Formation and occurrence of iodinated tyrosyl dipeptides in...

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Article

Formation and occurrence of iodinated tyrosyldipeptides in disinfected drinking water

Guang Huang, Ping Jiang, Lindsay K Jmaiff Blackstock, Dayong Tian, and Xing-Fang LiEnviron. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06276 • Publication Date (Web): 28 Feb 2018

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1

Formation and Occurrence of Iodinated Tyrosyl Dipeptides 2

in Disinfected Drinking Water 3

Guang Huang,1,¶

Ping Jiang,1,¶

Lindsay K. Jmaiff Blackstock,1 Dayong Tian,

1,2 Xing-Fang Li

1* 4

5

1. Division of Analytical and Environmental Toxicology, Department of Laboratory 6

Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta 7

Edmonton, AB Canada T6G 2G3 8

2. College of Chemical and Environmental Engineering, Anyang Institute of Technology, 9

Anyang 455000, Henan, P. R. China 10

11

Corresponding author: Xing-Fang Li, [email protected], 1-780-492-5094 12

13

14

Page 1 of 24

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Environmental Science & Technology

Abstract 15

Iodinated disinfection byproducts (I-DBPs) are highly toxic, but few precursors of I-DBPs 16

have been investigated. Tyrosine containing biomolecules are ubiquitous in surface water. Here 17

we investigated the formation of I-DBPs from the chloramination of seven tyrosyl dipeptides 18

(i.e., tyrosylglycine, tyrosylalanine, tyrosylvaline, tyrosylhistidine, tyrosylglutamine, 19

tyrosylglutamic acid, and tyrosylphenylalanine) in the presence of potassium iodide. High 20

resolution mass spectrometry (HRMS) and tandem mass spectrometry (MS/MS) analyses of the 21

benchtop reaction solutions found all seven precursors formed both I- and Cl- substituted tyrosyl 22

dipeptide products. Iodine substitutions occurred on the 3- and 3,5-positions of the tyrosyl-23

phenol ring while chlorine substituted on the free amino group. To reach the needed sensitivity to 24

detect iodinated tyrosyl dipeptides in authentic waters, we developed a high performance liquid 25

chromatography (HPLC)-MS/MS method with multiple reaction monitoring mode and solid 26

phase extraction. HPLC-MS/MS analysis of tap and corresponding raw water samples, collected 27

from three cities, identified four iodinated peptides: 3-I-/3,5-di-I-Tyr-Ala and 3-I-/3,5-di-I-Tyr-28

Gly in the tap, but not the raw waters. The corresponding precursors, Tyr-Ala and Tyr-Gly, were 29

also detected in the same tap and raw water samples. This study demonstrates that iodinated 30

dipeptides exist as DBPs in drinking water. 31

32

33

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Introduction 34

Chemical disinfectants used in drinking water treatment plants (DWTPs) prevent 35

waterborne disease outbreaks.1 Disinfection byproducts (DBPs) are inevitably formed from 36

reactions between the disinfectant and dissolved organic matter (DOM). The first group of DBPs 37

(i.e., trihalomethanes, THMs) in chlorinated drinking water were discovered in 1974.2,3

Since 38

then, epidemiological studies have found associations between long term consumption of 39

chlorinated drinking water and an increased risk of bladder cancer.4,5

While 11 DBPs are 40

regulated by the United States Environmental Protection Agency, none of these regulated DBPs 41

cause bladder cancer in animals.6 Over 600 DBPs have been identified in drinking water 42

including: THMs, haloacetic acids (HAAs), haloacetonitriles (HANs),7 halonitromethanes 43

(HNMs), cyanogen chloride (CNCl),8 nitrosamines,

9 halobenzoquinones,

10 44

halohydroxybenzaldehydes, halosalicylic acids, and trihalophenols11

among many others. 45

Despite intense efforts made to discover new DBPs, the known halogenated DBPs only account 46

for approximately 30% of the total organic halogens (TOX) formed during chlorination.8 Health 47

concerns over DBP exposure have currently driven analytical identification of unknown DBPs of 48

toxicological relevance.12,13

49

The exploration for unknown iodinated DBPs (I-DBPs) has recently drawn increased 50

attention because of the high cytotoxicity and genotoxicity of iodoacetic acids (IAAs).14

Iodide 51

exists naturally in many surface and groundwater sources.14-17

Source waters can have elevated 52

iodide concentrations due to impacts by natural and anthropogenic drivers such as salt water 53

intrusion,18,19

geomorphological deposits,20

hydraulic fracturing activities21

or wastewater 54

discharge.22,23

Furthermore, to comply with regulatory guidelines, many DWTPs have switched 55

their disinfection method from chlorination to chloramination to reduce the formation of 56

regulated THMs and HAAs in finished water. Chloramination oxidizes iodide to the reactive 57

hypoiodous acid (HOI), whereas chlorination can further oxidize HOI to iodic acid (HIO3) that 58

does not react with DOM. As a result, chloramination promotes greater formation of I-DBPs than 59

chlorination.8,24-26

Among the discovered halogenated DBPs, in vitro27

and in vivo28

studies have 60

identified a trend in cyto- and genotoxicity: I- > Br- >> Cl-DBPs. For example, IAAs are one to 61

two orders of magnitude more cytotoxic and genotoxic than their chlorinated analogs.29

