Ascorbic Acid Assisted High Performance Liquid Chromatography

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Ascorbic Acid Assisted High Performance Liquid Chromatography Mass Spectrometry Differentiation of Isomeric C-chloro- and N-chloro- Tyrosyl Peptides in Water Ping Jiang, Guang Huang, Lindsay K Jmaiff Blackstock, Jianye Zhang, and Xing-Fang Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04262 • Publication Date (Web): 25 Nov 2017 Downloaded from http://pubs.acs.org on November 30, 2017

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Analytical Chemistry

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Ascorbic Acid Assisted High Performance Liquid Chromatography Mass Spectrometry

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Differentiation of Isomeric C-chloro- and N-chloro- Tyrosyl Peptides in Water

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Ping Jiang,† Guang Huang,† Lindsay K. Jmaiff Blackstock,† Jianye Zhang,§,† Xing-Fang Li†,*

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†

Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and

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Pathology, Faculty of Medicine and Dentistry, University of Alberta Edmonton, AB Canada

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T6G 2G3

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§

College of Chemistry and Molecular Engineering, Zhengzhou University, Henan, PR China, 450052

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

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ACS Paragon Plus Environment

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract We report a new method of ascorbic acid assisted high performance liquid

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chromatography (HPLC) with high resolution tandem mass spectrometry (HRMS/MS) for the

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differentiation of isomeric N-chloro (N-Cl) from phenol ring C-chloro (C-Cl) peptides produced

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in chlorination of water. Using the specific reductive nature of ascorbic acid we successfully

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identified the N-Cl isomers and C-Cl isomer, overcoming the difficulties that due to lack of

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standards, these isomers cannot be separated by HPLC-HRMS. Using the new approach, we

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identified 36 new chlorinated products including mono-, di-, tri-, and tetra-Cl- tyrosyl dipeptides

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in the reaction mixture based on retention time, accurate mass, 35Cl/37Cl isotopic pattern, and

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characteristic MS/MS fragments. The method was further applied to investigate competitive

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reactions when mixed tyrosyl dipeptides were chlorinated. Tyrosyl histidine was the most

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reactive tyrosyl dipeptide in the mixture. The chlorinated products formed are identical when the

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dipeptides are chlorinated separately or as a mixture. The formation conditions and stability of

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the chlorinated products were also examined. With increasing chlorine dose, the number of

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chlorine substituents on the tyrosyl dipeptides increased from products with one/two to three/four

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Cl atoms. Most of chlorinated products are stable up to 9 days. By chlorination of tyrosyl

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dipeptides spiked into raw water, we projected that chlorinated tyrosyl dipeptides can form

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during water treatment of raw water containing tyrosyl dipeptides even at low µg/L levels. This

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new method can be utilized for the discovery of a wide range of chlorinated peptide DBPs and

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the study of their formation and occurrence in water.

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Drinking water disinfection is essential to eliminate pathogenic microorganisms and

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prevent waterborne disease, but unintentionally generates disinfection by-products (DBPs).

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DBPs are a potential public health concern indicated by the consistent epidemiological

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association of increased bladder cancer incidence with long-term consumption of disinfected

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water.1,2 So far, over 700 carbonaceous DBPs (C-DBPs) have been identified,3-7 while only a

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small fraction, including trihalomethanes (THMs) and haloacetic acids (HAAs) have been

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regulated8,9 in drinking water. However, there is disagreement in tumor sites and potency7

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between the results from epidemiological and animal toxicological studies of regulated DBPs.

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Therefore, efforts have been made to identify and investigate DBPs of toxicological relevance.

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Nitrogenous DBPs (N-DBPs), including halonitriles, haloacetamides, and N-haloamines

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are of particular interest as they are more cyto- and genotoxic than the regulated C-DBPs.10 With

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quantitative structure toxicity relationship (QSTR) analysis, N-haloamines were predicted to

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have sufficient potency or selectivity to account for the epidemiologically associated adverse

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health effects.11 For example, endogenous N-chloramines derived from protein could enhance

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protein radical formation,12 which can further induce secondary lipid oxidation13 or DNA strand

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breaks.14 Furthermore, the presence of N-chloramines in water could be deleterious to water

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treatment efforts. For instance, N-chloramines can confound estimations of disinfectant levels in

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water treatment plants (WTPs) as they yield a similar response to chlorine during measurement.

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Compared to chlorine, N-chloramines are weaker disinfectants, thereby interfering with accurate

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estimations of pathogen deactivation and hindering disinfection efficacy.15,16 Additionally, N-

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chloramines have been found to propagate DBP formation, acting as indispensable intermediates

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for the formation of halonitriles and haloaldehydes.17 Due to the potential negative effects of N-


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haloamines on human health and water quality, research on exploring their formation and

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occurrence during water disinfection processes is of value.

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N-haloamines are generated through reactions between disinfectants and dissolved

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organic nitrogen (DON), such as amino acids, peptides, and proteins present in source water.

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Many studies have reported N-haloamine formation from reactions between HClO or NaClO

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with various amino acids.18-22 However, individual amino acids are both structurally less

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complex and far less abundant11,12 in source waters compared to combined amino acids (i.e.,

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peptides). Recently, our group used a non-targeted strategy to identify more than 600 peptides

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(100 to 1600 Da) in source water.23 We hypothesize that these peptides consisting of 2~7 amino

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acids could potentially pass through drinking water pre-treatment (e.g., coagulation, or filtration)

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and serve as precursors of halogenated peptides, formed during disinfection.

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Several studies have reported the chlorination of peptides in model solutions24-27 and

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wastewater,28,29 with N-chlorinated peptides, especially mono-Cl- peptides, observed as by-

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products. However, current research on the formation of N-chlorinated peptides has mainly

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focused on the chlorination of peptides containing glycine (Gly) or alanine (Ala) amino residues.

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Less has been observed for peptides containing other natural amino acids, for example tyrosine

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(Tyr). N-chloramine formation from Tyr containing peptides should be investigated as Tyr is one

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of most abundant amino acids detected in source waters.18 Furthermore, Tyr has been reported as

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one of the amino acids that could produce chlorinated compounds with high mutagenicity based

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on four different kinds of mutagenesis tests.30 Interestingly, endogenous chlorinated peptides can

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degrade to 3-Cl-Tyr or 3,5-di-Cl-Tyr, which can serve as biomarkers for confirming the role of

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oxidants in inflammatory pathologies.31,32 To supplement existing research on the formation of


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N-chloramine from peptides, we aim to explore the unknown chlorinated products resulting from

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reactions between tyrosyl dipeptides and free chlorine in water.

