3 FTFBSDI SUJDMF 5IF*OëVFODFPG1IPTQIBUF#VFSPOUIF ...downloads.hindawi.com/journals/jchem/2013/818943.pdf · mjuumf buufoujpo ibt cffo qbje up uif jo vfodf pg cv fs nbusjdft po uif

Hindawi Publishing CorporationJournal of ChemistryVolume 2013, Article ID 818943, 9 pageshttp://dx.doi.org/10.1155/2013/818943

Research Article�he In�uence of �hos�hate �u�er on the Formation ofN-Nitrosodimethylamine fromDimethylamine Nitrosation

Long Xu,1 Zhi Sun,1 QingMing Liu,1 Yong Dong Liu,1 Ru Gang Zhong,1 and FengchangWu2

1 College of Life Science & Bioengineering, Beijing University of Technology, Beijing 100124, China2 State Environmental Protection Key Laboratory for Lake Pollution Control, Chinese Research Academy of Environmental Sciences,Beijing 100021, China

Correspondence should be addressed to Yong Dong Liu; [email protected]

Received 17 July 2012; Revised 10 September 2012; Accepted 13 September 2012

Academic Editor: Luis F. Guido

Copyright © 2013 Long Xu et al.is is an open access article distributed under the Creative Commons Attribution License, whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Buffer solutions were widely used for almost all the investigations concerning N-nitrosodimethylamine (NDMA), a member ofpowerful mutagenic and carcinogenic compounds which are ubiquitous in the environment. However, whether or how the buffermatrixes in�uence NDMA formation is still unknown. e effect of buffer solutions on NDMA formation from the nitrosation ofdimethylamine (DMA) by nitrite (NaNO2) was investigated at pH 6.4 in four kinds of buffer solutions, that is, Na2HPO4/C6H8O7,Na3(C6H5O7)/C6H8O7, NaH2PO4/NaOH, and NaH2PO4/Na2HPO4. Our observations demonstrate an unexpected inhibitoryeffect of the buffer solutions on NDMA formation and the phosphate buffer plays a more signi�cant role in inhibiting NDMAformation compared to the citrate buffer. Moreover, the amount of the phosphate in the buffer was also found to greatly impactthe formation of NDMA. A further investigation indicates that it is the interaction between NaH2PO4 and reactant NaNO2 ratherthan DMA that leads to the inhibitory effect of phosphate buffer during the DMA nitrosation reaction. is study expands theunderstanding of the in�uence of buffer solution on nitrosamines formation through the nitrosation pathway and further gives ahint for water plants to reduce the formation of nitrosamines.

1. Introduction

N-Nitrosamines are undesired industrial and environmentalpollutants with rising concerns due to their widespreadobservation in foods, soil, industrial workplace environ-ments, and cosmetics [1–5] as well as due to their high car-cinogenic risks [6–8]. US Environmental Protection Agency(U.S. EPA) has prescribed six kinds of nitrosamines (Scheme1) as probable B2 carcinogenic compounds to humans.Nitrosodimethylamine (NDMA), as the simplest moleculein structure among all the nitrosamines, has been demon-strated to be one of the most carcinogenic, mutagenic, andteratogenic nitrosamines [9]. In the U.S. EPA IntegratedRisk Information Service (IRIS) database, NDMA has beenidenti�ed to have an estimated 10−6 lifetime cancer risklevel at a concentration as low as 0.7 ng/L [10]. Althoughno national regulation controlling NDMA has been estab-lished yet, the Ontario Ministry of the Environment [11]and the California Department of Public Health [12] have

amaximumallowable concentration (MAC) of 9 and 10 ng/L,respectively.

Such a low MAC of NDMA has attracted great interestin the investigation of the formation mechanisms of NDMA.Generally, there are two types of pathways contributingto the NDMA formation, that is, oxidation of UDMHor chlorinated unsymmetrical dimethylhydrazine (UDMH-Cl) derived from the dimethylamine (DMA) pathway andnitrosation of the DMA pathway. e former pathway wasproposed recently by Choi and Valentine [13, 14] and Mitchand Sedlak [15] based on their �ndings that NDMA emergedduring the chlorine and chloramine disinfection of drinkingwater and sewage, in which DMA was �rst chlorinated toformUDMHorUDMH-Cl, which then underwent oxidationto yield NDMA. e later nitrosation pathway involvinga reaction between nitrite and common organic nitrogenprecursors, such as a DMA, has been known for a whileand has commanded much attention because it could occuralmost everywhere especially in vivo. Until now, it has been

2 Journal of Chemistry

N

N

O N

N

O N

N

O

N

N

ON

N O

N

NO

N-Nitrosodimethylamine(NDMA)

C2H6N2O C3H8N2OC6H14N2O

C8H18N2O 4 10 2C H N O 2C12H10N O

N-Nitrosomethylethylamine(NDMEA)(NdnPA)

N-Nitrosodiphenylamine

(NDPHA)

N-Nitrosodiethylamine

(NDEA)

N-Nitrosodi-n-butylamine

(NdnBA)

N-Nitrosodi-n-propylamine

S 1: Structures of six possibly human carcinogenic N-nitrosamines (U.S. EPA).

known that a series of reaction factors impact the NDMAformation including pH value, reaction time, temperature,and catalysts. Mirvish [16] demonstrated that NDMA forma-tion via DMA nitrosation is a highly pH-dependent reactionand the reaction occurs most rapidly at pH 3.4. Choi andValentine [14] and Mitch and Sedlak [15] found that theNDMA formation via oxidation of the UDMH(-Cl) pathwayappears to increase with pH, sharply increasing to about pH8. Several catalysts such as formaldehyde, chloral, carbondioxide, hypochlorite, and fulvic acid [17–21] have beenproved able to catalyze the nitrosation reaction at a neutral oreven basic pH.Andrzejewski andNawrocki [22] even veri�edthe formation of NDMA from the reaction of potassiumpermanganate and DMA via the nitrosation pathway. Veryrecently, Padhye et al. [23] found that the most commonlyused activated carbon during the preconcentration steps ofNDMA analysis can catalyze transformation of DMA to yieldtrace levels of NDMA under ambient aerobic conditions.eir results show that some unexpected potent errors couldde�nitely be induced by reaction conditions and analyticalmethods. us, it is signi�cantly important to obtain moreinformation about the in�uence of various reaction condi-tions and analytical methods on the NDMA formation.

