Trace analysis of nitrosated foodstuffs for nitrosamides

Fd Chem. Toxic. Vol. 29, No. 3, pp. 167-172, 1991 Printed in Great Britain.All fights reserved

0278-6915/91 $3.00+ 0.00 Copyright © 1991 PergamonPress pie

TRACE ANALYSIS OF NITROSATED FOODSTUFFS FOR NITROSAMIDES P. MENDE, B. SPIEGELHALDERand R. PREUSSMANN German Cancer Research Center, Institute for Toxicology and Chemotherapy, Im Neuenheimer Feld 280, D-6900 Heidelberg, Germany (Received 9 July 1990; revisions received 19 November 1990) Abstract--An analytical procedure previously developed for the trace determination of nitrosamides was applied to a screening of nitrosated foodstuffs for nitrosoureas. Different types of foodstuffs were nitrosated both under chemical conditions using a high nitrite concentration, and under simulated gastric conditions. Methylating activity corresponding to N-nitroso-N-methylurea (MNU) was detected in most samples. Under chemical conditions, the yields spanned several orders of magnitude with processed fish and meat products being at the top, and plant products at the bottom of the scale. After nitrosation under simulated gastric conditions, the range of MNU activity was significantly smaller. No correlation exists between the yields determined under chemical and simulated gastric conditions.

INTRODUCTION

Diet is regarded as an important factor in the aetiology of human gastric cancer, with emphasis on the formation of direct alkylating nitrosamides from dietary precursors (Mirvish, 1983). This class of compounds includes, for example, nitrosoureas, nitrosoguanidines and nitrosourethanes. Because of their instability, nitrosamides probably do not persist in foods, but might be generated in vivo from amide precursors and nitrite under the acidic conditions of the stomach (Correa et al., 1975; Mirvish, 1971). This was confirmed by the finding t h a t / n vitro treatment of certain foodstuffs (e.g. fried bacon and fish products) with excessive nitrite resulted in the formation of N-methyl-N-nitrosourea (MNU; Mirvish et al., 1980). The main problem of nitrosamide analysis was the lack of suitable methods for trace analysis. In the present study, we used a test system based on the release of diazoalkanes which are trapped in a second step by the scavenger reagent N-nitroso-N-tertbutylglycine (NTBG). This method has been proven to be specific for non-polar alkali-labile nitrosamides (Mende et al., 1989) and provides the sensitivity required for the detection of nitrosamides formed under simulated gastric conditions.

MATERIALS AND METHODS

Materials. All chemicals used were of analytical grade. M N U and N-nitroso-N-butylurea (BNU) were synthesized from the corresponding alkylureas by nitrosation and recrystallized from methanol. The synthesis of NTBG and its ester derivatives has been described previously (Mende et al., 1989). Abbreviations: BNU = N-nitroso-N-butylurea; GLC = gas-liquid chromatography; MNU = N-nitroso-Nmethylurea; NTBG = N-nitroso-N-tert-butylglycine; TEA = thermal energy analysis.

Preparation o f food samples. Food samples purchased at local stores or from an importer were homogenized with dry ice in a mixer and stored at - 2 0 ° C before the experiments. Bacon, fish and meat were fried in a stainless-steel pan at about 170°C. Nitrosation o f foodstuffs under chemical conditions. The homogenized food sample (5 g) was mixed with 20 ml sodium nitrite solution (final concentration 100 mM) and the pH was adjusted to 1.0 with concentrated HCI. The mixture was incubated at 37°C for 1 hr. The nitrosation was stopped by the addition of I g solid ammonium sulphamate. To stabilize the nitrosourea compounds formed, the pH was adjusted to 3-4 with sodium hydrogen carbonate solution under vigorous stirring. Sticky samples were centrifuged before extraction, and the pellet was resuspended in 10ml water and centrifuged again. The solution was extracted twice with 10 ml n-hexane to remove fat, and the hexane phase was re-extracted with 10 ml water. The combined aqueous phases were absorbed on Extrelut (Merck AG, Darmstadt, F R G ) and eluted with dichloromethane to yield a final volume of 50 ml. The eluate was evaporated to dryness in vacuo and the residue was dissolved in 4 ml dichloromethane. Nitrosation o f foodstuffs under simulated gastric conditions. Nitrosation of food samples (5 g each) was carried out in 50 ml simulated gastric juice based on a 50mM-KCI/HC1 system and containing 70mMNaCI, 50 mM-KC1, 2.2 mM-D-glucose, 0.14 mMpepsin, 1.1 mM-lactic acid, 1.5 mM-thiocyanate and 0.14 mM-catechin (Gillatt et al., 1985). After adjusting the pH to 2.0 with HCI, the nitrosation was initiated by adding 5 ppm (72 g M) sodium nitrite. The mixture was incubated for 1 hr at 37°C with supplements of 1 ppm nitrite every 10 min. The sample workup was carried out as described above. Control samples. Each experiment was carried out with several controls. In the first control, nitrite was omitted and the sample was spiked with M N U (5/~g, or 0.1/zg in simulated gastric juice) after complete incubation. This sample was taken as an external