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Based on its structure, there are two types of chlorination sites on the tyrosine residue; the

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N-terminal amino group and the side chain phenol ring. Substitution on the amino group

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produces N-chloramines (i.e., N-chloro dipeptides) containing N-Cl bonds; while substitution on

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the ortho positions of the phenolic group can produce chlorinated dipeptides containing C-chloro

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(C-Cl) but not N-Cl bonds. This presents a challenge; owing to the multiple chlorination sites on

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tyrosyl dipeptides, a complex mixture of chlorinated peptide by-products could form during

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disinfection, some of which existing as isomers. Theoretically, for each tyrosyl dipeptide, the

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formation of eight chlorinated products could be predicted during chlorination. Using tyrosyl

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valine (YV) as an example, Scheme 1 illustrates these eight predicted chlorinated products,

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including two mono-Cl-, three di-Cl-, two tri-Cl-, and one tetra-Cl- YV isomers. Structures

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containing only C-Cl bonds (I and III), are termed C-Cl products. All other chlorinated

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structures contain at least one N-Cl bond and are termed N-Cl products.

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One of the challenges in the identification of possible chlorinated products from tyrosyl

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dipeptides is that their standards are not commercially available and not easily synthesized. High

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performance liquid chromatography with high resolution mass spectrometry (HPLC-HRMS) or

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tandem mass spectrometry (MS/MS) is a powerful technique33 commonly used to project the

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identity of unknowns using the accurate mass information provided by HPLC-HRMS with the

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characteristic fragments observed in MS/MS. However, the C-Cl or N-Cl substituted dipeptide

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isomers proposed in Scheme 1 have identical mass information and the mass spectrometry

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software proposed very similar mass fragmentation patterns, making them indistinguishable. The

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literature is also inconsistent on the characteristic peaks that should be used to confirm N-Cl


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products.31,32 Thus, extra efforts besides HPLC-MS/MS is required to differentiate the possible

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chlorinated dipeptide products, specifically, C-Cl substituted from N-Cl substituted isomers.

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Previous studies have shown that ascorbic acid can reduce N-Cl protein back to non-chlorinated

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amines,34 but whether the C-Cl bond is affected in the same way was not described. If C-Cl

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bonds are not susceptible to reduction by ascorbic acid, the specific reactivity with N-Cl bonds

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could potentially be utilized for the differentiation of N-Cl from C-Cl tyrosyl dipeptide DBP

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isomers.

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An additional challenge in the investigation of chlorinated tyrosyl dipeptides comes from

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the complexity of different amino acid combinations. Theoretically, the adjacent amino acid in

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the tyrosyl dipeptides could vary among the twenty, common natural amino acids. These can

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generally be divided into several subgroups based on their side chains: small, hydrophobic,

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nucleophilic, aromatic, acidic, neutral, or basic. When combinations of all amino acids with

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tyrosine coexist in water, they may compete with each other for the chlorination reaction. It is

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not feasible to carry out chlorination experiments for all possible tyrosyl dipeptides. To

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effectively cover a wide range of these amino acids, in total, we selected six representative

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tyrosyl dipeptides: tyrosyl alanine (small hydrophobic), tyrosyl valine (hydrophobic), tyrosyl

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phenylalanine (aromatic), tyrosyl glutamic acid (acidic), tyrosyl glutamine (neutral), and tyrosyl

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histidine (basic). Side chains of nucleophilic amino acids have known high reactivity towards

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chlorine, so no representative was selected from this subgroup, as it is unlikely chlorine would

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substitute on the neighbouring Tyr residue.

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Lastly, the formation of chlorinated tyrosyl dipeptides in water may not necessarily be

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environmentally relevant nor pose a risk to human health. Several factors could affect the

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occurrence of chlorinated tyrosyl dipeptides in treated drinking water. First, different chlorine


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doses have been reported to affect the generation of chlorinated dipeptides.34 What is observed in

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laboratory conditions may not reflect realistic chlorinated tyrosyl dipeptide formation, depending

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on the dose used at WTPs. Second, if the products are not stable they will not reach the consumer,

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and exposure is required for risk. Lastly, the abundance of dipeptides in the source water is

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environmentally variable, which could lead to different levels of chlorinated products. All the

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above factors should be evaluated to illustrate the potential risks from exposure to any new DBPs.

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In this paper, we aim to comprehensively investigate the formation of chlorinated tyrosyl

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dipeptides in water. The first objective is to confirm the possible chlorinated products generated

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through the chlorination of tyrosyl dipeptides. Based on the predicted chlorinated products, we

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utilized HPLC-HRMS or MS/MS combined with ascorbic acid experiments to differentiate the

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N-Cl from the C-Cl isomers. Our second objective is to explore the competitive reaction between

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different tyrosyl dipeptides. Representative tyrosyl dipeptides were chlorinated as a mixture. The

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residual percentage of each dipeptide has been used to illustrate their reactivity towards free

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chlorine. The third objective is to evaluate the possibility of the occurrence of chlorinated

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dipeptides in treated drinking water, by monitoring the change of the chlorinated dipeptides with

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different chlorine doses, chlorination time, and dipeptide starting concentrations in raw water

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samples.

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MATERIALS AND METHODS

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Chemicals and solvents

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Tyrosine (Tyr or Y) (98%), 3-Cl-tyrosine (3-Cl-Y) (97%), tyrosyl alanine (Tyr-Ala or

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YA), tyrosyl phenylalanine (Tyr-Phe or YF), and L-ascorbic acid (≥ 99%) were obtained from

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Sigma-Aldrich (St. Louis, MO, USA). Tyrosyl glutamic acid (Tyr-Glu or YE) (> 99%), tyrosyl


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histidine (Tyr-His or YH) (≥ 99%), tyrosyl glutamine (Tyr-Gln or YQ), and tyrosyl valine (Tyr-

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Val or YV) (>98%) were obtained from BACHEM (Torrance, CA, USA). Optima LC/MS grade

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water, methanol (MeOH), and acetonitrile (ACN) were purchased from Fisher Scientific

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(Nepean, ON, Canada). HPLC grade formic acid (FA, 49-51% in water) was purchased from

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Fluka. Sodium hypochlorite (NaClO) solution (reagent grade, 10-15% available chlorine) was

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obtained from Acros Organics (Fair Lawn, NJ, USA). The concentration of free chlorine in the

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NaClO solution was measured to be 120 mg/mL (1.7 mol/L) by a chlorine amperometric titrator

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(Autocat 9000, HACH).