62

Additionally, recently discovered phenolic/aromatic I-DBPs show significantly higher 63

developmental toxicity and growth inhibition than aliphatic I-DBPs.30

With elevated demand on 64

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limited fresh water sources coupled with the change from chlorination to chloramination in 65

compliance of regulatory guidelines, the potential for occurrence of I-DBPs in finished drinking 66

water is increasing.

67

So far, several I-DBPs have been identified in drinking water.14,31-34

In a study covering 68

the drinking water distribution systems of 23 cities in the United States, highly toxic IAAs and I-69

THMs were detected with concentrations ranging from sub µg/L to several µg/L levels.14

Polar I-70

DBPs, including mainly polar I-aromatic acids and I-phenols, have been detected in simulated 71

drinking water prepared using commercial natural organic matter standard reference 72

materials.32,33

Overall compared to chlorinated DBPs, far fewer studies have reported the 73

precursors or occurrence of I-DBPs. A primary reason may be that unlike chlorinated 74

compounds, iodinated organic compounds lack distinct isotopic patterns, making them more 75

difficult to identify at low concentrations in authentic water with mass spectrometry. To reveal 76

unknown I-DBPs, an effective approach is to examine the precursors in source water and monitor 77

their reactions with disinfectants for the potential generation of iodinated by-products. Currently, 78

humic substances are known precursors for polar iodinated compounds not only during water 79

disinfection processes,33

but also in other natural processes like photoiodination35

due to their 80

available carboxylic or phenolic groups. Phenol can generate I-phenols in drinking water with 81

the presence of iodide.36

However, studies on other biomolecules containing a phenol moiety as 82

precursors for I-DBPs are rare. 83

The amino acid tyrosine, or peptides and proteins consisting of tyrosine, could be 84

potential precursors for I-DBPs. Tyrosine’s structure contains two reactive sites. First, 85

electrophilic reactants have affinity towards the amino group. Reactions between amines and 86

disinfectants, such as HOCl, occur readily. Second, the phenol side chain of tyrosine can also be 87

attacked by electrophiles. Phenolic groups have been reported to be essential for the formation of 88

I-DBPs.33,36

In practice, the potential for large proteins (i.e., MW ≥ 10 KDa) containing tyrosine 89

residues to serve as I-DBPs precursor is unlikely. Typically, large proteins are effectively 90

removed prior to disinfection by common water treatments processes such as coagulation and/or 91

filtration,37

whereas lower molecular weight peptides can pass through filtration because of their 92

relatively small size and high solubility in water. The remaining small peptides could be broken 93

down into smaller peptides and amino acids in the DWTP by biological and oxidative 94

processes,38

potentially increasing small peptides in the water prior to disinfection. Previously 95

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our group identified over 600 chemical signatures matching with small peptides in the Human 96

Metabolome Database (HMDB) using our strategic workflow for non-target analysis of peptides 97

in drinking water.39

Many of these signatures correspond to tyrosine containing peptides. 98

Recently, our study showed that chlorination of tyrosyl dipeptides can generate chlorinated 99

tyrosyl-dipeptides.40,41

N-chlorinated tyrosylglycine (Tyr-Gly) and N-chlorinated tyrosylalanine 100

(Tyr-Ala) were detected in drinking water.40

However, potential reactions from chloramination of 101

water containing tyrosyl dipeptides and iodine has not be reported in the literature. 102

In this study, we aimed to investigate tyrosyl dipeptides as precursors of I-DBPs. We first 103

performed controlled laboratory reactions to demonstrate the generation of I-DBPs from the 104

chloramination of tyrosyl dipeptides in the presence of iodide. Chloramination was investigated 105

due to its frequent use and reports of increased formation of I-DBPs compared to chlorination. 106

Seven tyrosyl dipeptides including Tyr-Gly, Tyr-Ala, tyrosylvaline (Tyr-Val), 107

tyrosylphenylalanine (Tyr-Phe), tyrosylglutamic acid (Tyr-Glu), tyrosylglutamine (Tyr-Gln), and 108

tyrosylhistidine (Tyr-His) were selected to represent the 20 common amino acids. The simulated 109

reactions were carried out at both high and environmentally relevant concentrations of tyrosyl 110

dipeptides. The identities of the iodinated Tyr-Ala products were confirmed through comparison 111

of standards using high resolution mass spectrometry (HR-MS) and liquid chromatography with 112

tandem mass spectrometry (LC-MS/MS). The iodinated products of the other six tyrosyl 113

dipeptides, whose standards are not available, were identified based on the accurate mass and 114