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rapid extraction using absorption of the aqueous extract onto silica gel (Extrelut) followed by elution with dichloromethane. The dichloromethane extract could then be used for the nitrosamide test without further purification. The test system used for nitrosamide detection is composed of a small column filled with layers of silica gel and anhydrous potassium carbonate. The scavenger reagent (NTBG) is applied on the top of the silica-gel layer. During the passage of the potassium carbonate, nitrosamides decompose to their corresponding diazoalkanes, which rapidly react with NTBG. The NTBG esters formed are selectively eluted with an organic solvent, and the eluate is analysed directly by GLC/TEA. Traces of water and acidic compounds contained in the sample are trapped by the anhydrous potassium carbonate and do not interfere with the test system. This method is specific for nitrosoureas and all related compounds that release diazoalkanes upon treatment with alkali; other direct alkylating compounds do not react under the conditions of the test system (Mende et al., 1989). Figure la shows a typical chromatogram obtained from the nitrosation of pickled shrimp. Since nitrosamide activity was detected only after nitrosation, samples not treated with nitrite, but spiked with M N U and worked up in the usual manner, were included in each assay as external standards (Fig. lc). The recoveries for M N U ranged from 62 to 95%. The nitrosamide origin of the alkylated scavenger reagent was checked by several controls. In the first, NTBG was omitted during passage of the sample through the test column; this represents the nitrosamine background of the sample (Fig. lb). For some samples a second control was included in which the aqueous nitrosated extract was made strongly alkaline before the isolation procedure. Under these conditions, most nitrosamides rapidly decompose and hence no peaks derived from the alkylated scavenger reagent were observed. Fifty food samples were treated with nitrite and analysed for direct alkylating activity derived from

standard for quantification of M N U equivalents. An additional control was nitrosated as usual, but no scavenger reagent was used in the nitrosamide test in order to evaluate the nitrosamine background of the nitrosated sample. Analysis of nitrosamides by their alkylating activity. The nitrosamide content in nitrosated food samples was measured after releasing diazoalkanes by treatment with alkali and trapping them with the scavenger reagent (NTBG). The NTGB-esters formed were analysed using a gas-liquid chromatograph/ thermal energy analyser (GLC/TEA) as described in detail previously (Mende et al., 1989). Some modifications of the original system were established: the glass-wool layer to separate scavenger reagent and potassium carbonate was omitted, and coarse potassium carbonate was added on the top layer. Sample aliquots (1 ml each) of the nitrosated extract dissolved in dichloromethane were applied to the columns and eluted with dichloromethane--ether (9:1, v/v) to yield a final volume of 4 ml. Each determination was performed in triplicate. The eluates were concentrated to 1 ml under a stream of nitrogen and analysed by gas-liquid chromatography (Hewlett Packard gas chromatograph 5880, connected to an NO-specific chemiluminescence detector (TEA). An OV-17 column was used (1.2m x 2 m m i.d.; 10% on Chromosorb WHP) with temperature programming from 120-190°C (5°C/min). The injection port temperature was maintained at 200°C and the injection volume was 5-10#1. NTBG esters were used as reference compounds. RESULTS Most nitrosamides, including nitrosoureas, are sensitive to light, high pH and temperature. Therefore a simple procedure was developed to minimize the decomposition of nitrosoureas during the workup of samples of nitrosated foodstuffs. This method includes adjusting the pH after nitrosation to the optimal range of stability (Kakuda et al., 1980), and

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Fig. 1. Detection of nitrosourea activity by GLC/TEA in a sample of pickled fish (sample 39 in Table 1): (a) after treatment with 100 mM-nitrite at pH 1; (b) nitrosated control with N-nitroso-N-tert-butylglycine (NTBG) omitted during the nitrosamide test; (c) control without nitrite treatment, but spiked with 1 #g N-nitroso-N-methylurea and I/~g N-nitroso-N-butylurea. The arrows indicate the presence of the methyl ester of NTBG (at 4.8 min) and the butyl ester of NTBG (at 9.3 min).

