumption of acesulfame-K (ACE), a stable synthetic sweetener, and its complete excretion in urine, makes it an ideal urinary marker. Here we report the occurrence of ACE and its potential application in swimming pools and hot tubs. First, we developed a new method for achieving high-throughput analysis of ACE without preconcentration or large-volume injection. Analysis of more than 250 samples from 31 pools and tubs from two Canadian cities showed ACE in all samples. Concentrations ranged from 30 to 7110 ng/L, up to 570-fold greater than in the input tap water. The level of dissolved organic carbon was significantly greater in all pools and tubs than in the input water. Finally, we determined the levels of ACE over 3 weeks in two pools (110000 and 220000 U.S. gallons) and used the average ACE level to estimate the urine contribution as 30 and 75 L. This study clearly shows the human impact in pools and tubs. This work is useful for future studies of the human contribution to DBP formation, epidemiological assessment of exposure, and adverse health effects in recreational facilities.

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