Chapter 18 Methods for the Determination of N-Nitroso Compounds in Food and Biological Fluids

CHAPT ER

18 Methods for the Determination of N-Nitroso Compounds in Food and Biological Fluids Sidney S. Mirvish

Contents

1. Introduction 2. Physical and Chemical Properties of NOC 2.1 UV, nuclear magnetic resonance (NMR) and mass spectrometry (MS) 2.2 Synthesis, formation and decomposition of NOC 3. Health Effects 3.1 Human studies 3.2 Animal studies 4. Analytical Methods 4.1 Safety 4.2 Nomenclature 4.3 General considerations in NOC analysis 4.4 Determination of volatile nitrosamines 4.5 HPLC-TEA of nitrosamino acids 4.6 GC-TEA of methyl esters of nitrosamino acids 4.7 GC-TEA of hydroxyalkylnitrosamines 4.8 HPLC-photolysis-TEA of non-volatile NOC 4.9 Havery’s HPLC-TEA method for all types of NOC 4.10 Determination of total ANC 4.11 Determination of nitrosamides as ANC 4.12 Determination of alkylureas 4.13 HPLC-photolysis-TEA of nitrosamides 5. Occurrence of NOC in Foods 5.1 Occurrence of volatile nitrosamines 5.2 Occurrence of nitrosamino acids 5.3 Occurrence and identity of total ANC 5.4 Occurrence of alkylureas and alkylnitrosoureas 6. Conclusions and Future Trends

Comprehensive Analytical Chemistry, Volume 51 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00018-4

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1. INTRODUCTION N-Nitroso compounds (NOC) comprise nitrosamines [R1N(NO)R2] and nitrosamides [R1N(NO)COR2]. Nitrosamines include dialkylnitrosamines, e.g., N-nitrosodimethylamine (NDMA), and cyclic nitrosamines, e.g., N-nitrosopyrrolidine (NPYR) (Figure 1). In 1937 Freund [1] reported two cases of acute liver toxicity caused by exposure to NDMA in a laboratory. In one of these cases, the individual had cleaned up a spill in the open laboratory from a broken bottle of NDMA, became sick soon thereafter and later died from acute liver toxicity. NDMA is no longer synthesized on a large scale for reduction to the rocket fuel 1, 1-dimethnylhydrazine (Figure 2) [2]. In 1956, Magee and Barnes [3] reported that NDMA (50 mg/kg diet) induced liver cancer in rats. The first indication that nitrosamines were an environmental hazard was the discovery in 1965 when the presence of NDMA in spoiled herring was responsible for an outbreak of acute liver toxicity in Norwegian sheep [4]. NDMA still occurs in some fish products [5], where it could arise from the fish constituents trimethylamine and trimethylamine N-oxide. Because even low doses of NOC could induce cancer in humans [6], it is vital to minimize NOC levels in foods. This chapter will review the determination of the various types of NOC in foods, with some attention also to NOC in biological materials. This is a widely studied area, e.g., the Scifnder database contained over 900 references to ‘‘nitrosamine determination,’’ most of which involved food analysis. References are generally not discussed if the detection limits were relatively high, e.g., greater than 1–2 mg of volatile nitrosamines/kg food. Nitrosamines are carcinogenic because they are activated by cytochrome P450 isozymes that insert a hydroxy group on a carbon atom adjacent to the N-nitroso

Figure 1 Some common nitrosamines. NMOR, N-nitrosomorpholine; NPIP, N-nitrosopiperidine; NSAR, N-nitrososarcosine; NPRO, N-nitrosoproline; and NTCA, N-nitrosothiazolidine-4-carboxylic acid.

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Figure 2 Reduction of NDMA to 1,1-dimethylhydrazine.

Figure 3 Metabolic activation of NDMA by cytochrome P450 2E1 and conversion of NMU to alkylating agents. R1R2NH and R3OH ¼ DNA bases, e.g., guanine.

(NNO) group. The hydroxyalkyl group in the product is split off by hydrolysis. The resulting alkyldiazonium cation can alkylate DNA bases and thereby induce mutations and, eventually, cancer (Figure 3) [7,8]. Nitrosamides, e.g., N-nitrosomethylurea (NMU), do not require enzymic activation as they are converted directly to alkyldiazonium ion (Figure 3). People are exposed to exogenous NOC ingested in the diet or inhaled in cigarette smoke, and are also exposed to endogenous NOC. These are produced in vivo by acid-catalyzed nitrosation of amines and amides in the stomach (perhaps the major in vivo route), by bacteriacatalyzed nitrosation in the colon and achlorhydric (high pH) stomach, and by nitrosation by the nitrogen oxides N2O3 and N2O4 (which also produce nitramines) and peroxynitrite (ONOO) (Equations (1)–(4)). These agents arise from the oxidation of endogenous nitric oxide (NO) or, in the case of peroxynitrite (OONO), by the reaction of NO with superoxide (O 2 ) during inflammation (Equations (3) and (4)) [8,9]. 2R1 NHR2 þ N2 O3 ! 2R1 NðNOÞR2 þ H2 O

(1)

2R1 NHR2 þ N2 O4 ! R1 NðNOÞR2 þ R1 NðNO2 ÞR2 þ H2 O

(2)

NO þ O 2 ! ONOO

(3)

R1 NHR2 þ OONO ! R1 NðNOÞR2 þ HOO

(4)

As increasingly sensitive analytical methods are developed for NOC, an important question is to decide what level of NOC in foods should be considered

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harmful. This may eventually be decided by epidemiological studies correlating cancer incidence with the intake of individual foods with known NOC contents. Meanwhile, a conservative approach is to ensure that NOC intake in specific foods should not increase much the current daily intake of NOC, e.g., of about 1 mg/day for volatile nitrosamines [6]. For more details, including many of the original references, readers are referred to previous reviews on health effects of nitrate, nitrite and NOC by the National Academy of Sciences in 1981 [6] and by the author in 1995 [8]; and to reviews on NOC determination by Scanlan in 1973 [10], Tricker et al. in 1984 [11], Scanlan and Reyes in 1985 [12], Sen and Kubacki in 1987 [13], Massey in 1988 [14], Tricker and Kubacki in 1992 [15], Yeh and Ebeler in 1998 [16] and Biaudet et al. in 2000 [17].

2. PHYSICAL AND CHEMICAL PROPERTIES OF NOC 2.1 UV, nuclear magnetic resonance (NMR) spectrometry and mass spectrometry (MS) NOC can be determined in simple solutions by their UV absorption [18]. NDMA shows absorption maxima at 225 (7,100) and 331 (91) nm in water, 227 (5,900) and 343 (98) nm in 95% ethanol and 349 (97), 359 (117) and 370 (96) nm in ether (parentheses here and below give molar extinction coefficients). NMU shows absorption maxima in water at 235 (6,700), 390 (95) and 417 (64) nm; and in ether at 230 (7,700), 378 (87), 391 (136) and 410 (131) nm. UV absorption can detect 50 mg NOC/ml when the maximum at greater than 320 nm is used and 0.5 mg NOC/ml when the maximum below 240 nm is used. This method of determining NOC has been employed to study the kinetics of NOC formation from nitrite in simple solutions and effects of inhibitors and enhancers of this formation [19]. NMR spectrometry of nitrosamines [20] usually reveals the presence of syn and anti isomers, which occur because of the partial double-bond character of the N–N bond (Figure 4). These isomers can be separated by high pressure liquid chromatography (HPLC), but interconvert at raised temperatures [20]. MS is used to detect and determine NOC and to confirm the identity of NOC detected by other means. The mass spectra of aliphatic dialkylnitrosamines show prominent molecular ions with relative intensities exceeding 25% of the base peak [21]. An ion at m/z 30 due to NO is common. Peaks at M–17 (loss of OH) and M–31 (loss of NOH) are prominent, whereas peaks at M–30 (loss of NO) are rare.

Figure 4 Syn and anti forms of nitrosamines.

