Formation of N-nitroso-N-methylurea in various samples of smoked/dried fish, fish sauce, seafoods, and ethnic fermented/pickled vegetables following incubation with nitrite under acidic conditions.

In continuation of our previous studies on N-nitroso-N-methylurea (NMU) formation in cured meats following incubation with nitrite at gastric pH, we extended the investigation to other foods mentioned in the title of this paper. The main objective was to determine whether these foods have the potential to form NMU at pH's that can be found in the human stomach. This was done by nitrosating an aliquot (5 g for fish sauce, 10 g for the others) of each with 7.25 microM to 1.59 mM levels of sodium nitrite for 2 h at room temperature at pH 0.8--1.5 and measuring the amounts of NMU formed. Of the samples tested, fish sauce formed 2--712 ng of NMU, followed in decreasing order by herring (<0.3--688 ng); dried anchovy, shrimp, and other fishes (<0.3--134 ng); crab and lobster paté (<0.3--342 ng); sardines (6--59 ng); oysters and mussels (11--31 ng); dried squid (3--47 ng); kimchi (7--107 ng); and Japanese pickled radish (<0.3--72 ng). Incorporation of 200-2000 ppm of ascorbic acid in the fish sauce and other foods, prior to nitrosation, appreciably inhibited such NMU formation. Although previous researchers in China reported NMU formation in nitrosated samples of fish sauce, this is the first reported formation of NMU upon nitrosation of the other foods mentioned above, and the first reported inhibition of such formation by added ascorbic acid.

Investigation on the possible formation of N-nitroso-N-methylurea by nitrosation of creatinine in model systems and in cured meats at gastric pH.

N-Nitroso-N-methylurea (NMU) is a highly potent direct-acting carcinogen that has been shown to induce cancer in a number of animal species. Although previous research has indicated that nitrosation of creatinine (CRN), a common constituent of meats, dried fish, and seafoods, can form traces of NMU, there is uncertainty as to (1) the yield of NMU and (2) whether detectable amounts of NMU can be formed from cured meats following nitrosation under acidic conditions given the low residual levels of nitrite found in cured meats at the present time. Lack of sensitive and specific analytical methods most likely has hindered progress in research in these areas. An HPLC postcolumn denitrosation-thermal energy analyzer technique and a GC-MS confirmation technique were developed for the determination of NMU in cured meats. Both techniques are highly sensitive (0.5 and 0.03 ppb, respectively) and specific. The optimum pH for NMU formation from CRN ranged between pH 1 and pH 3, and the yields of NMU under variable reactant concentrations ranged between 0.00004 and 0.0046%. When 27 samples of various cured meats (10 g aliquots each) were acidified with HCl (final pH values of 0.8-2.5) and incubated at room temperature for 2 h, without any additional nitrite, 24 gave results below detectable levels but 3 formed 2-26 ng of NMU/10 g of meat. Incubation of the negative meats with additional nitrite (50-500 microg/g of meat) formed 0.6-176 ng of NMU/10 g of sample. Although the amounts of NMU formed were extremely small, this seems to be the first reported formation of NMU from cured meats with and without additional nitrite.

Rapid semi-quantitative estimation of N-nitrosodibutylamine and N-nitrosodibenzylamine in smoked hams by solid-phase microextraction followed by gas chromatography-thermal energy analysis.

A solid-phase microextraction (SPME) analytical method has been developed for the determination of N-nitrosodi-n-butylamine (NDBA) and N-nitrosodibenzylamine (NDBzA) in hams that is based on: (a) isolation of the compounds by steam distillation, (b) SPME from the distillate headspace using a polyacrylate coated silica fibre and (c) determination by gas chromatography-thermal energy analyzer technique or confirmation by gas chromatography-mass spectrometry. Recoveries of both NDBA and NDBzA from hams spiked at 5 to 160 micrograms/kg levels ranged between 41 to 112%. The overall method is fast, sensitive (detection limits, 1 to 3 micrograms/kg), precise (within 10%) and fairly accurate (average recoveries 86% and 70%, respectively). The results obtained by this technique for seven ham samples agreed fairly well with those obtained by an existing method (r2 = 0.97). The new method is solventless, environmentally friendly and useful for rapid monitoring purposes.