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Chlorination of tyrosine and tyrosyl dipeptides

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A chlorine stock solution (0.17 mol/L of free chlorine) was prepared by diluting the

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above NaClO solution with optima water. Each dipeptide (2.5 µmol; i.e., YA, YE, YF, YH, YQ,

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or YV), or tyrosine alone, was chlorinated by adding 5 µmol free chlorine (i.e., 30 µL chlorine

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stock solution) in 25 mL of optima water, resulting in a precursor to free chlorine molar ratio of

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1/2. The reaction mixture was kept in the dark for 1 h at room temperature (22 °C), then excess

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free chlorine was quenched by adding 1.25 mL of 50% FA.35 Ascorbic acid is a commonly used

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quenching reagent that reacts with N-chloramines34 (See section “identification of chlorinated

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tyrosyl dipeptides”), thus it is not suitable for this study.

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Identification of chlorinated tyrosyl dipeptides

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To identify the chlorinated dipeptides formed through the chlorination reaction, we

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developed an ascorbic acid assisted high performance liquid chromatography high resolution

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tandem mass spectrometry (HPLC-HRMS/MS) method (as shown in Scheme 2). In step 1, the

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quenched reaction solution was subjected to HPLC-HRMS analysis. After quenching, the


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chlorinated solutions were separated on an Accucore C18 column (150 × 2.1 mm i.d., 2.6 µm;

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pore size, 80 Å; Thermo Scientific, Waltham, MA, USA). The mobile phase consisted of solvent

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(A), water containing 0.1% FA; and solvent (B), acetonitrile (ACN): water = 5:95 containing 0.1%

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FA. A high-resolution quadrupole time-of-flight (QTOF) mass spectrometer (Sciex X500R)

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(with the resolution of 25,000-35,000) with a Turbo-V electrospray ionization (ESI) source was

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used in positive mode to obtain accurate mass measurements of the [M+H]+ ions. Details of the

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HPLC-HRMS conditions are presented in Supporting Information (SI) Section 1. In step 2, an

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aliquot (10 mL) of the above FA quenched chlorinated solution was reacted with 20 µmol

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ascorbic acid under darkness at room temperature. After reacting with ascorbic acid for 10 min,

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the solutions were analyzed by HPLC-HRMS again without extraction. The HPLC-HRMS full

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scan mass spectra for the chlorinated solution before and after the ascorbic acid were compared.

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In step 3, the quenched chlorination solution before the addition of ascorbic acid was analyzed

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using HPLC-MS/MS running with the information dependent acquisition (IDA) strategy. The

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IDA related parameters are presented in SI Section 2.

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Distribution of chlorinated dipeptides with chlorine dose

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Once the identity of the chlorinated tyrosyl-dipeptide was determined for each

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chromatographic peak, the retention time and peak intensity was used to investigate the

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competitive reaction between different tyrosyl dipeptides as well as the effect of chlorine dose on

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the generation of chlorinated dipeptides. Various amounts of the chlorine stock (i.e., 3, 7.5, 15,

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30, 45, 75, and 150 µmol of free chlorine) were added to solutions containing equal amounts of

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the six dipeptides (i.e., 2.5 µmol of each, in total 15 µmol) in 25 mL optima water. The lowest

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chlorine dose (i.e., 3 µmol or 4.3 mg/L) used was similar to the concentration of residual chlorine

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in treated drinking water (0.04-2 mg/L).9 All the solutions were kept in the dark at the room


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temperature and subjected to HPLC-HRMS analysis after 1 h. The identification of chlorinated

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dipeptides generated was obtained based on their retention time. The change in the intensity of

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the peak areas of each dipeptide and its corresponding chlorinated products were monitored

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versus chlorine doses.

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Distribution of tyrosyl dipeptides and the chlorinated products with chlorination time

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Equal amounts of the six dipeptides (YA, YE, YF, YH, YQ and YV) (i.e., 2.5 µmol of

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each) were mixed in either 25 mL of optima water or raw water in a 40 mL amber vial (i.e., with

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headspace). We believe that the targeted chlorinated products would not diffuse to the headspace

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because of their low volatility. Free chlorine (30 µmol, i.e., 180 µL of the chlorine stock solution)

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was added so the molar ratio between the total dipeptides and free chlorine was 1/2. The chlorine

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dose of 30 µmol (~43 mg/L) was higher than the typical total residual chlorine (0.04- 2 mg/L)9 in

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treated drinking water. This high chlorine dose was used to ensure the generation of all the

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possible chlorinated tyrosyl dipeptide products, so that their overall distribution could be

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investigated. A much lower chlorine dose within the range of 0.04-2 mg/L was used when the

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concentration of dipeptides was reduced in the section “chlorination of dipeptides in raw water”.

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To study the change of the chlorinated dipeptides with chlorination time, the reaction solution in

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the 40 mL amber vial was kept under dark at room temperature up to 9 days. An aliquot (1 mL)

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was taken daily from the reaction solution to monitor the formation of chlorinated dipeptides

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using HPLC-HRMS. The peak area of the chlorinated dipeptides was studied versus the

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chlorination time. This data also illustrate the stability of the chlorinated dipeptides in water. To

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assess the stability the chlorinated tyrosyl dipeptides at a low pH, the same experiment was

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performed again except that the reaction solution was quenched at 1 h by adding formic acid.


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Chlorination of dipeptides in raw water The concentration of peptides in raw water varies spatially and seasonally. To relate our

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research to the water treatment process, we explored the generation of chlorinated dipeptides

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from raw water spiked with various concentrations of dipeptides. Pre-cleaned 4 L amber glass

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bottles (rinsed 3x methanol followed by 3x optima water) were used to collect raw water by grab

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sampling from the Rossdale water treatment plant (Edmonton, Alberta, Canada) in April 2017.

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The water samples were filtered with a glass microfiber filter (47 mm × 1.5 µm, Waterman) and

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a nylon membrane disk filter (47 mm × 0.45 µm), then stored at 4 °C prior to analysis. The total

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organic carbon (TOC) of the filtered raw water was 4.3 mg/L (measured in the Natural

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Resources Analytical Laboratory at the University of Alberta).

