Journal of Chemistry

Volume 2013 (2013), Article ID 818943, 9 pages

http://dx.doi.org/10.1155/2013/818943

Research Article

The Influence of Phosphate Buffer on the Formation of N-Nitrosodimethylamine from Dimethylamine Nitrosation

Long Xu,¹ Zhi Sun,¹ Qing Ming Liu,¹ Yong Dong Liu,¹ Ru Gang Zhong,¹ and Fengchang Wu²

¹College of Life Science & Bioengineering, Beijing University of Technology, Beijing 100124, China

²State Environmental Protection Key Laboratory for Lake Pollution Control, Chinese Research Academy of Environmental Sciences, Beijing 100021, China

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

Academic Editor: Luis F. Guido

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

Abstract.

Buffer solutions were widely used for almost all the investigations concerning N-nitrosodimethylamine (NDMA), a member of powerful mutagenic and carcinogenic compounds which are ubiquitous in the environment. However, whether or how the buffer matrixes influence NDMA formation is still unknown. The effect of buffer solutions on NDMA formation from the nitrosation of dimethylamine (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 inhibitory effect of the buffer solutions on NDMA formation and the phosphate buffer plays a more significant role in inhibiting NDMA formation compared to the citrate buffer. Moreover, the amount of the phosphate in the buffer was also found to greatly impact the formation of NDMA. A further investigation indicates that it is the interaction between NaH₂PO₄ and reactant NaNO₂ rather than DMA that leads to the inhibitory effect of phosphate buffer during the DMA nitrosation reaction. This study expands the understanding of the influence of buffer solution on nitrosamines formation through the nitrosation pathway and further gives a hint for water plants to reduce the formation of nitrosamines.

1. Introduction

N-Nitrosamines are undesired industrial and environmental pollutants with rising concerns due to their widespread observation in foods, soil, industrial workplace environments, and cosmetics [1–5] as well as due to their high carcinogenic risks [6–8]. US Environmental Protection Agency (U.S. EPA) has prescribed six kinds of nitrosamines (Scheme 1) as probable B2 carcinogenic compounds to humans. Nitrosodimethylamine (NDMA), as the simplest molecule in structure among all the nitrosamines, has been demonstrated to be one of the most carcinogenic, mutagenic, and teratogenic nitrosamines [9]. In the U.S. EPA Integrated Risk Information Service (IRIS) database, NDMA has been identified to have an estimated 10⁻⁶ lifetime cancer risk level at a concentration as low as 0.7 ng/L [10]. Although no national regulation controlling NDMA has been established yet, the Ontario Ministry of the Environment [11] and the California Department of Public Health [12] have a maximum allowable concentration (MAC) of 9 and 10 ng/L, respectively.

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

Such a low MAC of NDMA has attracted great interest in the investigation of the formation mechanisms of NDMA. Generally, there are two types of pathways contributing to the NDMA formation, that is, oxidation of UDMH or chlorinated unsymmetrical dimethylhydrazine (UDMH-Cl) derived from the dimethylamine (DMA) pathway and nitrosation of the DMA pathway. The former pathway was proposed recently by Choi and Valentine [13, 14] and Mitch and Sedlak [15] based on their findings that NDMA emerged during the chlorine and chloramine disinfection of drinking water and sewage, in which DMA was first chlorinated to form UDMH or UDMH-Cl, which then underwent oxidation to yield NDMA. The later nitrosation pathway involving a reaction between nitrite and common organic nitrogen precursors, such as a DMA, has been known for a while and has commanded much attention because it could occur almost everywhere especially in vivo. Until now, it has been known that a series of reaction factors impact the NDMA formation including pH value, reaction time, temperature, and catalysts. Mirvish [16] demonstrated that NDMA formation via DMA nitrosation is a highly pH-dependent reaction and the reaction occurs most rapidly at pH 3.4. Choi and Valentine [14] and Mitch and Sedlak [15] found that the NDMA formation via oxidation of the UDMH(-Cl) pathway appears to increase with pH, sharply increasing to about pH 8. Several catalysts such as formaldehyde, chloral, carbon dioxide, hypochlorite, and fulvic acid [17–21] have been proved able to catalyze the nitrosation reaction at a neutral or even basic pH. Andrzejewski and Nawrocki [22] even verified the formation of NDMA from the reaction of potassium permanganate and DMA via the nitrosation pathway. Very recently, Padhye et al. [23] found that the most commonly used activated carbon during the preconcentration steps of NDMA analysis can catalyze transformation of DMA to yield trace levels of NDMA under ambient aerobic conditions. Their results show that some unexpected potent errors could definitely be induced by reaction conditions and analytical methods. Thus, it is significantly important to obtain more information about the influence of various reaction conditions and analytical methods on the NDMA formation.

