Determination of Heterocyclic Amines in Ready to Eat Chicken Kabab Commercially Available from Pakistani Markets

Three HCAs standards of 2-amino-3-methyl-imidazo [4,5-f] quinoline (IQ), 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) were acquired from Toronto Research Chemicals (Toronto, Canada). Chemicals and solvents such as acetonitrile (HPLC grade), methanol (HPLC grade) ethyl acetate (commercial grade) sodium hydroxide (ACS-grade) diatomaceous earth, millipore filter (25mm, 0.22 micron) and filter paper (47mm, 0.22µm) were purchased from Integrated Biosciences, Karachi-Pakistan.

Commercially available four brands (B, D, S and K) of ready-to-eat (RTE) Chicken Kabab samples were procured from local departmental stores. All samples were divided in two groups. Control group of RTE chicken kabab were processed for extraction after thawing at room temperature. Second group of samples was further cooked by using three high temperature cooking processes recommended by manufacturers of the RTE brands i.e. microwave treatment (70-160ºc) pan-frying (150-200ºc) and deep-frying (190-220ºc). Cooking duration, temperatures and respective quantification of HCAs during various cooking methods are shown in Table I.

For Extraction of HCAs from chicken kabab samples, the most commonly used method (Gross and Grueter, 1992) with little modifications (Messner and Murkovic, 2004) was adapted. Accordingly 1g of homogenized kabab sample was taken and mixed well in 12 ml of 1N NaoH. A homogenous suspension was obtained by magnetic stirring of 1h at 600-700 rpm. Mixture was passed through lab-based diatomaceous earth filled column. Twenty milliliter ethyl acetate (C2H5COOH) was passed through the Extrelut columns. Cartridges were washed with 2 ml 0.1 N HCL followed by 2 ml MeOH. It was followed by elution of resulting analytes with 2ml MeoH-25% ammonium hydroxide (19:1, ml/ml). Elute was collected.

After Extraction, analysis of HCAs was done by using a High Performance Liquid Chromatography (Perkin Elmer, Total Chrom) with UV/Vis. detector. Separation of HCAs on the basis of their polarity was done by a reversed phase Sharpsil-U C18 Column (250L ×4.6mm, 5µm, 100Aº). Mobile phase A was prepared by the addition of methanol /acetonitrile / water /acetic acid (8ml/14ml/76ml/2ml). pH of the solvent A was adjusted at 5.0 with the help of 25% ammonium hydroxide. Acetonitrile (ACN) was used as solvent B. A linear gradient (100%A, 0-15min; 75%A, 15-30 min.) was used. Injection volume of sample was adjusted at 10µl. Flow rate of mobile phases were adjusted at 0.7ml/min at 258nm.

The limit of detection and limit of quantitation of three HCAs standards IQ, MeIQx and PhIP were found in the range of 0.1-10ng, 0.1-100ng and 10-1000ng, respectively.

The present results showed that the level of heterocyclic amines (HCAs) in the thawed (control) ready-to-eat (RTE) chicken kabab of different brands ranged from 0.97 to 13.66 µg/g. Whereas high temperature cooking procedures, in general, enhanced the levels of HCAs so that they ranged from 1.62 to 22.21 µg/g and 0.71 to 35 µg/g for the different samples following deep frying and microwave cooking, respectively (Table I). Quantitative determination of three HCAs revealed that control (thawed) samples contained high level of PhIP (0.66-12.44 µg/g) followed by MeIQx (nd- 1.22 µg/g) and IQ (nd-0.2 µg/g).

As far as cooking of RTE chicken kabab samples are concerned the results indicated that microwave cooking produced the highest total HCAs content (0.71-35 µg/g) followed by deep-frying (1.62-22.21 µg/g) and pan-frying (1.11-8.57 µg/g). Deep frying was found to produce highest content of both MeIQx and PhIP up to 9.91 µg/g and 10 µg/g respectively. Pan-fried chicken samples were found to contain highest level of IQ (0.25-7.48 µg/g) followed by MeIQx (nd-2.46 µg/g) and PhIP (nd-2.46 µg/g) as shown in Table I.