N-Nitrosamines in Nitrite-Cured Chicken-Seal Salami.

The effect of inclusion of mechanically separated seal meat (MSSM) and seal protein hydrolyzate (SPH) on the formation of N-nitrosamines in chicken salami was investigated. Inclusion of MSSM (10 or 20%) or SPH (1 or 2%) in salami had little effect on the formation of N-nitrosodimethylamine (NDMA) in the products. While the control sample contained 0.33 µg/kg NDMA, other samples contained 0.37 to 0.52 µg/kg of NDMA. Smoking of the samples resulted in small but an insignificant difference in the content of NDMA in the products. Some products contained traces of N-nitrosopiperidine, N-nitrosopyrrolidine and N-nitrosomorpholine, which were perhaps formed from interaction of amines in spice mixes with nitrite. Boiling, microwaving and frying had little effect on the content of N-nitrosamines; fried products contained up to 0.79 µg/kg NDMA.

Rapid and sensitive determination of nitrite in foods and biological materials by flow injection or high-performance liquid chromatography with chemiluminescence detection.

A method is described for the determination of nitrite that is based on reduction of nitrite with potassium iodide followed by chemiluminescence detection of the liberated NO using a thermal energy analyzer. The method worked well both in the flow injection mode and upon interfacing with a reversed-phase high-performance liquid chromatography system. Limited data available indicated a good agreement between results obtained by the chemiluminescence method and those obtained by using a well established colorimetric procedure when they were applied for the determination of nitrite in a number of cured meats, human saliva, and baby foods. The chemiluminescence method is, however, much superior to the colorimetric one in its speed, versatility, as well as sensitivity (200 times more), and requires only a minimum of sample preparation. The detection limit of the new method is about 0.1 ng NaNO2 per injection or 5 micrograms/kg in cured meats and other substrates analyzed.

Absence of volatile N-nitrosamines in cooked nitrite-free cured muscle foods.

Nitrite-free cured pork systems were prepared using the preformed cooked cured-meat pigment (CCMP) and sodium ascorbate. Absence of volatile N-nitrosamines in cooked nitrite-free systems was confirmed using a gas chromatography-thermal energy analyzer (GC-TEA) methodology. Similar results were obtained when cod, cod surimi or mixtures containing pork with 15 or 50% cod or cod surimi were used. However, counterpart samples cured with sodium nitrite (156 ppm) and sodium ascorbate (550 ppm) produced N-nitrosodimethylamine at 1·0 ppb levels or less. Results demonstrate that nitrite-free curing of meat and meat/fish systems containing the preformed CCMP is successful in yielding products devoid of volatile N-nitrosamines.

A method for the determination of methyl carbamate and ethyl carbamate in wines.

A method is described for the simultaneous determination of methyl carbamate (MC) and ethyl carbamate (EC) in wines that is based on: (a) extraction of the sample with dichloromethane using an extraction tube or an alumina-Celite column, (b) concentration of the extract to a small volume, and (c) determination by gas-liquid chromatography-thermal energy analyser (N-mode). The method is highly sensitive (1-2 ng/ml), accurate (recoveries greater than 80%), and precise (CV, 5-10%). Nineteen of 27 samples of wines analysed contained traces (up to 2.7 ng/ml) of MC, and most contained EC (up to 70 ng/ml). Wines treated in the laboratory with 200 ppm dimethyl pyrocarbonate (DMPC)-a cold sterilant recently approved for use in wines-indicated that such a treatment may increase the MC contents of the wines to 10 ng/ml. Additional studies suggested that formation of MC in DMPC-treated wines is dependent on both pH and ammonia content of the wines. The identity of MC in a few selected samples was confirmed by gas-liquid chromatography-high resolution (10 K) mass spectrometry. The natural low levels of MC found in these wines are not considered to pose a risk to human health.

