Nitrosamine method

The procedure described here is an example of overall conversions by the nitrosamine method for the electrophilic substitution of 1 to 3 via the intermediate anion 2, as outlined in detail in a recent review article. ... 

Nitrosamines (Method 607). The nitrosamines are extracted with methylene chloride, treated with HC1, concentrated, and solvent exchanged to methanol for direct nitrogen-phosphorus or thermal energy analyzer (TEA) detection. Provision is made for Florisil or aluminum oxide column cleanup prior to GC analysis. The GC column liquid phase is 10 Carbowax 20 M plus 2 KOH. N-Nitrosodiphenylamine thermally degrades to diphenylamine in the GC and is measured as diphenylamine after prior removal of any diphenylamine occurring, as... 

Wet chemical methods determining titratable amine ate reported for products entering urethane (amine number as meq/g) or epoxy (AHEW = amine hydrogen equivalent weight) trade appHcations. For secondary amines /V-nitrosamine contaminants are reportable down to ppb using Thermoelectron Corporation thermal energy analy2er techniques. 

The efficient recovery of volatile nitrosamines from frankfurters, followed by gc with chemiluminescence detection, has been described (133). Recoveries ranged from 84.3 to 104.8% for samples spiked at the 20 ppb level. Methods for herbicide residues and other contaminants that may also relate to food have been discussed. Inorganic elements in food can be deterrnined by atomic absorption (AA) methods. These methods have been extensively reviewed. Table 8 Hsts methods for the analysis of elements in foods (134). 

By using modem production methods it is possible to reduce the amounts of 1,4-dioxane to a level that is barely detectable with the best current analytical methods. Free ethylene oxide is now below detectable levels. Furthermore, volatile and nonvolatile nitrosamines ( NDELA ) both seem to be below detection limits of ppb in the alkanolamide-based sulfosuccinates. A good overview of modern analytical methods for the detection of 1,4-dioxane and ethylene oxide as well as nitrosamines and formaldehyde is given in Ref. 60. 

Some of the methods used for decreasing nitrosamine formation are [41] ... 

To check that the NO exposure was necessary for nitrosamine formation, morpholine was gavaged to mice as usual, but the NO exposure was omitted. Five g mouse homogenate was worked up by the Iqbal method (after addition of 11 mg cis-DMM) and yielded neither NMOR nor cis-DMNM. Similarly, when 10 mg morpholine was added to 5 g homogenate prepared from an untreated mouse and... 

A migration test was developed to simulate human exposure patterns. 5 g of rubber material was cut into 1-2 mm stripes and then immersed in 20 ml of standard test solution of artificial saliva (4.2 g NaHC03, 0.2 g K2CO3, 0.5 g NaCl, 0.03 g NaNO, ad 1000 ml with aqua dest.j. After 24 h incubation at 40 C volatile nitrosamines were determined in an aliquot and determined after destination with a standard technique (GC-TEA-method). 

The conversion to indirect-firing is costly both in capital investment and in increased fuel costs nevertheless, much of the U.S. malting industry is converting to indirect-firing because it ultimately may be the most effective method for reducing nitrosamine levels in malt and in beer. 

Nonvolatile Nitrosamines In Tobacco. A method which we developed several years ago for the analysis of tobacco-specific nitrosamines (TSNA 31) involves extraction of tobacco with buffered ascorbic acid TpH 4.5) followed by partition with ethyl acetate, chromatographic clean-up on silica gel, and analysis by HPLC-TEA (Figure 9). Results obtained with this method for a large spectrum of tobacco products (Table IV), strongly support the concept that the levels of nitrate and alkaloids, and especially the methods for curing and fermentation, determine the yields of TSNA in tobacco products. Recent and as yet preliminary data from snuff analyses indicate that aerobic bacteria play a role in the formation of TSNA during air curing and fermentation. 

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. 

Reliable methods are available for determination of nitrosamines, especially volatile nitrosamines, in a variety of foods, environmental samples, commercial products, blood and animal tissues. Reviews of these methods are available (1, 2) and descriptions of some state-of-the-art procedures are included in papers on nitrosamine occurrence in this volume. This paper is not intended to be a comprehensive review of historical developments or of the many variations of procedures... 

Major emphasis in studies of N-nitroso compounds in foods has been placed upon volatile nitrosamines, in part because these compounds are relatively easy to isolate from complex matrices by virtue of their volatility. Procedures utilizing atmospheric pressure or vacuum distillation have been used by most investigators, with variations of the method of Fine e al. (2) being among the most popular. This procedure employs vacuum distillation of a mineral oil suspension of the sample with optional addition of water to improve nitrosamine recovery from low moisture content samples (6) The usual approach to prevention of nitrosamine formation during analysis involves adding sulfamic acid or ascorbate to destroy residual nitrite at an early stage of sample preparation. 

Beer Samples. The beer samples were examined as part of the American Society of Brewing Chemists (ASBC) and Association of Official Analytical Chemists (AOAC) collaborative studies of NDMA in beer. Duplicate samples were analyzed by the column extraction procedure and the ASBC distillation procedure (35). The AOAC procedure (36) was similar, except that a larger sample (50 vs. 25 g) was examined and sulfamic acid was added to minimize artifactual formation of nitrosamines. Both methods utilize N-nitrosodipropylamine (NDPA) as an internal standard. 

Radioisotope-labeled nitrosamines have proven valuable in development of analytical methods and for demonstrating efficiency of recovery of nitrosamines from tobacco products and smoke (37-39). The very high specific activity required for low part-per-billion determinations has discouraged most analysts from using this approach. Unless a radiochromatographic detector with adequate sensitivity is available, samples must be counted independently of the final chromatographic determination, and one of the advantages of internal standardization, correction for variation in volume injected, is lost. 

Direct Injection of Amines. In the course of developing methods for investigation of in vivo formation of NMOR and NPYR in rats treated with precursor amines and nitrite, it was necessary to determine the contamination levels of the amines by the nitrosamines. Spiegelhalder et al (32) reported the presence of nitrosamines in all secondary and tertiary amine samples which they examined, using vacuum steam distillation followed by extraction and GC-TEA determination. 

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). 

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. 

---