Analysis of mutagenic heterocyclic amines in cooked beef products by high-performance liquid chromatography in combination with mass spectrometry

Fd Chem. Toxic. Vol. 26, No. 6, pp. 501-509, 1988 Printed in Great Britain. All rights reserved

0278-6915/88 $3.00+0.00 Copyright © 1988 Pergamon Press plc

ANALYSIS OF MUTAGENIC HETEROCYCLIC AMINES IN COOKED BEEF PRODUCTS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY IN COMBINATION WITH MASS SPECTROMETRY R. J. TURESKY, H. BuR, T. HUYNH-B^, H. U. AESCHaACHr~ and H. MxtoN Nestl~ Research Centre, Nestec Ltd, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland (Received 23 September 1987; revisions received 9 March 1988)

Abstraet--Mutagenic activity detected in beef extracts and in fried beef heated for varying periods of time was purified and then analysed by high-performance liquid chromatography in combination with mass spectrometry (LC-MS). The major mutagenic component found in all of the beef products was identified as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQ~) followed by lesser amounts of 2-amino-3methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQ~). Identification and quantification of mutagens were achieved by the use of deuterium-labelled analogues. Measured levels of MeIQ~ and 4,8-DiMeIQ~ in different batches of beef extract were in the range I 1.7-52.2 and 0-11.2 ng/g, respectively, and in beef heated at 275°C for 5-15rain the values of MeIQx and 4,8-DiMeIQx were in the range 2.7-12.3 and 0-3.9 rig/g, respectively. The levels of IQ found in beef extracts were 0-36.8 ng/g and in fried beef the amounts were estimated at 0.3-1,9 ng/g. The method of purification is rapid, requiring only XAD-2 adsorption followed by an acid-base liquid partition against ethyl acetate and blue cotton treatmenl (trisulpho-copper-phthalocyanine) prior to LC-MS analysis. Because of the sensitivity of LC-MS, mutagens present in cooked beef can be detected at the low partsper-billion-level and as little as l0 g of cooked beef was required for analysis.

INTRODUCTION It is well established that mutagenic and potentially carcinogenic compounds are formed in foods such as beef and fish during typical cooking procedures. The heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-
Abbreviations:

cyclic amines have been formed in model systems by heating creatinine with sugars and amino acids (Grivas et al. 1985; J:~gerstad et al. 1984). All of the amines that have been tested to date are multipotential carcinogens to rodents when given as part of their diet (Ohgaki et al. 1986 & 1987; Sugimura, 1985). In order to fully assess the risk to human health posed by the daily consumption of foods containing these mutagens/carcinogens it is essential to quantify the amount of carcinogen to which man is chronically exposed. Early techniques used to identify and quantify IQ, MeIQ and MeIQ~ included high-performance liquid chromatography (HPLC) with a UV photodiode array detector (Turesky et al. 1983), gas chromatography-mass spectrometry (Nishimura, 1986) and mass spectrometry with a direct inlet probe (Felton et al. 1984; Hargraves & Pariza, 1983; Kasai et al. 1980). These methods of identification and quantification were problematic because they required large amounts of starting material, laborious purification procedures, and the estimation of the heterocyclic amine content was based on highly purified fractions containing minor percentages of the initial mutagenic activity. A more sensitive and selective method of analysis used to detect these mutagens is liquid chromatography in combination with electrochemical detection (Takahashi et al. 1985a,b). Because of the specificity of electrochemical detection towards these amines, the investigators were able to simplify the isolation scheme with a high recovery of mutagenic components and thus improved the accuracy of 501