MS/MS spectra. In the same chloramination reactions, chlorinated tyrosyl dipeptides also 115

formed, but these differed in substitution site from iodinated tyrosyl dipeptides. The rationale for 116

the difference between the formation of I-DBPs and Cl-DBPs was discussed. Factors that may 117

affect the formation of the I-DBPs in water, such as pH and stability, were also studied. Lastly, 118

using our new method of solid phase extraction (SPE) and LC-MS/MS with multiple reaction 119

monitoring (MRM) mode we confirmed the occurrence of iodinated tyrosyl dipeptides as DBPs 120

in authentic drinking water. 121

122

Experimental Section 123

Chemicals and Materials. Potassium iodide (KI), ascorbic acid, tyrosine (Tyr), 3-iodo-L-124

tyrosine (3-I-Tyr), 3,5-di-iodo-L-tyrosine (3,5-di-I-Tyr), 3-chloro-L-tyrosine (3-Cl-Tyr), Tyr-Gly, 125

Tyr-Ala, Phe-Gly, Tyr-Val, Tyr-His, Tyr-Gln, Tyr-Glu, and Tyr-Phe were purchased from 126

Page 5 of 24



Sigma-Aldrich (St. Louis, MO). Optima LC/MS grade water, methanol, and acetonitrile were 127

purchased from Thermo Fisher Scientific (Fair Lawn, NJ). 3-Iodo-Tyr-Ala (3-I-Tyr-Ala), and 128

3,5-di-iodo-Tyr-Ala (3,5-di-I-Tyr-Ala) were obtained from the Chinese Peptide Company 129

(Hangzhou, China). Ammonium chloride and sodium bicarbonate were obtained from Acros 130

Organics (Thermo Fisher Scientific). LC/MS grade formic acid (FA, 49−51%), sodium 131

hydroxide and sodium phosphate monobasic were obtained from Sigma-Aldrich (St. Louis, MO). 132

The concentration of free chlorine in the sodium hypochlorite solution was measured to be 120 133

mg/mL by a chlorine amperometric titrator (Autocat 9000, HACH). 134

Chloramination of Dipeptides with the Addition of Iodide. Seven dipeptides, including Tyr-135

Ala, Tyr-Gly, Tyr-Val, Tyr-His, Tyr-Gln, Tyr-Glu, and Tyr-Phe (0.1 mg of each, total dipeptides, 136

2.5 µmol), were mixed and dissolved in 100 mL of phosphate buffer (10 mM, pH=7) spiked with 137

2.0 mg/L KI. We used a higher concentration of I- than typically found in source waters (i.e., ≤ 138

0.2 mg/L) to facilitate formation and detection of iodinated DBPs. Controlled reactions were 139

performed in phosphate buffer (10 mM), including chloramination of dipeptides without iodide, 140

and dipeptides in the presence of iodide without chloramination. The phosphate buffer was 141

prepared by adding monobasic potassium phosphate (0.1200 g, 1 mmol) into 100 mL Optima 142

water, and adjusting the pH to 7.0 with sodium hydroxide solution (1 M). Note: the phosphate 143

buffer was prepared fresh from potassium phosphate because the commercial phosphate buffer 144

solution contains iodide (determined using ICP-MS, data not shown). Monochloramine (5.0 145

µmol) freshly prepared, was added into the dipeptide mixture solution at a molar ratio of 146

monochloramine/total dipeptides at 2/1. After the mixture reacted in dark at room temperature 147

for 24 h, an aliquot of 0.5 mL of 50% FA was added to quench the reaction. The quenched 148

solution was desalted and concentrated by solid phase extraction (SPE) with Waters Oasis HLB 149

cartridges (6 mL, 200 mg per cartridge) mounted in a VISIPREP SPE manifold (Supelco, 150

Bellefonte, PA) with flow control liners. The HLB cartridge was first rinsed twice with 6 mL of 151

methanol containing 0.25% FA, then rinsed twice by 6 mL of acidified water with 0.25% FA. 152

The sample was drawn through the cartridge under vacuum at a flow rate of approximately 8 153

mL/min. After sample loading, the cartridge was washed twice with 6 mL of acidified water 154

(0.25% FA) and then dried under vacuum for 10 min. The analytes on the cartridge were eluted 155

with 10 mL of methanol (0.25% FA), then the methanol extract was divided into two equal 156

portions. One portion was analyzed using the high resolution quadrupole time-of-flight (X500R 157

Page 6 of 24



QTOF System) instrument (Sciex, Concord, Ontario, Canada) with direct infusion to acquire 158

both MS and MS/MS spectra. The remaining 5 mL methanol extract was evaporated down to 159

100 µL under a gentle nitrogen stream, and then reconstituted with Optima water to a volume of 160

1100 µL water/methanol (v/v 10/1). After brief sonication and vortexing, a 20-µL aliquot of the 161

reconstituted SPE extract (1100 µL) was diluted with Optima water to 1000 µL. Finally, a 25-µL 162

aliquot of the 1000 µL solution was further diluted with Optima water to a volume of 1000 µL. 163