It is well known that the NDMA-forming reaction ishighly dependent on pH; thus, a speci�c buffer [24–33]solution has to be used to regulate the suitable pH value foralmost all the investigations concerning NDMA. However, tothe best of our knowledge, little attention has been paid tothe in�uence of buffer matrices on the formation of NDMA,in spite of its widespread employment during these studies.Only Schreiber and Mitch [34] reported that the elevatedphosphate buffer is capable of decreasing NDMA formationduring chloramination. Due to its widespread application,phosphate buffer is one of the most favorable buffer matrixes;

therefore it is of signi�cant importance to understand thein�uence of phosphate buffer on the formation of NDMA.Otherwise, as Padhye et al. [23] reported, without suchknowledge, nitrosamine analysis will continue to be sus-ceptible to this potential error. However, up to now, thislack of attention results in an uncertainty whether phosphatebuffer affects the NDMA formation and how it executes itsin�uence.

Taking into account what was previously mentioned,batch experiments on NDMA formation from the re-action of DMA and nitrite were conducted in the fourkinds of buffer solutions, that is, Na2HPO4/C6H8O7,Na3(C6H5O7)/C6H8O7, NaH2PO4/NaOH, and NaH2PO4/Na2HPO4. e results distinctly illuminated the in�uenceof buffer matrix on the formation of NDMA from thenitrosation of DMA by nitrite. Because of the widespreadapplications of buffer matrices in the nitrosamines research,this �nding will expand our understanding of the in�uenceof buffer matrices on N-nitrosamines formation and willremind the researcher to pay more attention to minimize thepotent errors in the later investigations.

As a preliminary work to identify the in�uence ofphosphate buffer on NDMA formation, more systematicalinvestigations are underway to further illustrate roles of morebuffer matrices on the formation of N-nitrosamines.

2. Experimental Part

2.1. Materials. Standard-grade NDMA (solution in meth-anol, 100 ng/𝜇𝜇L) was obtained from Dr. EhrenstorferGmbH and diluted using dichloromethane to desiredconcentrations. Dimethylamine hydrochloride (DMA⋅HCl,99%+) and sodium nitrite (NaNO2, 99%+) werepurchased from Sinopharm Chemical Reagent Co. Ltd.and Beijing Chemical Works, respectively. Methanol,

Journal of Chemistry 3

hexane, and dichloromethane were of Chromatographicpurity and were used during the extraction process.Deuterated N-nitrosodimethylamine (NDMA-d6, 1000 𝜇𝜇g/mL in methanol) was purchased from CambridgeIsotopes (Andover, MA) and diluted to the requiredconcentration. Buffer solutions (pH = 6.4) containing Na2HPO4/H3(C6H5O7), Na3(C6H5O7)/C6H8O7), NaH2PO4/NaOH, and NaH2PO4/Na2HPO4, which were all ofanalytical laboratory grade, were prepared using ultrapurewater (18.2MΩ cm at 25∘C) from a Millipore Mili-Q waterpuri�cation system. All chemicals were used without furtherpuri�cation.

2.2. Preparing the Solid Phase Extraction (SPE) Cartridges.Materials used for the Solid Phase Extraction (SPE) cartridgeswere purchased from Agela Technologies, Tianjin. Methodsfor preparing the SPE column were the same as previouslyreported [35].e SPE cartridge was kept airtight until it wasused for the extraction of NDMA in aqueous solution.

2.3. NDMA Formation. All glassware used in theseexperiments were washed with an ultrasonic methodand air-dried prior to use. NDMA formation experimentsfrom DMA (10mM) and NaNO2(4mM) were conducted ingraduated tubes at 90∘C placed in a temperature-controlledwater bath. e tubes were sealed with a Te�on-linedscrew cap and wrapped by black plastic to shield themfrom light, so as to avoid NDMA photolysis. Reactionswere performed at pH 6.4 maintained by four buffersolutions: Na2HPO4/C6H8O7, Na3(C6H5O7)/C6H8O7,NaH2PO4/NaOH, and NaH2PO4/Na2HPO4. Reactionsolutions were prepared by the addition of the desiredamount of dimethylamine stock solution and NaNO2 stocksolution into the buffer solution with the total volume of10mL.

Dependence of NDMA formation on reaction conditionswas assessed by varying reaction time, buffer species as well asthe form and concentration of buffer components. All batchexperiments were performed at least triplicated to ensure therepeatability of the results.

2.4. Mechanical Studies. Experiments were conducted to elu-cidate how the buffermatrices inhibitNDMA formation fromDMA and NaNO2. e DMA/NaNO2 ratio was de�nitelycontrolled as either 100 or 0.01 in the two sets of reactions.In one case, 1mM NaH2PO4 was added into the citratebuffer solution containing 0.4mMNaNO2 and 40mMDMA,whereas in the other case, 1mM NaH2PO4 was added into acitrate buffer solution containing 40mMNaNO2 and 0.4mMDMA⋅NaH2PO4 was added aer all the other componentswere completelymixed.en themixtures were treated as thesame procedure described in NDMA Formation section.