It is well known that the NDMA-forming reaction is highly dependent on pH; thus, a specific buffer [24–33] solution has to be used to regulate the suitable pH value for almost all the investigations concerning NDMA. However, to the best of our knowledge, little attention has been paid to the influence 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 elevated phosphate buffer is capable of decreasing NDMA formation during chloramination. Due to its widespread application, phosphate buffer is one of the most favorable buffer matrixes; therefore it is of significant importance to understand the influence of phosphate buffer on the formation of NDMA. Otherwise, as Padhye et al. [23] reported, without such knowledge, nitrosamine analysis will continue to be susceptible to this potential error. However, up to now, this lack of attention results in an uncertainty whether phosphate buffer affects the NDMA formation and how it executes its influence.

Taking into account what was previously mentioned, batch experiments on NDMA formation from the reaction of DMA and nitrite were conducted in the four kinds of buffer solutions, that is, Na₂HPO₄/C₆H₈O₇, Na₃(C₆H₅O₇)/C₆H₈O₇, NaH₂PO₄/NaOH, and NaH₂PO₄/Na₂HPO₄. The results distinctly illuminated the influence of buffer matrix on the formation of NDMA from the nitrosation of DMA by nitrite. Because of the widespread applications of buffer matrices in the nitrosamines research, this finding will expand our understanding of the influence of buffer matrices on N-nitrosamines formation and will remind the researcher to pay more attention to minimize the potent errors in the later investigations.

As a preliminary work to identify the influence of phosphate buffer on NDMA formation, more systematical investigations are underway to further illustrate roles of more buffer matrices on the formation of N-nitrosamines.

2. Experimental Part

2.1. Materials

Standard-grade NDMA (solution in methanol, 100 ng/μL) was obtained from Dr. Ehrenstorfer GmbH and diluted using dichloromethane to desired concentrations. Dimethylamine hydrochloride (DMA·HCl, 99%+) and sodium nitrite (NaNO₂, 99%+) were purchased from Sinopharm Chemical Reagent Co. Ltd. and Beijing Chemical Works, respectively. Methanol, hexane, and dichloromethane were of Chromatographic purity and were used during the extraction process. Deuterated N-nitrosodimethylamine (NDMA-d₆, 1000 μg/mL in methanol) was purchased from Cambridge Isotopes (Andover, MA) and diluted to the required concentration. Buffer solutions (pH = 6.4) containing Na₂HPO₄/H₃(C₆H₅O₇), Na₃(C₆H₅O₇)/C₆H₈O₇), NaH₂PO₄/NaOH, and NaH₂PO₄/Na₂HPO₄, which were all of analytical laboratory grade, were prepared using ultrapure water (18.2 MΩ cm at 25°C) from a Millipore Mili-Q water purification system. All chemicals were used without further purification.

2.2. Preparing the Solid Phase Extraction (SPE) Cartridges

Materials used for the Solid Phase Extraction (SPE) cartridges were purchased from Agela Technologies, Tianjin. Methods for preparing the SPE column were the same as previously reported [35]. The SPE cartridge was kept airtight until it was used for the extraction of NDMA in aqueous solution.

2.3. NDMA Formation

All glassware used in these experiments were washed with an ultrasonic method and air-dried prior to use. NDMA formation experiments from DMA (10 mM) and NaNO2 (4 mM) were conducted in graduated tubes at 90°C placed in a temperature-controlled water bath. The tubes were sealed with a Teflon-lined screw cap and wrapped by black plastic to shield them from light, so as to avoid NDMA photolysis. Reactions were performed at pH 6.4 maintained by four buffer solutions: Na₂HPO₄/C₆H₈O₇, Na₃(C₆H₅O₇)/C₆H₈O₇, NaH₂PO₄/NaOH, and NaH₂PO₄/Na₂HPO₄. Reaction solutions were prepared by the addition of the desired amount of dimethylamine stock solution and NaNO₂ stock solution into the buffer solution with the total volume of 10 mL.

Dependence of NDMA formation on reaction conditions was assessed by varying reaction time, buffer species as well as the form and concentration of buffer components. All batch experiments were performed at least triplicated to ensure the repeatability of the results.

