Concentration of nitrosamines

The concentrations of nitrosamines were reduced to undetectable levels by ultraviolet treatment of the amine solutions and were not increased by addition of 2 ppm NaN02> indicating that the nitrosamines were present originally in the amines and were not formed in the GC injection port. Similar concentrations were found when the amine samples were analyzed using the column extraction method. Direct injection is appropriate for analysis of relatively simple mixtures, if adequate precautions are taken ( ), but can result in significant artifact formation in more complex systems (42). 

The pH of a metalworking fluid must be kept above neutrality in order to prevent acid corrosion of the metal In vitro, acid catalyzed nitrosation is optimized at pH 3.5 (4 0) however, it has been shown that In the presence of other catalysts, aqueous solutions of amines and nitrite leads to significant yields of nitrosamines at room temperature over the pH range of 6.4 to 11.0 (41). Furthermore, C-nitro-containing, formaldehyde-releasing biocides, such as bronopol or tris nitro, exert their potential catalytic effect in alkaline solution. It would thus be desirable to determine the optimum pH for a metalworking fluid that would lead to the lowest concentration of nitrosamine possible. 

Concentration of nitrosamines in processed meats, including bacon... 

Nitrosamines in air may be analyzed by NIOSH Method 2522 (NIOSH, 1989). Air at a flow rate between 0.2 and 2 L/min is passed through a solid sorbent tube containing Thermosorb/N as an adsorbent. The sample volume should be between 15 and 1000 L. If high concentrations of nitrosamines are expected,... 

The main approaches that have been taken to ensure that the concentration of nitrosamines (and nitrosatable compounds) in a given rubber is as low as possible are as follows ... 

It was initially reported by Fan et al in 1976 that four of seven herbicides purchased in retail outlets had contained measurable concentrations of nitrosamines as detected with a thermal energy analyzer (43). Three of the herbicides consisted... 

Lignin samples were then suspended in unbuffered solutions containing varying concentrations of N-nitro-sodiethylamine and equilibrated for 25 h. The remaining concentration of nitrosamine was determined in the supernatant by polarography V. ... 

The effects of uv radiation on V/-nitroso compounds depend on the pH and the medium. Under neutral conditions and ia the absence of radical scavengers, these compounds often appear chemically stable, although the E—Z equiUbrium, with respect to rotation around the N—N bond, can be affected (70). This apparent stabiUty is due to rapid recombination of aminyl radicals and nitric oxide [10102-43-9] formed duting photolysis. In the presence of radical scavengers nitrosamines decay rapidly (71). At lower pH, a variety of photoproducts are formed, including compounds attributed to photoelimination, photoreduction, and photo-oxidation (69). Low concentrations of most nitrosamines, even at neutral pH, can be eliminated by prolonged kradiation at 366 nm. This technique is used ki the identification of /V-nitrosamines that are present ki low concentrations ki complex mixtures (72). 

Inhibition of Nitrosamine Formation. Nitrites can react with secondary amines and A/-substituted amides under the acidic conditions of the stomach to form /V-nitrosamines and A/-nitrosamides. These compounds are collectively called N-nitroso compounds. There is strong circumstantial evidence that in vivo A/-nitroso compounds production contributes to the etiology of cancer of the stomach (135,136), esophagus (136,137), and nasopharynx (136,138). Ascorbic acid consumption is negatively correlated with the incidence of these cancers, due to ascorbic acid inhibition of in vivo A/-nitroso compound formation (139). The concentration of A/-nitroso compounds formed in the stomach depends on the nitrate and nitrite intake. 

Nitrite concentration The kinetics of N-nitrosamine formation in vitro has been studied at length (, ) and, in moderately acidic media, the reaction rate is directly proportional to the concentration of the free amine (non-protonated) and to the square of the concentration of the undissociated nitrous acid. Therefore, it is not surprising that the amount of nitrite permitted in bacon has received considerable attention. Although, there have been suggestions that it is the initial and not the residual nitrite that influences N-nitrosamine formation in bacon (41), recent evidence seems to indicate that the lowest residual nitrite gives the least probability of N-nitrosamines... 

Recently, Robach et al. ( ) investigated the effects of various concentrations of sodium nitrite and potassium sorbate on N-nitrosamine formation in commercially prepared bacon. 