2.5. NDMA Analysis. e analysis of NDMA was performedvia a method using a ermo Fisher Scienti�c GC-MSequipped with EI source (J�W scienti�c DB1701 column30m by 0.25mm I.D. by 0.25 𝜇𝜇m �lm thickness) aer solid-phase extraction (SPE). Prior to extraction, the reaction

solution was cooled to room temperature and all sampleswere dosed with the deuterated NDMA-d6. Extractions wereperformed with SPE cartridges. In brief, the SPE methodfor extracting NDMA in aqueous solution mainly includesmethanol activation, sample addition, hexane leaching, andmethylene dichloride elution [35]. In every step, the SPEcartridge was drained to dry thoroughly, and the �ow ratewas maintained at about 2-3mL/min. Aer eluting, theextract was concentrated to 3mL with dichloromethane andthe determination of NDMA from the concentrated extractwas achieved by using an isotopic dilution method basedon the mass detection of molecular ion (m/z = 74) andthe characteristic molecular ion of NDMA-d6 (m/z = 80).NDMA was analyzed by both full scan and selected ionmonitoring (SIM). e GC/MS operating conditions wereset as follows: splitless injections of 1𝜇𝜇L at 200∘C for inlet,230∘C for transfer line, and 250∘C for ion source, respectively.Initial temperature of oven was set at 45∘C (5min) wandthen ramped at 10∘C/min to 200∘C (held for 5min); massspectrometry was performed in electron ionization mode.Helium carrier gas was maintained at 1.0mL/min and theretention time for NDMA was 6.9min. e instrument andmethod detection limits for NDMA by using SPE-GC/MSmethod are 1 and 5 ng/L, respectively.

2.6. Computational Details. Quantum chemistry calcula-tions were conducted to gain more insights into the effectof a phosphate buffer on the formation of NDMA in the“DMA + NO2

−” reaction system. All structures and energyoptimization calculations of reactants and products wereperformed with Gaussian03W using the B3LYP methodin conjunction with 6-311+G(d,p) basis set [36]. Minimaand transition states were optimized and characterized byharmonic vibrational frequency analysis. Intrinsic ReactionCoordinate (IRC) [37] calculations were performed to con-�rm that every transition state connected the correspondingreactant and product through the minimized-energy path-way.

3. Results and Discussion

e yield of NDMA formation from DMA nitrosationaffected by many reaction conditions included reactant con-centration, pH, and reaction time. us, before embarkingon this work, it is necessary to optimize some reactionconditions.

3.1. Effect of Reaction Time on NDMA Formation via DMANitrosation. In order to choose an appropriate reaction time,experiments to evaluate the effect of a time scale on NDMAformation were performed. In this case, NDMA concentra-tions of the reactions of 10mM DMA with 4mM NaNO2 atpH 6.4 phosphate buffer were monitored every 1 h up to 9 h.

Figure 1 shows the results of time-dependent NDMAformation and exhibits a nonlinear correlation between theNDMA concentration and time scale. e yield of NDMAappears to increase rather slowly within the �rst hour,whereas it increases dramatically from 1 to 4 h with the value


Time (h)

0 1 2 3 4 5 6 7 8 9 10

ND

MA

(m

g/L

)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

F 1: NDMA formation concentrations in the reaction of10mM DMA and 4mM NaNO2 at 90

∘C in the pH 6.4 phosphatebuffer with the reaction time from 1 to 9 h. e vertical barsrepresent the standard deviation (𝑛𝑛 𝑛 𝑛).

reaching approximately 0.026mM. us, the NDMA yieldat 4 h reaches around 0.26% of the initial amount of DMAconcentration. During 4 to 9 h, the NDMA concentration isalmost the same. Although a small enhancement of NDMAformation occurs at 6 h, there is little change when theexperimental errors were taken into account. us, the yieldof NDMA can be considered to reach the maximum at 4 hand be stable aer this time.erefore, 4 h was selected as thereaction time throughout all the experiments involved in thisstudy. Similarly, when discussing the formation of NDMAfrom TMA and DMA, Scanlan et al. [31] also chose 4 h asthe reaction time.

3.2. Effect of Buffer Matrix on NDMA Formation. To thebest of our knowledge, although the in�uence of sev�eral parameters on NDMA formation has already beenmade during these years, little research work has beensystematically performed on the in�uence of buffermatrices on NDMA formation. us, four commonlyused buffer matrices, that is, Na2HPO4/C6H8O7, Na𝑛(C6H5O7)/C6H8O7, NaH2PO4/Na2HPO4, and NaH2PO4/NaOH, have been investigated in this study.

First of all, it is necessary to rule out the in�uence ofbuffer matrix on the already formed NDMA. To achieve thisgoal, a standard 300 𝜇𝜇g/L NDMA was put into deionizedwater as control and into four buffer matrices studiedhere; then the NDMA concentrations were monitoredaer 4 h. e concentrations of the standard NDMA in�ve different solutions were shown in Figure 2. As shownin Figure 2, the average NDMA concentrations were278, 273, 276, 287, and 273 𝜇𝜇g/L for deionized water,Na2HPO4/C6H8O7, Na𝑛(C6H5O7)/C6H8O7, NaH2PO4/

NDMA (mg/L)

0 0.1 0.2 0.3 0.4 0.5

Diff

eren

t b

uff

er m

atri

ces

1

2

3

4

5

1: Deionized water

4/H2: Na2HPO 7)5O6H3(C7)5OH63(C7)/H6H3: Na3(C 5O e value of standard NDMA

4/NaOH4: NaH2PONa2HPO44/5: NaH2PO

F 2: Concentrations of 300 𝜇𝜇g/L standard NDMA aerstoring at 90∘C in ultrawater (blank), Na2HPO4/C6H8O7,Na𝑛(C6H5O7)/C6H8O7, NaH2PO4/NaOH, andNaH2PO4/Na2HPO4 buffer solutions (pH = 6.4) for 4 h. ehorizontal bars represent the standard deviation (𝑛𝑛 𝑛 𝑛).