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2.2 Synthesis, formation and decomposition of NOC NDMA and other simple nitrosamines are volatile liquids, whereas nitrosamides and more complex nitrosamines are solids. NOC are synthesized by the reaction of secondary amines (Equations (5)–(7)) or N-alkylamides (Equation (8)) with nitrous acid (HNO2, i.e., acidified nitrite, Equations (5) and (8)), with the gasses N2O3 and N2O4 (Equations (6) and (7)) and with peroxynitrite (OONO) (Equation (4)) [9]. Nitrosamine solutions in water decompose slowly in the presence of acid by the reverse of Equation (5) [22] and are best stored under alkaline conditions. In contrast, nitrosamides are rapidly decomposed by alkali. R1 NHR2 þ HNO2 Ð R1 NðNOÞR2 þ H2 O

(5)

R1 NHR2 þ N2 O3 Ð R1 NðNOÞR2 þ HNO2

(6)

R1 NHR2 þ N2 O4 Ð R1 NðNOÞR2 þ HNO3

(7)

R1 NHCOR2 þ HNO2 Ð R1 NðNOÞCONHR2 þ H2 O

(8)

The kinetics of NOC formation from nitrite [23] are relevant here because the ease of NOC formation increases with decreasing pH (down to pH 3.0–3.3 for secondary amines and with no pH limit for N-alkylamides), varies enormously for different NOC and may be an important factor in determining NOC concentration in foods, e.g., acidic foods are more likely to generate NOC than neutral foods. For secondary amines, the rate of nitrosation is proportional to nitrite concentration squared (Equation (9)). Most but not all tertiary amines, e.g., nicotine, are nitrosated far more slowly than secondary amines. The rate of nitrosation of N-alkylamides is proportional to the concentration of nitrite multiplied by that of hydrogen ion (Equation (10)). The rate constants, which determine the ease of nitrosation, vary by factors of more than 105 between different amines and between different amides. For secondary amines, nitrosation rate is largely governed by the acidic dissociation constant (pKa) of the amine, with the rate increasing as the pKa decreases. Rate ¼ k1 ½amine½nitrite2

(9)

Rate ¼ k2 ½amide½nitrite½Hþ 

(10)

3. HEALTH EFFECTS 3.1 Human studies After the initial report of the toxicity of NDMA (see Section 1) [1], several other cases were reported of acute liver toxicity due to exposure to NDMA vapor

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[24,25]. In a 1967 review on NOC, Magee and Barnes proposed that NOC may be significant causes of human cancer [26]. More recent laboratory and epidemiologic studies indicate that dietary NOC are etiological agents for human cancer of the oesophagus, nasopharynx, stomach, colon, brain and urinary bladder [8,27]. Salted dried fish and fish sauce are established risk factors for gastric cancer [28], perhaps due in part to NMU produced by the nitrosation of creatinine (CRN) and methylguanidine (see Section 4.12). The occurrence of polymorphic forms of enzymes that activate nitrosamines show correlations with the incidences of certain cancers. For example, increased incidences of nasopharyngeal cancer were observed in individuals with certain polymorphic forms of a DNA repair enzyme and of cytochrome P450s 2E1 and 2B6, which are known or suspected of being involved in nitrosamine metabolism [29]. In addition to dietary NOC, nitrosamines in cigarette smoke are a probable cause of cancer of the lung, oesophagus, nasal cavity and pancreas induced by smoking [30]. Certain meat and fish products are ‘‘processed,’’ which generally means preserved with sodium nitrite and, in some cases, sodium chloride. Fresh and processed red meat products are reported risk factors for colon cancer, and the risk for processed meat appeared greater than that for fresh meat [31,32]. Associations have been reported of processed meat with gastric and oesophageal cancer [27], and of preserved fish and vegetable products and smoked foods with gastric cancer [27,28]. These associations may be due to NOC present in or produced from nitrite in these foods, though a high sodium chloride level in fish products is probably an additional risk factor for gastric cancer. NOC in processed meat and fish products have often been measured as total apparent NOC (ANC, see Section 4.10) [33]. Hot dogs (frankfurters, a nitritepreserved meat product) contain ANC and, when fed to mice, increased ANC levels in the faeces [34]. Red meat had a similar effect in humans [35] and mice [34], but in mice the effect of red meat was weaker than that of hot dogs. In an epidemiological study in Europe, estimated faecal ANC excretion after the ingestion of meat was significantly (hazard ration, 1.42) correlated with the incidence of non-cardiac gastric cancer [36]. The estimated intake of NDMA showed no such correlation. Because nitrosoureas induce brain tumours in the offspring of pregnant rats treated with these compounds, it was proposed [37] that childhood brain cancer can be induced by in utero exposure to nitrosoureas (see Section 3.2). Childhood brain cancer was associated [38] with the consumption by pregnant women or their children of nitrite-preserved meat, which could produce NOC that, like nitrosoureas, are direct-acting mutagens [39]. Processed meat is also a risk factor (in addition to smoking) for pancreatic cancer [40] and chronic obstructive lung disease [41]. Processed meat is not a risk factor for prostate cancer [42]. Partly because exposure to ingested nitrate and nitrite can produce NOC, an International Agency for Research on Cancer Working Group concluded in 2006 that ‘‘ingested nitrate or nitrite under conditions that result in endogenous nitrosation is probably carcinogenic to humans’’ [43].

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3.2 Animal studies NDMA produced acute liver necrosis in rats and other animal species [1,7,25,26], and the acute toxicities in animals of many NOC have been recorded [18,44]. The dose of NDMA causing 50% lethality in rats is 40 mg/kg. After the initial report of the carcinogenicity of NDMA [3], it was found that most NOC are carcinogenic, inducing tumours in different organs of many rodent and other species, with organ specificity depending on the particular NOC and its dose schedule and mode of administration [7,18,26]. Nitrosamines often induce tumours at sites distant from the site of administration, probably because the sensitive tissues contain cytochrome P450 isozymes that can activate the nitrosamines. In rats and mice, most volatile nitrosamines are potent carcinogens, frequently inducing tumours of the liver and oesophagus in rats. Particular nitrosamines also induce tumours of the nasal cavity, kidney, pancreas and urinary bladder [7,18]. Dimethylnitramine (Me2NNO2) is also carcinogenic, but less so than NDMA [45]. The toxicology of non-volatile nitrosamines has hardly been investigated, except for tests showing that N-nitrosoproline (NPRO) was not carcinogenic in rats [45]. The only reported carcinogen among these compounds is N-nitrososarcosine (NSAR), which was a weak oesophageal carcinogen in rats [18]. Nitrosamines with high ether–water partition coefficients and high volatilities were more carcinogenic than related nitrosamines with the opposite characteristics [46], probably because carcinogens need to cross the lipidic plasma membrane of cells before they can alkylate DNA bases. This suggests that nitrosamino acids, which are expected to have low ether–water partition coefficients, are at most only weak carcinogens. Probably because alkylnitrosoureas are direct-acting mutagens (not requiring metabolic activation), they often, but not always, induce tumours at the site where exposure occurs [7]. After oral administration, NMU induced gastric cancer in guinea pigs [47], Nu-acetyl-N-methyl-N-nitrosourea induced glandular stomach cancer in rats [48] and NMU induced gastric cancer in Mongolian gerbils when their stomachs were infected with the bacterium Helicobacter pylori, a cofactor for human gastric carcinogenesis [49]. Alkylnitrosoureas can be readily produced by the acid-catalyzed nitrosation of alkylureas in the stomach [23,50]. Therefore, as first proposed by the author in 1972 [28,51], nitrosoureas could be factors in the etiology of human gastric cancer. When alkylnitrosoureas were injected into pregnant rats, the offspring developed high incidences of brain tumours [18].

4. ANALYTICAL METHODS 4.1 Safety Because of the carcinogenicity of NOC and the volatility of many nitrosamines [46], NOC should always be worked with in a fume hood while wearing gloves.

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Because fat-soluble nitrosamines readily penetrate most types of gloves [52,53], gloves should be removed and placed in the hood immediately after contact with nitrosamine solutions. Excess nitrosamines should be destroyed by reduction with aluminium–nickel alloy in alkaline solutions [54]. This reduces nitrosamines to hydrazines, which are further reduced to amines. Potassium ferroate (K3FeO4), a potent oxidant, has also been used to destroy nitrosamines [55]. Nitrosamides should be decomposed by treatment with aqueous alkali. The resulting mixture should be kept overnight in a chemical hood because volatile carcinogenic diazoalkanes are produced.

4.2 Nomenclature The most widely used nomenclature for NOC, which has been adopted by the International Agency for Research on Cancer, is to add the prefix ‘‘N-nitroso’’ to the name of the parent amine or amide. This system is used here. An alternative, simpler system is to name NOC as derivatives of nitrosamines or nitrosamides, e.g., as dimethylnitrosamine rather than as NDMA, but this system is hard to use with more complex NOC. One should refer to ‘‘nitrosamines,’’ not ‘‘N-nitrosamines,’’ because the ‘‘N’’ is superfluous.