Analytical methods for the determination and mass spectrometric confirmation of 1-methyl-2-nitroso-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid and 2-nitroso-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid in foods.

A method is described for the determination of the two title compounds that is based on: (a) extraction of the acidified sample with methanol, (b) removal of fats and lipids by partitioning of the extract with n-hexane, (c) clean-up on acidic alumina extraction cartridge, and (d) determination by a post-HPLC column chemical denitrosation-thermal energy analyser (TEA) technique or by conventional HPLC-TEA analysis after derivatization of the compounds with diazomethane. Confirmation was carried out by HPLC-mass spectrometry of the free acids and also by gas chromatography-mass spectrometry of the methyl esters. The formation of both 1-methyl-2-nitroso-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid and 2-nitroso-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid in nitrosated samples of several Japanese and Chinese pickled vegetables, one soy sauce, and two cheeses was demonstrated.

Recent studies in Canada on the occurrence and formation of N-nitroso compounds in foods and food contact materials.

We present data on the levels of both volatile and nonvolatile N-nitroso compounds in various smoked meats, including bacon, and in food contact materials (e.g., baby bottle rubber nipples and pacifiers). Evidence presented suggests that the formation of N-nitrosothiazolidine and N-nitrosothiazolidine 4-carboxylic acid in smoked meats and bacon and that of N-nitroso-N-methylaniline in Icelandic smoked mutton, can be minimized by changing or modifying the smoking methods. The presence of two other nonvolatile N-nitroso compounds in these products is also reported.

Evaluation of genotoxicity of N-nitrosodibenzylamine in Chinese hamster V79 cells and in Salmonella.

Health concerns have arisen due to the formation of N-nitrosodibenzylamine (NDBzA; CAS No. 5336-53-8) in pork processed in a new type of rubber netting. In view of the potent carcinogenicity of related nitrosamines (e.g. N-nitroso-n-dibutylamine and N-nitrosodiethylamine), NDBzA was evaluated for genotoxicity in vitro in both Chinese hamster V79 cells and in Salmonella. In V79 cells, concentrations up to 25 micrograms/ml were tested with and without activation by rat or hamster hepatocytes. Significant elevation of SCE frequency was seen only at 25 micrograms/ml in the presence of uninduced hamster hepatocytes. Mutation to 6-thioguanine resistance was observed at 25 micrograms/ml, in the absence of hepatocytes and in the presence of induced (Aroclor 1254) or uninduced hamster hepatocytes, but not with rat hepatocytes. With uninduced rat hepatocytes, a small but significant (p less than 0.05) increase in the mutation frequency was seen with 10 micrograms/ml NDBzA. In the Salmonella assay, using a pre-incubation protocol and concentrations up to 1000 micrograms/ml, NDBzA was negative in strain TA98, and in TA100 with rat S9, but was positive at the highest dose in TA100 with hamster S9, and more strongly with Aroclor 1254-induced hamster S9. When activated by uninduced rat or hamster hepatocytes, as opposed to S9, NDBzA was negative with all tester strains. Hamster hepatocytes activated more than rat in the V79 studies, and hamster S9 was more strongly activating in the Salmonella assay. These results indicate that NDBzA is weakly mutagenic to both Salmonella and V79 cells.

Mass spectrometric confirmation of the presence of N-nitrosopyrrolidine in instant coffee.

Traces of N-nitrosopyrrolidine (NPYR) may occur in some samples of both instant coffee and fine-ground roasted coffee. The identity of NPYR in 2 samples of instant coffee was confirmed by mass spectrometry as well as by liquid chromatography-thermal energy analysis. A 2-step cleanup procedure, involving fractionation on basic alumina followed by gradient elution on reverse-phase C18 cartridge, is described that allows full-scan mass spectrometric confirmation of NPYR in tested samples.

