Formation and occurrence of iodinated tyrosyl dipeptides in

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Formation and occurrence of iodinated tyrosyl dipeptides in disinfected drinking water Guang Huang, Ping Jiang, Lindsay K Jmaiff Blackstock, Dayong Tian, and Xing-Fang Li Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06276 • Publication Date (Web): 28 Feb 2018 Downloaded from http://pubs.acs.org on February 28, 2018

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Environmental Science & Technology

1

Formation and Occurrence of Iodinated Tyrosyl Dipeptides

2

in Disinfected Drinking Water

3 4 5 6

Guang Huang,

1,¶

Ping Jiang,

1,¶

Lindsay K. Jmaiff Blackstock,1 Dayong Tian,1,2 Xing-Fang Li1*

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

7

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

8

Edmonton, AB Canada T6G 2G3

9 10

2. College of Chemical and Environmental Engineering, Anyang Institute of Technology, Anyang 455000, Henan, P. R. China

11 12

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

13 14

ACS Paragon Plus Environment


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Abstract

15 16

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

17

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

18

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

19

(i.e.,

20

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

21

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

22

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

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

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

31

dipeptides exist as DBPs in drinking water.

tyrosylglycine,

tyrosylalanine,

tyrosylvaline,

tyrosylhistidine,

32 33


tyrosylglutamine,

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34


Introduction

35

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

36

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

37

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

38

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

39

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

40

chlorinated drinking water and an increased risk of bladder cancer.4,5 While 11 DBPs are

41

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

42

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

43

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

44

(HNMs),

45

halohydroxybenzaldehydes, halosalicylic acids, and trihalophenols11 among many others.

46

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

47

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

48

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

49

toxicological relevance.12,13

cyanogen

chloride

(CNCl),8

nitrosamines,9

halobenzoquinones,10

50

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

51

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

52

exists naturally in many surface and groundwater sources.14-17 Source waters can have elevated

53

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

54

intrusion,18,19 geomorphological deposits,20 hydraulic fracturing activities21 or wastewater

55

discharge.22,23 Furthermore, to comply with regulatory guidelines, many DWTPs have switched

56

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

57

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

58

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

59

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

60

chlorination.8,24-26 Among the discovered halogenated DBPs, in vitro27 and in vivo28 studies have

61

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

62

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

63

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

64

developmental toxicity and growth inhibition than aliphatic I-DBPs.30 With elevated demand on



65

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

66

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

67

water is increasing.

68

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

69

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

70

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

71

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

72

drinking water prepared using commercial natural organic matter standard reference

73

materials.32,33 Overall compared to chlorinated DBPs, far fewer studies have reported the

74

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

75

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

76

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

77

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

78

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

79

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

80

disinfection processes,33 but also in other natural processes like photoiodination35 due to their

81

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

82

the presence of iodide.36 However, studies on other biomolecules containing a phenol moiety as

83

precursors for I-DBPs are rare.

84

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

85

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

86

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

87

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

88

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

89

I-DBPs.33,36 In practice, the potential for large proteins (i.e., MW ≥ 10 KDa) containing tyrosine

90

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

91

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

92

filtration,37 whereas lower molecular weight peptides can pass through filtration because of their

93

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

94

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

95

processes,38 potentially increasing small peptides in the water prior to disinfection. Previously


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

97

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

98

in drinking water.39 Many of these signatures correspond to tyrosine containing peptides.

99

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

100

tyrosyl-dipeptides.40,41 N-chlorinated tyrosylglycine (Tyr-Gly) and N-chlorinated tyrosylalanine

101

(Tyr-Ala) were detected in drinking water.40 However, potential reactions from chloramination of

102

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

103

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

104

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

105

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

106

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

107

Seven

108

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

109

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

110

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

111

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

112

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

113

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

114

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

115

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

116

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

117

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

118

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

119

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

120

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

121

in authentic drinking water.

tyrosyl

dipeptides

including

Tyr-Gly,

Tyr-Ala,

tyrosylvaline

(Tyr-Val),

122 123

Experimental Section

124

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

125

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

126

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



127

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

128

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

129

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

130

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

131

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

132

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

133

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

134

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

135

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

136

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

137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

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

153

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

154

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

155

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

156

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

157

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


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158

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

159

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

160

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

161

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

162

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

163

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

164

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

165

QTRAP 5500).

166

Iodinated and chlorinated tyrosyl dipeptides were expected to form in chloramination

167

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

168

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

169

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

170

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

171

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

172

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

173

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

174

on the dipeptides, we used a recently published strategy41 utilizing the specific reductive nature

175

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

176

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

177

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

178

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

179

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

180

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

181

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

182

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

183

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

184

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

185

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

186

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

187

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

188

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



189

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

190

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

191

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

192

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

193

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

194

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

195

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

196

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

197

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

198

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

199

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

200

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

201

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

202

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

203

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

204

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

205

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

206

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

207

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

208

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

209

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

210

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

211

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

212

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

213

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

214

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

215

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

216

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

217

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

218

version 1.2 was used for data analysis.