This final diluted extract solution was analyzed with HPLC-MS/MS in MRM mode (Sciex 164

QTRAP 5500). 165

Iodinated and chlorinated tyrosyl dipeptides were expected to form in chloramination 166

reactions. We utilized different strategies to confirm the structures of these halogenated products. 167

First, to confirm the structures of iodinated Tyr-Ala products, we used the commercially 168

available standards 3-I-Tyr-Ala and 3,5-di-I-Tyr-Ala. The standards were analyzed with HR-MS 169

and HPLC-MS/MS in MRM mode to compare with the resulting reaction products. In addition, 170

the chloramination of Tyr was carried out to support the identification as described in SI Section 171

1. Second, based on the iodinated structures we identified for Tyr-Ala and Tyr, we predicted the 172

analogous iodinated products for other tyrosyl dipeptides. Lastly, to explore the chlorination site 173

on the dipeptides, we used a recently published strategy41

utilizing the specific reductive nature 174

of ascorbic acid to N-Cl- bonds, with details provided in SI Section 2. After the halogenated 175

compounds were identified, we evaluated the recovery of the tyrosyl dipeptides and their 176

iodinated products using commercially available standards (i.e., 7 tyrosyl dipeptides, 3-I-Tyr-Ala 177

and 3,5-di-I-Tyr-Ala). The details are described in SI Section 3. 178

Effect of pH on the Formation of Iodinated Dipeptides. To explore the effect of pH on the 179

generation of iodinated tyrosyl dipeptides, a series of chloramination reactions of seven 180

dipeptides (0.1 mg each, total dipeptides, 2.5 µmol) were performed in 100 mL of phosphate 181

buffer containing 2.0 mg/L KI at different pH, including pH 6.0, 7.0, 8.0 and 9.0, at a molar ratio 182

of monochloramine/total dipeptides of 2/1. The reaction solutions were kept in the dark at room 183

temperature for 24h. Next, all solutions were quenched and prepared with SPE as described 184

above. After SPE, the diluted extracts were analyzed by direct infusion on X500R QTOF system. 185

Stability of the Iodinated Tyrosyl Dipeptides. The 3-I- and 3,5-di-I-Tyr-dipeptides were 186

synthesized by chloramination of the seven corresponding tyrosyl dipeptides at pH 7.0. The 187

details of synthesis, extraction, and preparation of the extracts for LC-MS/MS analysis are 188

Page 7 of 24



described in SI Section 4. Phe-Gly was added to the extracts as an internal standard because it 189

does not react with other halogenated dipeptides, and no other compounds are converted to Phe-190

Gly. The final diluted extracts in Optima water were kept at 4 oC in the autosampler and 191

analyzed by HPLC-MS/MS. We determined the peak areas of the iodinated DBPs daily using 192

HPLC-MS/MS in MRM mode (Sciex QTRAP 5500) over 8 days. All the peak areas of iodinated 193

DBPs were normalized to that of Phe-Gly. The relative value of the normalized peak area to that 194

of the first injection was calculated and plotted versus time (days) to illustrate the stability of 195

each iodinated DBP. The residual tyrosyl dipeptides and the yields of 3-I/3,5-di-I-Tyr-Ala were 196

determined using the standards and are described in SI Section 5. 197

Sample Collection and SPE extraction. Water samples were collected by grab sampling in 4 L 198

amber glass bottles that were pre-cleaned with methanol and water. Tap water was collected 199

from City 1 in July 2017 and from City 2 and City 3 in June 2017. Raw water was collected from 200

City 1 in April 2017 and City 3 in June 2017. All the water samples were filtered through a glass 201

microfiber filter (47 mm × 1.5 µm, Waterman) and a nylon membrane disk filter (47 mm × 0.45 202

µm), then stored at 4 °C prior to analysis. 203

Two liters each of Optima water and authentic water samples including three tap waters (T1, 204

T2, and T3) from Cities 1, 2, and 3 and two raw waters from Cities 1 and 3 (R1 and R3) were 205

extracted using the same SPE procedures described above. The methanol extract was evaporated 206

down to 100 µL and then reconstituted with Optima water to a final volume of 1100 µL 207

water/methanol (v/v 10/1). 208

High Resolution Mass Spectrometry Analysis. A QTOF mass spectrometer (SCIEX X500R 209

QTOF) with a Turbo-V electrospray ionization (ESI) source was used to determine the accurate 210

masses for the parent and product ions (i.e., MS and MS/MS) of the seven dipeptides. The 211

methanol extract from the chloramination solution was investigated by direct infusion with the 212

flow rate at 7 µL/min. All the experiments were performed in positive mode with the following 213

parameters: ionspray voltage, 5500 V; declustering potential (DP), 80 V; temperature, 0 °C; gas 214