2.4. Mechanical Studies

Experiments were conducted to elucidate how the buffer matrices inhibit NDMA formation from DMA and NaNO₂. The DMA/NaNO₂ ratio was definitely controlled as either 100 or 0.01 in the two sets of reactions. In one case, 1 mM NaH₂PO₄ was added into the citrate buffer solution containing 0.4 mM NaNO₂ and 40 mM DMA, whereas in the other case, 1 mM NaH₂PO₄ was added into a citrate buffer solution containing 40 mM NaNO₂ and 0.4 mM DMANaH₂PO₄ was added after all the other components were completely mixed. Then the mixtures were treated as the same procedure described in NDMA Formation section.

2.5. NDMA Analysis

The analysis of NDMA was performed via a method using a Thermo Fisher Scientific GC-MS equipped with EI source (J&W scientific DB1701 column 30 m by 0.25 mm I.D. by 0.25 μm film thickness) after solid-phase extraction (SPE). Prior to extraction, the reaction solution was cooled to room temperature and all samples were dosed with the deuterated NDMA-d₆. Extractions were performed with SPE cartridges. In brief, the SPE method for extracting NDMA in aqueous solution mainly includes methanol activation, sample addition, hexane leaching, and methylene dichloride elution [35]. In every step, the SPE cartridge was drained to dry thoroughly, and the flow rate was maintained at about 2-3 mL/min. After eluting, the extract was concentrated to 3 mL with dichloromethane and the determination of NDMA from the concentrated extract was achieved by using an isotopic dilution method based on the mass detection of molecular ion (m/z = 74) and the characteristic molecular ion of NDMA-d₆ (m/z = 80). NDMA was analyzed by both full scan and selected ion monitoring (SIM). The GC/MS operating conditions were set as follows: splitless injections of 1 μL 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 (5 min) wand then ramped at 10°C/min to 200°C (held for 5 min); mass spectrometry was performed in electron ionization mode. Helium carrier gas was maintained at 1.0 mL/min and the retention time for NDMA was 6.9 min. The instrument and method detection limits for NDMA by using SPE-GC/MS method are 1 and 5 ng/L, respectively.

2.6. Computational Details

Quantum chemistry calculations were conducted to gain more insights into the effect of a phosphate buffer on the formation of NDMA in the “DMA + ” reaction system. All structures and energy optimization calculations of reactants and products were performed with Gaussian03W using the B3LYP method in conjunction with 6-311+G(d,p) basis set [36]. Minima and transition states were optimized and characterized by harmonic vibrational frequency analysis. Intrinsic Reaction Coordinate (IRC) [37] calculations were performed to confirm that every transition state connected the corresponding reactant and product through the minimized-energy pathway.

3. Results and Discussion

The yield of NDMA formation from DMA nitrosation affected by many reaction conditions included reactantconcentration, pH, and reaction time. Thus, before embarking on this work, it is necessary to optimize some reaction conditions.

3.1. Effect of Reaction Time on NDMA Formation via DMA Nitrosation

In order to choose an appropriate reaction time, experiments to evaluate the effect of a time scale on NDMA formation were performed. In this case, NDMA concentrations of the reactions of 10 mM DMA with 4 mM NaNO₂ at pH 6.4 phosphate buffer were monitored every 1 h up to 9 h.

Figure 1 shows the results of time-dependent NDMA formation and exhibits a nonlinear correlation between the NDMA concentration and time scale. The yield of NDMA appears to increase rather slowly within the first hour, whereas it increases dramatically from 1 to 4 h with the value reaching approximately 0.026 mM. Thus, the NDMA yield at 4 h reaches around 0.26% of the initial amount of DMA concentration. During 4 to 9 h, the NDMA concentration is almost the same. Although a small enhancement of NDMA formation occurs at 6 h, there is little change when the experimental errors were taken into account. Thus, the yield of NDMA can be considered to reach the maximum at 4 h and be stable after this time. Therefore, 4 h was selected as the reaction time throughout all the experiments involved in this study. Similarly, when discussing the formation of NDMA from TMA and DMA, Scanlan et al. [31] also chose 4 h as the reaction time.

Figure 1: NDMA formation concentrations in the reaction of 10 mM DMA and 4 mM NaNO₂ at 90°C in the pH 6.4 phosphate buffer with the reaction time from 1 to 9 h. The vertical bars represent the standard deviation ().

3.2. Effect of Buffer Matrix on NDMA Formation

To the best of our knowledge, although the influence of several parameters on NDMA formation has already been made during these years, little research work has been systematically performed on the influence of buffer matrices on NDMA formation. Thus, four commonly used buffer matrices, that is, Na₂HPO₄/C₆H₈O₇, Na₃(C₆H₅O₇)/C₆H₈O₇, NaH₂PO₄/Na₂HPO₄, and NaH₂PO₄/NaOH, have been investigated in this study.