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Table 1. Analysis of nitrosated foodstuffs for nitrosoureas MNU or BNU* activity (ppb) under Sample no.

Country of origin

Sample type

Chemical conditions

Simulated gastric conditions

Fresh fish and meat Green herring Red perch Minced meat (beef)

Germany Germany Germany

440 100 140

--5.6

4 5 6 7 8 9 10 I1 12 13 14

Processed fish and meat Fried red perch Fried white scale fish chin-huay Broiled mackerel Smoked sardines Fried beef Smoked bacon Smoked bacon Smoked bacon Smoked and fried bacon Smoked and fried bacon Smoked and fried bacon

Germany Thailand Japan Japan Germany Germany Germany Germany Germany Germany Germany

780 270 250 110 950 160 110 370 3150 4840 1790

----10.3 --4.2 --3.5

15 16 17 18 19 20 21 22 23 24 25 26

Dried fish Dried sardelles Dried sardelles Dried sardelles Dried sardelles Dried sardelles Dried fish (unkngwn) Salted and dried See-Chon fish Salted and dried white scale fish Dried turbot Dried halibut Salted and dried cod Dried bonito Hana Katsuo

Japan Tanzania Thailand Thailand Thailand Tanzania Thailand Korea Iceland Iceland Iceland Japan

1060 1240 1460 1650 1620 1520 3020 170 2040 2450 2200 1990

8.3 ---11.0 -11.1 8.4 -ND 6.3 17.5

27 28 29 30 31 32 33 34 35 36

Plant products Soy sauce Tamari Spice cake Dried vegetable Salted cabbage Boiled broccoli Boiled cauliflower Boiled red kidney beans Raw sauerkraut Dried fava beans (Vicia faba) Red wine

Japan India India China Germany Germany USA Germany Germany Italy

15 130 40 170 20 4 -20 ND 35

4.2 --ND 6.4 1.4 3.8 2.6 ---

37 38 39 40 41 42 43 44 45 46 47 48

Miscellaneous Fish sauce Anchovy fish sauce Pickled Ziganid fish Pickled Gcuramy fish Pickled shrimp Minced shrimp Dried shrimps Dried squid Preservedcrab meat Oyster sauce Oyster sauce Dried shark fins

Thailand Thailand Philippines Thailand Thailand Hong Kong Thailand Thailand Thailand Hong Kong Thailand China

100 35 140 (20) 40 (190) 30 (25) 130 (25) 150 70 20 25 40 80

----(ND) - - (ND) - - (ND) 14.6 (ND) -------

1 2 3

= not determined ND = not detectable (< 1 ppb) *BNU levels are given in brackets for those samples in which BNU activity was detected. Nitrosation was carried out for 1 hr at 37°C under chemical conditions (100 mM-nitrite, pH 1) or simulated gastric conditions (0.072 mM-nitrite, pH 2). -

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nitrosamides. The nitrosation was performed both u n d e r c h e m i c a l c o n d i t i o n s w i t h a large excess o f nitrite ( 1 0 0 m M ) at p H 1, a n d in s i m u l a t e d gastric juice w i t h 5 p p m (0.072 mM) nitrite at p H 2. A t p H 1, the M N U yield w a s m a x i m a l after 1 h r o f i n c u b a t i o n , w h e r e a s at l o n g e r i n c u b a t i o n times the c o n c u r r e n t acidic d e c o m p o s i t i o n o f the n i t r o s a m i d e s ( S n y d e r a n d Stock, 1980) b e c a m e significant. L o w e r yields, inc r e a s i n g o v e r a n i n c u b a t i o n p e r i o d o f 4 hr, were o b t a i n e d at p H 2 a n d 3 ( n o t s h o w n ) . A f t e r n i t r o s a t i o n w i t h h i g h nitrite c o n c e n t r a t i o n s , alkali-labile m e t h y l a t i n g activity w a s o b s e r v e d in

nearly all f o o d s a m p l e s , w i t h M N U e q u i v a l e n t s s p a n n i n g several o r d e r s o f m a g n i t u d e ( T a b l e 1). L o w M N U activity w a s f o u n d in fresh fish a n d m e a t , a n d in p l a n t - b a s e d f o o d items. I n c o n t r a s t , except f o r a scale fish ( s a m p l e 22), M N U yields f r o m dried fish were generally high. T h e frying o f m e a t , fish a n d b a c o n resulted in a d r a m a t i c increase o f M N U yield after n i t r o s a t i o n , as is s h o w n b y c o m p a r i s o n o f s a m p l e s 2, 3 a n d 10 w i t h s a m p l e s 4, 8 a n d 13, respectively ( T a b l e 1). T h e m e t h y l ester o f N T B G w a s the p r e v a l e n t p r o d u c t in all positive s a m p l e s , indicating the

