Nitrosamines measurement

Toxic food contaminants might also be formed within the food itself or within the human digestive tract by reactions of some food ingredients and additives (e.g. nitrosamines). Measures required to prevent contamination include ... 

Raman spectroscopy has been utilized in the quant detn of nitramines and nitrosamines (Vol 1, A177-R Ref 61), and ultra-violet spectroscopy is employed for the quant measurement of TNT in HBX compn (Refs 39, 61 63)... 

No accurate rate measurements have yet been carried out, but qualitative experiments showed that the exchange was acid-catalysed so that it is likely that the protonated form of the nitrosamine (LIII) acts itself as a nitrosating agent. This would account for all the earlier cross-nitrosations that have been observed without necessitating the prior formation of nitrous acid, nitrosyl chloride or any other carrier of NO+. 

The iji vitro experiments, using the S-9 fraction from livers of uninduced Fisher 344 rats, was complicated by the fact that it became apparent that formaldehyde production was a poor measure of the extent of metabolism. The reason for that was that the S-9 fraction apparently catalyzed the oxidation of formaldehyde to formate. Consequently, determination of formaldehyde in an S-9 catalyzed reaction consistently gave low values of nitrosamine metabolism. Many workers use semicarbazide to suppress formaldehyde loss. We found, however, that semicarbazide is not a neutral bystander. 

The trialkyltrlazenes are essentially protected diazo-nlum ions. They decompose cleanly and quantitatively to the dlazonlum ions and the corresponding amines over a wide pH range (M). Good kinetic data were obtained over the range of pH 6.9 - 8.3. In more acid solutions, the reactions are too rapid to measure by conventional kinetics. The decomposition reaction is subject to general acid catalysis. Thus, the trialkyltrlazenes will be a useful tool for the study of the reactive intermediates produced by the metabolism of dialkyl-nitrosamines. 

We recently completed a new survey of 25 beers representing the same labels from similar markets in Oregon and found a mean value of 0.2 ppb, which is more than an order of magnitude lower than the original mean value. The reduction is a direct result of measures taken by the industry to reduce nitrosamine levels in beer. Reduction of nitrosamine levels in beer will be further discussed in the final section of this paper. 

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 tobacco, we found the highest NDELA values for fresh and aged snuff with 6.8 and 3.2 ppm, respectively. The fermentation process appears to increase NDELA, as was also observed for VNA concentrations. There was clear evidence that tobaccos which had not been treated with MH-30, and cigarette smoke obtained from these tobaccos, were free of NDELA, whereas all MH-30 treated tobaccos and cigarette smoke derived from them showed measurable quantities of this nitrosamine (19). 

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. 

Confirmation of identity of nitrosamines via an independent detection system is desirable since a higher level of confidence is achieved if a different physical property or structural characteristic is measured. Mass spectrometry (MS) has been used... 

Perfluoroalkane-225 (PGR, Gainesville, FL) was admitted through a glass inlet system to provide reference peaks. Analytical and reference peaks for the nitrosamines studied are shown in Table I. Sample and reference peaks were scanned alternately at a repetition rate of approximately 1 sec and were monitored on an oscilloscope. When the nitrosamine peak appeared, the oscillographic recorder chart drive was engaged and remained on until the peak disappeared. Nitrosamine quantities were estimated by comparing the sum of sample peak heights measured from the chart (usually 10 to 20 values) with values derived from injection of standard solutions. 

Eisenbrand and Preussman [21] have described a colorimetric technique in which nitrosamines are cleaved to nitrosyl bromide and secondary amines, and the liberated NO+ ion is measured colorimetrically after reacting with N-l-naphthalenyl-1,2-ethanediamine. 

Bradley, M.O., Dysart, G., Fitzsimmons, K., Harback, P., Lewin, J. and Wolf, G. (1982). Measurement hy filter elution of DNA single- and double-strand breaks in rat hepatocytes Effects of nitrosamines and y-irradiation. Cancer Res. 42 2569-2597. 

An improved HPLC-photohydrolysis-colorimetry method was validated for twenty-eight reference nitrosamines. These were separated by HPLC and photolytically cleaved by UV radiation. The resulting nitric oxide was oxidized and hydrolyzed to nitrite ions, which were derivatized into an azo dye with Griess reagent and measured spectrophoto-metrically. The method was applied to separate and detect hitherto unknown nonvolatile nitrosamines in biological fluids and food extracts591. 

The barriers to rotation of esters deserve mention here, especially in comparison to amide barriers. The H NMR spectra of some nitrites (45) were measured in 1957 (83). The temperature had to be lowered to - 58°C at 30 MHz to see the separate signals of propyl nitrite. The barriers to rotation were ca. 10 kcal/mol. This result may be rationalized by considering the lesser electron-donating ability of the alkoxy relative to the dialkylamino group. The dipolar canonical form (46) of nitrite esters is not as stable as that of nitrosamines. 

An EPA method was created for measuring NDMA and six additional nitrosamines in drinking water (EPA Method 521) [55]. This method uses GC/ chemical ionization (CI)-MS/MS and enables the measurement of NDMA and six other nitrosamines (N-nitrosomethylethylamine, N-nitrosodiethylamine, N-nitroso-di-n-propylamine, N-nitroso-di- -butylamine, N-nitrosopyrrolidine, and N-nitrosopiperidine) in drinking water at detection limits ranging fi om 1.2 to 2.1 ng/L. A liquid chromatography (LC)/MS/MS method [56] can also be used to measure nine nitrosamines, including N-nitrosodiphenylamine, which is thermally unstable and cannot be measured using the EPA Method. 

NDMA (and other nitrosamines) can dramatically increase in concentration in distribution systems (relative to finished water at the drinking water-treatment plant). For example, an initial level of 67 ng/L in drinking water-treatment plant effluent was shown to increase to 180 ng/L in the distribution system [53]. As a result, measurements taken at water-treatment plants may substantially underestimate the public s exposure to this carcinogen. 

In the second, >100 DBFs were identified in indoor chlorinated and brominated pools, including many nitrogenous DBFs (haloamides, halonitriles, haloanilines, haloamines, haloanisoles, and halonitro-compounds), likely due to the nitrogen-containing precursors from swimmers (urine, sweat, etc.) [143]. Trichloramine and THMs were also measured in the pool air [143]. Nitrosamines have been measured in chlorinated pools and hot tubs, up to a maximum of429 ng/L... 

GC/MS(/MS) is also popular for quantifying DBFs. Selected ion monitoring (SIM) or multiple reaction monitoring (MRM) mode are used with GC/MS and GC/ MS/MS, respectively, to maximize the sensitivity and provide low detection limits. Some EFA Methods utilize GC/MS, including EFA Method 524.2, which uses GC/ EI-MS for THM analysis [155], and EFA Method 521, which uses for GC/CI-MS/ MS for nitrosamine analysis [55]. In addition, many priority unregulated DBFs have been measured using GC/MS in a U.S. Nationwide Occurrence Study [11,12]. 

N-nitrosation by oxides of nitrogen at neutral and alkaline pH has an important bearing on assessing human exposure to N-nitroso compounds, particularly as this route has been almost totally disregarded in the past. Those analytical techniques which have relied on a basic pH to inhibit nitrosation need further study to ensure the validity of the findings. For the same reason, measurements of N-nitrosamines in engine exhausts and tobacco smoke are likely to be particularly artifact prone. 

---