NaOH, and NaH2PO4/Na2HPO4 buffer, respectively. It isclear that all the values of the NDMA concentrations inthe solutions are slightly lower than the 300 𝜇𝜇g/L originalstandard NDMA. is decrease results from the SPEextraction recovery. However, the results show that theNDMA concentrations in the four buffer solutions arealmost the same as that of deionized water. us, it can beconcluded that the four types of buffer solutions employedin this study wholly have little effect on the standard NDMA.erefore, it is reasonable to preclude the in�uence of buffermatrix on the already formed NDMA.

Now it is time to see whether the buffer matrix hasan in�uence on NDMA formation during the reactionof NaNO2 and DMA. e NDMA concentrationsformed from the reactions of 10mM DMA with 4mMNaNO2 in Na2HPO4/C6H8O7, Na𝑛(C6H5O7)/C6H8O7,NaH2PO4/Na2HPO4, and NaH2PO4/NaOH buffer solutionswere shown in Figure 3. e results of Figure 3 demonstratean obvious difference among the NDMA formationconcentrations in the four buffer solutions, whose values are1.18, 1.66, 1.16, and 2.42mg/L for Na2HPO4/C6H8O7,Na𝑛(C6H5O7)/C6H8O7, NaH2PO4/Na2HPO4, andNaH2PO4/NaOH buffer solutions, respectively. e NDMAyield for the four buffers ranged from 0.26% to 0.54% ofthe initial amount of DMA concentration and the largestdifferent yield induced from the different buffer reached0.28% which is almost in the same level of the lowest yield.�y comparing the yields of NDMA in the �rst column withthat of the second, that is, in the Na2HPO4/C6H8O7 andNa𝑛(C6H5O7)/C6H8O7 buffer solutions, the NDMA yieldin the buffer solution involved an amount of phosphate was


Different buffer matrices

ND

MA

(m

g/L

)

0

0.5

1

1.5

2

2.5

3

4Na2HPO /78O6HC 78OH6CNa3 )/76H(C 5O

Na2HPO44/NaH2PO4/NaH2PO

NaOH

F 3: NDMA formation concentrations in thereaction of 10mM DMA and 4mM NaNO2 in theNa2HPO4/C6H8O7, Na3(C6H5O7)/C6H8O7, NaH2PO4/NaOH,and NaH2PO4/Na2HPO4 buffer solutions (pH = 6.4). Reaction time= 4 h. Temperature = 90∘C. e vertical bars represent the standarddeviation (𝑛𝑛 𝑛 3).

relatively lower than that in the citrate buffer by around28.9%. Similar results can also be found when a comparisonbetween the yields of NDMA in Na3(C6H5O7)/C6H8O7and NaH2PO4/Na2HPO4 buffers was made. It indicates thatthe phosphate component of the buffer plays a signi�cantrole in inhibiting NDMA formation compared to thecitrate buffer. Unfortunately, the concentration of NDMAin the NaH2PO4/NaOH buffer (the third column) israther high, with the value of 2.42mg/L, which seemscompletely contrary to the above conclusion. However,further analysis of the concentration of the phosphate listedin Table 1 demonstrates that the �nal concentrations ofphosphate in Na2HPO4/C6H8O7, NaH2PO4/Na2HPO4,and NaH2PO4/NaOH buffer solutions are approximately0.14, 0.2, and 0.05M, respectively. e �nal concentrationof phosphate in the NaH2PO4/NaOH buffer is 2.8 timeslower than that of the other groups. erefore, the higherNDMA formation is possibly attributed to the less amountof phosphate in the NaH2PO4/NaOH buffer. It suggeststhat the amount of phosphate in the buffer may be a criticalfactor for NDMA formation during the nitrosation of DMAby nitrite. is conclusion is in a good agreement with theresult reported by Schreiber and Mitch [34] that the elevatedphosphate concentration can signi�cantly decrease NDMAformation during DMA chloramination with the decreasebeing ∼50% when the concentration increased from 5 to50mM.

Based on the results obtained it can be concluded thatthe phosphate buffer has an inhibitory effect for NDMAformation. However, which ingredient of the phosphatebuffer is an inhibitory and how it inhibits NDMA formationwill be discussed in the next section.

3.3. Role of Buffer Components in the NDMA Formation. Inorder to examine which ingredient of buffer components

ND

MA

(m

g/L

)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 10 20 30 40

4NaH2PONa3 )76H(C 5O

Amounts of the addition of components (mmol⋅10−3)

F 4: NDMA formation in the presence of variable amountsof NaH2PO4 and Na3(C6H5O7) addition; 4 h reaction; 90∘C. epH was maintained at 6.4 with Na3(C6H5O7)/C6H8O7 buffer. evertical bars represent the standard deviation (𝑛𝑛 𝑛 3).

leads to the decrease of NDMA formation, experiments wereconducted in which various concentrations of NaH2PO4or Na3(C6H5O7) were added into a pH 6.4 citrate buffercontaining 10mM DMA and 4mM NaNO2. Citrate bufferwas selected as the buffer matrix because it appears to havelittle in�uence on NDMA formation. It should be noted thatH2PO4

− is the main existing form of the added NaH2PO4in pH 6.4 buffer solution, which was accounted for thedissociation constants of phosphoric acid (pKa1 = 2.16, pKa2= 7.21, pKa3 = 12.32).

e NDMA concentrations at various dosages ofNaH2PO4 or Na3(C6H5O7) were listed in Figure 4. Basedon the results of Figure 4, either addition of NaH2PO4or Na3(C6H5O7) causes a decrease of NDMA formation.Moreover the decrease of NDMA caused by NaH2PO4was more signi�cant than that caused by Na3(C6H5O7)with the same concentration. For instance, the NDMAconcentration decreased from 1.66mg/L at 0mmolNaH2PO4 to approximately 1.31mg/L at 0.01mmolNaH2PO4, while 0.01mmol Na3(C6H5O7) decreased theNDMA concentration from 1.66mg/L to approximately1.49mg/L. erefore, it can be concluded that both ofNaH2PO4 and Na3(C6H5O7) are able to inhibit NDMAformation, but the inhibitory ability of NaH2PO4 issomewhat stronger than that of Na3(C6H5O7). is �ndingis well consistent with the conclusion obtained from theabove section. Furthermore, it is worth noting that theNDMA-inhibiting ability of NaH2PO4 and Na3(C6H5O7)was also shown in a dose-dependent manner. As shown inFigure 4, with the amount of NaH2PO4 addition increasedfrom 0 to 0.03mmol, the concentration of NDMA formationdecreased from 1.66 to 1.17mg/L while it is changed from


T 1: Buffer solution (pH = 6.4) preparation.