4.3 General considerations in NOC analysis To detect contamination during the analysis, reagent blanks should be included with each series of analyses. Contamination with nitrosamines can occur by contact with rubber tubing and stoppers, and from solvents. Artifact formation due to nitrosation by nitrite (present in the sample or formed from atmospheric nitrogen oxides) is best avoided by adding sulfamic acid (SA) or, less commonly, ascorbic acid under acidic conditions before the work-up. Solvents containing alcohols should be avoided in case they form nitrite esters, even though such esters are destroyed by SA [33]. Nitrite reacts with SA to produce nitrogen and reacts with ascorbic acid to produce NO. A 50-fold excess of SA reacts completely with nitrite within 2–3 min at room temperature [56]. Artifactual nitrosamines could also occur by transnitrosation from nitrosothiols (RSNO) directly or due to nitrosation by nitrogen oxides produced by decomposition of the nitrosothiols [57]. Because both nitrosamines [58,59] and nitrosamides [60] undergo photolysis fairly readily, bright lights in the laboratory should be avoided or replaced by yellow lights. Loss of volatile nitrosamines by evaporation should be minimized. An NOC that is not present in the sample should be included as an internal standard wherever possible. An amine, e.g., dibutylamine, can be added that yields a nitrosamine not present in the sample. The appearance of this nitrosamine indicates artifactual nitrosation [61]. Low results can occur due to decomposition of NOC during the analysis, e.g., by slow acid hydrolysis back to nitrite and amines or amides (the reverse of Equations (5) and (8)) [22]. Nitrosamine (but not nitrosamide!) solutions are best stored by making them alkaline and then keeping them at 151C.

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Several of the preliminary clean-up methods used for secondary amines that are NOC precursors (NOCP) and for nitrosamines that also contain amino groups involve adsorption of the protonated amines from acidic solution by the acidic form of cation exchange resin and elution of the free amines from the resin under basic conditions.

4.4 Determination of volatile nitrosamines Volatile nitrosamines are usually determined by gas chromatography (GC) with detection by thermal energy analysis (TEA). A review by Hotchkiss in 1981 [62] summarizes early methods of analysis and should be consulted for the relevant references. The analytical strategy includes the addition of di-n-propyl nitrosamine, which does not occur naturally, as an internal standard and of SA and a mineral acid (the ‘‘stopping solution’’) to prevent artifactual nitrosation. The first step in an early method was atmospheric or vacuum distillation from slurries, or from slurries with added mineral oil and aqueous alkali, followed by vacuum distillation. The distillate was extracted with dichloromethane. The extract was dried over sodium sulfate and concentrated to about 4 ml with a Kuderna–Danish evaporative concentrator. Final concentration of 0.25–1.0 ml was achieved with a gentle stream of nitrogen. The food matrix can also be extracted directly with dichloromethane by adsorption on a column of Celite and elution with the same solvent. This avoids the distillation step but can rapidly contaminate the GC column with fats. In early studies, the final step for determining volatile nitrosamines was GC-MS [63,64]. After Fine et al. [63] introduced the TEA in 1975, GC-MS was mostly replaced by GC-TEA, but has been re-introduced in several recent studies. Packed (rather than capillary) column GC is adequate for many purposes. GC conditions have included the use of 10% Carbowax 1540 plus 5% KOH as column packing, with the GC programmed from 80 to 1801C (perhaps the most widely used system), 25% Carbowax 20M plus 2% NaOH with the GC run at 1701C (for NDMA), and of Carbowax 20M-terephthalic acid. Capillary column GC can provide improved separations [65]. In GC-TEA, the nitrosamine is passed after the GC through a pyrolyzer at 4501C to decompose the nitrosamine to NO, which is passed into a Thermal Energy Analyser (Thermo-Orion, Beverly, MA, USA). A common practice is to mount the pyrolyzer on top of the GC apparatus. An oil pump maintains the pressure of the system below 0.7 torr (mm Hg). In the TEA, NO reacts with ozone to form NO2 in an excited state. This decomposes to give NO2 and photons of infrared light, which are determined with a photomultiplier. This method (often called ‘‘determination of NOC by chemiluminescence’’) readily detects 25 pmol of nitrosamine. Nitrosamines can also be assayed with the ‘‘Sievers purge system and NO chemiluminescence detector’’ (General Electric Analytical, Boulder, CO), which determines NO by the same method as that used in the TEA and has been widely employed to follow NO formation in biological systems. This system includes a compact one-piece reaction vessel, which contains a single sodium hydroxide trap to remove acids. The sensitivity of this system appears similar to that of the TEA.

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The GC-TEA peaks are identified by comparing their retention times with those for nitrosamine standards run on the same day. Identity should be confirmed by GC-MS. The TEA system usually provides clear well-defined peaks because it only detects compounds that yield NO. Hence this system can be used without elaborate prior purification, whereas classical MS detects all volatile compounds. Compounds other than nitrosamines that are detected by GC-TEA include nitramines, some pyrroles and some aliphatic, but not aromatic, C-nitro compounds [62,66]. Nitrosamines can be distinguished from nitramines by UV irradiation, which destroys nitrosamines far more readily than it does nitramines [67]. The decomposition of nitrosamines by HBr in acetic acid [68] has been used to help identify them [67]. The presence of NDMA and NPYR has been confirmed by their oxidation to the corresponding nitramines with pentafluoroperoxybenzoic acid, a clean-up with Celite and acid alumina, and GC-TEA or GC-electron capture detection [66]. Use of this method confirmed the identity of NDMA and NPYR peaks derived from bacon, malt, non-fat dried milk powder and beer [66]. Collaborative studies with a reproducibility of, generally, less than 15% were performed for the analysis by the same method of volatile nitrosamines in cheese, cured meat, malt and beer [62]. In 1990 a collaborative study [69] was carried out by 10 laboratories on a method for determining nitrosamines in hot dogs containing minced fish or meat spiked with 0, 3 or 5 mg/kg each of NDMA, NPYR and N-nitrosomorpholine (NMOR). Instead of distillation, this method involved adsorption on a mixture of Celite and anhydrous sodium sulfate with elution by dichloromethane, passage of the eluate through a second column containing acidified Celite (to remove amines), with elution by pentane–dichloromethane 95:5 (to remove fats) and then by pure dichloromethane, concentration of the latter eluate to about 4 ml in a Kuderna–Danish apparatus and then to 1 ml on a microSnyder column, and analysis by GC-TEA. The results showed good agreement between the different laboratories. A similar method has been used to determine NDMA in beer [70]. The Pensabene group [71,72] reported a similar method for determining N-nitrosodibenzylamine, a contaminant derived from rubber and found in hams processed in elastic rubber netting. Ham samples were ground in a mortar and pestle with anhydrous sodium sulfate, Celite, propyl gallate and an internal standard. The mixtures were packed in columns and eluted with dichloromethane, which was passed through silica gel columns with elution by ether– dichloromethane 3:7 or through a Sep-Pak column (solid phase extraction (SPE)), and were analysed by GC-TEA [69]. Because of the poor volatility of Nnitrosodibenzylamine, the GC apparatus was linked to the TEA by a line heated to 2751C. N-Nitrosodibenzylamine (3–130 mg/kg) occurred in 12 of 18 hams. The detection limit was 1 mg/kg. Identity was confirmed by GC-MS. The method could be used to analyse 10 volatile nitrosamines in addition to the test compound. Pensabene et al. [73] also reported the analysis of nitrosamines in hams using supercritical fluid extraction (SFE) with CO2 instead of SPE. Results were similar of the two methods, but the supercritical fluid method enabled 20–24 samples/day to be analysed compared to 8–12 samples/day for SPE.

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Several recent studies determined volatile nitrosamine levels in foods and cigarettes by GC-MS rather than GC-TEA, presumably because analytical laboratories are more likely to have MS than chemiluminescence facilities, MS instruments are simpler to use and cheaper than earlier, and MS can both measure the amount of nitrosamine and confirm its identity. MS is now fast, accurate, specific, sensitive and quantitative. However, TEA usually requires less elaborate clean-up of samples than MS because of the high specificity for compounds that yield NO. As an example of the use of MS, the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and Nu-nitrosonornicotine were determined in cigarettes by adding 13C-NNK as an internal standard and using automated extraction with ethyl acetate in a Dionex ASE 200 accelerated solvent extractor [74]. The extracts were washed with aqueous sodium hydroxide, passed through extraction columns and analysed by GC-MS. Two fragment ions were measured for each nitrosamine. Other examples of this approach induce studies on NDMA levels in human faeces [75] and on NDMA and N-nitrosodiethylamine (NDEA) levels in meat after the addition of sodium chloride and sodium ascorbate, which lowered the nitrosamine levels, or after baking the product, which increased these levels [76]. The latter study was performed using a clean-up by combined distillation-solvent extraction [77], followed by capillary GC at 50–1501C coupled to MS. This study is discussed in Section 5.1. Reverse-phase HPLC-TEA methods are generally unsuitable because cold traps are needed to remove organic solvents after the HPLC and they become blocked with ice. However, Eerola et al. [78] used HPLC-MS to determine volatile nitrosamines in dry sausages. Homogenized sausages (10 g) were minced with 300 mg of propyl gallate, 10 ng of N-nitrosodipropylamine (as internal standard), sodium sulfate, Celite and dichloromethane. The filtered extract was applied to a silica column, which was developed with dichloromethane–pentane 1:3 and then with dichloromethane–ether 7:3. The latter eluate was subjected to HPLC on a Spherisorb-ODS column, which was eluted with a water–methanol gradient, with monitoring by UV absorption at 230 and 254 nm. The eluate was passed continuously into a mass spectrometer with a HPLC interface operated in the positive ionization mode. Analysis was carried out by tandem MS using atmospheric pressure chemical ionization in the selected reaction monitoring (SRM) mode. This soft ionization method is a less energetic process than electron ionization. The MH+ ions were used to determine NDMA, NPYR, NDEA, N-nitrosopiperidine and N-nitrosodipropylamine. Monitored product ions were formed by loss of the nitroso or the alkyl group from the parent ion. In 1989 Belardi and Pawliszyn [79] described a solid phase microextraction (SPME) technique that has become widely used for concentrating organic compounds from water and foods. An advantage of this method is that no environmentally damaging organic solvents are used. In 1997 Sen et al. [80] applied this method to the semi-quantitative analysis of N-nitrosodibutylamine and N-nitrosodibenzylamine in smoked hams packed in elastic rubber netting. The hams were steam-distilled and SPME fused silica fibres coated with a liquid

