Ascorbic Acid Assisted High Performance Liquid Chromatography Mass Spectrometry Differentiation of Isomeric C-Chloro- and N-Chloro-Tyrosyl Peptides in 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-35/Cl-37 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 mu g/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.