Buffer type Na2HPO4/C6H8O7,0.1M

Na3(C6H5O7)/C6H8O7,0.1M

NaH2PO4/NaOH,0.05M

NaH2PO4/Na2HPO,0.2M

Volume (mL) 13.85/6.15(20, total)

2.0/18.0(20, total)

5.0/1.26(dilute to 20, total)

5.3/14.7(20, total)

1.66 to 1.39mg/L over the same range for Na3(C6H5O7).In comparison to the addition of 0.03mmol NaH2PO4or Na3(C6H5O7), the NDMA concentration shows littlevariation with the addition of 0.04mmol. It indicates thatthe addition of around 0.03mmol reaches an inhibitorysaturation for the reagent concentrations applied in thisstudy.

e results of these experiments clearly verify the in-hibitory effects of NaH2PO4 buffer on NDMA formation.ey also indicate that the choice of phosphate buffer cande�nitely impact on the analytical results in nitrosaminemeasurements.

3.4. Proposed Mechanism of NaH2PO4 Affecting NDMA For-mation during DMA Nitrosation. To illustrate the inhibitionmechanism of NaH2PO4 on NDMA formation, two kindsof experiments were designed to conduct in a citrate bufferto determine which reactant (NaNO2 or DMA) can interactwith NaH2PO4 in the DMA nitrosation reaction. In the �rstexperiment, the concentration of NaNO2 was chosen to be40mM which is far greater than that of DMA (0.4mM),while in the second one, in contrast, the concentration ofDMA was 40mM which is far greater compared to that ofNaNO2 (0.4mM). e concentration ratio of DMA/NaNO2for the two experiments was controlled to be 0.01 and 100,respectively. Since the concentration ofNaH2PO4 was chosento be 1mM, it means that NaH2PO4 is in excess compared toDMA and NaNO2 for the two experiments, respectively. eresults for the two reactions are presented in Figures 5(a) and5(b), respectively.

As shown in Figure 5(a), the yield of NDMA with thepresence of NaH2PO4 was 577 𝜇𝜇g/L while the control exper-iment (marked as blank) without the NaH2PO4 addition was580 𝜇𝜇g/L. e NDMA concentrations are approximately thesame which reveals that there is no signi�cant differencein NDMA formation caused by the NaH2PO4 addition forthe �rst experiment. It also indicates that NaH2PO4 doesnot react or react slowly with DMA otherwise DMA wouldbe completely exhausted because the concentration ratio ofDMA/NaNO2 is only 0.01, which would lead to the declineof NDMA concentration.

In the second experiment (DMA/NaNO2 ratio controlledat 100), NaH2PO4 was found to reduce NDMA formation(Figure 5(b)). e NDMA concentration in the experimentalgroup was around 53𝜇𝜇g/L, whereas that in the control exper-iment was 70 𝜇𝜇g/L. is result shows that NDMA formationwas almost lower by 24% aer adding NaH2PO4. Since theconcentration of DMA was in large excess over NaNO2 inthe second experiment, it can be inferred that it is the mutualinteraction between NaNO2 or its derivative and NaH2PO4that resulted in the decrease of NDMA formation.

0

100

200

300

400

500

600

700

0

10

20

30

40

50

60

70

80

(b)(a)

Blank Blank4NaH2PO 4NaH2PO

F 5: (a) Amounts of NDMA formed aer 2 h from the reactionof a large excess of NaNO2 (40mM) with DMA (0.4mM). (b)Amounts ofNDMA formed from the reaction between a large excessofDMA(40mM) andNaNO2 (0.4mM).e reaction solutionswerebuffered at pH 6.4 citrate buffer at 90∘C with the concentrationof NaH2PO4 being 1mM. e vertical bars represent the standarddeviation (𝑛𝑛 𝑛 3).

Based on the above two experiments, it can be concludedthat the inhibition mechanism of NaH2PO4 on NDMA for-mation during the DMAnitrosation reaction results from theinteraction between NaNO2 or its derivative and NaH2PO4.

3.5. eoretical Work on the Inhibition Mechanism ofNaH2PO4. e experiments have shown that phosphateexhibits inhibitory effect on the formation of NDMA. Basi-cally, the possibility of reaction between amine and phos-phate accounting for the inhibition can be excluded, becausethe nucleophilic amine (DMA) is expected to resist thephosphate anion. erefore, we focused on the interactionbetween phosphate and nitrosating agent N2O3 which isviewed as the effective nitrosating agent in the solventnitrosation reactions [18]. e nitrosating agent N2O3 isproduced via the protonation of the nitrite ion and furtherdehydration of two HNO2 molecules (1), and this has beenwell established in previous studies [38, 39]

H+ +NO2− ⟶ HNO2

2HNO2 ⟶ N2O3 +H2O(1)

It should be noted that H2PO4− is the main existing form

of the added NaH2PO4 in pH 6.4 buffer solution; therefore,only H2PO4

− anion was considered in the calculations. eresults show that H2PO4

− reacts easily with N2O3. erefore,H2PO4

− is proposed to be responsible for the inhibition ofNDMA formation through scavenging the nitrosating agent


+

NDMA

DMA

DMA

2.565

1.299

1.306

1.707

1.197

1.244 1.177

1.298

2.363

1.909

1.194 1.226

1.292

2.532

1.882

1.151 1.353

2.3151.933

1.179 1.235

2.1062.129

TS1

TS2

TS3

TS5

TS4

N2O32HNO2

HNO2HNO2

DMA: (CH3)2N–HNDMA: (CH3)2N–NO

H2PO4− HNO2

H2PO4−

H2PO4−

2OH

2OH

−HPO4NO

Phosphate-buffered ‘‘DMA+ NO2−” system

F 6: Possible reactions and transition structures (distances in angstroms) involved in the phosphate-buffered “DMA + NO2−” reaction

system.