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phase 75 m thick of poly(dimethylsiloxane) or polyacrylate were equilibrated with the head-space (HS) over the distillate. The fibres were inserted into needles and injected into the injector port (maintained at 2201C) of a GC-TEA apparatus to desorb the nitrosamines. A glass lining in the injector port guided the SPME fibre needle into a capillary column, which was coated with Supelcowax-10. The GC was run with a temperature gradient of up to 2201C. Recoveries of the test nitrosamines were 41–112%. Detection limits were 1–3 mg/kg. In 2001, Ruiz et al. [81] described an SPME method for the analysis of volatile compounds in foods. The SPME fibre was injected directly into a food using a specially designed extraction device. The fibre was equilibrated for 30 min and desorbed in the GC injector port at 2801C. The gas in the injector port was then passed rapidly via a valve into the GC apparatus [82]. The transfer line from the GC to the MS instruments was maintained at 2801C. Ventanas et al. [83] applied this method to the analysis of volatile nitrosamines in gelatin gels. GC was performed on a capillary column lined with 5% phenylmethylsilicone (HP-5) bonded-phase fused silica. Analysis was carried out by MS using the selected ion-monitoring mode. In a study of Andrade et al. [84], volatile nitrosamines in sausages were determined by HS analysis using SPME (HS-SPME) on fused silica fibres coated with polyacrylate or polydimethylsiloxane-divinylbenzene, followed by GC-TEA. The method was stated to be simple, rapid and of adequate accuracy and sensitivity. Volatile nitrosamines in sausages were determined by vacuum steam distillation followed by extraction with active carbon and micellar electrokinetic chromatography, with confirmation by MS [85]. NDMA was detected in 17% of Chilean fish-meal samples, even though the detection limit of 11 mg/kg was relatively high. The method included extraction with ethyl acetate, column chromatography on silica gel and GC linked to a nitrogen–phosphorus-specific detector [5]. In 2003 Sanches-Filho et al. [86] described the analysis of volatile nitrosamines in sausages by vacuum steam distillation, adsorption from the distillate onto activated carbon, elution with acetone–dichloromethane and capillary electrophoresis (micellar electrophoretic chromatography) using a fused silica capillary and a potential of 10 kV. The method separated and detected several added nitrosamines, but was only applied to two unspiked samples. These contained peaks with the retention times of NDMA, NMOR, NPYR and NDEA. Cardenes et al. [87] recently described a sensitive method for determining volatile nitrosamines after dichloromethane extraction from aqueous extracts at neutral pH and denitrosation by heating with HBr–HOAc–water for 10 min at 1001C to give the corresponding secondary amines. Conversion to dansyl derivatives (Figure 5) was achieved by reacting the amines for 30 min at 401C with dansyl chloride in bicarbonate buffer or by a fast microwave-assisted procedure that took less than 5 min and would be especially useful for an automated system. The dansyl derivatives were separated by HPLC on a C-18 column. Cigarette smoke condensate contained 20–80 ng NMOR and 0–5 ng NPYR–cigarette. However, this method would determine the corresponding secondary amines in addition to the nitrosamines. Perhaps prior extraction of the

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Figure 5

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Formation of dansyl derivatives of secondary amines.

nitrosamines from acidified solutions would prevent interference from the amines. Eerola et al. [78] reviewed several earlier papers that used the dansylation method to determine nitrosamines and noted that the corresponding amines would have been determined as nitrosamines.

4.5 HPLC-TEA of nitrosamino acids In 1984, Tricker et al. [11] described an HPLC-TEA method for determining nonvolatile nitrosamines in cured meats. Cured meat (5 g samples) was blended with ammonium sulfamate and sodium sulfate in 2 M phosphoric acid. Pipecolic acid and a test nitrosamine were added to detect artifact formation (indicated by the appearance of N-nitrosopipecolic acid) and measure recoveries. The resulting slurry was extracted with hexane (to remove fats), which was discarded, and then with ethyl acetate, which was concentrated. Nitrosamino acids in the concentrate were passed through a Bond-Elut aminopropyl column and subjected to HPLC on a cyano-based column with hexane–acetone–acetic acid (81:18:1) as the mobile phase. The HPLC eluate was passed successively through (a) the TEA pyrolyzer run at 4801C to liberate NO, (b) cold traps of dry ice-isopropanol and liquid nitrogen to freeze out the solvents and (c) the TEA apparatus. Recoveries were 59–88% for 250 mg/kg each of the added nitrosamines NSAR, NPRO, N-nitrosothiazolidine-4-carboxylic acid (NTCA), N-nitrosohydroxyproline, N-nirosothiazolidine and three N-nitroso derivatives of dipeptides with N-terminal proline. The detection limit for each nitrosamine was 5–10 mg/kg. To prevent ice formation in the cold traps, which blocked the gas flow, Sen et al. [13] used an organic buffer containing 2% water as the HPLC solvent. By this means they could separate the syn and anti isomers of NSAR and NPRO on a silica column. However, this method sometimes showed high responses in blank runs and seems not to have been pursued.

4.6 GC-TEA of methyl esters of nitrosamino acids This is now the preferred method for determining nitrosamino acids such as NPRO, because it is more rugged, more sensitive and simpler to use than HPLCTEA [13]. The analysis of poorly volatile nitrosamines, including nitrosamino acids, was reviewed by Massey [14], Sen and Kubacki [13] and Tricker et al. [11].

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In 1981, Ohshima and Bartsch [88] reported that NPRO occurred in the urine of a subject who ingested nitrate and the amino acid l-proline. NPRO formation is a useful test because it may indicate the propensity for the in vivo (probably gastric) formation of carcinogenic nitrosamines. Gastric NPRO formation should depend on the gastric concentrations of proline, nitrite and inhibitors and promoters of nitrosation, and on gastric pH. The test is considered safe because NPRO was not carcinogenic in tests on rats using doses of up to 36 g NPRO/kg body weight [45,89,90]. Since 1981 many studies, including those from the Ohshima–Bartsch group [91] and the author’s group [92–94], have reported on nitrosamino acid levels in human urine. In such studies, N-nitrosopipecolic acid (NPIC) is added to urine sample as an internal standard, as well as SA and HCl. The nitrosamino acids are converted to their methyl esters with diazomethane (CH2N2) [88] or by heating for 75 min at 701C with boron trifluoride in methanol [95], and are then analysed by GC-TEA. Packed GC columns that have been used in the author’s laboratory include 10% Carbowax 20M on 60–80 mesh Chromosorb W [88] and 3% OV-225 on base-washed 80/100 Supelcoport. Figure 6 shows the separation of NPRO from NPIC on the latter column. NSAR is often detected in human urine in addition to NPRO [93,96]. When diazomethane is used as the methylating agent, NTCA and 2-methyl-NTCA are also detected [97,98]. These nitrosamines arise by nitrosation of the product

NPIC

NPRO

millivolts

300

200

100

2

4

6

8

minutes

Figure 6 GC-TEA tracing of TMS derivatives of NPRO extracted from human saliva and of the internal standard NPIC.

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Figure 7 Formation of NTCA and 2-methyl-NTCA.

formed by the condensation of formaldehyde (in the case of NTCA) or acetaldehyde (in the case of 2-methyl-NTCA) with cysteine (Figure 7). Their level in urine is about twice that of NPRO and their formation proceeds up to 500 times faster than that of NPRO. NTCA and 2-methyl-NTCA are not detected when BF3–methanol is used for the methylation, apparently because they are hydrolyzed by this reagent [99]. Nevertheless, BF3–methanol is easier to use and safer than diazomethane. Also, NTCA and 3-methyl-NTCA are unlikely to be good predictors of the in vivo formation of carcinogenic nitrosamines, because their gastric formation presumably depends on the levels of their precursors, formaldehyde, acetaldehyde and cysteine, in addition to those of nitrite, other nitrosating agents and nitrosation promoters and inhibitors. Therefore, the author’s group used BF3–methanol in their studies. Outram and Pollock [100–102] reported that N-nitrosodialkanoic acids (free and bound as amides to amino acids and peptides) were formed during the nitrosation of gastric juice and of dipeptides [101]. These products were detected by GC-TEA after methylation with diazomethane and probably arose via the genesis of carboxyalkylating agents from nitrosated amino acids and peptides (Figure 8) [100]. The structures of representative compounds were confirmed by elemental analysis [102] and by NMR spectrometry [102] and MS [100]. The authors estimated that 80% of the total nitrosamines detected in nitrosated gastric juice by their GC-TEA method were N-nitrosodialkanoic acids.