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502

quantification. Most recently, liquid chromatography in combination with mass spectrometry (LC-MS) has been used to detect IQ and MelQ formed in fried fish at levels approaching I ppb (Edmonds et al. 1986; Yamaizumi et al. 1986). The application of LC-MS has also been used for the detection and quantification of the heterocyclic aromatic amines 3-amino- 1,4-dimethyl-5H-pyrido[4,3-b]indole (TrpP-I) and 3-amino-l-methyl-SH-pyrido[4,3-b]indole (Trp-P-2) formed in heated tryptophan (Milon et al. 1987). In this report we present our results for the quantification of IQ, MelQ~ and DiMelQx formed in heated beef products by means of LC-MS using stable isotopically-labelled internal standards. The isolation scheme used prior to LC-MS is rapid and should be applicable to the analysis of other fused tricyclic amines which may be formed in protein-rich heated foods. MATERIALSAND METHODS Chemicals. IQ, MelQ~ and 2-amino-3trideuteriomethyl-8-methylimidazo[4,5-f]quinoxaline ([2H3]MelQx) were purchased from Toronto Research Chemicals, Ontario, Canada. 2-amino-3trideuteriomethylimidazo[4,5-f]quinoline ([2H3]IQ) was prepared as previously described (Kasai et aL 1980a) by reacting iodomethane-d 3 (Aldrich Chemicals, Steinheim, FRG) with 2-aminoimidazo[4,5-f]quinoline, a gift supplied by Dr S. R. Tannenbaum (Department of Applied Biological Sciences, MIT, Cambridge, MA, USA), 4,8DiMelQ~, 2-amino-3-trideuteriomethyl-4,8-dimethylimidazo-[4,5-f]quinoxaline ([2H3]4,8-DiMelQ~), 7,8DiMeiQx and 2-amino-3-trideuteriomethyl-7,8-di methylimidazo[4,5-f]quinoxaline([2H3] 7,8-DiMelQx) were synthesized via reaction pathways which differed from those already described (Grivas, 1985; Lovelette et al. 1987; Olsson & Grivas, 1986). The main step was a conveniently high yielding N-methylation, and in particular N-trideuteriomethylation, avoiding the excess use of costly labelled reagents. The 4,8-DiMelQx analogues were prepared from the precursor 5-chloro-2-methyl-4-nitroaniline (Aldrich Chemicals). A chlorine exchange with ammonia (85% yield) followed by reduction of the nitro group and subsequent condensation with methylglyoxal (36%), N-acetylation (87%), N-methylation or N-trideuteriomethylation (97%) with amide hydrolysis (96%) afforded a mixture of 2,7- and 3,7-dimethylquinoxalin-2-amines in a ratio of 60:40. Nitration of the above key intermediate with subsequent isomer separation, followed by nitro reduction and cyanogen treatment (Grivas, 1985) were performed to obtain 4,8-DiMelQ~ and its trideuteriolabelled analogue. The similar key compounds required for the synthesis of the 7,8-DiMelQ~ analogues, namely 2,3-dimethylquinoxalin-6-methylamine and 2,3-dimethylquinoxalin-6-trideuteriomethylamine, were obtained from 4-nitro1,2-phenylenediamine after condensation with butane-2,3-dione (96%) followed by nitro reduction with subsequent N-acetylation (91%), N-methylation or N-trideuteriomethylation (85%) and amide hydrolysis (96%). Complete experimental details for