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The chlorination of dipeptides was conducted at three different concentration levels. We

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spiked 0.25, 2.5, or 25 nmol of each dipeptide into 25 mL of raw water, resulting in dipeptide

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concentrations of 0.01, 0.1, or 1 µM. At each concentration level, the molar ratio of total

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dipeptides to free chlorine was controlled at 1/2 unless otherwise defined. Therefore, the chlorine

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doses are 0.4 µg/L, 4 µg/L, and 0.04 mg/L, respectively, which are within the low end of the

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typical chlorine concentration range of real water samples. As a control, identical experiments

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were performed in optima water. Before each chlorination experiment, three blanks including

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raw water, raw water with dipeptides, and raw water with NaClO were analyzed with the HPLC-

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MRM method. All the above reaction solutions were kept in the dark at room temperature. After

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1 h, 1 mL of each solution was analyzed using HPLC-MS/MS (Sciex QTRAP 5500) in multiple

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reaction monitoring (MRM) mode to measure the unreacted tyrosyl dipeptides and the

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chlorinated products during chlorination in raw water. Detailed instrument conditions can be

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found in SI Section 3.


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RESULTS AND DISCUSSION

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Differentiation of C-chloro- from N-chloro-tyrosyl dipeptides

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Tyrosyl dipeptides have two possible sites: the free amino group and the phenol ring, for

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chlorine substitution during chlorination, forming a mixture of N-Cl tyrosyl dipeptides (with N-

239

Cl bonds) and C-Cl tyrosyl dipeptides (without N-Cl bonds), respectively. Some of the products

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are isomers (Scheme 1). No standards are commercially available for the chlorinated tyrosyl

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dipeptides. HPLC-HRMS alone cannot differentiate these isomers. To overcome this problem,

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we have developed a new ascorbic acid assisted HPLC-HRMS/MS method for the identification

243

of the chlorinated dipeptides produced in chlorination of tyrosyl dipeptides. Scheme 2 describes

244

the three steps of this method. Step 1 uses HPLC to separate the compounds in the reaction

245

mixture resulting from chlorination of each tyrosyl dipeptide solution, coupled with HRMS

246

detection to determine the exact mass of the chlorinated compounds and generate a

247

corresponding molecular formula. In step 2, we utilized the specific reduction of N-Cl bonds by

248

ascorbic acid to determine if the chlorine substitution was on the phenol ring (C-Cl) or on the

249

amino group (N-Cl). To validate our experimental design, we confirmed that phenol C-Cl bonds

250

are not reduced by ascorbic acid (SI Section 4 and Figure S1). In step 3, we used the MS/MS

251

spectra to confirm the isomeric structures of chlorinated tyrosyl dipeptide. This 3-step method is

252

demonstrated below using tyrosyl valine (YV) as an example.

253

In step 1, the HPLC-HRMS analysis of the blank, YV standard solution, and chlorinated

254

YV solution prior to the addition of ascorbic acid yielded total ion chromatograms (TICs), as

255

shown in Figure 1. Six major peaks were detected in the TIC of the chlorinated YV solution

256

without ascorbic acid, compared to the blank. Peak 1 has identical retention time and mass


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257

spectrum compared to the YV standard (Figure 1(a-b)), identifying peak 1 (m/z 281) as the

258

remaining unreacted YV after chlorination. In Figure 1(a) The mass spectra show the [M+H]+

259

ions of m/z 315 (peaks 2 and 4), 349 (peaks 3 and 5), and 383 (peak 6). The mass difference (∆m)

260

of peaks 2 and 4 from peak 1 is 34 Da, which matches with the mass gain from the substitution

261

of one proton by one Cl atom, indicating that peaks 2 and 4 correspond to mono-Cl-YV

262

(theoretical m/z 315.1106). Similarly, peaks 3 and 5 have ∆m of 68 Da compared to YV,

263

corresponding to di-Cl-YV (theoretical m/z 349.0716), whereas peak 6 (∆m of 102) corresponds

264

to tri-Cl-YV (theoretical m/z 383.0327). The isotopic patterns of the mass spectra Figure 1(c-g))

265

also agree with the theoretical patterns of compounds containing one, two, or three Cl atoms. For

266

example, when the compound contains one Cl atom, a pair of peaks with m/z = M (containing

267

35

268

/37Cl abundance, the ratio of the peak intensity of M/M+2 should be 3/1. In Figures 1(c) and (e),

269

the ratio of peak heights of m/z 315 and 317 is 3/1. Similarly, when the compound contains two

270

Cl atoms, a group of peaks with m/z = M (containing two 35Cl ), M+2 (containing one 35Cl and

271

one 37Cl), and M+4 (containing two 37Cl) are supposed to appear during MS. Their theoretical

272

relative intensity of M/M+2/M+4 should be 9/6/1. Figures 1(d) and (f) show a ratio of the peak

273

heights of m/z 349, 351, and 353 is 9/6/1, matching the theoretical isotopic pattern of compound

274

that containing two Cl atoms. Mass accuracy for all peaks is within 5 ppm, strongly supporting

275

the proposed molecular formula of peaks 2-6 (shown on Figures 1(b-f)).

276

Cl) and M+2 (containing 37Cl) would show up in the mass spectrum. Based on the natural 35Cl

Having achieved the HPLC-HRMS differentiation of the chlorinated dipeptides with

277

different number of Cl atoms, in step 2 we identified the positions where the Cl atoms were

278

attached. As predicted in Scheme 1, the Cl atom can either substitute on the phenol ring or the

279

amino group, which can lead to the formation of C-Cl or N-Cl bond. To differentiate the


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Page 14 of 31

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chlorinated dipeptides containing C-Cl only from those containing N-Cl, we used the specific

281

reductive nature of ascorbic acid to reduce the N-Cl bond back to N-H, leaving the C-Cl bond

282

intact. Figure 2 shows the extracted ion chromatograms of YV, mono-, di-, and tri-Cl- YV

283

(m/z=281.1496, 315.1106, 349.0716 and 383.0327) from the TICs of chlorinated YV solution

284

before (Figure 2(a) and after (Figure 2(b)) the addition of ascorbic acid. The mono-Cl-YV

285

products (peaks 2 and 4) on Figure 2 correspond to either 3-Cl-YV or N-Cl-YV as predicted in

286

Scheme 1 (I or II). In Figure 2(b), peak 2 remained after the addition of ascorbic acid,

287

suggesting it only contains C-Cl bond. The other mono-Cl-YV isomer (peak 4) disappeared

288

indicating peak 4 is a mono-Cl-YV product containing an N-Cl bond. Thus, peak 2 is 3-Cl-YV

289

(I), while peak 4 represents N-Cl-YV (II).