Percentage quantification of three HCAs (IQ, MeIQx and PhIP) have revealed that highest %-age of PhIP was found for microwave cooking (100%) followed by pan-frying (84%) and deep-frying (50%). Highest %-ages of IQ in microwave, pan and deep-fried samples were found as 100%, 95.05% and 22.4%, in RTE chicken kabab samples cooked for the duration of 5min., 3min. and 2min. respectively. Deep-frying was found to produce highest %-age content of MeIQx up to 77.78% followed by microwave cooking (54%) and pan-frying (42.24%).

IQ has been detected as dominating HCA in microwave treated samples (nd-35 µg/g) followed by pan-fried (0.25-7.48 µg/g) and deep-fried (nd-2.3 µg/g) RTE chicken kabab samples. IQ contents emerged up to 0.25 µg/g, 1.44 µg/g, 4.85 µg/g and 4.97 µg/g at the cooking temperatures of 150-160°c, 160-180°c, 180-190°c and

Table I. Quantitative determination of IQ (2-amino-3-methyl-imidazo[4,5-f] quinolone), MeIQx (2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline) and PhIP (2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine) and total HCAs (µg/g) in B, D, S and K brands of ready-to-eat chicken kabab cooked for different durations by microwave, pan-frying and deep-frying.

N.A, not applied; ND, not detected.

190-200°c, respectively in the pan-fried RTE chicken products. This subsequent rise in the HCAs level with the gradual increase in temperature indicates that production of HCAs can be controlled by lowering cooking temperature.

A detailed quantitative analysis of HCAs in the four commercially available brands (B, D, S and K) indicated that microwave cooking produced highest total HCAs contents in the K brand (35 µg/g) followed by S (8.36 µg/g), D (7.92 µg/g) and B (6.53 µg/g) brands. Deep-frying, however, produced highest total HCAs content in the B brand (22.21 µg/g) followed by D (7.38 µg/g), S (6.44 µg/g) and K (1.62 µg/g) brands. Whereas, pan-frying, produced highest total HCAs content in the D brand (8.57 µg/g) followed by S (8.48 µg/g), K (5.86 µg/g) and B (5.58 µg/g) brands (Table I).

The present results agree well to another study, wherein microwave cooking has been found to produce highest HCAs (18.09 ng/g) followed by pan-frying (6.98 ng/g) (Oz et al., 2010). The present findings are in conformity to another study which revealed highest detected %-age of PhIP (24%) among HCAs followed by MeIQx (23%) and MeIQ (12%) (Jahurul et al., 2010). PhIP contents have been documented in the range of 38.2 ng/g to 46.9 ng/g in the chicken samples cooked by frying (Busquets et al., 2004; Keating and Bogen, 2004). Recently, Khan et al. (2019) have found highest IQ contents in chicken drumsticks (~ 6.79 ng/g), followed by fried crispy burgers (~4.08 ng/g) and the beef burgers (3.64 ng/g).

Per day estimated dietary consumption of 96, 84, 66 and 63ng of HCAs has been associated with bladder, kidney, colon and rectal cancers, respectively (Augustsson et al., 1999). An increased dietary intake of HCAs exceeding 41.4 ng/day could enhance the chances of developing colorectal cancer (Rohrmann et al., 2009).

An investigation on dietary median and/or average intake of HCAs/capita/day indicated a relatively higher intake of HCAs up to 1393 ng/day in United States (Keating and Bogen, 2004), followed by Spain (934 ng/day) (Busquets et al., 2004) Malaysia (553.7 ng/g) (Jahurul et al., 2010) Germany (103 ng/d) (Rohrmann and Becker, 2002) Sweden (77 ng/d) (Augustsson et al., 1999) and Singapore (49·95 ng/d) (Wong et al., 2005).

The thawed samples were found to harbor HCAs from 0.97 to 13.66 µg/g. After deep-frying and microwave cooking, the RTE chicken kabab samples attained levels of heterocyclic amines (HCAs) ranging from 1.62 to 22.21 µg/g and from 0.71 to 35µg/g, respectively. Conclusively, high thermal processing of the RTE chicken kabab increased their HCAs contents. Keeping this in view, it is speculated that dietary intake of HCAs in this country is highest worldwide (up to 1.4mg/d). A profound link, thereby, can be established between the dietary intake of high thermally processed food and increased incidence of cancer cases in Pakistani population. The higher HCAs contents in RTE chicken Kabab is a point of serious health concern of Pakistani community and draws significant attention of public health authorities to take necessary measures.

The authors declare there is no conflict of interest.