the synthesis of 4,8-DiMeIQx and 7,8-DiMeIQx will be forwarded on request. Radiolabelled standards 2[14C]IQ (11 mCi/mmol) and 2-[t4C]MelQ~ (50 mCi/ mmol) were purchased from Toronto Research Chemicals. Bacterial-grade beef extract was purchased from Difco Laboratories, Detroit, MI, USA, and food-grade beef extracts were purchased from several food suppliers. Blue cotton was purchased from Funakoshi Pharmaceutical Co., Tokyo, Japan and XAD-2 resin (0.3-1.0-mm particle diameter) was purchased from Serva, Heidelberg, FRG. Dimilume30 scintillation fluid was purchased from Packard Instruments Co., Zurich. Isolation o f heteroeyclic amines. The extraction scheme is summarized in Fig. 1. Ten grams of beef extract were dissolved in 20 ml water and added to 60 ml stirring methanol. The mixture was then centrifuged at 10,000 g for 10 min to remove precipitated protein, The protein precipitate was dissolved in 20 ml water and then added to another 60 ml stirring methanol. The protein was once again removed by centrifugation. The supernatants, containing the mutagens, were pooled and concentrated by rotary evaporation at 37°C to a final volume of approximately 20 ml. The pH of the solution was adjusted to pH 8.5 with 1 N-NaOH and then the mutagens were adsorbed on 7 g XAD-2 resin which had been prewashed in sequence with acetone, methanol, water and packed into a glass column (20 c m x 1 cm ID, Felton et al. (1984). The top of the column was packed with a small plug of glass wool to stabilize the resin. The beef solution was passed through the column at a flow rate of 2 ml/min and the resin was then washed with 70ml distilled water. The mutagenic heterocyclic amines were eluted from the resin by successive washes of 70 ml acetone followed by 70ml methanol. These two organic washes were pooled and rotary evaporated to dryness. The extract was then dissolved in 25 ml water and acidified to pH 2.0 with dilute hydrochloric acid. Non-mutagenic neutral and acidic material was removed by extraction with ethyl acetate (three times with 20 ml). The pH of the aqueous phase was adjusted to pH 12 with concentrated NaOH and the heterocyclic amines were extracted into ethyl acetate (three times with ethyl acetate-water, 2.5:1, by vol.). The extract was dried over sodium sulphate and then rotary evaporated to dryness. Final purification was achieved by adsorbing the heterocyclic amines onto blue cotton (Hayatsu et al. 1983) with modifications. The residue was dissolved in 10 ml distilled water and poured into a glass column (10 cm x I cm ID) which contained 0.5 g packed blue cotton. The solution was passed through the column at a flow rate of 2 ml/min and then the blue cotton was washed with 25 ml distilled water. The mutagens were desorbed from the blue cotton by washing the column with 50ml methanol-ammonium hydroxide (50: 1, by vol.). The eluant was evaporated to dryness and resuspended in 0.5ml of methanol-water (1:1, by vol.). Samples were then analysed by LC-MS. Preparation o f fried beef. Fresh chopped beef was purchased at a local supermarket and 100-g batches were moulded into patties (8-10cm in diameter, 1.5-1.8 cm in thickness). The patties were heated for 5, 10 or 15 min on a commercially available two-sided

LC-MS analysis of heterocyclic amines

503

Cooked beef

I_

Deuterium- LabeLLed internal standards IO, MeZOx and DiMeIOx

Methanol extraction

X A D - 2 adsorption

Acid-base partition against ethyL acetate

CeLLuLose trisuLpho - copper- phthaLocyanine adsorption (bLue c o t t o n )

LC-MS

Fig. 1. Purification scheme for heterocyclic amines found in cooked beef products.

fryer which contained a heating element on both sides. The initial temperature of the heating surface was 275°C. The cooked patties were finely chopped and then homogenized in 250 ml distilled water, using a Waring blender. Thirty gram equivalents of starting material were used for analysis. The protein was removed by precipitation in 200 ml methanol followed by centrifugation as previously described. The protein pellet was re-homogenized in 75 ml water and precipitated a second time in 200 ml methanol. The heterocyclic amines were isolated as described for the beef extract samples. Recovery experiments. The quantification of heterocyclic amines was achieved by means of measuring the recovery of deuterium-labelled internal standards in the purified extracts by LC-MS. Deuteriumlabelled IQ and MeIQ~ were added to Difco beef extract, which had been dissolved in water, at a concentration of 50 ng/g and both DiMeIQ~ derivatives were added at 10 ng/g. These values were chosen as they are the concentrations that have been estimated by electrochemical detection to be present in Difco beef extract (Takahashi et al. 1985a,b). All deuterium-labelled internal standards were added to food-grade beef extract at a concentration of 10 ng/g. Deuterium-labelled standards were added to fried beef during the homogenization of the meat samples at a concentration of 5 ng/g. The efficiency of each purification step was also measured by spiking meat samples with radioactive IQ and MeIQx at the appropriate concentrations. Radioactivity was quantified by liquid scintillation counting using an LKB 1217 counter. Mutation assays. The Ames reversion assay was