290

To support the identification of 3-Cl-YV and N-Cl-YV, we conducted parallel reactions

291

of chlorinated tyrosine with ascorbic acid. As shown in SI Figure S2, similar to the dipeptide

292

YV, the chlorination of tyrosine produced two mono-Cl-Y compounds. The changes before and

293

after the addition of ascorbic acid to mono-Cl-Y are identical to those for the mono-chlorinated

294

YV. The early eluting mono-Cl-Y has been confirmed as 3-Cl-Y using its authentic standard.

295

The late eluting peak has identical m/z to 3-Cl-Y but is diminished after reacting with ascorbic

296

acid, confirming it as N-Cl-Y. The results of mono-Cl-Y support the identification of mono-Cl-

297

YV (i.e., 3-Cl- and N-Cl-YV).

298

We applied the same strategy to the two di-Cl-YV isomers (peaks 3 and 5) on Figure 2,

299

which correspond to two out of the three possible structures proposed in Scheme 1 (III-V). Peak

300

3 did not react with ascorbic acid (Figure 2), indicating it only contains C-Cl bonds, confirming

301

peak 3 as 3,5-di-Cl-YV. Unlike peak 3, peak 5 can react with ascorbic acid, so it contains at least

302

one N-Cl bond. Based on the proposed structures in Scheme 1, peak 5 could be either compound


14

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303

IV (3,N-di-Cl-YV) or V (N,N-di-Cl-YV). Similarly, peak 6 on Figure 2 is a tri-Cl-YV product

304

that can react with ascorbic acid. Thus, it could be either 3,5,N-tri-Cl-YV or 3,N,N-tri-Cl-YV as

305

predicted in Scheme 1(VI-VII).

306

In summary, through the ascorbic acid experiment, we have confirmed peaks 2-4 on

307

Figure 2 as 3-Cl-YV, 3,5-di-Cl-YV, and N-Cl-YV. In addition, we have identified peaks 5 and 6

308

as N-Cl peptides, but the number of N-Cl bonds they contain remains unknown. Further

309

experimentation is required to accurately assign structures to peaks 5 and 6.

310

Step 3 further elucidates the structures of peaks 5 and 6 using tandem mass spectrometry.

311

Figure 3(a) is the MS/MS spectrum collected using IDA for m/z=349.1 at tR=17.2 min (i.e.,

312

peak 5, Figure 2(a)). Fragment ions of m/z=141 and 153, which are specific to the phenol ring

313

C-Cl moieties with 35Cl isotope, were observed for peak 5 in Figure 3(a), identifying peak 5 as

314

3,N-di-Cl-YV. The proposed fragmentation scheme is shown in SI Figure S3. Additional

315

fragment ions in Figure 3(a) also match with those predicted by PeakView software and are

316

tabulated in SI Table S2. In addition, we have observed the m/z=143 and 155 on the MS/MS

317

spectrum of m/z=351.1, which is an isotopic peak of m/z=349. The fragmentation pattern mass

318

shift (2 Da) is due to the fragment ions containing the 37Cl isotope SI Figure S4(a). Similarly,

319

Figure 3(b) is the MS/MS spectrum of m/z=383.1 at tR=19.8 min (i.e., peak 6 on Figure 2(a)).

320

The fragment ions of m/z 175 and 186 are characteristic fragments of a compound with two Cl

321

atoms substituted on the phenol ring, identifying peak 6 as3,5,N-tri-Cl-YV. For the isotopic peak

322

m/z=385.0, fragments such as m/z 177 and 188 containing 37Cl were detected (SI Figure S4(b).

323 324

Using the above 3-step method, we are able to prove that peaks 1-6 in Figure 2(a) are YV, 3-Cl-YV, 3,5-di-Cl-YV, N-Cl-YV, 3,N-di-Cl-YV, and 3,5,N-tri-Cl-YV. Additional support


15


Page 16 of 31

325

for the assignments of peaks 1-6 was observed in Figure 2. Recall that ascorbic acid

326

dechlorinates the N-Cl bond, reducing it back to an N-H bond.34,36 As a result, N-Cl-YV will

327

revert to YV, 3,N-di-Cl-YV to 3-Cl-YV, and 3,5,N-tri-Cl-YV to 3,5-di-Cl-YV. Only with current

328

assignments for peaks 1-6, can we expect to see the increase in peaks 1-3 and the disappearance

329

of peaks 4-6 simultaneously as shown in Figure 2(b) after the addition of ascorbic acid.

330

In summary, we achieved the identification of two mono-Cl-, two di-Cl-, and one tri-Cl-

331

YV products formed from the chlorination of the dipeptide YV, using the ascorbic acid assisted

332

HPLC-HRMS and MS/MS method at YV to Cl molar ratio of 1/2 chlorination conditions. Using

333

the same strategy, we found one more product, which is the tetra-Cl-YV (i.e., 3,5,N,N-tetra-Cl-

334

YV) at an increased chlorination condition of YV to Cl molar ratio of 1/5 (SI Figure S5). In total,

335

for an individual tyrosyl dipeptide, such as YV, we identified 6 chlorinated products. Using our

336

3-step strategy, we also confirmed the formation of 6 chlorinated products from the chlorination

337

of each tyrosyl dipeptide (YA, YQ, YE, YH, and YF), all the essential XICs and MS/MS spectra

338

for identifying the mono-, di-, and tri-Cl- products were provided in SI Figure S6-S10. The

339

application of the method to water research is further demonstrated in the formation and

340

occurrence of chlorinated tyrosyl peptides in drinking water.

341

Formation of chlorinated dipeptides as a function of dipeptide/free chlorine molar ratio

342

Having established the method for identification of tyrosyl dipeptides and their

343

chlorinated products, we studied competitive reactions between the tyrosyl dipeptides with

344

various doses of chlorine. When a mixture of six tyrosyl dipeptides was chlorinated, with

345

increasing chlorine to total dipeptide molar ratio, the intensity of the residual dipeptides

346

decreases rapidly (SI Figure S11), indicating an increase in reaction rate. At every chlorine dose,


16

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347

YH had the lowest residual percentage, indicating YH was the most reactive dipeptide studied.