The inhibition of formation of NPYR and NDMA in fried bacon by the use of cure-solubilized a-tocopherol (500 mg/kg) has been demonstrated by Fiddler et al. (50). Walters et al. (53) also reported reduced levels of N-nitrosamines in the vapors during the frying of bacon in fat containing a-tocopherol. It has also been shown that a-tocopherol is dispersed quite effectively during frying of bacon slices therefore, application to bacon may be made by spray or dip to overcome the problem of water insolubility (51). Controlled addition of this antioxidant may be an effective and practical way of reducing the concentration of N-nitrosamines in cooked... 

In a similar study, Gray et al. (60) investigated the possible formation of N-nitrosamines in heated chicken frankfurters which been prepared with various levels of nitrite (0-156 mg/kg). As expected, apparent N-nitrosamine levels increased with increasing concentrations of nitrite, but did not exceed 4 yg/kg except for two samples which contained 8 and II yg/kg of NMOR. The presence of these relatively high levels of NMOR was confirmed by mass spectrometry and raised the question as to its mode of formation. It was shown to be due to the morpholine present in the steam entering the smokehouse, as this amine is commonly used as a corrosion inhibitor in steam process equipment ( ). The detectable levels of NMOR in the Canadian study ( ) were also attributed in part to the use of morpholine as an anti-corrosion agent in the steam supply (62). 

The formation of nitrosamines in aprotic solvents has applicability to many practical lipophilic systems including foods (particularly bacon), cigarette smoke, cosmetics, and some drugs. The very rapid kinetics of nitrosation reactions in lipid solution indicates that the lipid phase of emulsions or analogous multiphase systems can act as "catalyst" to facilitate nitrosation reactions that may be far slower in purely aqueous media (41, 53, 54). This is apparently true in some cosmetic emulsion systems and may have important applicability to nitrosation reactions in vivo, particularly in the GI tract. In these multiphase systems, the pH of the aqueous phase may be poor for nitrosation in aqueous media (e.g., neutral or alkaline pH) because of the very small concentration of HONO or that can exist at these pH ranges. 

In Tobacco. At the time of harvesting, fresh tobacco leaves do not contain measurable amounts of nitrosamines (<5 ppb). However, these compounds are formed during curing, aging and fermentation. Their concentrations depend primarily on the content of proteins, alkaloids, agricultural chemicals and nitrate in the tobacco, as well as on the processing conditions which lead to the reduction of the nitrates. 

In Smoke. We compared the gas chromatograms of nitrosamines in matching aliquots of mainstream smoke derived from Burley type cigarettes that were identical except for the degree of nitrate fertilization during cultivation (Figure 2). This comparison supports the concept that the nitrate concentration in tobacco is a determining factor for the nitrosamine yields in the smoke. The data in Table II confirm this concept. These studies have... 

Reliable analytical methods are available for determination of many volatile nitrosamines at concentrations of 0.1 to 10 ppb in a variety of environmental and biological samples. Most methods employ distillation, extraction, an optional cleanup step, concentration, and final separation by gas chromatography (GC). Use of the highly specific Thermal Energy Analyzer (TEA) as a GC detector affords simplification of sample handling and cleanup without sacrifice of selectivity or sensitivity. Mass spectrometry (MS) is usually employed to confirm the identity of nitrosamines. Utilization of the mass spectrometer s capability to provide quantitative data affords additional confirmatory evidence and quantitative confirmation should be a required criterion of environmental sample analysis. Artifactual formation of nitrosamines continues to be a problem, especially at low levels (0.1 to 1 ppb), and precautions must be taken, such as addition of sulfamic acid or other nitrosation inhibitors. The efficacy of measures for prevention of artifactual nitrosamine formation should be evaluated in each type of sample examined. 

Figure 1 shows narrow range high resolution scans of the molecular ion region of NDMA, recorded near the maximum of the GC peaks, present in one of the beer samples prepared in the AOAC collaborative study. The peak at m/z 74.0480 represents approximately 0.15 ng of NDMA injected on the column, corresponding to a concentration of 0.6 yg/kg of beer. Use of high resolution MS permitted confirmation of the identity and amount of nitrosamine without additional cleanup of the concentrate prepared by the AOAC method. Sample quantity requirements were comparable to those of the TEA. 