4.7 GC-TEA of hydroxyalkylnitrosamines Volatile hydroxyalkylnitrosamines can be determined directly by GC-TEA if the molecular weight is not too large and high temperatures are used for the GC. Thus the author’s group used GC-TEA with a column of 10% Carbowax-20MTPA on Chromosorb GHP heated to 1901C to separate and determine underivatized N-nitroso-2-, 3-, 4- and 5-hydroxymethylpentylamine, which are metabolites of N-nitrosomethylpentylamine, a potent oesophageal carcinogen in the rat [103]. Most studies have determined hydroxyalkylnitrosamines by GC-TEA of their trimethylsilyl (TMS) derivatives. For example, there is evidence that the inductions of lung cancer by cigarette smoke is in part due to the lung

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Figure 8 Proposed origin of N-nitrosodialkanoic acids.

Figure 9 Tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and Nu-nitrosonornicotine (NNN), NNK metabolite 4-(methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL) and two internal standards, iso-NNAL and NPPA, used for NNAL analysis.

carcinogen NNK [30]. NNK is metabolized to give 4-(methylnitrosamino)-1-(3pyridyl)-1-butanol (NNAL), which is excreted in the urine together with its b-Oglucuronide (Figure 9). The urinary level of NNAL plus its glucuronide has been determined in many studies. A standard method [65,104] involves hydrolysis of the glucuronide with b-glucuronidase, adsorption of the NNAL on Celite columns, elution with dichloromethane, transfer to methanol–HCl, passage through cation exchange cartridges with elution by a water–methanol–NH4OH mixture, evaporation to dryness, conversion to TMS derivatives by treatment with N,O-bis-TMS-trifluoroacetamide and capillary GC-TEA. An internal standard, 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL), is added before b-glucuronidase treatment and a second nitrosamine, N-nitroso-3picolylamine, is added before the silylation step as an injection standard. A recent method for NNAL in urine involved HPLC on a molecularly imprinted polymer (MIP) specific for NNAL [105,106]. To prepare this maternal, highly cross-linked polymer is synthesized in the presence of a template molecule that mimics the analyte and forms cavities that are sterically and chemically

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complementary to the analyte. The HPLC eluate is then analysed for NNAL by electrospray ionization MS-MS. This system requires a less elaborate clean-up than previous methods.

4.8 HPLC-photolysis-TEA of non-volatile NOC Photolysis has been used to detect non-volatile NOC after their separation by HPLC. The resulting NO is determined by TEA. This system has been applied to nitrosamides (see Section 4.13). A 1989 study by Conboy and Hotchkiss [107] used an ion-suppression system (10 mM trifluoracetic acid at pH less than 2) or an ion-pair system (tetrabutylammonium dihydrogen phosphate at pH 7) with water–acetonitrile gradients. The HPLC eluate was passed through a borosilicate coil into which was bubbled a stream of helium while the column was irradiated with a mercury-vapor UV lamp. NO produced from the NOC was swept thorough a series of cold traps and determined by TEA. Peak widths of 30–60 s were achieved for mixtures containing 2.4–14 ng each of eight NOC, including NPRO, hydroxy-NPRO, NTCA and four nitrosamides. The method was applied to the analysis of human urine and gastric juice. The detection limit was 2–5 mg/L.

4.9 Havery’s HPLC-TEA method for all types of NOC In 1990 Havery described an analytical system in which aqueous NOC solutions were separated by HPLC and then decomposed to form NO, which was determined by TEA ([108] and D.C. Havery, personal communication). The HPLC used a Zorbax-ODS column and different water–acetonitrile gradients depending on the NOC. The HPLC eluate was mixed with HI, which reacted with the NOC in a post-column reactor to liberate NO, which was analysed by TEA. Oxygen flow rate in the TEA was set to give a pressure of 0.35 torr when the HPLC was disconnected. After connecting the HPLC system, the TEA pump pulled the HPLC eluate and the reagents through the post-column reactor. In this reactor (a glass coil heated to 701C), the HPLC eluate was mixed with helium carrier gas, 10% KI in water (delivered with a separate pump) and 10% H2SO4 in acetic acid (drawn in by the TEA pump). The TEA vacuum gauge then read 0.9–1.0 torr. NOC reacted with HI formed from the KI to generate NO. The NO was swept by the helium through several cold traps to remove acids and water, and then passed into the TEA. Although the system is complicated, it could detect all the principal types of NOC. This method was used by Sen et al. [109,110] to determine nitrosoureas (see Section 4.13).

4.10 Determination of total ANC The method generally used for total ANC depends on the finding that HBr, but not HCl, reacts with NOC to produce NO (Equations (11) and (12)) [68]. The ANC method was introduced in 1976–1978 by the Walters group [111,112]. Solutions of ANC, generally but not necessarily in water, are treated with SA under acidic

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conditions to destroy any nitrite present. SA also eliminates a slight response due to nitrate [13]. The resulting solution is injected into a mixture containing HBr, which reacts with NOC to liberate NO, which is determined by TEA. If SA is not added, the method can be used to determine nitrite (which apparently is also reduced to NO by HBr), preferably after separating the nitrite by HPLC [113]. R1 NðNOÞR2 þ HBr ! R1 NHR2 þ NOBr

(11)

2NOBr ! 2NO þ Br2

(12)

In the U.K. Massey et al. reported ANC levels in beer, cured meat and other foods [114–116]. Beer was treated with SA and passed through an anion exchange column to remove nitrite and nitrate before the ANC determination [115]. For cured meat and other foods [114], 0.1–1.0 g minced food was shaken for 30 s with ethyl acetate, the mixture was treated with acetic acid and HBr, and NO evolution was monitored by TEA to measure ANC. This method is similar to the ANC method described below. ANC levels in ethyl acetate extracts of human gastric juice were positively correlated with gastric pH [117]. In 1993 Xu and Reed [118] examined the relationship between gastric ANC levels in un-extracted fasting gastric juice and gastric pH. They obtained two peaks of ANC concentration, at pH 1.2–2.5 and at pH 6.2–8.3. Mean ANC levels for the two pH ranges were 1.45 and 3.37 mM. This result suggests that both acid-catalyzed nitrosation at pH 1.2–2.5 and bacteria-mediated nitrosation at pH 6.2–8.3 produce gastric ANC. Volatile nitrosamines and nitrosamino acids generally constitute about 1–2% and 20% of the ANC in foods and biological fluids [117]. The remaining 80% of the ANC could be the most important fraction for carcinogenesis. It is not certain that all these ANC, which have mostly not been separated and identified (for an exception where an NOCP was identified, see Ref. [39]) truly are NOC. The various studies on ANC analysis used different solvents to extract the ANC from foods. The ANC method used but the author’s group [33,34] is based on similar but more complicated methods for the analysis of ANC in human gastric juice developed by Xu and Reed [119] and Pignatelli et al. [120]. Samples of diet or mouse faeces are dried to constant weight for 36 h at 1 torr and 01C. The samples (up to 1 g) are soaked for 30 min in water, vortexed and centrifuged. Aliquots of the supernatant are incubated with SA and HCl for 15 min and injected into a mixture of ethyl acetate, acetic acid, HCl and HBr that is refluxing in a four-neck round-bottom flask at less than 0.7 torr and 281C. The oil pump in the TEA maintains the vacuum. A stream of argon sweeps the liberated NO through six wash-bottles containing agents that remove acids and water, and then into the TEA. The method accurately measures ANC, even in crude extracts, but does not separate individual compounds. NPRO (0.1 nmol) is injected periodically as a standard. To determine ANC precursors (ANCP), aliquots are nitrosated with 110 mM nitrite at pH 1.5–2.0, SA is added and dilutions (usually 1/100) are analysed for ANC. Dilutions are made in 1% SA to remove nitrite derived from atmospheric nitrogen oxides.