348

Previous research has shown histidine to be more reactive than other individual amino acids

349

during chlorination.37

350

Figure 4 and SI Figures S12-S15 show that the chlorine dose affects both the type of the

351

chlorinated products generated and their relative abundance. Generally, as the chlorine dose

352

increases, the distribution of chlorinated products shifts to form more dipeptides with a higher

353

number of Cl-substitutions, indicating that the chlorination follows a stepwise mechanism. For

354

example, for five tyrosyl dipeptides (YA, YE, YF, YQ, and YV), the intensity of mono-Cl-

355

dipeptides formation, i.e., 3-Cl- (Figure 4(a) and SI Figure S12-15 (a)) or N-Cl- ((Figure 4(b)

356

and SI Figure S12-15 (b)) increases along the x-axis to a maximum at chlorine dose of 30 or 45

357

µmol, but are not detected when the chlorine dose was further increased. And at a higher

358

chlorine dose (≥ 45 µmol), the tetra-Cl-YV (i.e., 3,5,N,N-tetra-Cl-YV) signal begins to appear

359

(Figure 4(b) and SI Figure S12-15(b)). Because of the high reactivity of YH during

360

chlorination, we observed maximum formation of 3-Cl- or N-Cl- YH at approximately 2-fold

361

less chlorine dose (Figure 4(c-d)) compared to the mono-Cl- products formed from the other

362

dipeptides.

363

To facilitate complete pathogen inactivation in varying raw water conditions, different

364

chlorine doses are applied during water treatment. Our results confirmed that various chlorinated

365

products are generated from the chlorination of tyrosyl dipeptides over a wide range of chlorine

366

doses. When tyrosyl peptides exist in water as a mixture, those containing histidine are projected

367

to be more reactive towards generating chlorinated products.

368

Distribution of tyrosyl dipeptides and the chlorinated products with chlorination time


17


369

Page 18 of 31

To explore the possibility of the occurrence of chlorinated dipeptides in treated drinking

370

water, we studied the distribution of these chlorinated dipeptides with chlorination reaction time.

371

The chlorination was performed on a dipeptide mixture (15 µmol in total) and a chlorine dose of

372

30 µmol. Under these conditions, five chlorinated dipeptides (3-Cl-, 3,5-di-Cl-, N-Cl-, 3,N-di-Cl-,

373

and 3,5,N-tri-Cl-) formed from each tyrosyl dipeptide immediately after chlorination, but the

374

distribution of products changed with reaction time. Here we discuss the change of chlorinated

375

YV products with chlorination time as an example. The change over time in the distribution of

376

the chlorinated products formed from the other five tyrosyl dipeptides is similar to YV (SI

377

Figure S16-20). Figure 5(a) shows that the phenol ring C-Cl dipeptides including 3-Cl-YV and

378

3,5-di-Cl-YV continue formation rapidly, up to 9 days. The N-Cl dipeptides, including N-Cl-YV,

379

3,N-di-Cl-YV, and 3,5,N-tri-Cl-YV all increase slightly in the first 4-6 hours then decrease

380

gradually with reaction time (Figure 5(b)). By the 9th day, the intensities of all the N-Cl

381

dipeptides dropped below 50% of their maximum formation. Two reasons may cause the loss of

382

the N-Cl YV products. Firstly, the chlorination of YV to N-Cl YV is a reversible reaction.38

383

Therefore some N-Cl dipeptide products reverted back to N-H dipeptides, such as N-Cl-YV

384

hydrolyzing back to YV. Indeed, we observed an elevation of YV from 1h to 9 days.

385

Alternatively, the N-Cl YVs may degrade to other by-products, such as chloronitriles and

386

chloroaldehydes.39,40 Such degradation of N-Cl dipeptides can be accelerated with lower pH.39,40

387

For example, when the same experiment was carried out, except the reaction solution was

388

acidified with formic acid to pH=2, a more rapid decrease in the N-Cl dipeptides was observed

389

(SI Figure S21). Generally, it takes approximately 5 days for the intensity of N-Cl dipeptides to

390

decay to 50%. This length of time is comparable to the reported half-lives (2-10 days) of other

391

chlorinated dipeptides that do not contain tyrosine39 and much longer than the half lives39 of N-


18

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392

chlorinated amino acids (~0.1-5 h) reported previously. The change of chlorinated peptides with

393

chlorination time was repeated in raw water and results with the same trends were obtained (SI

394

Figures S22-27).

395

In all, over increasing chlorination time, the chlorinated products would gradually shift

396

from N-Cl dipeptides to phenol ring C-Cl dipeptides. Owing to the high stability of C-Cl peptides

397

and the higher stability of N-Cl peptides compared to their corresponding chlorinated amino

398

acids, the formation and occurrence of chlorinated tyrosyl dipeptides is highly likely during the

399

water treatment process.

400

Formation potential of chlorinated dipeptides from chlorination of raw water

401

The chlorination of a mixture of six tyrosyl dipeptides was also performed in raw water

402

to mimic the practical water treatment process to evaluate their occurrence potential. Each

403

dipeptide was spiked in the raw water at equal molar concentration and three concentration levels

404

(1µM, 0.1 µM, or 0.01 µM) were studied.

405

We first explored the generation of chlorinated YV from the chlorination of the dipeptide

406

mixture. No chlorinated YV can be detected in any of the blanks (SI Figure S28). The types and

407

the intensity of the chlorinated YV detected in optima and raw water were comparable (Figure

408

6(a-d) and SI Figure S29 (a-d)) at higher dipeptide spike concentrations (1µM or 0.1 µM). At

409

lower concentrations, fewer chlorinated dipeptide products were detected (Figure 6(a-e)).

410

Specifically, when the starting concentration of each dipeptide was decreased from 1 µM to 0.1

411

µM, the number of detected chlorinated products decreased from three (3-Cl-YV, N-Cl-YV, and

412

3,N-di-Cl-YV (Figure 6(a-c))) to two (3-Cl-YV and N-Cl-YV (Figure 6(d-e))).