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Fig. 2. Gas chromatograms of a salted and dried fish (sample 21 in Table 1), nitrosated under simulated gastric conditions: (a) nitrosated sample, N-nitroso-N-tert-butylglycine (NTBG) added during nitrosamide test; (b) nitrosated control, NTBG omitted during nitrosamide test; (c) control without nitrite treatment, but spiked with 0.I #g N-nitroso-N-methylurea. The methylester of NTBG is detected at a retention time of 4.8 min. formation of MNU. Significant butylating activity corresponding to BNU was also found in two pickled fish (samples 39 and 40), and two shrimp products (samples 41 and 42). In some samples (mustard, smoked Japanese Sanma fish and yoghurt) not included in Table l, the NTBG esters could not be analysed because of too many interfering peaks. From mustard, no spiked M N U could be recovered, perhaps because of rapid decomposition. Selected food samples were additionally nitrosated in simulated gastric juice with a low nitrite concentration (5 ppm). Under these conditions, the M N U activity of most samples ranged from 3 to 11 ppb (equivalent to 6-25 nmol MNU/litre in the assay). Only two samples (dried bonito fish from Japan and minced shrimp from Hong Kong) exceeded this value. Unlike the nitrosation experiments performed under chemical conditions, no other alkylnitrosourea activity was found. Typical chromatograms of the

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Fig. 3. Dependence of the formation of N-nitroso-Nmethylurea (MNU) activity on the nitrite concentration. Dried fish (sample 21 in Table 1) was nitrosated for 1 hr at pH 2 either in water (©) or in simulated gastric juice (O) with the indicated initial nitrite concentrations. In the case of simulated gastric juice, nitrite (one fifth of the initial amount) was supplemented every 10 min.

nitrosated sample, nitrosated control and untreated control spiked with M N U are shown in Fig. 2. The additional peak observed at 5.7 min is derived from an impurity of the scavenger reagent that diminishes by repeated recrystallization of NTBG from etber/hexane. To obtain a correlation between the nitrosation experiments performed under chemical and under simulated gastric conditions, the dependence of M N U formation on the nitrite concentration was measured (Fig. 3). Under chemical conditions, M N U activity increased logarithmically with increasing nitrite concentration. At the same nitrite concentration, the M N U activity was about one order of magnitude greater when the nitrosation was carried out in water than when it took place in simulated gastric juice.

DISCUSSION In previous studies, the nitrosation of food samples was carried out under conditions using high nitrite concentrations and long incubation times. Nitrosoureas were detected indirectly by identification of their denitrosation products (Mirvish et al., 1980 and 1982) or by measuring their alkylating activity (Groenen and Busink, 1988), but these methods had low sensitivity and specifity. The method presented in the current study also determines the direct alkylating activity, but with exclusive specifity for alkali-labile compounds. Direct conclusions on the structure of the nitrosamides cannot be drawn, because only the part of the molecule responsible for the alkylating activity is attached to the scavenger reagent. In addition, nitrosamides with polar groups are not detected because their NTBG-derivatives are retained in the test column by the polar silica-gel layer. Besides these limitations, the procedure is easy to perform and the alkylation of the scavenger is not subject to interference by other components of the sample. Although only few grams of the sample are required

