Abstract: Heterocyclic Aromatic amines (HCAs) are formed during cooking of meat at temperatures above 150°C. In this study, different cooking methods (microwave, oven, hot plate, pan-frying and barbecuing) and levels (rare, medium, well-done and very well-done) were evaluated for HCAs content in beef and lamb chops. The HCAs were detected in barbecued and pan-fried beef samples. The cooked samples were analyzed for nine HCAs, Including 2-amino-3-methylimidazo [4, 5-f] quinoline (IQ), 2-amino-3-methylimidazo [4, 5-f] quinoxaline (IQx), 2-amino-3,4-dimethylimidazo [4, 5-f] quinoline (MeIQ), 2-amino-3, 8-dimethylimidazo [4, 5-f] quinoxaline (MeIQx), 2-amino-3, 4, 8-trimethylimidazo [4,5-f] quinoxaline (4,8-DiMeIQx), 2-amino-3, 7, 8-trimethylimidazo [4, 5-f] quinoxaline (7, 8-DiMeIQx), 2-amino-1-methyl-6-phenylimidazo [4, 5-b] Pyridine (PhIP), 2-amino-9H-pyrido [2, 3-b] indole (AαC) and 2-amino-3-methyl-9H-pyrido [2, 3-b] indole (MeAαC). Results showed that the HCAs could not be detected in samples cooked with microwave, oven and hot plate to all various doneness degrees. The highest total amount of HCAs was found 9.78 and 3.06 ng g^-1 in beef and lamb chops, respectively. No AαC and MeAαC were detected in any samples analyzed.

INTRODUCTION

Heterocyclic Aromatic amines (HCAs) are chemical compounds that are formed during cooking proteinaceous foods such as meat or fish, at temperatures above 150°C (Knize et al., 1997a). To date, >25 different mutagenic and/or carcinogenic HCAs have been isolated and identified in foods and/or model systems (Alaejos et al., 2008). In comparison to other known food mutagens, HCAs are over 100 fold more mutagenic than aflatoxin B₁ and over 2000 fold more mutagenic than benzo [a] pyrene (Warzecha et al., 2004). The International Agency for Research on Cancer (IARC) regards some of the HCAs as possible human carcinogens (MeIQ, MeIQx and PhIP as class 2B carcinogens) and one of them as a probable human carcinogen (IQ, class 2A) (Skog, 2004).

Concentrations of HCAs depend on meat type, cooking duration, cooking temperatures, cooking equipment and methods, pH, water activity, carbohydrates, free amino acids and creatine (Felton et al., 1997; Pais et al., 1999; Oz and Kaya, 2006; Oz et al., 2007). In addition, it has been determined that heat and mass transfer, lipid, lipid oxidation and antioxidants have some effect on the accumulation of HCAs (Jagerstad et al., 1998). Human exposure to HCAs is influenced not only by the type of food and cooking method but also by portion size and intake frequency (Skog, 2004).

The aim of this study was to investigate the effects of different cooking methods (microwave, oven, hot plate, pan-frying and barbecuing) and levels (rare, medium, well and very well) on the formation of HCAs in beef and lamb chops.

MATERIALS AND METHODS

Chemicals and solvents were of High Performance Liquid Chromatography (HPLC) or analytical grade. Water was from a Milli-Q water purification system (Millipore, Bedford, Massachusetts, USA). All solutions were passed through a 0.45 μm filter (Milex, Massachusetts, USA). HCA standards were purchased from Toronto Research Chemicals (Downsview, Ontario, Canada). Total 4, 7, 8-TriMeIQx was used as the internal standard. The stock standard solutions were prepared according to Oz et al. (2007). Muscles of Longissimus dorsi used for both beef and lamb chops were obtained from a local market. Pre-cooking experiments were done to determine the cooking level of the samples. Based on the results of these experiments, the cooking time of the chops cooked

with different methods was given according to their cooking levels in Table 1. For the microwave experiment, a kitchen type microwave was used (Arcelik, Turkey). Beef and lamb chops were cooked at automatically selected temperatures for beef and lamb, respectively. For the oven experiment, again a kitchen type oven was used (Arcelik, Turkey). For grilling, hot plate was used. The pan-frying process was carried out with a Teflon-coated pan. Before cooking in oven, hot plate and pan-frying, cooking surfaces were preheated up to 200°C and then the chops were cooked. Temperatures were measured by using a digital thermocouple (part no. 0560 9260, Testo 926, Lenzkirch, Germany) with surface probe (0603 1992, Testo 926, Lenzkirch, Germany). For the charcoal barbecue, a bed of charcoal was prepared and ignited. When all flames had subsided, the bed was leveled by raking. No salt, spice, food additive and frying fat or oil were used in cooking procedures. All meat samples were turned over once a minute during the cooking time. After the cooking, the cooked samples were cooled at room temperature. The chops were homogenized using a kitchen blender to produce a uniform sample for analyses and frozen at -18°C until analyzed for heterocyclic aromatic amines. They were thawed in a refrigerator at 4°C for 12-24 h prior to use. The raw samples were analyzed for moisture, total lipids and pH according to Gokalp et al. (1999). HCAs, however were only tested in cooked samples.