19


413

Page 20 of 31

When 0.25 nmol (0.01 µM) of each dipeptide in raw water was chlorinated, the types of

414

chlorinated YV detected in the raw or optima water remained the same, with only N-Cl-YV

415

(Figure 6(f)) being detected. However, the signal-to-noise ratio (S/N) of the N-Cl-YV peak, of 5

416

in raw water was less intense compared to the S/N of 21, observed in optima water (SI Figure

417

S29 (f)). The lower yield of N-Cl-YV may be caused by inadequate free chlorine in the raw

418

water. This indicates that other NOM in the raw water are more reactive than the dipeptides,

419

consuming some available free chlorine. We confirmed this by increasing the ratio of the total

420

dipeptides to free chlorine to 1/10 in the raw water and detected the N-Cl-YV peak with S/N=44

421

(SI Figure S30). Experiments at even lower spiked concentrations were not conducted because it

422

is difficult to control the low volume of Cl solution required to be added under laboratory

423

conditions. For tyrosyl dipeptides including YA, YE, YF, and YQ, the generation of chlorinated

424

dipeptides obtained are similar to YV. Thus, for these five tyrosyl dipeptides, we only presented

425

the results for YV. However, for YH, more chlorinated products were observed during the

426

spiking experiments. For example, when 0.1 µM of YH was spiked in the optima water or raw

427

water, five chlorinated YH products were detected (SI Figure S31).

428

In general, despite that raw water had higher free chlorine demand than optima water, the

429

generation of chlorinated by-products are comparable. In raw water, amino acid concentrations

430

usually range from 0.02 to 10 mg/L,39 while peptides typically exist at concentrations 4-5 times

431

more abundant than that of amino acids.41 In our study, we investigated the threshold to trigger

432

the formation of chlorinated dipeptides. We observed at least one chlorinated product (N-Cl-

433

tyrosyl dipeptide) at spiked concentrations as low as 0.25 nmol of each dipeptide. By mass, this

434

is equivalent to 0.18 mg/L total dipeptides, which is an environmentally relevant concentration.

435

When the dipeptide concentration was increased to 2.5 nmol or higher, other by-products such as


20

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436

phenol ring C-Cl dipeptides could be detected with the current HPLC-MRM method. In practice,

437

it is highly likely that chlorinated dipeptides can be generated during the drinking water

438

treatment process and be detected using our method.

439

Conclusions

440

In this study, we developed a 3-step ascorbic acid assisted HPLC-HRMS/MS method for

441

the successful differentiation of N-chlorinated tyrosyl dipeptides from C-chlorinated isomers.

442

Over 30 new chlorinated tyrosyl dipeptide type DBPs have been identified. This method could

443

be applied to the investigation of different haloamine DBPs with multiple chlorination sites and

444

expand the discovery research of new DBPs. This study has broadened our understanding of the

445

potential formation of N-Chlorinated tyrosyl dipeptides as chlorination by-products. We found

446

these products have potential to form in raw water under drinking water treatment plant

447

conditions and have sufficient stability to potentiate exposure to municipal water consumers with

448

drinking water residence time less than 9 days. Due to the prioritization of N-haloamines as

449

potentially toxic DBPs by QSTR analysis, coupled with the previous illustrated potential

450

cytotoxicity attributed to endogenous N-chloramines, a toxicological evaluation of the 36 newly

451

identified chlorinated tyrosyl dipeptide products should be conducted. We hope thisstudy of

452

chlorinated tyrosyl dipeptide formation and stability under different chlorination conditions may

453

help inform water treatment operators and regulators in future efforts to reduce potential risk to

454

human health.

455

ACKNOWLEDGEMENTS

456 457

This work was supported by the Natural Science and Engineering Research Council of Canada (NSERC) and the University of Alberta.


21


Page 22 of 31

458

REFERENCES

459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501

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(7) Richardson, S. D.; Plewa, M. J.; Wagner, E. D.; Schoeny, R.; DeMarini, D. M. Mutat, Res., Rev. Mutat. Res. 2007, 636, 178-242. (8) United States Environmental Protection Agency (US EPA). National primary drinking water regulations: stage 2 disinfectants and disinfection byproducts rule; final rule. Fed Regist. 2006, 71, 388-493 (9) Health Canada. Guideline for Canadian drinking water quality, Ontario, Canada. 2017. Available from http://www.hc-sc.gc.ca/ewh-semt/alt_formats/pdf/pubs/water-eau/sum_guideres_recom/sum_guide-res_recom-eng.pdf [Accessed 22 Aug 2017] (5) (10) Plewa, M. J.; Simmons, J. E.; Richardson, S. D.; Wagner, E. D. Environ. Mol. Mutagen. 2010, 51, 871-878. (11) Bull, R. J.; Reckhow, D. A.; Rotello, V.; Bull, O. M. Kim, J. Use of Toxicological and Chemical Models to Prioritize DBP Research; American Water Works Association: Denver, USA, 2006, pp 384. (12) Hawkins, C. L.; Davies, M. J. Biochemical Journal 1999, 340, 539-548. (13) Hazell, L. J.; Davies, M. J.; Stocker, R. Biochem. J. 1999, 339, 489-495. (14) Hawkins, C. L.; Davies, M. J. Chem. Res. Toxicol. 2002, 15, 83-92. (15) Scully, F. E.; Hartman, A. C.; Rule, A.; Leblanc, N. Environ. Sci. Technol. 1996, 30, 14651471. (16) Amiri, F.; Mesquita, M. M. F.; Andrews, S. A. Water Res. 2010, 44, 845-853. (17) Joo, S. H.; Mitch, W. A. Environ. Sci. Technol. 2007, 41, 1288-1296. (18) How, Z. T.; Linge, K. L.; Busetti, F.; Joll, C. A. Water Res. 2016, 93, 65-73. (19) How, Z. T.; Linge, K. L.; Busetti, F.; Joll, C. A. In Disinfection by-Products in Drinking Water, Thompson, C.; Gillespie, S.; Goslan, E., Eds., 2016, pp 267-276. (20) Hong, H. C.; Wong, M. H.; Liang, Y. Arch. Environ. Contam. Toxicol. 2009, 56, 638-645. (21) Conyers, B.; Scully, F. E. Environ. Sci Technol. 1997, 31, 1680-1685. (22) Brosillon, S.; Lemasle, M.; Renault, E.; Tozza, D.; Heim, V.; Laplanche, A. Chemosphere 2009, 77, 1035-1042. (23) Tang, Y. N.; Xu, Y.; Li, F.; Jmaiff, L.; Hrudey, S. E.; Li, X. F. J. Environ. Sci. 2016, 42, 259-266. (24) Armesto, X. L.; Canle, M.; Garcia, M. V.; Losada, M.; Santaballa, J. A. Gazz. Chim. Ital. 1994, 124, 519-523.