Nitrosamides in foodstuffs for analysis, a lower determination limit of 5 ng M N U per sample is achieved. In the first part of our investigation, we nitrosated a wide range of foodstuffs for screening experiments under drastic (chemical) conditions with high nitrite concentrations. Nitrosated processed fish products have been associated with an elevated risk of gastric cancer (Haenszel and Correa, 1975). Strong alkylating activity related to the formation of nitrosamides was found in processed fish products (Yano, 1981). As is shown by the results in Table 1, the M N U activity of dried and salted fish products was about seven times higher than in fresh foods, but it must be taken into consideration that these calculations are done on a dry-weight basis. In contrast, frying of fish, meat and bacon seems to provide a source for nitrosourea precursors. The weight reduction during the heating process is responsible only in part for the increased M N U activity, since weights of fresh and fried foodstuffs differed by a factor of 2-4, whereas M N U activity increased up to 44-fold. The formation of the M N U precursor creatinine has been identified to be responsible for this effect (Mirvish et al., 1982). Fresh meat and fish contain very little creatinine, but it is formed in large amounts during heating, especially under dehydrating conditions. For example, analysis of raw lyacon yielded 0.1 g creatinine/kg, whereas fried bacon contained 3 g/kg (Mirvish et al., 1982). These findings are consistent with the increased M N U content of fried bacon samples found in our investigation (Table 1). In a previous investigation (Groenen and Busink, 1988), certain dairy products, raw sauerkraut and red wine exhibited strong alkylating alkali-labile activity in the 4-(4-nitrobenzyl)pyridine test after nitrosation under quasi-gastric conditions. As can be seen from Table 1, this activity is not due to the formation of simple alkylnitrosoureas, since the M N U equivalents in raw sauerkraut and red wine were extremely low even after nitrosation with excessive nitrite. The same is true for fava beans, which have been speculated to form a nitrosourea; in this case, the source of the alkylating activity was identified as a stable cthydroxy nitroso compound (Yang et al., 1984). In the present study, the levels of M N U activity in plant products were low in comparison with those in other foodstuffs, as one expects from the lack of creatinine in plants. However, Kumar et al. (1990) reported > 1 0 0 0 p p b M N U in a nitrosated dried Brassica vegetable from India. Therefore, precursors other than creatinine must be responsible for the M N U formation in nitrosated plant products. Work is under progress to identify these compounds. The occurrence of BNU activity in nitrosated foodstuffs has not been reported previously, although similar precursor alkylureas have been found in foodstuffs after nitrosation-denitrosation (Mirvish, 1982). It is interesting to note that----except for shrimp sauce--BNU activity was found exclusively in pickled food samples. At present, however, there is no information on the precursors of butylating activity. Data from nitrosation experiments performed under drastic (chemical) conditions are of limited value, since the conditions prevailing in the gastric tract are entirely different. Nitrosation under

171

simulated gastric conditions should therefore provide information for a more accurate risk assessment on the endogenous formation of nitrosamides from foodstuffs. Several model systems have been used previously in studies on the nitrosation of foodstuffs (Groenen and Busink, 1988; Waiters et al., 1976; Yano, 1981) and drugs (Gillatt et al., 1985). However, the use of artificial gastric juice at a given pH and nitrite concentration does not take into account the individual differences in gastric juice acidity, ~aH variations during digestion (Waiters et al., 1976), and microbial reduction of food nitrate to nitrite, nor does it take into consideration the possible nitrosation catalysed by the gastric flora. Therefore, the results obtained by such model systems would only reflect one of numerous possible nitrosation conditions prevalent in the gastric tract. As shown in the present study, only traces of M N U activity were detected from foodstuffs nitrosated in simulated gastric juice. This is obviously due to the fact that at low nitrite concentrations the major part of the nitrosating agent is trapped by the simulated gastric juice itself. This is demonstrated by the enormous differences in activities after nitrosation in gastric juice with those after nitrosation in water (Fig. 3): in simulated gastric juice 11 ppb M N U was detected from a fish product, in contrast with 100 ppb formed after nitrosation in water. In both cases 5 ppm nitrite was added initially. However, endogenous nitrosation may contribute to a significant burden of nitrosamides from foodstuffs that are consumed in high amounts or in cases of high gastric nitrite concentrations. Acknowledgement--We gratefully acknowledge Mrs G.

Walter for technical assistance in sample workup and performing nitrosamide tests.

REFERENCES

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nitrosoureas in aqueous solutions. Journal of Organic Chemistry 45, 1990-1994. Waiters C. L., Dyke C. S. and Saxby M. J. (1976) Nitrosation of food amines under stomach conditions. In Environmental N-Nitroso Compounds: Analysis and Formation. Edited by E. A. Walker, P. Bogovski, L. Griciute and W. Davis, IARC Scientific Publication no. 14. pp. 181-193. International Agency for Research on Cancer, Lyon. Yang D., Tannenbaum S. R., Biichi G. and Lee G. C. M. (1984) 4-Chloro-6-methoxyindole is the precursor of a potent mutagen (4-chloro-6-methoxy-2-hydroxy-lnitroso-indolin-3-one oxime) that forms during nitrosation of the fava bean (Vicia faba). Carcinogenesis 5, 1219-1224. Yano K. (1981) Alkylating activity of processed fish products treated with sodium nitrite in simulated gastric juice. Gann 72, 451-454.