used to monitor mutagenic activity, by the standard plate test with Salmonella typhimurium strain TA98 (Maron & Ames, 1983). Liver S-9 fraction obtained from male Sprague-Dawley rats induced with Aroclor 1254 was used for metabolic activation of meat extracts at a concentration of 10% S-9 mix which was equivalent to 1.Stag S-9 protein/plate. The beef extracts and fried beef were quantified for activity after partial purification by XAD-2 resin treatment. Linear regression analysis was performed using the least-squares method over the non-toxic portion of the dose-response curve. Each assay was performed in duplicate using five data points with three plates per point. The spontaneous revertant rate was 45-60 colonies/plate. Chromatography. The HPLC system consisted of a Kontron 600 pump and a Waters Wisp 710B injector. Chromatography was performed under two conditions. The first analysis was performed with a Supelco LC-CN column (25 cm x 3.6 ram ID, packed with 5-#m particles). The mobile phase was a mixture containing 13% acetonitrile-methanol (4: l, by vol.) and 87% 0.1 M-ammonium acetate. The pH was adjusted to 6.8 with triethylamine to optimize peak shape and resolution and the flow rate was 1.4 ml/min. The retention times for MelQ~, 7,8-DiMeIQ~, 4,8-DiMeIQ~ and IQ were, respectively, 10.8, 12.3, 13.9 and 16.1 rain. The second analysis was performed with a Supelco LC-18-DB column (25 c m x 4.6 mm ID, packed with 5-#m partides). Separation of the amines was achieved by using a linear gradient from 13% acetonitrile-methanol (4: l, by vol.) and 87% 0.1 M-ammonium acetate pH 6.8 to 30% acetonitrile-methanol (4: l, by

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Table I. Recoveryof radioactivemutagens[t4C]lQand [14C]MelQxon partialpurificationfromcookedbeef products Amount of % Recovery* of mutagen Purification Beef product added (ng/g) stage ['4C]IQ [I*C]MelQ, Bacterial-grade 50 XAD-2 81.2(84.8,77.5) 77.5(82.3,72.7) beef extract Acid-base partition 75.8(78.9,72.7) 69.3 (73.1,65.5) Blue cotton 67.8(72.7,62.9) 65.4(69.1,61.7) Food-grade 15 XAD-2 76.3(77.3,75.2) 78.5(78.8,78.2) beef extract Acid-base partition 75.3(76.0,74.5) 66.7(68.2,65.1) Blue cotton 66.0(67.6,64.4) 61.6(62.2,60.9) Food-grade 5 XAD-2 74.9(74.6,69.4) 64.8(67.3,62.3) beef extract Acid-base partition 71.1(74.1,68.0) 63.5(66.9,60.1) Blue cotton 65.8 (70.4,61.2) 55.2(58.1,52.3) Fried ground beeff 5 XAD-2 81.5(81.9,81.0) 72.1 (78.8,65.4) Acid-base partition 75.1 (77.6,72.6) 64.7 (68.8,60.5) Blue cotton 74.1 (76.1,72.1) 63.8 (67.6,60.0) *The values are the mean of two independentexperiments(individualresults given in parentheses). ~'Recoveryof radioactivitywas determinedin fried beef patties which were cooked for 15rain at 275°C.

vol.) and 70% 0.1 M-ammonium acetate pH 6.8 over 15 rain at a flow rate of 1.5 ml/min. The retention times for IQ, MelQx, 7,8-DiMelQx and 4,8-DiMelQ~ were, respectively, 10.5, 11.6, 14.3 and 15.8min. M a s s spectrometry. A Nermag R10-10C quadrupole system was coupled to the HPLC system via a thermospray interface (Vestee, USA). The temperature of the ion source was 265°C and the temperature of the thermospray tip was 202°C. The MS was operated in the positive ionization mode with the filament off at an ion pressure of 0.25 Torr. The ions monitored for each chemical were those corresponding to the protonated molecular ions for each mutagen and deuterium-labelled internal standards: IQ, m/z 199; [2H3]IQ, m/z 202; MelQ~, m/z 214; [2H3]MelQ~, m/z 217; DiMelQx m/z 228 and [2H3]DiMelQ~, m/z 231. RESULTS The isolation scheme, shown in Fig. 1, provided sufficient purification of the mutagens from cooked beef products to enable their detection and quantification by LC-MS. The method of isolation is