N2O3. Detailed discussion is given below to support thishypothesis. Reactions involved in the phosphate-buffered“DMA + NO2

−” reaction system are illustrated in Figure 6.Corresponding energy data are collected in Table 2.

As shown in Figure 6, the fate of N2O3 is related to threedifferent pathways for its further reactions: (1) reaction withDMA to formNDMA; (2) hydrolysis to regenerateHNO2; (3)elimination by H2PO4

− to give HPO4NO−.

Nitrosation of DMA by N2O3 occurs in a concerted stepwith a �ve-membered cyclic transition state (TS1 in Figure 6).NDMA forms via releasing nitrous acid (HNO2). Activationenthalpy (Table 2) was calculated to be 6.87 kcal/mol, andthe reaction is exothermic by 15.90 kcal/mol. NDMA formseasily via the N2O3-induced nitrosation of DMA, and this isconsistent with previous studies [40, 41]. In addition, N2O3undergoes hydrolysis (TS3-involved pathway in Figure 6) toreproduce HNO2, and the activation energy (12.80 kcal/mol)is approximately twice as high as that (6.87 kcal/mol) of itsreactionwithDMA to formNDMA.Moreover, the hydrolysis

is slightly endothermic by 1.50 kcal/mol. erefore, N2O3 ismore favored to be consumed by DMA to produce NDMAthan to be hydrolyzed into HNO2.

A very interesting �nding is the reaction between N2O3and the phosphate buffer (HPO4

2−). Figure 6 shows that thereaction of H2PO4

− with N2O3 occurs with a �ve-memberedcyclic transition state TS2, and a new species (HPO4NO

−)is generated via releasing HNO2. Corresponding activationenthalpy (Table 2) was calculated to be 8.40 kcal/mol, whichis very close to the activation enthalpy (6.87 kcal/mol) ofthe N2O3-induced nitrosation of DMA. is indicates thatthe reaction of N2O3 and DMA to give NDMA competeswith its reaction with H2PO4

− to produce HPO4NO−. Note

that HPO4NO− is an ON+ carrier which may also have

nitrosating ability. erefore, the nitrosation of DMA byHPO4NO

− (TS4-involved pathway in Figure 6) was studied,however, the activation energy (22.05 kcal/mol) is muchhigher than that (6.87 kcal/mol) of the N2O3-induced nitro-sation. Consequently, HPO4NO

− is a weak nitrosating agent


T 2: Calculated activation energies and reaction energies (in kcal/mol) for possible reactions involved in the phosphate-buffered “DMA+ NO2

−” reaction system at the B3LYP/6-311+G(d,p) level.

Reactions Δ𝐻𝐻≠ Δ𝐺𝐺≠ Δ𝐻𝐻 Δ𝐺𝐺DMA + N2O3 → NDMA + HNO2 6.87 8.24 −15.90 −18.05N2O3 + H2PO4

− → HPO4NO−+ HNO2 8.40 10.83 −2.59 −2.37

DMA + HPO4NO− → NDMA + H2PO4

− 22.05 24.97 −28.39 −28.52N2O3 + H2O → 2HNO2 12.80 15.85 1.50 −0.06HPO4NO

− + H2O → H2PO4− + HNO2 25.41 28.63 −8.77 −8.15

when compared with N2O3. In addition, Lewis et al. [42]proposed that the hydrolysis of HPO4NO

− is rapid whichcan account for the inhibitory effect of phosphate for theN2O3-induced nitrosation of DMA. However, the calculatedresult does not support this conclusion because the activationenergy predicted for the hydrolysis of HPO4NO

− (TS5-related pathway in Figure 6) is the highest (25.41 kcal/mol,Table 2) among the reactions of interest.

To conclude, the inhibited formation of NDMA inour experiment resulted from the fast elimination of thenitrosating agent N2O3 by H2PO4

− to give HPO4NO−, and

HPO4NO− is a “stable” species whose further reaction with

DMA and hydrolysis are relatively slow in the studied “DMA+ NO2

−” system.

4. Conclusions

In this paper, batch experiments on NDMA formation fromthe reaction of DMA and nitrite were systematically con-ducted in the four kinds of buffer solutions, that is,Na2HPO4/C6H8O7, Na3(C6H5O7)/C6H8O7, NaH2PO4/NaOH, and NaH2PO4/Na2HPO4. Experimental dataindicated that the phosphate component of the buffer plays asigni�cant role in inhibiting NDMA formation compared tothe citrate buffer and the inhibitory effect was proved to bein a dose-dependent manner. Moreover, it is the interactionbetween NaH2PO4 and reactant NaNO2 rather than DMAthat leads to the inhibitory effect of phosphate bufferduring the DMA nitrosation reaction. eoretical resultsfound that a �ve-membered ring intermediate compoundformed during the reaction of H2PO4

− with N2O3, and thenitrosation ability of this intermediate was weaker than thatof N2O3, which leads to the reduced amount of NDMAformation. is theoretical result supports the experimentalconclusion and further points out why phosphate bufferinhibits the formation of NDMA during the nitrosation ofDMA. is study discovered the inhibition of phosphatebuffer solutions on NDMA formation and gave a hint toreduce the carcinogenic nitrosamines.