Determination of non-volatile N-nitrosamines in baby bottle rubber nipples and pacifiers by high-performance liquid chromatography-thermal energy analysis.

A method is described for the determination of non-volatile N-nitrosamines in baby bottle rubber nipples and pacifiers. It consists of extraction of the sample with dichloromethane in the presence of ascorbyl palmitate (an inhibitor of artifactual formation of nitrosamines), clean-up on silica or basic alumina, and final analysis by high-performance liquid chromatography-thermal energy analysis, a technique which is highly specific for N-nitroso compounds. The method worked well for the determination of four rubber-related non-volatile nitrosamines, namely, N-nitrosomethylphenylamine, N-nitrosoethylphenylamine, N-nitrosodicyclohexylamine, and N-nitrosodibenzylamine (recoveries from spiked samples greater than 80%; detection limit, ca. 5 micrograms/kg for each). Eighteen out of twenty four samples analyzed were found to contain varying levels (mean, 41 micrograms/kg; range, 8-146 micrograms/kg) of N-nitrosodibenzylamine. The identity of the compound was confirmed by gas chromatography-thermal energy analysis as well as by gas chromatography-mass spectrometry analyses.

The analysis and significance of bound N-nitrosoproline in nitrite-cured meat products.

An alkaline hydrolysis method is described for the release of bound N-nitrosoproline (NPRO) from cured meats that is more efficient than an enzymic method reported previously. The bound NPRO contents of 17 cured meats analysed ranged between non-detectable and 578 micrograms/kg (mean 143 micrograms/kg). Administration of defatted meat powder, prepared from such meats, to rats led to increased excretion of free NPRO in the urine that could not be inhibited by concurrent administration of ascorbic acid. The significance of these findings with regard to the use of such cured meats in in vivo N-nitrosation studies is discussed.

Review of methodologies for the determination of nonvolatile N-nitroso compounds in foods.

An attempt has been made to briefly review methods available for the determination of total N-nitroso compounds, N-nitrosamides, N-nitrosamino acids, and miscellaneous other nonvolatile N-nitroso compounds in foods and beverages, giving special emphasis to the progress made during the past five years. It appears that a wide variety of acceptable methods are available for N-nitrosamino acids and simple hydroxylated N-nitrosamines, but none of them has yet been adequately validated. Only limited progress has been made for the analysis of N-nitrosamides, N-nitroso sugar amino acids, and other N-nitroso compounds. Various mass spectrometric methods available for the determination and confirmation of these compounds are also discussed.

Improved method for determination of volatile nitrosamines in baby bottle rubber nipples and pacifiers.

A method is described for determining volatile nitrosamines in baby bottle rubber nipples and pacifiers. The method consists of overnight soaking of the sample with dichloromethane in the presence of propyl gallate, an inhibitor for artifactual formation of nitrosamines; extraction with dichloromethane using an ambient temperature column procedure; distillation from aqueous alkali; extraction of the aqueous distillate with dichloromethane and concentration of the extract using a Kuderna-Danish concentrator; and final analysis by gas chromatography with detection by thermal energy analyzer. Major improvements over other published methods include a simpler extraction, a more efficient distillation and concentration to minimize losses, and incorporation of propyl gallate inhibitor as well as of a test to detect unsuitable dichloromethane solvent that otherwise might lead to artifactual formation. The method is highly accurate (approximately 90% recovery) and precise (av. CV = 4.7% at greater than 9 ppb levels) and is applicable to determination of nitrosamines in both rubber- and plastic-based products.

Determination of N-nitrosodimethylamine in nonfat dry milk: collaborative study.