rapid, requiring only a protein precipitation step followed by XAD-2 adsorption, an acid/base liquid partition against ethyl acetate and blue cotton treatment. The use of ~4C-labelled IQ or MeIQx revealed that the recovery of radioactivity from each purification step was approximately 80-90% and the percentage recovery was independent of the amount of mutagen added to the meat product (Table 1). Analysis by LC-MS of the deuterium-labelled internal standards in the purified extracts revealed similar results. The DiMeIQ~ isomers, in addition to IQ and MeIQx, were recovered from beef extracts and fried beef at values which were consistently between 40-70%, The thermospray mass spectrum of McIQ, is shown in Fig. 2. The thermospray LC-MS ionization process produces abundant pseudo-molecular ions for these chemicals and mass-spectra are observed in which the base peaks are detected at (M + H) +. These heterocyclic amines are compounds which are stable towards the ionization process and do not undergo notable fragmentation. Thus, single-ion monitoring of the (M + H) + ion is quite suitable for the analyses of these amines in complex mixtures. Because the

214

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Fig. 2. Thermospray mass spectrum of MeIQ~ and its trideuterio-labelled analogue.

LC-MS analysis of heterocyclic amines deuterium-labelled standards are three atomic mass units greater than the respective mutagens, they can serve as both internal chromatographic markers and as measures of percentage recovery for the quantification of the mutagens. Linear responses were obtained for calibration curves over the concentration range corresponding to 0.5-25ng mutagen/5 ng deuterium-labelled internal standard (Yamaizumi et al. 1986; R. J. Turesky et al. unpublished data, 1987) and thus substantiates the use of stable isotopes as a method of quantification. An LC-MS chromatogram of purified bacterialgrade beef extract analysed without isotopicallylabelled internal standards is shown in Fig. 3a. Prominent peaks are detected at m/z values 199 and 214 which correspond to the protonated molecular ions

lO0r"

of IQ and MeIQ~. A smaller but detectable peak is also observed at m/z 228, corresponding to 4,8-DiMeIQ~. The deuterium-labelled channels were free from interferences demonstrating that accurate measures of recovery of isotopically-labelled standards can be determined. Fig. 3b shows the corresponding LC-MS chromatogram of bacterial-grade beef extract in which deuterium-labelled standards had been added to the beef extract prior to fractionation. All of the internal standards are readily detectable in the chromatogram. Table 2 summarizes the estimates of the quantities of heterocyclic amines found in cooked beef products along with the mutagenic activities observed after partial purification by elution through an XAD-2 column. Under our test conditions the specific mu-

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Fig. 3. LC-MS analysis of purified bacterial-grade beef extract (a) without or (b) with isotopically-labelled internal standards. Profiles are for MH + ions with arrows marking the expected elution positions for IQ, [~H3]IQ, MeIQ~, [~-I3]MeIQx,DiMeIQx and [2H3]DiMeIQ~ isomers. [2H~]IQ and [2H3]MeIQxwere added to beef extract at levels of 50 ng/g, and [2H3]DiMeIQ~ isomers were added at 10 ng/g. Analysis was performed with a C-18 reverse-phase column as described in the Materials and Methods section.

506

R. J. TURF.SKY et al. Table 2. Estimates of mutagens formed in cooked beef products Mutagen content

Beef product Bacterial-grade beef extract Food-grade beef extract I Food-grade beef extract II Fried beef heated for--5 rain --10 min --15 rain

(ng/g)

IQ

MelQ~

4,8-DiMelQ~

36.8 _+ 12.0

52.2 _+ 12.8

11.2 9- 3.6

23,410

5.6 + 0.6

29.2 _+7.2

3.2 _+0.5

9,250

--

11.7 + 3.9

--

0.3 0.3 1.9

2.7 4.2 12.3

--3.9

7,8-DiMelQ~

Mutagenic activity* (no. of revertants/gram equivalent of beef)

3,680 --0.7

750 908 2,320

*Mutagenic activity was determined by linear regression analysis of XAD-2 residues over the non-toxic portion of the dose-response curve using the S, typhimurium TA98 tester strain. Quantification of mutagens was achieved by measuring the percentage recovery of deuterium-labelled internal standards in the purified extracts by LC-MS. Values are means +_SD for four analyses for beef extracts, and for duplicate analyses for fried beef.