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219

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

220

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

221

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

222

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

223

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

224

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

225

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

226

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

227

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

228

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

229

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

230

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

231

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

232

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

233

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

234

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

235 236

Results and Discussion

237

Identification of Halogenated Products from Chloramination of Dipeptides

238

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

239

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

240

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

241

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

242

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

243

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

244

was detected at m/z 253.1180 [M+H]+ with mass accuracy of 1.1 ppm. The other peaks labeled in

245

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

246

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

247

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

248

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

249

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



250

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

251

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

252

the [M+H]+ of mono-chlorinated, di-chlorinated, and mono-chlorinated and mono-iodinated Tyr-

253

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

254

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

255

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

256

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

257

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

258

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

259

intensity are described in SI Table S2.

260

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

261

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

262

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

263

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

264

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

265

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

266

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

267

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

268

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

269

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

270

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

271

hydroxyl group, the other is the amino group.41-43 Thus, halogenated tyrosyl dipeptides detected

272

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

273

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

274

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

275

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

276

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

277

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

278

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

279

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

280

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


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281

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

282

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

283

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

284

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

285

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

286

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

287

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

288

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

289

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

290

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

291

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

292

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

293

chloramination of water containing iodine.

294

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

295

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

296

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

297

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

298

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

299

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

300

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

301

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

302

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

303

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

304

were detected.

305

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

306

nature of ascorbic acid41 specific to N-Cl- bonds (as briefly described in SI Section 2) to confirm

307

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

308

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

309

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

310

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

311

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



312

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

313

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

314

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

315

Ala.40

316

The hard-soft-acid-base (HSAB) theory45 can account for observed trend: during

317

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

318

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

319

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

320

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

321

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

322

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

323

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

324

occurred during chloramination.

325

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

326

dissolved organic nitrogen (DON), 0.37 mg/L as N in surface water).46 To relate our study to an

327

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

328

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

329

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

330

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

331

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

332

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

333

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

334

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

335

chlorination.40 Therefore, we will focus on 3-I-, 3,5-di-I-dipeptides formation during

336

chloramination in the following sections.

337

Effect of Sample pH on the Formation of Iodinated Dipeptides.

338

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

339

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

340

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

341

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

342

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


Page 12 of 24

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343

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

344

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

345

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

346

at pH 6.0 (Eq. 1),22,47 and subsequently the HOCl can oxidize HOI to iodate (IO3-) (Eq. 2),

347

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

348

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

349

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

350

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

351

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

352

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

353

group at alkaline pH, leading to hydrolysis.48,49 Consequently, because of the hydrolysis, a

354

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

355

reported at alkaline pH.48,49 Over the pH range studied, similar results were obtained for the

356

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

357

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

358

condition for the formation of iodinated dipeptides.

359

NH2Cl + H2O → HClO + NH3

(1)

360

2HClO + HIO → IO3-+ 2Cl- + 3H+

(2)

361

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

362

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

363

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

364

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

365

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

366

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

367

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

368

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

369

drinking water samples.

370

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

371

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

372

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

373

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



374

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

375

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

376

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

377

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

378

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

379

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

380

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

381

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

382

paired MRM transitions matching with the commercially available or laboratory synthesized

383

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

384

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

385

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

386

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

387

peptides containing other amino acids.50 In the tap water samples, we detected two dipeptides

388

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

389

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-

390

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

391

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

392

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

393

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

394

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

395

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

396

to understanding the potential health effects of DBPs.

397 398

ASSOCIATED CONTENT

399

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

400

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

401

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

402

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

403

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

404


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Page 15 of 24


405

AUTHOR INFORMATION

406

Corresponding Author

407

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

408

E-mail: [email protected]

409

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

410 411

Author Contributions

412

¶

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

413 414

ACKNOWLEDGEMENT

415

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

416

Engineering Research Council of Canada and Alberta Health.

417 418

References

419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443

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45. Pearson, R.G. Hard and soft acids and bases. J. Am. Chem. Soc. 1963, 85, 3533-3539. 46. Westerhoff, P.; Mash, H., Dissolved organic nitrogen in drinking water supplies: a review. J. Water Supply Res. T. 2002, 51, (8), 415-448. 47. Hua, G.; Reckhow, D. A. DBP formation during chlorination and chloramination: effect of reaction time, pH, dosage, and temperature. J. Am. Water Works Ass. 2008, 100, (8), 82-95. 48. Hua, G., Reckhow, D. Effect of alkaline pH on the stability of halogenated DBPs. J. Am. Water Works Ass. 2012, 104 (2), 49-50. 49. Zhu, X.; Zhang, X. Modeling the formation of TOCl, TOBr and TOI during chlor(am)ination of drinking water. Water Res. 2016, 96, 166-176. 50. Peake, E.; Baker, B. L.; Hodgson, G. Hydrogeochemistry of the surface waters of the Mackenzie River drainage basin, Canada—II. The contribution of amino acids, hydrocarbons and chlorins to the Beaufort Sea by the Mackenzie River system. Geochim. Cosmochim. Ac. 1972, 36, (8), 867-883.

549 550


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552 553 554

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

555

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

556

reaction for 24 h.

557



558

559 560

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

561

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

562

the presence of iodine.

563


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564

565 566

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

567

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

568

presence of iodine.

569



570

571 572

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

573

presence of iodine under different phosphate-buffered pH conditions.

574

575 576

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

577

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

578

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


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Formation and occurrence of iodinated tyrosyl dipeptides in

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