The acidic reagents vary widely In their ability to lower NDPA levels In trifluralin The concentration of the acid Is critical to produce the desired effect In some Instances, the acid promoted additional nitrosamine formation, e g 10% hydrochloric acid, 40% phosphoric acid, ascorbic acid, etc Hydrochloric acid and hydrogen chloride gas were the most efficient at destroying NDPA Impurity ... 

In a recent comprehensive study, Feelish and coworkers (Bryan et al, 2004) determined the concentrations of RSNOs, N-nitrosamines (RNNOs) N02 , N03, heme nitrosyl (NO-heme) in plasma, RBCs, as well as brain, heart, liver, kidney, lung, and aortic tissues of rats. Furthermore, the levels of these analytes were monitored under conditions of eNOS inhibition, hypoxia and redox state. The emerging picture was that RSNOs were detected in all of the tissues examined in comparable levels to NO-... 

Surfactants that form micelles have also been shown to accelerate the formation of nitrosamlnes from amines and nitrite (33.) A rate enhancement of up to 80 0-fold was observed for the nitrosation of dihexylamine by nitrite in the presence of the cationic surfactant decyltrimethylammonium bromide (DTAB) at pH 3.5. A critical micelle concentration (CMC) of 0.08% of DTAB was required to cause this effect, which was attributed to a micelle with the hydrocarbon chains buried in the interior of the micelle. The positively-charged ends of the micelle would then cause an aggregation of free nitrosatable amine relative to protonated amine and thus lead to rate enhancements. Since surfactants are commonly used in water-based fluids (25-50% lubricating agent or 10-2 0% emulsifier in concentrates), concentrations above the CMC of a micelle-forming surfactant could enhance the formation of nitrosamines. 

The massive contamination of NDE1A in alkaline synthetic fluids (3%) found by Fan et al Q) cannot be explained by known nitrosation kinetics of di- or triethanolamine. Instead, more powerful nitrosation routes, possibly involving nitrogen oxide (N0X) derivatives (e.g., N02> N O t) may be responsible for the amounts of NDE1A in these products (34). In fact, a nitrite-free commercial concentrate was shown to accumulate NDE1A up to about 10 0 days at which time the levels dropped dramatically (19). Inhibition of N0X contaminants may be an effective route to the inhibition of nitrosamine formation in metalworking fluids. 

Inhibitors of nitrosation generally function by competing with the amine for the nitrosating agent. An inhibitor would thus react with nitrite at a faster rate than with amines. The inhibition reaction has recently been reviewed ( 35). The ability of ascorbate to act as a potent inhibitor of nitrosamine formation has resulted in the use of the vitamin in nitrite-preserved foods and pharmaceuticals. Furthermore, the effectiveness of ascorbate in inhibiting nitrosamine formation is dependent on (1) the concentration of ascorbate (an excess is required) (2) pH (ascorbate is nitrosated 240 times more rapidly than ascorbic acid) (3) the reactivity of the amine toward nitrosation and (4) the extent of oxygenation of the system. 

Nitrosamine levels in concentrates containing nitrite have been shown to increase during storage ( 39). Since a concentrate could contain up to 45% triethanolamine and 18% nitrite (7), the concentrate provides an ideal situation in which reaction may occur. In this study, conducted over a 5 to 7 month period, the concentration of NDE1A increased from 400 to 800 ppm. 

A review of the literature has revealed that several factors associated with metalworking fluids may enhance or control the formation of nitrosamines in metalworking fluids. If nitrite is present in the concentrate with appropriate amines the nitrosamine levels can reach the part-per-hundred level Yet even nitrite-free metalworking fluid concentrates have been shown to contain part-per-million quantities of nitrosamines. 

As detailed by Gatehouse and Tweats (1987), the nature and concentration of solvent may have a marked effect on the test result. Dimethysolphoxide is often used as the solvent of choice for hydrophibic compounds. However, there may be unforeseen effects, such as an increase in mutagenicity of some compounds, for example, / -phcnylenediamne (Burnett et al., 1982) or a decrease in mutagenicity of others, such as simple aliphatic nitrosamines (Yahagi et al., 1977). It is essential to... 

---