Extraction of HCAs: HCA content was determined by the method described by the method described by Gross and Gruter (1992) with some modifications. One gram sample of cooked meat was dissolved in 12 mL of 1 M NaOH. The suspension was homogenized by magnetic stirring for 1 h at 500 rpm. The alkaline solution was mixed with 13 g diatomaceous earth and then poured into empty LRC-PRS cartridges. The extractions were made with ethyl acetate and the eluate was passed through coupled diatomaceous earth extraction cartridges.

Table 1:	Cooking time for the beef and lamb chops in different cooking methods (min)

Non-polar HCAs were eluted with 6 mL of 0.01 M HCl, 15 mL MeOH: 0.1 M HCl (60:40) and 2 mL pure water.

The eluate in beaker was kept in refrigerator. For the polar HCAs, C₁₈ cartridges (100 mg) were coupled with the system and the polar HCAs were eluted 20 mL ammonium acetate and then 2 mL water. The cartridges were dried under nitrogen and then 800 μL of MeOH: NH₃ (9:1) was added and transferred to vials. To continue the non-polar HCAs, 500 μL 32% NH₃ and 25 mL pure water were added to the beaker removed from refrigerator. C₁₈ cartridges (500 mg) were coupled with the system and were cleaned with 5 mL MeOH and pure water. Then, the cartridges were dried under nitrogen and then 800 μL of MeOH: NH₃ (9:1) was added and transferred to vials. After polar and non-polar HCAs extraction, 100 μL MeOH was added to vials and vials were stored at -20°C until running.

HPLC analysis: The samples were analyzed on an Agilent 1100 HPLC with UV-DAD detector (Agilent, Waldbronn, Germany).

For the analysis of HCAs, a reversed-phase material (Semi Micro ODS-80 TS column, 5 μm, 250 x 2 mm i.d.) from Tosoh Bioscience GmbH (Stuttgart, Germany) was used with the following HPLC conditions: solvent A was methanol/acetonitrile/water/acetic acid (8/14/76/2, v/v/v/v) at pH 5.0 (adjusted with 25% ammonium hydroxide) and solvent B was acetonitrile. A linear gradient (0% B, 0-12 min; 0-30% B, 12-20 min; 30% B, 20-25 min) was used. Flow rate of the mobile phases was 0.3 mL min^-1 and the injection volume was 3 μL. Each injection volume contained 0.5 μL of internal standard. Extraction recoveries were determined by spiking the samples.

Statistical analysis: In the present study, a completely randomized design has been employed (two replicates).

RESULTS AND DISCUSSION

Table 2 shows the percentage of the average moisture, fat and pH values of raw meats. It was determined by Oz (2006) that the moisture content, fat% and pH values of the muscle of L. dorsi varied between 74.69-75.51, 1.26-2.48 and 5.57-5.61%, respectively.

The average recoveries of the HCAs varied between 26 and 75% for the PRS method. In the studies using PRS extraction method, Felton et al. (1994) and Knize et al. (1994, 1995) found that the average recoveries were 26-80, 46-71 and 30-68%, respectively.

Table 2:	The moisture, fat percentage and pH values of raw meat samples

Table 3:	HCAs in beef chops (ng g-1)
NDa: Not Detected

Table 4:	HCAs in lamb chops (ng g-1)
NDa: Not Detected

LOD (Limit Of Detection = 3) and LOQ (Limit Of Quantification = 10) values for the present study have been detected as 0.003 and 0.01 ng g^-1 according to Signal/Noise, respectively.

The HCAs content of beef chops: HCA amounts of the samples, extracted according to the PRS method are shown in Table 3. HCAs were not detected in chops microwave-cooked, oven and hot plate cooked for no cooking levels and in chops pan-fried for only rare and medium cooked levels. However, the total HCA amount increased with increasing degree of doneness. Total HCA amounts were detected as 0.92 and 1.64 ng g^-1 for well and very well-done pan-fried samples, respectively. On the other hand, the total HCA amount of barbecued samples increased until to medium cooking and then gradually decreased. The highest total HCAs amount was detected as 9.78 ng g^-1 in medium barbecued chops (Table 3).