First of all, it is necessary to rule out the influence of buffer matrix on the already formed NDMA. To achieve this goal, a standard 300 μg/L NDMA was put into deionized water as control and into four buffer matrices studied here; then the NDMA concentrations were monitored after 4 h. The concentrations of the standard NDMA in five different solutions were shown in Figure 2. As shown in Figure 2, the average NDMA concentrations were 278, 273, 276, 287, and 273 μg/L for deionized water, Na₂HPO₄/C₆H₈O₇, Na₃(C₆H₅O₇)/C₆H₈O₇, NaH₂PO₄/NaOH, and NaH₂PO₄/Na₂HPO₄ buffer, respectively. It is clear that all the values of the NDMA concentrations in the solutions are slightly lower than the 300 μg/L original standard NDMA. This decrease results from the SPE extraction recovery. However, the results show that the NDMA concentrations in the four buffer solutions are almost the same as that of deionized water. Thus, it can be concluded that the four types of buffer solutions employed in this study wholly have little effect on the standard NDMA. Therefore, it is reasonable to preclude the influence of buffer matrix on the already formed NDMA.

Figure 2: Concentrations of 300 μg/L standard NDMA after storing at 90°C in ultrawater (blank), Na₂HPO₄/C₆H₈O₇, Na₃(C₆H₅O₇)/C₆H₈O₇, NaH₂PO₄/NaOH, and NaH₂PO₄/Na₂HPO₄ buffer solutions (pH = 6.4) for 4 h. The horizontal bars represent the standard deviation ().

Now it is time to see whether the buffer matrix has an influence on NDMA formation during the reaction of NaNO₂ and DMA. The NDMA concentrations formed from the reactions of 10 mM DMA with 4 mM NaNO₂ in Na₂HPO₄/C₆H₈O₇, Na₃(C₆H₅O₇)/C₆H₈O₇, NaH₂PO₄/Na₂HPO₄, and NaH₂PO₄/NaOH buffer solutions were shown in Figure 3. The results of Figure 3 demonstrate an obvious difference among the NDMA formation concentrations in the four buffer solutions, whose values are 1.18, 1.66, 1.16, and 2.42 mg/L for Na₂HPO₄/C₆H₈O₇, Na₃(C₆H₅O₇)/C₆H₈O₇, NaH₂PO₄/Na₂HPO₄, and NaH₂PO₄/NaOH buffer solutions, respectively. The NDMA yield for the four buffers ranged from 0.26% to 0.54% of the initial amount of DMA concentration and the largest different yield induced from the different buffer reached 0.28% which is almost in the same level of the lowest yield. By comparing the yields of NDMA in the first column with that of the second, that is, in the Na₂HPO₄/C₆H₈O₇ and Na₃(C₆H₅O₇)/C₆H₈O₇ buffer solutions, the NDMA yield in the buffer solution involved an amount of phosphate was relatively lower than that in the citrate buffer by around 28.9%. Similar results can also be found when a comparison between the yields of NDMA in Na₃(C₆H₅O₇)/C₆H₈O₇ and NaH₂PO₄/Na₂HPO₄ buffers was made. It indicates that the phosphate component of the buffer plays a significant role in inhibiting NDMA formation compared to the citrate buffer. Unfortunately, the concentration of NDMA in the NaH₂PO₄/NaOH buffer (the third column) is rather high, with the value of 2.42 mg/L, which seems completely contrary to the above conclusion. However, further analysis of the concentration of the phosphate listed in Table 1 demonstrates that the final concentrations of phosphate in Na₂HPO₄/C₆H₈O₇, NaH₂PO₄/Na₂HPO₄, and NaH₂PO₄/NaOH buffer solutions are approximately 0.14, 0.2, and 0.05 M, respectively. The final concentration of phosphate in the NaH₂PO₄/NaOH buffer is 2.8 times lower than that of the other groups. Therefore, the higher NDMA formation is possibly attributed to the less amount of phosphate in the NaH₂PO₄/NaOH buffer. It suggests that the amount of phosphate in the buffer may be a critical factor for NDMA formation during the nitrosation of DMA by nitrite. This conclusion is in a good agreement with the result reported by Schreiber and Mitch [34] that the elevated phosphate concentration can significantly decrease NDMA formation during DMA chloramination with the decrease being ~50% when the concentration increased from 5 to 50 mM.