1 (spray gas, N2), 25 arbitrary units; gas 2 (heat conduction gas, N2), 0 arbitrary units; curtain gas 215

(N2), 25 arbitrary units; accumulation time, 0.25 s; collisionally activated dissociation (CAD) gas, 216

7 arbitrary units; and scan range, 100−800 m/z unless otherwise indicated. SCIEX OS software 217

version 1.2 was used for data analysis. 218

Page 8 of 24



HPLC-MS/MS (MRM) Method. An Agilent 1290 series LC system consisting of a binary 219

pump and an autosampler with temperature control (Agilent, Waldbronn, Germany) was used for 220

LC separation with a Luna C18(2) column (100 × 2.0 mm i.d., 3 µm; Phenomenex, Torrance, CA) 221

at room temperature (22 °C). The mobile phase consisted of solvent (A) water containing 0.1% 222

FA and solvent (B) acetonitrile containing 0.1% FA. The flow rate of the mobile phase was 170 223

µL/min, and the injection volume was 20 µL. A gradient program was performed as follows: 224

linearly increased B from 5% to 70% in 15 min; kept B at 95% for 9 min; changed B to 5% for 225

column equilibration at 24.01−33 min. The autosampler was kept at 4 oC. 226

HPLC-MS/MS with MRM mode was performed using a triple quadrupole ion-trap tandem 227

mass spectrometer (Sciex QTRAP 5500) to confirm the iodinated dipeptides in both the 228

chloraminated dipeptide solutions and the authentic water samples. The MS instrumental 229

parameters were optimized as follows: ion-spray voltage, 5500 V; source temperature, 500 °C; 230

gas 1, 45 arbitrary units; gas 2, 40 arbitrary units; curtain gas, 30 arbitrary units; accumulation 231

time for each ion pair, 50 ms. The MRM ion pairs and the optimized values of DP, collision 232

energy (CE), and cell exit potential (CXP) are listed in supporting information (SI) Table S1. 233

Analyst software version 1.5.2 for Sciex QTRAP 5500 was used for data analysis. 234

235

Results and Discussion 236

Identification of Halogenated Products from Chloramination of Dipeptides 237

Seven tyrosyl dipeptides were chosen for the investigation of the formation of iodinated 238

tyrosyl dipeptides during chloramination due to their possible existence in surface water.39,40

239

Figure 1 is a full scan high resolution mass spectrum of the chloraminated solution containing 240

seven tyrosyl dipeptides and iodide. The peaks marked with blue lines have the mass-to-charge 241

ratio (m/z) values that match with the theoretical m/z of the tyrosyl dipeptides within 5 ppm, 242

indicating they are the remaining dipeptides. For example, in Figure 1, the remaining Tyr-Ala 243

was detected at m/z 253.1180 [M+H]+

with mass accuracy of 1.1 ppm. The other peaks labeled in 244

red in Figure 1 represent newly formed halogenated dipeptides resulting from chloramination. In 245

the example of Tyr-Ala, a new pair of peaks with m/z of 379.0134 and 504.9114 differing from 246

the m/z of Tyr-Ala by 125.9 and 251.8 Da appeared. These observed mass differences 247

correspond to the substitution of one (-1) or two protons (-2) by one (+127) or two iodine atoms 248

(+254), resulting in the formation of mono-iodinated and di-iodinated Tyr-Ala (theoretical mass 249

Page 9 of 24



m/z 379.0149 and 504.9116, respectively). Similarly, a group of peaks with m/z 287.0791, 250

321.0402 and 412.9764 in Figure 1 differing from Tyr-Ala by ~34.0, 68.0 and 159.9, represents 251

the [M+H]+

of mono-chlorinated, di-chlorinated, and mono-chlorinated and mono-iodinated Tyr-252

Ala, respectively. For each of the other six tyrosyl dipeptides studied, five corresponding 253

halogenated products including mono-I-, di-I-, mono-Cl-, di-Cl-, and mono-I-mono-Cl- were 254

detected after chloramination in the presence of iodide. A total of 35 new DBPs have been 255

detected in Figure 1. Among them, the iodinated product signals are much more intense than the 256

chlorinated products. The details of the halogenated tyrosyl dipeptides, including structures, 257

theoretical m/z, measured m/z, molecular formula, mass accuracy (∆m, ppm), S/N, and peak 258

intensity are described in SI Table S2. 259

For the controlled reactions, chloramination of tyrosyl dipeptides without iodide resulted 260

in no formation of iodinated dipeptides. N-Cl-/N,N-di-Cl-tyrosyl dipeptides can be observed 261

(data not shown) and this formation from chlorination has been previously reported in detail.40