Gross et al. (1989) measured that highest total HCA (IQ, 4,8-DiMeIQx, MeIQx and PhIP) amount in beef fried for 10 min at 250°C was 2.6 ng g^-1. Total HCA (MeIQx, PhIP and 4,8-DiMeIQx) amount was found as 29.9 and 58.8 ng g^-1 in pan-fried beef steak at 190°C for 6 and 13 min, respectively (Gross, 1990). Murkovic and Pfannhauser (2000) reported that amount of MeIQx, IQ, 4, 8-DiMeIQx and PhIP increased with cooking temperature and time. Pais et al. (2000) reported total HCA values (MeIQx, DiMeIQx and PhIP) in well-done beef chops to be 3.0 and 2.8 ng g^-1 in pork chops. Their values ranged from 9.77-185 ng g^-1 in chops depending on the restaurants that the samples were taken from and the types of muscle that the chops were taken from. Skog et al. (1997) determined that total HCA content was <1 and 2 ng g^-1 in various meat samples cooked at 150 and 175°C, respectively.

Murkovic et al. (1998) found a total amount of 33.4 ng g^-1 for HCAs (10.2 ng g^-1 IQ, 2.46 ng g^-1 MeIQ, 13.2 ng g^-1 MeIQx, 2.26 ng g^-1 4,8-DiMeIQx and 5.28 ng g^-1 PhIP) in beef fried at up to 180°C for 20 min. Felton et al. (1994) reported that HCA content of beef fried 200-250°C for 12 min varied between none and 1 ng g^-1 for IQ, none and 5.1 ng g^-1 for MeIQx, 0.1 and 1.2 ng g^-1 for 4, 8-DiMeIQx and 0.7 and 13.3 ng g^-1 for PhIP. Johansson and Jagerstad (1994) could not detect IQ, MeIQ and PhIP in beef barbecued for 10 min but 1 ng g^-1 MeIQx and 0.2 ng g^-1 4, 8-DiMeIQx were found in the same samples. In another barbecuing study, MeIQx and PhIP content varied between 0.2 and 1.8 ng g^-1 and between 1.8 and 18.4 ng g^-1, respectively (Knize et al., 1998).

However, IQ, MeIQ and 4, 8-DiMeIQx content were found below the detectable levels by the same researches. Although no IQ and MeIQ were detected in beef fried at 175-200°C for 11.2 min, 0.7 ng g^-1 MeIQx and 0.6 ng g^-1 PhIP were found (Busquets et al., 2004).

The HCAs content of lamb chops: Table 4 shows the HCA content of lamb chops. HCAs could not be detected in lamb chops cooked in microwave, oven and hot plate for all cooking levels like in beef samples. No HCAs could be detected in pan-fried lamb at rare, medium and well-done levels. However, the total HCA amount of 0.62 ng g^-1, all of which was IQ was found in very well-done pan-fried lamb samples (Table 4). The total HCA content of barbecued lamb chops for rare, medium, well-done and very well-done was 1.78, 2.99, 1.38 and 3.06 ng g^-1, respectively (Table 4). It was determined that the highest total HCA amounts in lamb chops belonged to very well-done barbecued samples. Although, IQ and MeIQ could not be detected in lamb grilled at 175-200°C for 11 min, 1.3 ng g^-1 MeIQx, 1.8 ng g^-1 4, 8-DiMeIQx and 5.8 ng g^-1 PhIP were found by Busquets et al. (2004).

Skog et al. (1997) determined that the maximum content of MeIQx, 4, 8-DiMeIQx and PhIP of lamb meat fried at 150-225°C for 9 min was 0.4, 0.6 and 1.5 ng g^-1, respectively and could not detect IQ and MeIQ in any lamb samples. Wakabayashi et al. (1993) could not detect IQ and MeIQ in grilled mutton, too. However, they found 1.01 ng g^-1 MeIQx, 0.67 ng g^-1 4, 8 DiMeIQx and 42.5 ng g^-1 PhIP. Knize et al. (1997b) detected 1.6 ng g^-1 MeIQx and 11 ng g^-1 PhIP in grilled lamb.

CONCLUSION

In conclusion, pan-frying and barbecuing of samples are more effective than the other cooking methods in terms of the formation of HCAs. In the present study, no AαC and MeAαC have been detected in the samples.

ACKNOWLEDGEMENTS

This study has been supported by The Scientific and Technological Research Council of Turkey (TUBITAK)-Agriculture, Forestry and Veterinary Research Grant Committee (TOVAG-105O165).