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(25) Furness-Green, S. M.; Inskeep, T. R.; Starke, J. J.; Ping, L.; Greenleaf-Schumann, H. R.; Goyne, T. E. J. Chromatogr. Sci. 1998, 36, 227-236. (26) Armesto, X. L.; Canle, M.; Fernandez, M. I.; Garcia, M. V.; Rodriguez, S.; Santaballa, J. A. J. Chem. Soc.,Perkin Trans. 2 2001, 608-612. (27) Pereira, W. E.; Hoyano, Y.; Summons, R. E.; Bacon, V. A.; Duffield, A. M. Biochim. Biophys. Acta 1973, 313, 170-180. (28) Fox, T. C.; Keefe, D. J.; Scully, F. E.; Laikhter, A. Environ. Sci.Technol 1997, 31, 19791984. (29) Keefe, D. J.; Fox, T. C.; Conyers, B.; Scully, F. E. Environ. Sci. Technol. 1997, 31, 19731978. (30) Horth, M. F. H. Water Supply 1986, 4, 103-126. (31) Domigan, N. M.; Charlton, T. S.; Duncan, M. W.; Winterbourn, C. C.; Kettle, A. J. J. Biol. Chem. 1995, 270, 16542-16548. (32) Choe, J. K.; Richards, D. H.; Wilson, C. J.; Mitch, W. A. Environ. Sci. Technol. 2015, 49, 13331-13339. (33) Richardson, S. D.; Postigo, C. Compr. Anal. Chem 2017, In press. (34) Raftery, M. J. Anal. Biochem. 2007, 366, 218-227. (35) Zhao, Y.; Anichina, J.; Lu, X.; Bull, R. J.; Krasner, S. W.; Hrudey, S. E.; Li, X.-F. Water Res. 2012, 46, 4351-4360. (36) How, Z. T.; Linge, K. L.; Busetti, F.; Joll, C. A. Environ. Sci. & Technol. 2017, 51, 48704876. (37) Hawkins, C. L.; Pattison, D. I.; Davies, M. J. Amino Acids 2003, 25, 259-274. (38) Jersey., J. A.; Johnson. J. D. In Report number 265 from the Water Resource Research Institution of the University of North Carolina: Raleigh, NC, 1992. (39) How, Z. T.; Kristiana, I.; Busetti, F.; Linge, K. L.; Joll, C. A. J. Environ. Sci. 2017, 58, 2-18. (40) Antelo, J. M.; Arce, F.; Franco, J.; Forneas, M. J.; Sanchez, M. E.; Varela, A. Int. J. Chem. Kinet. 1986, 18, 1249-1258. (41) Hureiki, L.; Croue, J. P.; Legube, B. Water Res. 1994, 28, 2521-2531.

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Page 24 of 31

537

FIGURE CAPTION

538

Scheme 1. Theoretically predicted chlorinated YV products generated in the chlorination of YV.

539

Scheme 2. Experimental design for the identification of chlorinated tyrosyl dipeptides.

540 541 542

Figure 1. (a) Total ion current chromatograms (TICs) of the blank, 10 µM of YV solution, and 100 µM YV solution after chlorination; (b-g) mass spectra of peaks 1-6, showing isotopic patterns of YV and the chlorinated products.

543 544

Figure 2. Extracted ion current chromatograms (XICs) of YV, mono-, di-, and tri-Cl-YV before (a) and after (b) the addition of ascorbic acid.

545

Figure 3. MS/MS spectra of (a) m/z 349.1 of peak 5 (b) m/z 383.1 of peak 6.

546 547 548

Figure 4. Effect of chlorine dose on the formation and distribution of dipeptides and the chlorinated dipeptides during chlorination: (a) YV and phenol ring C-Cl YV; (b) N-Cl YV; (c) YH and phenol ring C-Cl YH; and (d) N-Cl YH.

549 550 551

Figure 5. Change of YV and chlorinated YV with chlorination time: (a) YV and phenol ring CCl YV and (b) N-Cl YV. Conditions: 2.5 µmol of each tyrosyl dieptide (YA, YE, YF, YH, YQ, and YV) 25 mL of optima water; chlorine dose: 30 µmol. Total dipeptides/chlorine = 1/2.

552 553 554 555

Figure 6. Detection of chlorinated YV products generated in the chlorination of raw water containing various amounts of dipeptides. The molar ratio of the total dipeptides to free chlorine was kept at 1/2. The raw water samples contained mixed peptides each at (a-c) 25 nmol (1 µM); (d-e) 2.5 nmol (0.1 µM); (f) 0.25 nmol (0.01 µM).

556 557 558 559 560


24

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561 562

Scheme 1. Theoretically predicted chlorinated YV products generated in the chlorination of YV.

563 564

565 566

Scheme 2. Experimental design for the identification of chlorinated tyrosyl dipeptides.


25


Page 26 of 31

567 568 569 570

Figure 1. (a) Total ion current chromatograms (TICs) of the blank, 10 µM of YV solution, and 100 µM YV solution after chlorination; (b-g) mass spectra of peaks 1-6, showing isotopic patterns of YV and the chlorinated products.


26

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571 572 573

Figure 2. Extracted ion current chromatograms (XICs) of YV, mono-, di-, and tri-Cl-YV before (a) and after (b) the addition of ascorbic acid.

574 575

Figure 3. MS/MS spectra of (a) m/z 349.1 of peak 5 (b) m/z 383.1 of peak 6.

576 577


27


Page 28 of 31

578 579 580 581

Figure 4. Effect of chlorine dose on the formation and distribution of dipeptides and the chlorinated dipeptides during chlorination, demonstrated in (a) YV and phenol ring C-Cl YV; (b) N-Cl YV; (c) YH and phenol ring C-Cl YH; and (d) N-Cl YH.


28

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582 583 584 585

Figure 5. Change of YV and chlorinated YV with chlorination time: (a) YV and phenol ring CCl YV and (b) N-Cl YV. Conditions: 2.5 µmol of each tyrosyl dipeptide (YA, YE, YF, YH, YQ, and YV) 25 mL of optima water; chlorine dose: 30 µmol. Total dipeptides/chlorine = 1/2.

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Figure 6. Detection of chlorinated YV products generated in the chlorination of raw water containing various amounts of dipeptides. The molar ratio of the total dipeptides to free chlorine was kept at 1/2. The raw water samples contained mixed peptides each at (a-c) 25 nmol (1 µM); (d-e) 2.5 nmol (0.1 µM); (f) 0.25 nmol (0.01 µM).

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TOC Graphic

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Ascorbic Acid Assisted High Performance Liquid Chromatography

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