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Relative to the response for NPRO, the ANC method gave yields of close to 100% for simple nitrosamines, including NDMA and NMOR, 77% for 1-methyl-3nitro-1-nitrosoguanidine (a carcinogen and direct-acting mutagen) and only 16% for NMU [33]. The specificity of the ANCP method was tested by treating various nitrogen compounds with nitrite under the conditions used to assay ANCP [33]. ANC yields were less than 1% for peptides with nitrogen only in the peptide (–NHCO–) bond and for diethylamine, 6–12% for alkylureas, 45–97% for the readily nitrosated [23] secondary amines proline, morpholine and piperazine, 67% for 1-deoxy-N-1-fructosylvaline, 27–88% for tryptophan, up to 31% for tryptophanyl alanine, up to 5% for alanyl tryptophan and less than 2% for histidine [33,39]. The low ANC yield for simple peptides not containing tryptophan and for diethylamine is attributed to their slow rate of nitrosation [23]. These results indicate that most of the hot dog ANCP are nitrosamines derived from readily nitrosated amines. The ANCP in hot dogs did not include significant amounts of tryptophan because the concentration of free tryptophan in hot dogs was far less than that of the ANCP and because ANC produced from tryptophan were far less stable than ANC derived from hot dog ANCP [39]. The ANCP in hot dogs were purified by adsorption from aqueous extracts onto silica gel, elution with acetonitrile and then methanol, adsorption of the methanol eluate on cation exchange resin in its protonated form, desorption with 1N NH4OH and HPLC on a lead (Pb++) carbohydrate column run at 901C [39]. After nitrosation, addition of SA, and neutralization, the material eluted from the resin with ammonium hydroxide (the ‘‘ammonia eluate’’) was directly mutagenic in the Ames test with Salmonella typhimurium TA-100, producing four times the number of background mutations [39]. Ammonia eluate that was not nitrosated showed twice the background mutations. These results suggest that the hot dog ANCP-derived ANC might indeed be carcinogenic. This might be especially relevant for the colon because feeding red meat and hot dogs, which contain both ANC and ANCP, increased the faecal excretion of ANC in humans [35] and mice [34], and colonic ANC levels are similar to those in the faeces [34]. ANCP-rich fractions from the HPLC of hot dog extract were converted to TMS derivatives and examined by GC-MS. One fraction was identified as the TMS derivative of 1-deoxy-N-1-glucosyl glycine (Figure 10) [39]. This suggests that N-1glucosyl amino acids and peptides are significant ANCP in meat products. Such compounds are very weak bases [121] and hence [23] would be rapidly nitrosated, as was the case for the hot dog ANCP [33]. Facile nitrosation of N-glycerylamines was indicated by the ready conversion of N-2-deoxy-2-fructosyl valine to NOC

Figure 10 1-Deoxy-N-1-glucosyl glycine.

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[33] and the low pKa predicted for these compounds. In fact, one N-glucosyl dipeptide had a pKa of only 5.6 [121], relevant because a low pKa for secondary amines is generally associated with a high rate of nitrosation [23]. HI has been used instead of HBr to produce NO from NOC. Thus Havery’s method for determining NOC used HI to generate NO [108] (see Section 4.9). Fiddler et al. [122] employed HI to assay ANC in acetonitrile extracts of cured meat products by a modification of Havery’s procedure. Also, Cox et al. [123] used HI in a method for determining ANC in human urine. After removal of nitrate and nitrite by treatment with anion exchange resin and sulfanilamide, urine extracts were mixed with HI–sulfuric acid and the liberated NO was determined by TEA. In 1932 cuprous chloride (CuCl) was reported to react with nitrosamines to give NO and the parent amines [124]. This reaction was later recommended as a means of purifying secondary amines [125]. In 2005, Wang et al. [126] described a method for determining ANC using cuprous chloride to produce NO from the ANC. As in the HBr method, SA was added before the TEA. ANC formation from NPRO was linear from 4 pmol to 2 nmol. The cuprous chloride method could be used with aqueous or organic solvents, except for dichloromethane and chloroform, and was applied successfully to the analysis of ANC in meat products and sauces. Excessive foaming interfered in the analysis of beer, shampoos and lotions. Equations (13)–(15) show the proposed reactions. Nitrite esters (RONO), oximes (RCH¼NOH) and C-nitro and C-nitroso compounds gave negative responses. Inorganic nitrite and nitrite esters were determined if SA was omitted. S-Nitroso-N-acetylpenicillamine and S-nitrosoglutathione were determined in yields of 3.3% and 32%, respectively, after SA was added. Nitrate increased the baseline, probably due to a very slow release of NO. Interference by nitrate could be prevented by absorbing the nitrate on anion exchange resin before the NOC analysis. CuCl þ HCL $ ½CuCl2  þ Hþ

(13)

R1 R2 NNO þ Hþ $ ½R1 R2 NHNOþ

(14)

½R1 R2 NHNOþ þ ½CuCl2  ! R1 R2 NH þ NO þ CuCl2

(15)

The author’s group (M.P. Lisowyj and S.S. Mirvish, unpublished results) used the HBr method to confirm and extend the findings of Wang et al. [126] that nitrosothiols could be determined as ANC. Solutions (1.0 nmol/mL) of glutathione and dithiothreitol were nitrosated and treated with SA as in the NOCP method [33] and then analysed for ANCP. After the solutions were stored in ice for 0–10, 60–70 and 90–100 min, percent ANC yields based on the response for NPRO were 47%, 32% and 18% for glutathione, and 42%, 20% and 20% for dithiothreitol. Analysis of S-nitroso-N-acetylpenicillamine as ANC after addition of SA gave apparent yields of 180% compared to that for NPRO. In contrast to the instability of these S-nitroso compounds, the ANCP in hot dogs were stable when stored for several hours in ice (unpublished results), indicating that the ANCP in hot dogs could include at most only a small proportion of nitrosothiols.

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4.11 Determination of nitrosamides as ANC Pignatelli et al. [120] reported only a 25% yield for NMU compared to that for NPRO when NMU was determined as ANC. Xu and Reed [119] reported a broad peak for NMU when HBr alone was used for denitrosation, but a sharp peak when both HBr and HCl were employed. In our study on ANC determined by the HBr method [33], 1-methyl-3-nitro-1-nitrosoguanidine showed a narrow peak (a short elution time), whereas NMU gave broad peaks 13–15 min wide, with molar yields less than 20% of those for NPRO. When the temperature of the refluxing ethyl acetate was raised from the standard 281C, the width of the NMU peaks was reduced to 3.5–4.0 min and the response rose to 32% (at 331C) and 39% (at 361C) of that for NPRO [33]. In contrast, Sen et al. [127] obtained nearly quantitative recoveries by the Havery method [108] when NMU was denitrosated with HI at 701C (see Section 4.13). Sen et al. [128] also reported that inorganic nitrite could be determined by the method for ANC if SA was omitted.

4.12 Determination of alkylureas Krull et al. [129] used an HPLC method with detection by UV absorption to determine 1-nitroso-1,3-bis-(2-chloroethyl)-urea in blood with a detection limit of 100–200 mg/L). Conboy and Hotchkiss [107] later used a similar method, but with detection by TEA. The detection limit was 2–5 mg/L when this method was applied to the analysis of N-nitrosotrimethylurea in porcine gastric fluid. Dried fish and nitrite-preserved meat products are likely risk factors for the etiology of gastric cancer [28] and alkylnitrosoureas and related NOC can induce gastric cancer in laboratory animals (see Section 3.2). Because alkylureas could readily be converted to alkylnitrosoureas in the stomach, the author’s group studied the occurrence of alkylureas in aqueous extracts of marine animal products and fried bacon [130]. Methylurea was not detected in the absence of nitrosation, but was detected after the aqueous extracts were ‘‘nitrosated– denitrosated,’’ i.e., were treated with very large amounts of nitrite at pH 1 to convert precursors to alkylnitrosoureas, and were then stored at pH 0 to convert the alkylnitrosoureas to alklylureas. This treatment destroys urea itself, which reacts with nitrite to give CO2 and nitrogen (the van Slyke reaction) [131,132]. The resultant alkylureas were extracted with n-butanol. Because alkylureas show pKa values of about 0.7 (the value for methylurea [133]), they were further purified by adsorption of the protonated alkylureas on columns of cation exchange resin at pH 1 and elution of the deprotonated ureas at pH 4. Purification was followed by a colorimetric method involving the addition of semidine (N-phenyl-pphenylenediamine), ferric chloride and HCl, which gives a purple colour with alklylureas and alkylnitrosoureas, with a detection limit of 10 nmol/sample [134,135]. Subsequent purification by paper chromatography and HPLC followed by MS enabled us to identify and determine alkylureas in the nitrosated– denitrosated products. n-Propylurea, 3-butenylurea and a hydroxybutylurea (not fully identified) were detected after nitrosation–denitrosation of extracts of certain crab and lobster species.

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Figure 11 Nitrosation of CRN and methylguanidine to give methylurea and NMU.

