Analysis of heterocyclic amines in cooked foodstuffs using liquid chromatography-mass spectrometry
- Autores: Mohammad Rizwan Khan
- Directores de la Tesis: Lluís Puignou i Garcia (dir. tes.)
- Lectura: En la Universitat de Barcelona ( España ) en 2009
- Idioma: español
- Tribunal Calificador de la Tesis: José Carlos Barbosa (presid.), Santiago Hernández Cassou (secret.), Josep Guasch Torres (voc.)
- Materias:
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- Resumen
Heterocyclic amines (HAs) are substances with a high mutagenic and carcinogenic potential. They are produced in a wide variety of cooked or processed meats and fish and are known to be carcinogenic in rodents. The International Agency for Research on Cancer (IARC, 1993) has classified some of HAs as probably carcinogenic to humans (Group 2A) and possibly carcinogenic to humans (Group 2B). From these evidences now the HAs are suspected to be human carcinogens. So it is important to quantify the amounts of these mutagens present in a variety of cooked foods in order to estimate intakes and risks to human health. Because of the relatively low amounts of HAs formed in food matrices, the challenge has been to develop rapid analytical methods that unequivocally identify HAs in these complex matrices at the low ppb level.
The thesis first introduces the framework of the work followed by the introduction of HAs, in which their chemistry and formation, their occurrence in food, their biological activities including mutagenicity and carcinogenicity, and general aspects of the analytical methodologies have been discussed. The experimental work was focused in the study of pressurised liquid extraction as a new technique for the analysis of HAs, the preparation of laboratory reference material and to establish the occurrence of HAs in several food samples. The main objectives achieved were: 1) To develop a simple and fast method based on pressurised liquid extraction (PLE) for the analysis of HAs applicable to thermally processed meat products. By using a mixed organic solvent as the extractant, at high pressure and temperature, the sample can be injected directly, without the need for any further clean-up. To demonstrate the suitability of the new method, it was used to analyse a laboratory reference material. Following this, several cooked meat samples, including roast beef loin, grilled beef fillet and fried beef chop, were analysed to further demonstrate the applicability of the method. The application of PLE method for the analysis of HAs from meat samples has been reported for the first time. The applied method allows obtaining good recoveries comparable to the most commonly applied solid-phase extraction (SPE) (45%-85%), without the need of further clean-up steps and increasing high sample throughput. In addition, the selected extraction method contributes advantages to reducing sample manipulation and total extraction time (nearly four times lower as compared with conventional SPE technique), and hence increasing the possibilities of process automation. As a consequence, the proposed method can be successfully selected as a good strategy for the analysis of HAs in a large variety of cooked meat samples.
2) To prepare and characterise a laboratory reference material (LRM) based on fried chicken for the analysis of HAs. To our knowledge, this is the first report on the preparation of a LRM where HAs have been naturally generated. The chicken meat was cooked at controlled conditions (average cooking temperature of 210 ºC for 3 minutes/side), ground, diluted (optimum proportions: 50/30, water/cooked meat), homogenised, lyophilised, sieved, homogenised, bottled and, labelled. The characterisation of uncooked and cooked freeze-dried LRM was carried out in term of major components (glucose, creatine, free and total amino acids, total nitrogen, fat, ashes, and moisture) and good values were obtained in comparison with previous studies. Homogeneity of LRM was studied by a single factor ANOVA, the F-calculated values (0.15-4.06) of the samples were inferior than theoretical value (4.26) and the comparison of coefficient variation (CV%) in both between-bottle and within-bottle was obtained less than 11.8% at a confidence level of 95%. Therefore, it can be considered that the material was homogeneous at the concentration level of the HAs. Stability of the LRM was also carried out and no statistical differences were observed in the content of the studied HAs in between-bottle and within-bottle at different storage temperatures (-18, +4, +25, and +40 ºC) and times (1, 3, 6 and 9 months) by means of HAs determination and analysis of the results. Consequently, the material can be considered homogeneous and stable and can be proposed for intercomparision exercises in order to control analytical processes involved in monitoring HAs in foodstuffs.
3) To study the occurrence of HAs in several offal and fish products which are most commonly consumed in Spanish population and other Western countries. The objective of this study was to determine for the fist time the formation or inhibition of twelve HAs in several offal (animal visceral) dishes as affected by various cooking additives. Beef liver, lamb kidney and beef tongue, has been selected because of their variation in the concentrations of creatine, amino acids and sugars in raw tissues and prepared according to common recipes from Spain. In order to establish the effect of additives, offal products cooked without ingredients under the same cooking conditions were also analysed. The concentrations of HAs found in the analysed thermally processed offal products are low as compared with the concentrations found in other type of cooked meat products. Due to low levels of HAs, the offal products could be proposed to elaboration of healthier meat derived products. From this study, it has also been concluded that by using cooking practices that comprise food ingredients the concentrations of HAs can be kept low even if prolonged thermal treatment are applied. The contents of HAs from these foodstuffs could be used to assess the human intake of HAs in Spain and contributes to look for a simple cooking practices that reduce the risk of exposure to HAs, and thus to improve food quality and safety.
Concerning fish, in this study ten fried fish and mollusc (salmon, tuna, hake, sardine, angler fish, cod, sole, swordfish, squid, and cuttlefish) have been analysed for HAs. The levels of creatine, glucose and free amino acids in the raw samples have also been quantified as a source of complementary information. From this study, thermally processed fish products seem to be an important source of HAs since the concentration levels have been found in the same order than the levels reported in meat or even higher in the case of swordfish (150.3 ng g-1). However, other cooked sample contains amounts between 10 ng g-1 and 50 ng g-1. DMIP, PhIP and the co-mutagens harman and norharman were found in all samples analysed at relative high concentrations. The dependence of HA levels on the type of fish, the amount of amino acids, creatine, and glucose in the raw seafood and the cooking weight loss has also been investigated chemometrically by principal component analysis and related tools. As a result, it has shown that the type of fish is the most differentiating parameter on the formation of HAs such as DMIP, PhIP, and norharman. Besides variables such as amounts of certain amino acids, creatine or weight loss were found so crucial since the complex and multi-parametric nature of their influence on amine formation.