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

Buffer type
Na₂HPO₄/C₆H₈O₇, 

0.1 M
Na₃(C₆H₅O₇)/C₆H₈O₇, 

0.1 M
NaH₂PO₄/NaOH, 

0.05 M
NaH₂PO₄/Na₂HPO, 

0.2 M

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)

Figure 3: NDMA formation concentrations in the reaction of 10 mM DMA and 4 mM NaNO₂ in the Na₂HPO₄/C₆H₈O₇, Na₃(C₆H₅O₇)/C₆H₈O₇, NaH₂PO₄/NaOH, and NaH₂PO₄/Na₂HPO₄ buffer solutions (pH = 6.4). Reaction time = 4 h. Temperature = 90°C. The vertical bars represent the standard deviation ().

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

3.3. Role of Buffer Components in the NDMA Formation

In order to examine which ingredient of buffer components leads to the decrease of NDMA formation, experiments were conducted in which various concentrations of NaH₂PO₄ or Na3 (C₆H₅O₇) were added into a pH 6.4 citrate buffer containing 10 mM DMA and 4 mM NaNO₂. Citrate buffer was selected as the buffer matrix because it appears to have little influence on NDMA formation. It should be noted that is the main existing form of the added NaH₂PO₄ in pH 6.4 buffer solution, which was accounted for the dissociation constants of phosphoric acid (pKa₁ = 2.16, pKa₂ = 7.21, pKa₃ = 12.32).

The NDMA concentrations at various dosages of NaH₂PO₄ or Na3 (C₆H₅O₇) were listed in Figure 4. Based on the results of Figure 4, either addition of NaH₂PO₄ or Na3 (C₆H₅O₇) causes a decrease of NDMA formation. Moreover the decrease of NDMA caused by NaH₂PO₄ was more significant than that caused by Na3 (C₆H₅O₇) with the same concentration. For instance, the NDMA concentration decreased from 1.66 mg/L at 0 mmol NaH₂PO₄ to approximately 1.31 mg/L at 0.01 mmol NaH₂PO₄, while 0.01 mmol Na₃(C₆H₅O₇) decreased the NDMA concentration from 1.66 mg/L to approximately 1.49 mg/L. Therefore, it can be concluded that both of NaH₂PO₄ and Na₃(C₆H₅O₇) are able to inhibit NDMA formation, but the inhibitory ability of NaH₂PO₄ is somewhat stronger than that of Na₃(C₆H₅O₇). This finding is well consistent with the conclusion obtained from the above section. Furthermore, it is worth noting that the NDMA-inhibiting ability of NaH₂PO₄ and Na₃(C₆H₅O₇) was also shown in a dose-dependent manner. As shown in Figure 4, with the amount of NaH₂PO₄ addition increased from 0 to 0.03 mmol, the concentration of NDMA formation decreased from 1.66 to 1.17 mg/L while it is changed from 1.66 to 1.39 mg/L over the same range for Na₃(C₆H₅O₇). In comparison to the addition of 0.03 mmol NaH₂PO₄ or Na3 (C₆H₅O₇), the NDMA concentration shows little variation with the addition of 0.04 mmol. It indicates that the addition of around 0.03 mmol reaches an inhibitory saturation for the reagent concentrations applied in this study.

Figure 4: NDMA formation in the presence of variable amounts of NaH₂PO₄ and Na₃(C₆H₅O₇) addition; 4 h reaction; 90°C. The pH was maintained at 6.4 with Na₃(C₆H₅O₇)/C₆H₈O₇ buffer. The vertical bars represent the standard deviation ().

The results of these experiments clearly verify the inhibitory effects of NaH₂PO₄ buffer on NDMA formation. They also indicate that the choice of phosphate buffer can definitely impact on the analytical results in nitrosamine measurements.

3.4. Proposed Mechanism of NaH₂PO₄ Affecting NDMA Formation during DMA Nitrosation

To illustrate the inhibition mechanism of NaH₂PO₄ on NDMA formation, two kinds of experiments were designed to conduct in a citrate buffer to determine which reactant (NaNO₂ or DMA) can interact with NaH₂PO₄ in the DMA nitrosation reaction. In the first experiment, the concentration of NaNO₂ was chosen to be 40 mM which is far greater than that of DMA (0.4 mM), while in the second one, in contrast, the concentration of DMA was 40 mM which is far greater compared to that of NaNO₂ (0.4 mM). The concentration ratio of DMA/NaNO₂ for the two experiments was controlled to be 0.01 and 100, respectively. Since the concentration of NaH₂PO₄ was chosen to be 1 mM, it means that NaH₂PO₄ is in excess compared to DMA and NaNO₂ for the two experiments, respectively. The results for the two reactions are presented in Figures 5(a) and 5(b), respectively.