Large amounts of creatine and phosphocreatine (which converts adenosine diphosphate to adenosine triphosphate in vivo) occur in muscle tissues of vertebrates, including fishes. When foods containing creatine are dehydrated or heated, the creatine loses water to give CRN, which is also the urinary metabolite of creatine. In 1971, Archer et al. [136] discovered that nitrosation of CRN produces CRN-5-oxime and 1-methylhydrantoin-5-oxime (Figure 11) [136]. In 1982 the author’s group discovered that the precursor of methylurea in dried bonito fish was CRN [137]. Yields of methylurea after nitrosation–denitrosation of CRN, CRN-5-oxme and 1-methylhydantoin-5-oxime were 0.04%, 0.04% and 7.0%. On this basis it was proposed that CRN produces methylurea by the sequence shown in Figure 11 and that the first two of these nitrosations were the ratelimiting steps. A later study of these reactions included the separate determination by HPLC of the syn and anti isomers of the two intermediate oximes [138]. Reaction rates were proportional to nitrite concentration squared as in Equation (9). Nitrosation of creatine produced NSAR [136] but not methylurea [137]. The extremely high CRN concentration of 4 g/kg in both dried fish and fried bacon, the lengthy storage time at ambient temperature for dried fish and the high temperature and low water content during the frying of bacon could make methylurea formation in these products significant, despite the slow nitrosation of CRN. Nitrosation of methylguanidine gave a 35% yield of NMU at a rate less than 1% of that for NMU formation from methylurea (Figure 11) [51,139]. This is relevant here because methylguanidine occurs at levels of 60–1,900 mg/kg in fresh beef and in various species of fish [140]. Bredereck et al. [141] reported in 1964 that urea reacts with the diethylacetal of dimethylformamide to form its dimethylaminomethylene derivative (Figure 12). In 1979, Kawabata et al. [142,143] applied this method to the detection of alklylureas in foods. For this purpose, they used successive chromatography on Dowex 50 (H+ form), cation exchange resin, Dowex IX anion exchange resin (OH form) and basic alumna, conversion of alkylureas to

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Figure 12 Formation of dimethylaminomethylene derivatives of alkylureas.

their dimethylaminomethylene derivatives (Figure 12) and GC of the derivatives on an OV-17 column with flame ionization detection. GC-MS using the same GC conditions confirmed the identities of the alkylurea derivatives. Mean recoveries of eight alkylureas, added to salted dried fish samples, were 71–90%. The detection limit for these alkylureas was 10–100 mg/kg food.

4.13 HPLC-photolysis-TEA of nitrosamides Nitrosamides cannot be determined by pyrolysis to give NO because most nitrosamides are poorly volatile and decompose on heating to produce nitrogen [144]. Shuker and Tannenbaum [145] reported in 1983 on a method for determining nitrosamides based on their photochemical decomposition to give nitrite. Because nitrosamides yield NO as the primary product of photolysis [60], nitrite is presumably produced by oxidation of NO to N2O3 and N2O4, which react with water to give nitrite. In the method of Shuker and Tannenbaum, nitrosamides were separated by HPLC on a C-18 cartridge eluted with pH-6 buffer-acetonitrile mixtures. The HPLC eluate was passed through 15 m of 0.25 mm Teflon tubing wound round the outside of a water-cooled cylindrical glass jacket, which surrounded a high-intensity-discharge metal halide lamp, that emits light at 380–420 nm and is used in light-houses. Teflon tubing is transparent to visible light and hence is suitable for studies on nitrosamides, which absorb light and therefore are decomposed at 380–430 nm. The apparatus was enclosed in a box lined with aluminum foil. Nitrosamides were completely decomposed to give nitrite after exposure to the light for 2 min. Nitrite in the eluate was analysed colorimetrically with the Griess reagent [146]. The method was used to determine methylnitronitrosoguanidine, NMU, N-nitroso derivatives of bile salts such as taurocholic acid, N-methyl-N-nitrosoacetamide and N-nitrosocimetidine, all of which are or are related to nitrosamides. Deng et al. [147–149] reported the occurrence of NMU when Oriental fish sauces were nitrosated under mild conditions (1 h, 5 mM nitrite, pH 2, 371C), followed by addition of SA. Under these conditions, most methylurea, but very little CRN, would be converted to NMU. Aqueous extracts of nitrosated fish sauce were mixed with sodium chloride, deproteinized with tungstophosphoric acid and extracted with acetone–dichloromethane 1:5. The extracts were subjected to HPLC on various columns with elution by aqueous trifluoroacetate–acetonitrile mixtures. NMU in the eluates was determined by photolysis to liberate NO, which was converted to and measured as nitrite [107]. Maximum NMU yields were obtained after nitrosation at pH 1 (the lowest pH tested) and

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pH 2 [148], as expected if the NMU precursor was methylurea [23,51], but NMU yields were not reported. NMU was not detected in the fish sauce in the absence of nitrosation. After nitrosated fish sauce extracts were subjected to HPLC, MS analysis of the eluates confirmed that NMU was present [150]. NMU was also detected in the stomachs of pigs that were intubated with fish sauce and nitrite and of humans who ingested these materials [151]. Sen et al. [109,110] detected NMU after CRN, Oriental fish sauce and other marine products were incubated with up to 1.6 mM nitrite. Aqueous extracts were incubated with ascorbic acid and SA at pH 1.0–1.5 to prevent artifactual nitrosation, saturated with sodium chloride and kept at pH less than 6–7. The workers avoided exposing samples to strong light and raised temperatures, and avoided delays in processing. The extracts were subjected to a series of additional extractions and clean-ups on C-18 and silica Sep-Pak columns, and were then analysed for NMU by HPLC with post-column chemical denitrosation followed by TEA according to Havery’s method (Section 4.9) [108], or were analysed by GC-MS. The HPLC-TEA method could detect 0.1–1.0 ng and GC-MS could detect 2.5 pg of NMU. In nitrosated cured meat, the authors could detect 0.5 mg/kg of NMU by HPLC-TEA and 0.03 mg/kg of NMU by GC-MS. NMU was not detected in un-nitrosated samples. Because methylurea is very readily converted to NMU [23,51], the procedures of Deng et al. [148] and Sen et al. [109,110], both of which involved nitrosation under mild conditions followed by analysis for NMU, in reality probably are methods for determining methylurea.

5. OCCURRENCE OF NOC IN FOODS 5.1 Occurrence of volatile nitrosamines Figure 1 shows the structures of many of the nitrosamines that can occur in foods. In general, NOC appear to accumulate in foods that are fermented and/or stored for long periods at ambient temperatures, e.g., beer and fish sauce. In the late 1960s, fried bacon was found to contain up to 100 mg/kg (ppb) of volatile nitrosamines, especially NDMA and NPYR [152,153]. In 1972 a group that included the author found that ascorbic acid (vitamin C) inhibited nitrosamine formation in chemical systems [19]. It was then shown that ascorbate inhibits the production of volatile nitrosamines in fried bacon [152]. As a result, the U.S. Food and Drug Administration and, later, government agencies in other countries mandated that nitrite levels in bacon be dropped from 150 to 120 mg/kg and that 500 mg/kg of ascorbate or its non-vitamin isomer, erythorbate, be added to bacon [154]. The effect of this mandate was to lower volatile nitrosamine levels in fried bacon to below 20 mg/kg [152,153]. These levels are considered safe because they do not add much to the total dietary burden of volatile nitrosamines. In 1979, German beer was found to contain a mean NDMA level of 2.7 mg/kg (maximum, 68 mg/kg) [155]. This probably arose from a reaction of dimethylamine, which occurs in malt (germinated barley) and beer [156], with nitrogen oxides in spent gas from the natural gas kilns in which the malted barley was