262

For the tyrosyl dipeptides mixed with iodide without chloramination, no iodinated or chlorinated 263

products were formed. Only the tyrosyl dipeptide starting materials could be detected (data not 264

shown). Compared with the results in Figure 1, it is evident that the iodinated tyrosyl dipeptides 265

are produced only from chloramination in the presence of iodide. In principle, during 266

chloramination of tyrosyl dipeptides in the presence of iodide, both iodination and chlorination 267

can occur via electrophilic substitution. Iodination involves hypoiodous acid (HOI) that is 268

oxidized from iodide, while chlorination involves monochloramine (H2N-Cl). Tyrosyl dipeptides 269

have two nucleophilic sites: one is the ortho- position of aromatic ring that is activated by the 270

hydroxyl group, the other is the amino group.41-43

Thus, halogenated tyrosyl dipeptides detected 271

in Figure 1 could be halogenated either on the aromatic ring or the amino group. In this study, 272

we have developed methods to elucidate the structure of each halogenated dipeptide. We found 273

that iodine substitutes on the aromatic ring to form aromatic I-DBPs while chlorine substitutes on 274

the amino group during chloramination of tyrosyl dipeptides in the presence of iodine. 275

To identify the structures of the discovered iodinated tyrosyl dipeptides, we further 276

performed LC-MS/MS analysis in MRM mode and QTOF/MS in product ion mode. We first 277

tackled the determination of some of the iodinated tyrosyl dipeptides (mono-I- and di-I- Tyr-Ala) 278

using authentic standards. Figure 2 shows that the mono-I- and di-I-Tyr-Ala generated in the 279

chloraminated solution match the retention time, paired MRM transitions, and paired MRM 280

Page 10 of 24



transition intensity ratios with those of the 3-I- and 3,5-di-I-Tyr-Ala standards. These results 281

support the generation of iodinated peptides via iodine substitution on the phenol ring. The 282

fragments in the high-resolution MS/MS spectra (Figure 3) also match with those from the 283

standards. SI Table S3 presents the MS/MS fragments of iodinated Tyr-Ala, including measured 284

accurate m/z, molecular formula and mass accuracy (∆m, ppm). Because the iodination occurred 285

on the phenol ring of the tyrosyl residue, chloramination of tyrosine was also conducted to 286

confirm the iodination pathway (SI Section 1). Similarly, 3-I- and 3,5-di-I-Tyr were identified as 287

the iodinated products, supported by comparing their extracted ion chromatograms (EIC) and 288

MS/MS spectra to those of the commercially available standards (Figure S1-S2 and SI Table 289

S4). Other studies have also observed the formation of 3-I- and 3,5-di-I- products from the 290

chloramination of L-tyrosyl-L-arginine with the addition of iodide.44

These results together 291

confirmed that iodination of tyrosyl peptides occur on the phenol ring of tyrosine during 292

chloramination of water containing iodine. 293

Based on this generic rule of iodination, we projected that the iodinated products for the 294

other six tyrosyl dipeptides are 3-I- and 3,5-di-I- products despite that their standards are not 295

commercially available. The MS/MS fragments confirm our prediction. For example, 296

characteristic fragments either m/z 232.9 or 272.9 for 3-I-Tyr-Ala, and both m/z 232.9 and 387.8 297

for 3,5-di-I-Tyr-Ala were detected on the MS/MS spectrum of the chloraminated tyrosyl 298

dipeptide solution (Figure 3). These four characteristic fragments have been observed in the 299

MS/MS spectra of the iodinated products corresponding to the six other tyrosyl dipeptides (SI 300

Figures S3-S8). Thus, for all the tyrosyl dipeptides, chloramination in the presence of iodide 301

generated 3-I- and 3,5-di-I-Tyr-products, which are all aromatic I-DBPs. No peaks 302

corresponding to substitution of iodine on the nucleophilic amino group of the tyrosyl dipeptides 303

were detected. 304

Because of the lack of standards for the chlorinated tyrosyl dipeptides, we used the reductive 305

nature of ascorbic acid41

specific to N-Cl- bonds (as briefly described in SI Section 2) to confirm 306

the structures of mono-Cl-, di-Cl-, and mono-I-mono-Cl. For the chloraminated solution 307

quenched by ascorbic acid, the peaks of mono-Cl- and di-Cl-Tyr-Ala disappeared, and mono-I-308

mono-Cl-Tyr-Ala became weaker (SI Figure S9). Similar decreased signals for the mono-Cl-, 309

di-Cl- and mono-I-mono-Cl- products of other tyrosyl dipeptides were also observed (SI Figures 310

S10-S15). The disappearance of the chlorinated tyrosyl dipeptide peaks in the presence of 311

Page 11 of 24



ascorbic acid provides support that chlorine substitutes on the amino group of Tyr-Ala under 312

chloramination conditions. The chlorinated Tyr-Ala formed in the chloramination solution are 313

consistent with N-Cl-/N,N-di-Cl-Tyr-Ala identified in our previous study of chlorination of Tyr-314

Ala.40

315

The hard-soft-acid-base (HSAB) theory45

can account for observed trend: during 316

chloramination, iodine substitutes on phenolic group of tyrosyl dipeptides and chlorine 317

substitutes on amino group of tyrosyl dipeptides. Monochloramine is a harder acid compared to 318

