Ascorbic Acid Assisted High Performance Liquid Chromatography Mass Spectrometry Differentiation of Isomeric C-Chloro- and N-Chloro-Tyrosyl Peptides in Water

Abstract

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.

Formation, Identification, and Occurrence of New Bromo- and Mixed Halo-Tyrosyl Dipeptides in Chloraminated Water

“…32 This was determined by both MS/MS fragmentation data and a previously published threestep ascorbic acid strategy used to differentiate N-Cl and C-Cl isomers. 33 To support the proposed N-Br-and N,N-di-Brtyrosyl dipeptide structure, the three-step ascorbic acid strategy was performed on the brominated dipeptides. As shown in Figure S2, both N-Br-and N,N-di-Br-tyrosyl dipeptide peaks disappear after ascorbic acid treatment, providing evidence that the bromine substituted on the amine group.…”

A Rapid Visual Detection of Ascorbic Acid Through Morphology Transformation of Silver Triangular Nanoplates

“…Therefore, quantitative detection of AA has great importance in pharmaceutical analysis, food industries, and clinical diagnosis. Recently, common analysis techniques for AA including fluorescence [4][5][6][7], chromatography [8,9], electrochemical [10][11][12], and colorimetric [13][14][15] have been extensively developed. Among these strategies, colorimetric methods have tremendous potential in rapid on-site analysis of AA due to the low cost, rapidity, high practicality and simplicity.…”

Cytotoxicity of Halogenated Tyrosyl Compounds, an Emerging Class of Disinfection Byproducts

“…With its two reactive sites, the amino acid tyrosine (Tyr) has been shown to be reactive to chlorination and can form highly stable reaction products, 11 which was also observed with tyrosyl dipeptides. 12 Halogenated tyrosyl compounds have been identified as DBPs in treated tap water 13−15 and in lettuce and spinach washed with treated water. 16 Formation of these compounds is influenced not only by the identity of the peptides present, but also by the presence of free halogen ions, including iodide and bromide.…”

“…Thus, these molecules form a rich precursor pool from which DBPs may be formed under water treatment conditions. With its two reactive sites, the amino acid tyrosine (Tyr) has been shown to be reactive to chlorination and can form highly stable reaction products, which was also observed with tyrosyl dipeptides . Halogenated tyrosyl compounds have been identified as DBPs in treated tap water − and in lettuce and spinach washed with treated water .…”