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dried. After 1979, NDMA levels in beer were lowered by reducing the flame temperature, by adding elemental sulfur to the flames and especially by the use of indirect heating, in which the heated gasses travel through pipes and are only in indirect contact with the malted barley. The heated gases contain nitrogen oxides that are derived from the reaction of nitrogen with oxygen in the flames and can nitrosate amine. These techniques lowered NDMA levels in beer to less than 0.4 mg/L [70,156,157]. In 1981, the U.S. National Academy of Sciences [6] listed the following values for NOC concentration in foods. In England, fried bacon contained 1–20 (occasionally up to 200), 0.1–0.5 and 0.1–0.25 mg/kg of NPYR, NDMA and N-nitrosopiperidine. Cured meat in the United States and United Kingdom contained less than 1 mg/kg of volatile nitrosamines. In the U.K., 80% of uncooked and fried fish samples contained detectable NDMA, with about onethird of the samples showing 1–10 mg/kg of NDMA and the rest showing lower levels. Japanese broiled salted dried fish and shellfish showed up to 310 and up to 13 mg/kg of NDMA and NPYR, with the highest values in dried squid. In 1986, levels in mg/kg of home-cooked fried bacon were reported to be 17, 4, 9 and 0.7 for NPYR, NDMA, N-nitrosothiazolidine and N-nitrosopiperidine [158]. The fried-out fat contained somewhat higher levels of these nitrosamines. In the United States, in 1981, the mean daily intake of volatile nitrosamines in mg/person was estimated to be 17 from cigarettes (due largely to NNK and Nu-nitrosonornicotine), up to 1.0 in beer, 0.41 in cosmetics and 0.17 in cured meat [6]. The nitrosamines in the last three items were mostly NDMA and NPYR. In Germany, before corrective measures were adopted for beer, the percentages of dietary intake of NDMA due to beer, meat and meat products, cheese and other items were 24, 9, 1 and 66 [159]. In the U.K., the total intakes of volatile nitrosamines in cured meat, fish, cheese and all other foods were 0.43, less than 0.01, less than 0.01 and 0.08 mg/person/day [160]. In Chinese foods, most nitrosamine levels were similar to those in the U.K. but NDMA levels were 5–130 mg/kg in dried shrimp and 3–26 mg/kg in shrimp meat. In Spain, the mean dietary intake of NDMA was estimated in 2006 to be only 0.11 mg/day, mostly derived from processed meat and cured cheese [161]. Rywotycki [76] recently reported mean nitrosamine levels for raw beef of 8 mg/kg (range, 6–11) each for NDMA and NDEA. These results are much higher than those listed above for fried bacon and cured meat. NDEA has not usually been detected in meat products. Most analyses have reported more NPYR than NDMA, whereas here NPYR was not mentioned. However, few results have been reported previously for raw meat. A problem with this study may have been that, apparently, few of the checks listed in Section 4.3 were performed.

5.2 Occurrence of nitrosamino acids Tricker et al. [11] reported the occurrence of nitrosamino acids in smoked cured meat from Germany. The detection limit was 5–10 mg/kg. The principal nitrosamino acids detected and their highest observed concentrations in mg/kg

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were NSAR, 410; NPRO, 360; N-nitrosohydroxyproline, 560 and NTCA, 1,620. The highest nitrosamino acid levels were found in Icelandic smoked mutton.

5.3 Occurrence and identity of total ANC In the U.K., Massey et al. [114–116] reported ANC levels in mmol/kg of 0.2–1.0 for dried milk, dried soups, coffee, tea and cocoa/chocolate; 0.2–18 for beer; 14–32 for canned cured meat; 11–136 for raw bacon and 8–55 for fried bacon. (The British groups recorded their results as mg of NNO group/kg of food. These results are re-expressed here as mmol/kg by dividing mg NNO by 44.) In a U.S. study by Haorah et al. [33], mean NOC and NOCP values in mmol/kg were 5.5 and 2,700 for hot dogs, 0.5 and 660 for fresh meat, 3.7 and 1,300 for summer sausage, 6 and 1,100 for canned corned beef; 5.8 and 5,800 for salted, dried fish, 2.7 and 6,600 for all sauces (mostly soy sauces), 0.7 and 950 for ketchup, and 29.6 (a very high value) and 7,800 for a Chinese ground bean sauce [33]. In the study by Fiddler et al. [122] on acetonitrile extracts of cured meat, mean results for hot dogs, bacon, fermented salami, dried beef, canned corned beef, canned ham and netted ham were 1.9, 3.5, 9.0, 8.1, 54.6, 5.5 and 6.4 mmol/kg of ANC (these results were presented as mg NPRO/kg and are recalculated here). Note the high results for canned corned beef. In this study, 10 g comminuted meat was mixed with 15 ml acetonitrile to prepare the extracts. In contrast, the Haorah study [33] reported results for water extracts and stated that acetonitrile extracts contained very little ANC. Unlike the Fiddler et al. [122] study, Haorah et al. [33] evaporated the meat samples to dryness before adding acetonitrile, so that the acetonitrile extracts contained little water. Hence the two sets of results are probably comparable. Mean total ANC excretion by healthy subjects was 1.3 mmol/day in the urine and 1.3–3.2 mmol/day in the faeces [117]. The total ANC level in normal gastric juice was 1.3–1.6 mmol/L [117]. Nitrosamino acids and volatile nitrosamines constituted 16% and 0.03% of the ANC in human urine. N-Nitroso derivatives of proline and peptides with N-terminal proline, together with other similar derivatives, constituted 15–20% of the total ANC in meat products [117]. The remaining 80% of the ANC in hot dogs remain to be identified, though the identification of 1-deoxoy-N-1-glucosylglycine in hot dog extracts (Section 4.10) suggests that it and similar glycosyl amino acids and peptides are the major components of these ANC.

5.4 Occurrence of alkylureas and alkylnitrosoureas In the study of methylurea levels in nitrosated–denitrosated dried fish and fried bacon [130] (Section 4.12), methylurea levels after nitrosation–denitrosation were 350 mmol (25 mg) per kg for both foods. In the study on NMU determination in nitrosated food extracts [109] (Section 4.13), NMU levels were up to 140 ng/kg for fish sauces, up to 34 mg/kg for crab and lobster paste, and somewhat lower for other marine animal products. I emphasize that these figures do not indicate actual occurrences, but rather show yields in the foods after nitrosation– denitrosation (for methylurea) or after nitrosation alone (for NMU).

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6. CONCLUSIONS AND FUTURE TRENDS In most of the principal methods for determining NOC, the NOC are converted to NO, which is measured by TEA. Decomposition of NOC to give NO is achieved by heating volatile nitrosamines to 4501C (pyrolysis) after GC, by treatment of NOC or ANC with HBr [33], HI [108] or cuprous chloride [126], or by photochemical methods [107,145,147–149]. Analyses of volatile nitrosamines were mostly performed by MS until the TEA was introduced in 1978. TEA has since been adopted as the detection step for most methods of NOC analysis. Because TEA is relatively specific for NOC, it generally gives simpler results (clearer tracings) than MS. However, MS has been a useful method for confirming the identity of GC-TEA peaks. Also, many recent studies have used MS rather than TEA to determine nitrosamines, partly because of the greater availability of MS compared to chemiluminescence (TEA and Sievers) instruments in analytical laboratories. In the author’s opinion, the most likely classes of NOC to be causes of human cancer are the simple volatile dialkyl and cyclic nitrosamines (‘‘simple’’ ¼ without other functional groups), which may be causes of oesophageal, nasopharyngeal, laryngeal and bladder cancer [8]; NMU formed by the nitrosation of CRN and, possibly, methylguanidine, which may be causes of stomach and brain cancer; and (with less evidence) N-nitrosoglucosyl amino acids and related compounds, which might be causes of colon cancer. Future analysis of food should focus on these classes of NOC. Following NPRO excretion in urine may continue to be a valuable method for monitoring in vivo nitrosation. The carcinogenicity of non-volatile nitrosamines, including nitrosamines derived from amino acids with secondary amino groups, has mostly not been investigated, except that NPRO is clearly not carcinogenic. Therefore, some of these nitrosamines should at least be tested for bacterial mutagenicity in the Ames test. This would be useful because the Ames test for mutagenicity is relatively cheap and its results are fairly well correlated with carcinogenicity [162]. Individual ANC in crude food extracts have mostly not been identified. This raises the question of whether ANC truly measures NOC, especially since it was reported [126] and confirmed here (see Section 4.10) that some nitrosothiols can also be determined as ANC. It may not matter much if the ANC include small properties of nitrosothiols, if ANC levels are used mainly to indicate the potential for NOC formation, i.e., to indicate the presence of nitrosating agents that could react with NOCP to form NOC. Nevertheless, research is urgently needed to identify the major ANC in foods. The author recommends that, for uniformity, future ANC results should be reported as mmol ANC/kg as in reference [33] and not as mg NNO group/kg [114,115] or as mg NPRO/kg [122]. Most of the results for NOC analysis were published in the early 1980s, e.g., those referred to in [6], and it appears that the monitoring of foods and beverages for their NOC content has slackened off in recent years. However, it is clearly vital to continue this effort by periodic analysis, probably in most cases by government agencies, in case NOC levels rise due to changes in agricultural or

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manufacturing practices. These analyses should be preformed with at least some of the precautions listed in Section 4.3.

ACKNOWLEDGEMENTS I thank the Editor of this book (Yolanda Pico) and the Reviewer of this article for their advice and encouragement, Donald C. Havery (U.S. Food and Drug Administration) for information about his studies and Nilesh W. Gaikwad (Eppley Institute for Research in Cancer) for advice about MS techniques. This review was written while the author’s research was funded by grant RO3-CA-11753 from the National Cancer Institute, a contract with the Division of Cancer Prevention, National Cancer Institute and a grant to Stephen I. Rennard from the Institute for Science and Health. Several of the techniques used in these projects are summarized here. I thank Michal P. Lisowyj and Michael E. Davis for laboratory assistance with these projects, Michal P. Lisowyj for help in finding references and preparing the figures, and Darcy C. Jackson and Diane C. Torrey for help in typing and arranging the manuscript.

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