Figure 5: (a) Amounts of NDMA formed after 2 h from the reaction of a large excess of NaNO₂ (40 mM) with DMA (0.4 mM). (b) Amounts of NDMA formed from the reaction between a large excess of DMA (40 mM) and NaNO₂ (0.4 mM). The reaction solutions were buffered at pH 6.4 citrate buffer at 90°C with the concentration of NaH₂PO₄ being 1 mM. The vertical bars represent the standard deviation ().

As shown in Figure 5(a), the yield of NDMA with the presence of NaH₂PO₄ was 577 μg/L while the control experiment (marked as blank) without the NaH₂PO₄ addition was 580 μg/L. The NDMA concentrations are approximately the same which reveals that there is no significant difference in NDMA formation caused by the NaH₂PO₄ addition for the first experiment. It also indicates that NaH₂PO₄ does not react or react slowly with DMA otherwise DMA would be completely exhausted because the concentration ratio of DMA/NaNO₂ is only 0.01, which would lead to the decline of NDMA concentration.

In the second experiment (DMA/NaNO₂ ratio controlled at 100), NaH₂PO₄ was found to reduce NDMA formation (Figure 5(b)). The NDMA concentration in the experimental group was around 53 μg/L, whereas that in the control experiment was 70 μg/L. This result shows that NDMA formation was almost lower by 24% after adding NaH₂PO₄. Since the concentration of DMA was in large excess over NaNO₂ in the second experiment, it can be inferred that it is the mutual interaction between NaNO₂ or its derivative and NaH₂PO₄ that resulted in the decrease of NDMA formation.

Based on the above two experiments, it can be concluded that the inhibition mechanism of NaH₂PO₄ on NDMA formation during the DMA nitrosation reaction results from the interaction between NaNO₂ or its derivative and NaH₂PO₄.

3.5. Theoretical Work on the Inhibition Mechanism of NaH₂PO₄

The experiments have shown that phosphate exhibits inhibitory effect on the formation of NDMA. Basically, the possibility of reaction between amine and phosphate accounting for the inhibition can be excluded, because the nucleophilic amine (DMA) is expected to resist the phosphate anion. Therefore, we focused on the interaction between phosphate and nitrosating agent N₂O₃ which is viewed as the effective nitrosating agent in the solvent nitrosation reactions [18]. The nitrosating agent N₂O₃ is produced via the protonation of the nitrite ion and further dehydration of two HNO₂ molecules (1), and this has been well established in previous studies [38, 39]

It should be noted that is the main existing form of the added NaH₂PO₄ in pH 6.4 buffer solution; therefore, only anion was considered in the calculations. The results show that reacts easily with N₂O₃. Therefore, is proposed to be responsible for the inhibition of NDMA formation through scavenging the nitrosating agent N₂O₃. Detailed discussion is given below to support this hypothesis. Reactions involved in the phosphate-buffered “DMA + ” reaction system are illustrated in Figure 6. Corresponding energy data are collected in Table 2.

Table 2: Calculated activation energies and reaction energies (in kcal/mol) for possible reactions involved in the phosphate-buffered “DMA + ” reaction system at the B3LYP/6-311+G(d,p) level.

Reactions

DMA + N₂O₃ → NDMA + HNO₂
6.87
8.24
−15.90
−18.05

N₂O₃ + H₂ → HPO₄+ HNO₂
8.40
10.83
−2.59
−2.37

DMA + HPO₄ → NDMA + H₂
22.05
24.97
−28.39
−28.52

N₂O₃ + H₂O → 2HNO₂
12.80
15.85
1.50
−0.06

HPO₄ + H₂O → H₂ + HNO₂
25.41
28.63
−8.77
−8.15

Figure 6: Possible reactions and transition structures (distances in angstroms) involved in the phosphate-buffered “DMA + ” reaction system.

As shown in Figure 6, the fate of N₂O₃ is related to three different pathways for its further reactions: reaction with DMA to form NDMA; (2) hydrolysis to regenerate HNO₂; (3) elimination by to give .