HOI due to the lower polarizability and smaller diameter of chlorine in H2N-Cl than iodine in 319

HOI. The amino group is a harder base in contrast to phenolic group due to its higher electron 320

density and smaller diameter. The hard acid H2N-Cl shows strong interaction with the hard base 321

of the amino group, while the soft acid HOI tends to interact with soft base of phenolic group. 322

This supports the observed difference for chlorine and iodine substitution on dipeptides that 323

occurred during chloramination. 324

The typical concentration of dipeptides in surface water is low (e.g., concentration of 325

dissolved organic nitrogen (DON), 0.37 mg/L as N in surface water).46

To relate our study to an 326

environmentally relevant concentration of dipeptides, we also performed chloramination of a low 327

concentration mixture of the seven tyrosyl dipeptides (i.e., 0.02 mg of each dipeptide = 0.16 328

mg/L as N in phosphate buffer at pH 7.0) in the presence of iodide. Under these conditions, 3-I-, 329

3,5-di-I-, and N-Cl-dipeptides were clearly formed, but no N,N-di-Cl-dipeptides or N-Cl-3-I-330

dipeptides were detected. This indicates that 3-I-, 3,5-di-I-, N-Cl-dipeptides can be potentially 331

generated from the chloramination of real raw water and may exist in our drinking water due to 332

the wide use of chloramination as the disinfectant in drinking water treatment plants. Our 333

previous study has confirmed the occurrence of N-Cl-dipeptides in drinking water treated with 334

chlorination.40

Therefore, we will focus on 3-I-, 3,5-di-I-dipeptides formation during 335

chloramination in the following sections. 336

Effect of Sample pH on the Formation of Iodinated Dipeptides. 337

In an aqueous reaction solution containing tyrosyl dipeptides, H2N-Cl and iodide, iodide is 338

first oxidized to HOI by H2N-Cl. HOI can then substitute on the aromatic ring of the tyrosyl 339

dipeptides to form iodinated dipeptides. Sample pH is a key parameter affecting the formation of 340

the iodinated dipeptides, because the stability of H2N-Cl and iodinated DBPs are pH dependent. 341

A series of chloramination reactions of seven tyrosyl dipeptides were performed in 100 mL of 342

Page 12 of 24



phosphate buffer at pH 6.0, 7.0, 8.0, and 9.0. Figure 4 shows the abundance of iodinated Tyr-Ala 343

first increased when the pH increased from 6.0 to 7.0, then decreased when changing the pH 344

from 7.0 to 9.0. As previously reported, H2N-Cl can be hydrolyzed to hypochlorous acid (HOCl) 345

at pH 6.0 (Eq. 1),22,47

and subsequently the HOCl can oxidize HOI to iodate (IO3-) (Eq. 2), 346

decreasing the concentration of HOI. Therefore, the formation of iodinated Tyr-Ala at pH 6.0 347

was less than pH 7.0. At alkaline pH 7.0–9.0, although over 96% of HOI (pKa=10.4) remains in 348

the neutral form, the decreased formation of iodinated dipeptides from pH 7 to pH 8 and 9 are 349

likely due to their hydrolysis. The iodine-carbon bond (I-C) in the organic iodine compounds is 350

susceptible to break to form a carbenium ion due to the large diameter and relatively low 351

electronegativity of iodine. The carbenium ion can be attacked by a nucleophile, like hydroxyl 352

group at alkaline pH, leading to hydrolysis.48,49

Consequently, because of the hydrolysis, a 353

decrease of the total organic iodine in drinking water disinfected by chloramination has been 354

reported at alkaline pH.48,49

Over the pH range studied, similar results were obtained for the 355

iodinated products generated from the other six dipeptides (i.e., Tyr-Gly, Tyr-Val, Tyr-His, Tyr-356

Gln, Tyr-Glu, and Tyr-Phe) (SI Figure S16-S21). Overall pH 7 was found to be the optimal 357

condition for the formation of iodinated dipeptides. 358

NH2Cl + H2O → HClO + NH3 (1) 359

2HClO + HIO → IO3-+ 2Cl

- + 3H

+ (2) 360

Stability of the Iodinated Dipeptides. Having confirmed formation of iodinated tyrosyl 361

dipeptides from chloramination in bench top experiments, we next investigated the stability of 362

these compounds to assess the potential formation of iodinated tyrosyl dipeptides in drinking 363

water treatment process. SI Figure S22 shows that more than 80% of I-Tyr-peptides remained in 364

the synthesized standard solution in Optima water after 8 days, indicating that the I-Tyr-peptides 365

are stable in Optima water under the laboratory conditions. These stability results indicate the 366

potential existence of iodinated dipeptides in chloraminated drinking water containing iodine. 367