tagenic activities for IQ, MeIQx, 4,8-DiMeIQx and 7,8-DiMeIQ~ were measured in the Ames reversion assay as, respectively, 317,000, 77,000, 70,000 and 73,900 revertants/#g when activated by hepatic S-9 obtained from rats pretreated with Aroclor 1254. The mutagenic activity of the beef products correlated well with the estimated beterocyclic amine content. Two batches of food-grade beef extract containing, respectively, very potent and weak mutagenic activity were found to have significant amounts of MeIQx. However, IQ was detectable only in the batch of beef extract that exhibited the high level of mutagenic activity. It should be noted that MeIQx does not account for all of the mutagenic activity observed in food-grade extract II. The absence of IQ or the DiMeIQ~ isomers suggests there are other prominent mutagenic components present in this batch of extract. These heterocyclic amines were readily detected in fried beef prepared under typical cooking procedures. As is shown in the chromatograms of Fig. 4a,b, MeIQ~ was the principal mutagen formed in beef that had been fried for 5 rain. The formation of these amines increased with the heating time and was paralleled with higher levels of mutagenic activity (Table 2). In addition to IQ, MeIQ~ and 4,8-DiMeIQ~, the 7,8-DiMeIQ~ isomer also appears to have been formed in trace quantities in beef heated for 15min at 275°C. Thus, both the chemical and biological data demonstrated that MeIQ~ was responsible for a major proportion of the mutagenic activity found in cooked beef products. DISCUSSION

The application of HPLC in combination with mass spectrometry has permitted the development of a simplified scheme for the isolation of mutagenic components in cooked foods. One can selectively monitor a specific molecular weight ion with LC-MS and thus interferences that are associated with the less specific UV and electrochemical detection systems are eliminated. LC-MS analysis with deuterium-labelled analogues employed for quantification has already been used to estimate the amounts of IQ and MelQ formed in broiled fish (Edmonds et al. 1986; Yamaizumi et al. 1986). In this report, we have extended the use of LC-MS using

deuterium-labelled internal standards as a means of quantifying the mutagens present in cooked beef products. Our pre-fractionation scheme for the mutagenic components is rapid and does not require an HPLC purification step prior to LC-MS analysis as was used in previous analyses (Edmonds et al. 1986; Yamaizumi et al. 1986). It was discovered that partial purification of heterocyclic amines by XAD-2 column elution in combination with blue cotton treatment and a solvent partition sufficiently reduced background ions to a level that permitted accurate estimates of mutagen content. The fractionation scheme is rapid and requires as little as 10 g cooked beef. The amounts of IQ, MeIQx and 4,8-DiMeIQx found in bacterial-grade beef extract are in excellent agreement with those estimates previously reported by means of electrochemical detection (Takahashi et al. 1985a,b). These three heterocyclic amines were reported to account for 76% of the mutagenic activity in Difco beef extracts (Takahashi et al. 1985a,b). Whereas they accounted for approximately 70% of the total mutagenic activity in the analysis reported here. IQ, MeIQx and 4,8-DiMeIQx were also found in food-grade beef extracts in appreciable amounts and were thought to be responsible for significant proportions of the total mutagenicity. The amounts of IQ and MeIQx which investigators have detected in cooked ground beef vary greatly. There are several factors that contribute to these variations and these include non-standardized purification schemes, different cooking temperatures and cooking methods in addition to different meat composition. Felton et aL (1984) reported the formation of MeIQ~ (1.0 #g/kg) and IQ (0.02/~g/kg) in ground beef which had been fried at 250°C for 6 min per side. 4,8-DiMeIQx was also found, but accurate estimates were not made. In another study, MeIQ~ was detected at a concentration of 0.45 #g/kg in ground beef heated at 191°C for 2rain per side (Hargraves & Pariza, 1983). In a third study, the amount of IQ formed in fried beef heated at 240°C for 5 min per side was estimated at 0.53 gg/kg in a 10.6%-fat content sample and 20.1#g/kg in a 27.5%-fat content sample (Barnes et al. 1983). In this study we detected significant levels of mutagenic activity in fried ground beef heated at 275°C. Mutagenic activity increased with the heating time and