Nitrosation of DMA by N₂O₃ occurs in a concerted step with a five-membered cyclic transition state (TS1 in Figure 6). NDMA forms via releasing nitrous acid (HNO₂). Activation enthalpy (Table 2) was calculated to be 6.87 kcal/mol, and the reaction is exothermic by 15.90 kcal/mol. NDMA forms easily via the N₂O₃-induced nitrosation of DMA, and this is consistent with previous studies [40, 41]. In addition, N₂O₃ undergoes hydrolysis (TS3-involved pathway in Figure 6) to reproduce HNO₂, and the activation energy (12.80 kcal/mol) is approximately twice as high as that (6.87 kcal/mol) of its reaction with DMA to form NDMA. Moreover, the hydrolysis is slightly endothermic by 1.50 kcal/mol. Therefore, N₂O₃ is more favored to be consumed by DMA to produce NDMA than to be hydrolyzed into HNO₂.

A very interesting finding is the reaction between N₂O₃ and the phosphate buffer (). Figure 6 shows that the reaction of with N₂O₃ occurs with a five-membered cyclic transition state TS2, and a new species () is generated via releasing HNO₂. Corresponding activation enthalpy (Table 2) was calculated to be 8.40 kcal/mol, which is very close to the activation enthalpy (6.87 kcal/mol) of the N₂O₃-induced nitrosation of DMA. This indicates that the reaction of N₂O₃ and DMA to give NDMA competes with its reaction with to produce . Note that is an ON⁺ carrier which may also have nitrosating ability. Therefore, the nitrosation of DMA by (TS4-involved pathway in Figure 6) was studied, however, the activation energy (22.05 kcal/mol) is much higher than that (6.87 kcal/mol) of the N₂O₃-induced nitrosation. Consequently, is a weak nitrosating agent when compared with N₂O₃. In addition, Lewis et al. [42] proposed that the hydrolysis of is rapid which can account for the inhibitory effect of phosphate for the N₂O₃-induced nitrosation of DMA. However, the calculated result does not support this conclusion because the activation energy predicted for the hydrolysis of (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 in our experiment resulted from the fast elimination of the nitrosating agent N₂O₃ by to give , and is a “stable” species whose further reaction with DMA and hydrolysis are relatively slow in the studied “DMA + ” system.

4. Conclusions

In this paper, batch experiments on NDMA formation from the reaction of DMA and nitrite were systematically conducted in the four kinds of buffer solutions, that is, Na₂HPO₄/C₆H₈O₇, Na₃(C₆H₅O₇)/C₆H₈O₇, NaH₂PO₄/NaOH, and NaH₂PO₄/Na₂HPO₄. Experimental data indicated that the phosphate component of the buffer plays a significant role in inhibiting NDMA formation compared to the citrate buffer and the inhibitory effect was proved to be in a dose-dependent manner. Moreover, it is the interaction between NaH₂PO₄ and reactant NaNO₂ rather than DMA that leads to the inhibitory effect of phosphate buffer during the DMA nitrosation reaction. Theoretical results found that a five-membered ring intermediate compound formed during the reaction of with N₂O₃, and the nitrosation ability of this intermediate was weaker than that of N₂O₃, which leads to the reduced amount of NDMA formation. This theoretical result supports the experimental conclusion and further points out why phosphate buffer inhibits the formation of NDMA during the nitrosation of DMA. This study discovered the inhibition of phosphate buffer solutions on NDMA formation and gave a hint to reduce the carcinogenic nitrosamines.

Acknowledgments

The authors thank James Tarzia P. Eng., Beijing University of Technology for English language suggestion. This research was supported by National Natural Science Foundation of China (no. 20903006), Beijing Natural Science Foundation (no. 2092008), Beijing Nova Program (no. 2008B09), and Environmental Protection Public Project (no. 2010467032). They thank the High Performance Computing (HPC) Center in Beijing University of Technology and Beijing Computing Center for providing the high-performance computing clusters.

References

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

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

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

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

F. De Vocht, I. Burstyn, K. Straif et al., “Occupational exposure to NDMA and NMor in the European rubber industry,” Journal of Environmental Monitoring, vol. 9, no. 3, pp. 253–259, 2007.

W. Lijinsky and S. S. Epstein, “Nitrosamines as environmental carcinogens,” Nature, vol. 225, no. 5227, pp. 21–23, 1970.

R. N. Loeppky and C. J. Micheljda, Nitrosamines and Related N-Nitroso Compounds: Chemistry and Biochemistry, American Chemical Society, Washington, DC, USA, 1994.

United States Environmental Protection Agency (USEPA) IRIS database, 2002

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

N. M. Vieno, H. Härkki, T. Tuhkanen, and L. Kronberg, “Occurrence of pharmaceuticals in river water and their elimination in a pilot-scale drinking water treatment plant,” Environmental Science & Technology, vol. 41, no. 14, pp. 5077–5084, 2007.