Ten laboratories participated in a collaborative study of a method for the determination of N-nitrosodimethylamine (NDMA) in nonfat dry milk. NDMA is eluted with dichloromethane from a mixture of Celite, acidic sulfamic acid, and nonfat dry milk (all packed in a chromatography column), concentrated in a Kuderna -Danish concentrator, and finally analyzed by a GC-thermal energy analyzer technique. Ten samples were studied: 6 were naturally contaminated (NDMA levels 0.38-3.56 ppb) and 4 were spiked with known levels (0.96 and 3.2 ppb) of NDMA. The coefficients of variation (CV) of the complete data for the naturally contaminated samples (excluding the 2 samples containing the lowest levels) were 8.5% and 22.5% for repeatability and reproducibility, respectively. The corresponding CVs for the spiked samples were 14.4% and 20.4%, respectively. The percent recoveries of the added NDMA in the spiked samples (at the 2 levels indicated above) were 101.6 +/- 3.2 (omitting 1 outlier) and 95 +/- 2.1, respectively. The method has been adopted official first action.

On-line combination of high-performance liquid chromatography and total N-nitroso determination apparatus for the determination of N-nitrosamides and other N-nitroso compounds, and some recent data on the levels of N-nitrosoproline in foods and beverages.

A simple high-performance liquid chromatographic chemiluminescence detection method has been developed for the quantitative determination of N-nitrosamides and is based on post-column denitrosation of the compounds with hydrogen bromide-acetic acid or hydrogen iodide-acetic acid, followed by detection of the liberated NO by a chemiluminescence detector (TEATM). The method was used for the simultaneous analysis of DiazaldR, N-nitroso-N-methylurea, N-methyl-N'-nitro-N-nitrosoguandine and streptozotocin standards, and for N-methyl-N-nitrosourea extracted from model-system reaction mixtures and spiked, cured meats. The minimum detection limit of N-methyl-N-nitrosourea was about 0.5 ng. The method for the determination of N-nitrosarcosine and N-nitrosoproline in foods and beverages has been improved by replacing diazomethane with a mixture of BF3-methanol as the esterifying reagent and by simplifying the clean-up technique for purifying the prepared methyl esters. The average levels of N-nitrosoproline detected in 11 malts, 37 beers, 11 samples of fried bacon and 18 cured-meat products were found to be 24.1 micrograms/kg (5-113), 1.7 micrograms/kg (undetectable-6.0), 19.0 micrograms/kg (0.9-67.5), and 3.2 micrograms/kg (undetectable-21.6), respectively.

Volatile N-nitrosamines in baby bottle rubber nipples and pacifiers. Analysis, occurrence and migration.

A simple direct extraction method has been developed for rapid determination of volatile N-nitrosamines in rubber nipples and pacifiers. It consists of overnight extraction of the sample with dichloromethane in the presence of ascorbyl palmitate for N-nitrosation inhibition, filtration of the extract and rinsing of the samples with dichloromethane, concentration of the extract using a Kuderna-Danish concentrator, and final analysis by gas-liquid chromatography-Thermal Energy Analyser. The method gave excellent (80-100%) recoveries of various volatile N-nitrosamines that had been added to cut nipples or pacifiers at 20-80 micrograms/kg levels, and gave comparable or lower values than those obtained with another published method. The method is recommended for rapid screening purposes. A survey of 30 samples of various nipples and pacifiers indicated the presence of the following N-nitrosamines: N-nitrosodimethylamine (up to 70 micrograms/kg), N-nitrosodiethylamine (up to 88 micrograms/kg), N-nitrosodi-n-butylamine (up to 2796 micrograms/kg), N-nitrosopiperidine (up to 180 micrograms/kg), and N-nitrosomorpholine (up to 86 micrograms/kg). A more recent study, however, indicated a general decline in the levels of various volatile N-nitrosamines in these products. These N-nitrosamines were shown to migrate easily from the rubber products to liquid infant formula, orange juice and simulated human saliva.