LC-MS analysis of heterocyclic amines 100 r

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time(rain)

Fig. 4. L C - M S analysis o f purified beef which had been fried for 5 rain (a) without or (b) with isotopically-labelled internal standards. All deuterium-labelled internal standards were added to the fried beef at a value o f 5 ng/g. Analysis was performed with C-18 reverse-phase column as described in the t e x t

was paralleled by increased levels of heterocyclic amine content (Table 2). Assuming the interaction of activity amongst heterocyclic amines in fried beef partially purified by XAD-2 is additive in nature, the contribution of the sum of the aminoimidazoquinolines and aminoimidazoquinoxalines to the total mutagenicity in beef heated for 5, 10 and 15 rain at 275°C increased with time and was, respectively, 40, 46 and 80% (Table 2). MeIQ., was the principal mutagen detected at each time-point. Comparable levels of mutagenic potency were observed in ground beef that had been heated at 300°C for the same periods of time and MeIQ, was responsible for a

significant proportion of the total mutagenicity (Knize et al. 1985). One notable difference in our study was the formation of 4,8-DiMeIQ~. We did not readily detect 4,8-DiMeIQ~ in fried beef, as judged by LC-MS, until the meat patties had been cooked for 15min at 275°C. In contrast, Knize et al. (1985) reported that 4,8-DiMeIQ~ was the second major mutagenic component, following MeIQ~, in fried beef heated for 6 rain per side at 200, 250 and 300°C. A prominent mutagen which is formed in broiled fish is the methylated homologue of IQ, 2-amino3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) (Kasai et al. 1980; Sugimura & Sato, 1983). Unfortunately a

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sample of synthetic MelQ was not available, but our prefractionation scheme is generally applicable to fused tricyclic heteroaromatic amines such as MeIQ. There was no detection of a peak in the LC-MS chromatograms with a m/z value of 213 (the protonated molecular ion of MeIQ) which suggests that MelQ must be present in cooked beef products below the limits of detection, 0.1 ppb. Recently another complex heterocyclic amine, 2-amino-l-methyl6-phenylimidazo[4,5-b]pyridine(PhIP), has been isolated from beef cooked at high temperatures (Felton et al. 1986b). The mutagenic potency of PhIP is much weaker than those of the aminoimidazoquinolineand aminoimidazoquinoxaline series of chemicals. However, because of its abundance, PhIP may make a large contribution to the total amount of genotoxic substances formed in fried beef. PhIP contains the 2-amino-N-methylimidazo moiety which is common to IQ and MeIQ~, but PhIP is not a fused tricyclic and thus may have a lower affinity for the copperphthalcyanine pigment of blue cotton (Hayatsu et al. 1983). The purification scheme used was specific for fused tricyclics, it is not known whether PhIP or other heterocyclic amines with dissimilar chemical structures to that of IQ and MeIQ~ would be recovered from the blue cotton purification step. It would be of interest to develop a rapid methodology which could be used to accurately quantify the formation of PhlP and other mutagens that are formed in cooked beef prepared by typical cooking practices (Felton et al. 1984 & 1986a). The aminoimidazoquinolines and aminoimidazoquinoxalines are a class of chemical carcinogens which are present in protein-rich heated foods. IQ, MelQ and MelQ~ have been shown to be multipotential carcinogens in at least one in vivo rodent assay system (Ohgaki et al. 1986 & 1987; Sugimura, 1985). Because of the greater abundance of MelQ~ formed in cooked beef (Felton et al. 1984; Hargraves & Pariza, 1983; this study) and fish products (Kato et al. 1986), MelQ~ may be the most significant of all these heterocyclic amines as a potential initiator for carcinogenesis in humans. With the development of LC-MS, accurate methods of measuring the presence of these amines in the diet are available and a better assessment of exposure and human health risk towards these chemicals can be made. Acknowledgement--We wish to thank C. Julmi for conduc-

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