Therefore, we further determined these iodinated peptides in authentic raw and chloraminated 368

drinking water samples. 369

Determination of Iodinated Dipeptides in Authentic Water Samples. Detection of iodinated 370

dipeptides has challenges, as they are present in finished water at low concentrations due to the 371

low concentration of dipeptides and iodide in raw water. Also, sample matrices may interfere 372

with the detection of iodinated DBPs. To overcome these problems, we have developed a new 373

Page 13 of 24



LC-MS/MS method using MRM mode. Optimized parameters for the MRM ion transitions of 374

the iodinated dipeptides, including DP, EP, CE, and CXP, are described in SI Table S1. The 375

iodinated tyrosyl dipeptides formed after chloramination of tyrosyl dipeptides in the presence of 376

iodide were used as standards, while commercially available standards of 3-I- and 3,5-di-I-Tyr-377

Ala were also used. We analyzed the standard dipeptide chloramination solution (Std); treated 378

water, including T1- finished water (City 1), T2- tap water (City 2), and T3- tap water (City 3); 379

raw water, including R1- (City 1) and R3 - (City 3); and a B- blank (Optima water). Criteria for a 380

positive detection included retention time, paired MRM transitions, and intensity ratio of the 381

paired MRM transitions matching with the commercially available or laboratory synthesized 382

standards. Using these three criteria, neither dipeptides nor their iodinated products were 383

detected in the blank (Figure 5). In both raw water samples, two tyrosyl dipeptides: Tyr-Ala and 384

Tyr-Gly were detected, as shown in Figure 5a and SI Figure S23(a). This is consistent with 385

previous reports that peptides containing Gly and Ala are more abundant in surface water than 386

peptides containing other amino acids.50

In the tap water samples, we detected two dipeptides 387

and the corresponding four iodinated products: Tyr-Ala, 3-I-Tyr-Ala, 3,5-di-I-Tyr-Ala (Figure 388

5(b-c)), Tyr-Gly, 3-I-Tyr-Gly, and 3,5-di-I-Tyr-Gly (SI Figure S23(b-c)). The mono- and di-389

iodinated compounds detected were consistent with the major species found to form at low 390

concentrations and were the most stable of all iodinated tyrosyl dipeptides investigated. The 391

confirmation of iodo-tyrosyl DBPs in authentic water is significant, because other aromatic I-392

DBPs have shown significantly higher developmental effects than Cl-DBPs.30

The new 393

analytical method and occurrence evidence provide necessary tools and information for further 394

studies of toxicity and a survey for a wide range of aromatic iodinated dipeptides, to contribute 395

to understanding the potential health effects of DBPs. 396

397

ASSOCIATED CONTENT 398

Supporting Information: The Supporting Information is available free of charge on the ACS 399

publication website at DOI: Experimental procedures for standard preparation, structural 400

identification, stability, and recovery studies. Mass spectrometry data for the identification of 401

halogenated dipeptides. The effect of pH on iodinated dipeptide formation and additional 402

materials in Sections 1-5, Tables S1-S4, and Figures S1-S23. 403

404

Page 14 of 24



AUTHOR INFORMATION 405

Corresponding Author 406

* Tel: (780) 492-5074, Fax: (780) 492-7800 407

E-mail: [email protected] 408

ORCID: Xing-Fang Li: 0000-0003-1844-7700 409

410

Author Contributions 411

¶ G.H. and P.J. contributed equally to this study. 412

413

ACKNOWLEDGEMENT 414

The authors acknowledge the support of this study by the grants from the Natural Sciences and 415

Engineering Research Council of Canada and Alberta Health. 416

417

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549

550

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551

552 553

Figure 1. Full scan high resolution mass spectrum of the extract of the mixed seven dipeptides 554

(2.5 µmol) in the presence of potassium iodide (2.0 mg/L) and monochloramine (5.0 µmol) after 555

reaction for 24 h. 556

557

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558

559

Figure 2. EIC chromatograms of Tyr-Ala, 3-I-Tyr-Ala, and 3,5-di-I-Tyr-Ala in the standard 560

solution and in the reaction mixture containing seven tyrosyl dipeptides after chloramination in 561

the presence of iodine. 562

563

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564

565

Figure 3. MS/MS spectra of N-Cl-3-I-Tyr-Ala, 3-I-Tyr-Ala, and 3,5-di-I-Tyr-Ala identified in 566

the reaction mixture containing seven tyrosyl dipeptides after chloramination for 24 h in the 567

presence of iodine. 568

569

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570

571

Figure 4. Formation of 3-I-Tyr-Ala, and 3,5-di-I-Tyr-Ala from chloramination of Tyr-Ala in the 572

presence of iodine under different phosphate-buffered pH conditions. 573

574

575

Figure 5. LC-MS/MS chromatograms of (a) Tyr-Ala, (b) 3-I-Tyr-Ala, and (c) 3,5-di-I-Tyr-Ala 576

detected in tap waters T1 (City 1), T2 (City 2), and T3 (City 3) and raw waters R1 (City 1) and 577

R3 (City 3), compared with corresponding standards (Std) and blank (B). 578

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