Acknowledgments

e authors thank James Tarzia P. Eng., Beijing Universityof Technology for English language suggestion.is researchwas supported by National Natural Science Foundation ofChina (no. 20903006), Beijing Natural Science Foundation(no. 2092008), Beijing Nova Program (no. 2008B09), and

Environmental Protection Public Project (no. 2010467032).ey thank the High Performance Computing (HPC) Centerin Beijing University of Technology and Beijing ComputingCenter for providing the high-performance computing clus-ters.

References

[1] L. Padhye, Y. Luzinova, M. Cho, B. Mizaikoff, J. H. Kim, and C.H. Huang, “PolyDADMAC and dimethylamine as precursorsof N-nitrosodimethylamine during ozonation: reaction kineticsand mechanisms,” Environmental Science & Technology, vol. 45,no. 10, pp. 4353–4359, 2011.

[2] L. Padhye, U. Tezel, W. A. Mitch, S. G. Pavlostathis, and C.H. Huang, “Occurrence and fate of nitrosamines and theirprecursors in municipal sludge and anaerobic digestion sys-tems,” Environmental Science & Technology, vol. 43, no. 9, pp.3087–3093, 2009.

[3] M. Izquierdo-Pulido, J. F. Barbour, and R. A. Scanlan, “N-nitrosodimethylamine in Spanish beers,” Food and ChemicalToxicology, vol. 34, no. 3, pp. 297–299, 1996.

[4] S. Haruta, W. Chen, J. Gan, J. Simunek, A. C. Chang, andL. Wu, “Leaching risk of N-nitrosodimethylamine (NDMA)in soil receiving reclaimed wastewater,” Ecotoxicology andEnvironmental Safety, vol. 69, no. 3, pp. 374–380, 2008.

[5] F. De Vocht, I. Burstyn, K. Straif et al., “Occupational exposureto NDMA and NMor in the European rubber industry,” Journalof Environmental Monitoring, vol. 9, no. 3, pp. 253–259, 2007.

[6] W. Lijinsky and S. S. Epstein, “Nitrosamines as environmentalcarcinogens,” Nature, vol. 225, no. 5227, pp. 21–23, 1970.

[7] R. N. Loeppky and C. J. Micheljda, Nitrosamines and RelatedN-Nitroso Compounds: Chemistry and Biochemistry, AmericanChemical Society, Washington, DC, USA, 1994.

[8] United States Environmental Protection Agency (USEPA) IRISdatabase, 2002.

[9] Y. Goto, T. Matsuda, K. Ito et al., “Mutagenicities of N-nit-rosodimethylamine and N-nitrosodiethylamine in Drosophilaand their relationship to the levels of O-alkyl adducts in DNA,”Mutation Research, vol. 425, no. 1, pp. 125–134, 1999.

[10] N.M. Vieno, H. Härkki, T. Tuhkanen, and L. Kronberg, “Occur-rence of pharmaceuticals in river water and their eliminationin a pilot-scale drinking water treatment plant,” EnvironmentalScience & Technology, vol. 41, no. 14, pp. 5077–5084, 2007.

[11] Safe Drinking Water Act 2002, Ontario Regulation 169/03,Schedule 2. 2002.

[12] CaliforniaDrinkingWater: NDMA-Related Activities, CaliforniaDepartment of Public Health, 2009.

[13] J. Choi and R. L. Valentine, “Formation of N-nitrosod-imethylamine (NDMA) from reaction of monochloramine:


a new disinfection by-product,” Water Research, vol. 36, no. 4,pp. 817–824, 2002.

[14] J. Choi and R. L. Valentine, “N-Nitrosodimethylamine forma-tion by free-chlorine-enhanced nitrosation of dimethylamine,”Environmental Science & Technology, vol. 37, no. 21, pp.4871–4876, 2003.

[15] W. A. Mitch and D. L. Sedlak, “Formation of N-nitrosodimethylamine (NDMA) from dimethylamine duringchlorination,” Environmental Science & Technology, vol. 36, no.4, pp. 588–595, 2002.

[16] S. S. Mirvish, “Formation of N-nitroso compounds: chemistry,kinetics, and in vivo occurrence,” Toxicology and Applied Phar-macology, vol. 31, no. 3, pp. 325–351, 1975.

[17] D. L. H. Williams, Nitrosation Reactions and the Chemistry ofNitric Oxide, Elsevier, Amsterdam, e Netherlands, 2004.

[18] L. K. Keefer and P. P. Roller, “N-nitrosation by nitrite ion inneutral and basic medium,” Science, vol. 181, no. 4106, pp.1245–1247, 1973.

[19] S. Weerasooriya and C. Dissanayake, “e enhanced formationof N-nitrosamines in fulvic acid mediated environment,” Envi-ronmental Toxicology and Chemistry, vol. 25, no. 1, pp. 57–62,1989.

[20] C. L. Lv, Y. D. Liu, R. G. Zhong, and Y. Wang, “eoreticalstudies on the formation of N-nitrosodimethylamine,” Journalof Molecular Structure, vol. 802, no. 1–3, pp. 1–6, 2007.

[21] C. L. Lv, Y. D. Liu, and R. G. Zhong, “eoretical investigationof N-nitrosodimethylamine formation from dimethylaminenitrosation catalyzed by carbonyl compounds,” e Journal ofPhysical Chemistry A, vol. 113, no. 4, pp. 713–718, 2009.

[22] P. Andrzejewski and J. Nawrocki, “N-nitrosodimethylamine(NDMA) as a product of potassium permanganate reactionwith aqueous solutions of dimethylamine (DMA),” WaterResearch, vol. 43, no. 5, pp. 1219–1228, 2009.

[23] L. Padhye, P. Wang, T. Karan�l, and C. H. Huang, “Unexpectedrole of activated carbon in promoting transformation of sec-ondary amines to N-nitrosamines,” Environmental Science &Technology, vol. 44, no. 11, pp. 4161–4168, 2010.