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

California Drinking Water: NDMA-Related Activities, California Department of Public Health, 2009.

J. Choi and R. L. Valentine, “Formation of N-nitrosodimethylamine (NDMA) from reaction of monochloramine: a new disinfection by-product,” Water Research, vol. 36, no. 4, pp. 817–824, 2002.

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

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

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

D. L. H. Williams, Nitrosation Reactions and the Chemistry of Nitric Oxide, Elsevier, Amsterdam, The Netherlands, 2004.

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

S. Weerasooriya and C. Dissanayake, “The enhanced formation of N-nitrosamines in fulvic acid mediated environment,” Environmental Toxicology and Chemistry, vol. 25, no. 1, pp. 57–62, 1989.

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

C. L. Lv, Y. D. Liu, and R. G. Zhong, “Theoretical investigation of N-nitrosodimethylamine formation from dimethylamine nitrosation catalyzed by carbonyl compounds,” The Journal of Physical Chemistry A, vol. 113, no. 4, pp. 713–718, 2009.

P. Andrzejewski and J. Nawrocki, “N-nitrosodimethylamine (NDMA) as a product of potassium permanganate reaction with aqueous solutions of dimethylamine (DMA),” Water Research, vol. 43, no. 5, pp. 1219–1228, 2009.

L. Padhye, P. Wang, T. Karanfil, and C. H. Huang, “Unexpected role of activated carbon in promoting transformation of secondary amines to N-nitrosamines,” Environmental Science & Technology, vol. 44, no. 11, pp. 4161–4168, 2010.

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

U. Tezel, L. Padhye, C. H. Huang, and S. G. Pavlostathis, “Biotransformation of nitrosamines and precursor secondary amines under methanogenic conditions,” Environmental Science & Technology, vol. 45, no. 19, pp. 8290–8297, 2011.

H. Delalu and A. Marchand, “The Investigation of the conditions for the formation of formaldehyde dimethylhydrazone (FDMH) from unsymmetrical dimethylhydrazine (UDMH) oxidation by chloramine. 1. Kinetics of the unsymmetrical dimethylhydrazine oxidation by chloramines,” Journal de Chimie Physique et de Physico-Chimie Biologique, vol. 84, p. 991, 1987.

M. A. Zolfigol, M. Bagherzadeh, A. G. Choghamarani, H. Keypour, and S. Salehzadeh, “N-nitrosation of secondary amines under mild and heterogeneous conditions,” Synthetic Communications, vol. 31, no. 8, pp. 1161–1166, 2001.

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

J. M. Kemper, S. S. Walse, and W. A. Mitch, “Quaternary amines as nitrosamine precursors: a role for consumer products?” Environmental Science & Technology, vol. 44, no. 4, pp. 1224–1231, 2010.

J. L. Roux, H. Gallard, and J. P. Croue, “Formation of NDMA and halogenated DBPs by chloramination of tertiary amines: the influence of bromide ion,” Environmental Science & Technology, vol. 46, no. 3, pp. 1581–1589, 2012.

R. A. Scanlan, S. M. Lohsen, D. D. Bills, and L. M. Libbey, “Formation of dimethylnitrosamine from dimethylamine and trimethylamine at elevated temperatures,” Journal of Agricultural and Food Chemistry, vol. 22, no. 1, pp. 149–151, 1974.

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

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

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

Q. Liu, Y. D. Liu, and R. G. Zhong, “A comparative investigation of the nitrosodimethylamine formation from the nitrosation of dimethylamine and trimethylamine by nitrite under the neutral condition,” in Proceedings of the IEEE International Conference on Bioinformatics and Biomedical Engineering, pp. 1–4, Piscataway, NJ, USA, 2011.

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-row elements,” Journal of the American Chemical Society, vol. 104, no. 19, pp. 5039–5048, 1982.

C. Gonzalez and H. B. Schlegel, “An improved algorithm for reaction path following,” The Journal of Chemical Physics, vol. 90, no. 4, pp. 2154–2161, 1989.

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

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

S. S. Mirvish, “The etiology of gastric cancer. Intragastric nitrosamide formation and other theories,” Journal of the National Cancer Institute, vol. 71, no. 3, pp. 629–647, 1983.

Z. Sun, Y. D. Liu, C. L. Lv, and R. G. Zhong, “Theoretical investigation of the isomerization of N₂O₃ and the N-nitrosation of dimethylamine by asym-N₂O₃, sym-N₂O₃, and trans-cis N₂O₃ isomers,” Journal of Molecular Structure, vol. 908, no. 1–3, pp. 107–113, 2009.

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