[24] M. Kohashi, N. Keitoku, Y.Matsu-Ura, and T.Watanabe, “Effectof ceramics radiation on nitrosodimethylamine production,”Bioscience, Biotechnology, and Biochemistry, vol. 59, no. 12, pp.2193–2197, 1995.

[25] U. Tezel, L. Padhye, C. H. Huang, and S. G. Pavlostathis,“Biotransformation of nitrosamines and precursor secondaryamines under methanogenic conditions,” Environmental Sci-ence & Technology, vol. 45, no. 19, pp. 8290–8297, 2011.

[26] H. Delalu and A. Marchand, “e Investigation of the con-ditions for the formation of formaldehyde dimethylhydrazone(FDMH) from unsymmetrical dimethylhydrazine (UDMH)oxidation by chloramine. 1. Kinetics of the unsymmetri-cal dimethylhydrazine oxidation by chloramines,” Journal deChimie Physique et de Physico-Chimie Biologique, vol. 84, p. 991,1987.

[27] M. A. Zol�gol, M. Bagherzadeh, A. G. Choghamarani, H.Keypour, and S. Salehzadeh, “N-nitrosation of secondaryamines under mild and heterogeneous conditions,” SyntheticCommunications, vol. 31, no. 8, pp. 1161–1166, 2001.

[28] Z. Chen and R. L. Valentine, “Modeling the formation of N-nitrosodimethylamine (NDMA) from the reaction of NaturalOrganic Matter (NOM) with monochloramine,” EnvironmentalScience & Technology, vol. 40, no. 23, pp. 7290–9297, 2006.

[29] J. M. Kemper, S. S.Walse, andW. A.Mitch, “Quaternary aminesas nitrosamine precursors: a role for consumer products?” Envi-ronmental Science & Technology, vol. 44, no. 4, pp. 1224–1231,2010.

[30] J. L. Roux, H. Gallard, and J. P. Croue, “Formation of NDMAand halogenated DBPs by chloramination of tertiary amines:the in�uence of bromide ion,” Environmental Science & Tech-nology, vol. 46, no. 3, pp. 1581–1589, 2012.

[31] R. A. Scanlan, S. M. Lohsen, D. D. Bills, and L. M. Libbey,“Formation of dimethylnitrosamine from dimethylamine andtrimethylamine at elevated temperatures,” Journal of Agricul-tural and Food Chemistry, vol. 22, no. 1, pp. 149–151, 1974.

[32] J. Luh and B. J. Marinnas, “Bromide ion effect on N-nitrosodimethylamine formation by monochloramine,” Envi-ronmental Science & Technology, vol. 46, pp. 5085–5092, 2012.

[33] B. Xu, C. Qin, C. Y. Hu, and N. Y. Gao, “Degradation kineticsand N-Nitrosodimethylamine formation during monochlo-ramination of chlortoluron,” Science of the Total Environment,vol. 417-418, pp. 241–247, 2012.

[34] I. M. Schreiber and W. A. Mitch, “Nitrosamine formationpathway revisited: the importance of chloramine speciation anddissolved oxygen,” Environmental Science & Technology, vol. 40,no. 19, pp. 6007–6014, 2006.

[35] Q. Liu, Y. D. Liu, and R. G. Zhong, “A comparative investigationof the nitrosodimethylamine formation from the nitrosationof dimethylamine and trimethylamine by nitrite under theneutral condition,” in Proceedings of the IEEE InternationalConference on Bioinformatics and Biomedical Engineering, pp.1–4, Piscataway, NJ, USA, 2011.

[36] W. J. Pietro, M. M. Francl, W. J. Hehre, D. J. Defrees, J. A. Pople,and J. S. Binkley, “Self-consistent molecular orbital methods.24. Supplemented small split-valence basis sets for second-rowelements,” Journal of the American Chemical Society, vol. 104,no. 19, pp. 5039–5048, 1982.

[37] C. Gonzalez and H. B. Schlegel, “An improved algorithm forreaction path following,” e Journal of Chemical Physics, vol.90, no. 4, pp. 2154–2161, 1989.

[38] M. M. Fickling, A. Fischer, B. R. Mann, J. Packer, andJ. Vaughan, “Hammett substituent constants for electron-withdrawing substituents: dissociation of phenols, aniliniumions and dimethylanilinium ions,” Journal of the AmericanChemical Society, vol. 81, no. 16, pp. 4226–4230, 1959.

[39] T. Turney and G. Wright, “Nitrous acid and nitrosation,”Chemical Reviews, vol. 59, no. 3, pp. 497–513, 1959.

[40] S. S. Mirvish, “e etiology of gastric cancer. Intragastricnitrosamide formation and other theories,” Journal of theNational Cancer Institute, vol. 71, no. 3, pp. 629–647, 1983.

[41] Z. Sun, Y. D. Liu, C. L. Lv, and R. G. Zhong, “eoretical inves-tigation of the isomerization of N2O3 and the N-nitrosation ofdimethylamine by asym-N2O3, sym-N2O3, and trans-cis N2O3isomers,” Journal of Molecular Structure, vol. 908, no. 1–3, pp.107–113, 2009.

[42] R. S. Lewis, S. R. Tannenbaum, andW.M. Deen, “Kinetics of N-nitrosation in oxygenated nitric oxide solutions at physiologicalpH: role of nitrous anhydride and effects of phosphate andchloride,” Journal of the American Chemical Society, vol. 117, no.14, pp. 3933–3939, 1995.

Submit your manuscripts athttp://www.hindawi.com

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation http://www.hindawi.com Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttp://www.hindawi.com

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation http://www.hindawi.com Volume 2014


CatalystsJournal of

Documents

3 FTFBSDI SUJDMF 5IF*OëVFODFPG1IPTQIBUF#VFSPOUIF ...downloads.hindawi.com/journals/jchem/2013/818943.pdf · mjuumf buufoujpo ibt cffo qbje up uif jo vfodf pg cv fs nbusjdft po uif