Determination of 10 Heterocyclic Aromatic Amines in Roast Meat by HPLC

Transcription

1 Advanced Materials Research Online: ISSN: , Vols , pp doi: / Trans Tech Publications, Switzerland Determination of 10 Heterocyclic Aromatic Amines in Roast Meat by HPLC Weiwei Wang 1, a, Yang Wen 2,b and Fei Zhao 3,c 1 College of Life and Environmental Sciences, Minzu University of China, Beijing , China 2 School of Environment and Natural Resources, Renmin University of China, Beijing , China 3 Chemical Engineering College, Beijing Institute of Petrochemical Technology, Beijing , China a wang.2w@foxmail.com, b @qq.com, c @qq.com Keywords: roast meat products; heterocyclic aromatic amines (HAAs); solid-phase extraction (SPE); high performance liquid chromatography (HPLC) Abstract. HAAs content was investigated in five meat products (including roasted chicken, mutton, beef, pork, fish) using solid-phase extraction-high performance liquid chromatography ( SPE-HPLC). The linear range was between 0.05 ~ 16.0µg ml -1, the detection limits (S/N=3) were in the range of 0.13 ~ 0.42ng g -1, and the recoveries were in the range of ~ %, while RSD was from 1.96 ~ 8.77%. Introduction Heterocyclic aromatic amines are mutagenic and carcinogenic compounds that are formed naturally during cooking of high-protein foods such as meat and fish [1]. According to the chemical structure, it can be classified into Aminoimidazo Azaarens (AIAs) and Amino-carboline Congeners (ACs) [2]. HAAs content in processed meat products, although only ng g -1 levels[3], but most of the heterocyclic amines have been verified to be carcinogenic to rodents and primates and can induce their liver, breast, colon and other target organs produce tumors. Some have much higher mutagenic activity than typical mutagens/carcinogens such as aflatoxin B1, AF-2 and benzo[a] pyrene. The International Agency for the Research of Cancer (IRAC) has classified MeIQ, 8-MeIQx, PhIP, AαC, MeAαC, Trp-P-1, Trp-P-2, Glu-P-1 and Glu-P-2 as possibly carcinogenic to humans (Group 2B), and recognized IQ as probably carcinogenic to humans (Group 2A), and proposed to reduce exposure to these compounds [4]. HAAs usually contain a planar structure, composed of three fused aromatic rings, at least one nitrogen atom in the ring structure, one amino ring outside and a maximum of four methyl groups as alternative. However, there are some exceptions, such as PhIP and DMIP only have two rings, while Harman and Norharman without the exocyclic amino group, IFP in the ring structure containing one oxygen atom [5]. So far, nearly 30 kinds of HAAs have identified from the high-temperature cooking of meat products [6]. Materials and Methods Samples. Meat products, which are determined in the experiment, were purchased from a barbecue stand of Beijing local farmer's market and kept at 4 until analysis. Instrument. A Supelco Visiprep SPE vacuum manifold was used for solid-phase extraction, Filter units (0.45 µm) was utilized for aqueous buffer and disposable syringe-tip filter units (0.45 µm) for sample cleanup (Supelco, Bellefonte,USA). Bond Elut PRS cartridge (100 mg) and Bond Elut C 18 cartridges (100 mg and 500 mg) were from Varian (Harbor City, USA). Diatomaceous earth was from Merck (Darmstadt, Germany).HPLC analysis was performed using the Agilent 1100 HPLC systems. Reagents. 10 kinds of heterocyclic amines standard (purity> 99%, were purchased in Canada Toronto Research Chemicals Corporation), its name, abbreviation and nature are shown in Table 1. All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans Tech Publications, (ID: , Pennsylvania State University, University Park, USA-12/05/16,09:23:37)

2 370 Environmental Protection and Resources Exploitation Methanol, ethyl acetate, triethylamine (HPLC grade, U.S. Tedia company), acetonitrile (HPLC grade, U.S. Roe company), other reagents (AR, Beijing Chemical Plant), test water was ultrapure water. Table 1 Chemical names, abbreviations and property of pure heterocyclic aromatic amines(haas) Chemical name Abbreviation Property 2-amino-3-methylimidazo[4,5-f]quinoine IQ polar 2-amino-3,4-dimethylimidazo[4,5-f]quinoine MeIQ polar 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline MeIQx polar 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine PhIP polar 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine Trp-P-1 nonpolar 3-amino-1-methyl-5Hpyrido[4,3-b]indole Trp-P-2 nonpolar 2-amino-6-methyldipyrido[1,2-a:3,2 -d]imidazole Glu-P-1 nonpolar 2-amino-dipyrido[1,2-a:3,2 -d]imidazole Glu-P-2 nonpolar 2-amino-dipyrido[1,2-a:3,2 -d]imidazole AaC nonpolar 2-amino-3-methyl-9Hpyrido[2,3-b]indol MeAaC nonpolar Determination Method. A method for the quantitative determination of 10 kinds of HAAs which are classified into suspected or potential carcinogens by IARC in roasted chicken, mutton, beef, pork and fish by HPLC-UVD. Sample Preparation. The procedures for sample preparation and clean-up were modified from the method developed by Gross and his co-workers [7]. The main differences included: (1) The internal standard method was used in the present study for quantitation and the IS was added into the sample homogenate before clean-up procedures to make up the loss of HAAs during SPE steps; (2) the present method analyze both the polar and nonpolor HAAs in one run, two final eluents containing nonpolar and polar HAAs were combined, dried with nitrogen stream and redissolved in 50µl of MeOH for HPLC analysis. Chromatographic Condition. The mobile phases were: A, acetonitrile; B, mol L -1 triethylammonium phosphate (TEPA), ph 3.2. A convex gradient were used (time %A %B): 0 min, 5:95; 5 min, 10:90; 10 min, 35:65; 15 min, 40:60; 20 min, 45:55. The optimizing excitation and emission fluorescence wavelength were determined with ultraviolet detection at 263 nm by scanning the wavelength spectrum of 10 kinds of standard HAAs between 210 ~ 400 nm. The chromatographic conditions were as follows: sample amount 25µL, flow rate 1. 0 ml min - 1 and column temperature 30. Standard Curve. Using methanol to prepare the mixed standard solution of 0.05, 0.5, 1, 2, 4, 8, 16 µ g ml -1 respectively, sample under the selected chromatographic conditions, corresponding to HPLC the measured peak area of the Y as the ordinate, using the standard sample of the concentration of X (g ml -1 ) as abscissa drawing standard curve, the correlation coefficient of the standard curve can reach more than The results show that, every component showed good linear relationship in the specific range (Table 2). And the actual samples of single kind of heterocyclic amines compounds content in general 0 ~ 100 ng g -1, visible, the linear range of the method can meet the requirements of actual samples. Table 2 Linear ranges, regression equations, correlation coefficients and LOD of 10 HAAs Linear range Correlation HAAs (µg ml -1 Regression equation LOD(ng g ) -1 ) Coefficient IQ 0.05~16.0 Y=1.108X MeIQ 0.05~16.0 Y=2.452X MeIQx 0.05~16.0 Y=1.697X PhIP 0.05~16.0 Y=1.692X Trp-P ~16.0 Y=0.467X Trp-P ~16.0 Y=0.636X Glu-P ~16.0 Y=0.386X Glu-P ~16.0 Y=1.329X AαC 0.05~16.0 Y=2.825X MeAαC 0.05~16.0 Y=2.683X

3 Advanced Materials Research Vols Recovery and Precision. Accurately weigh meat-like three copies, each 4 g, were added to a concentration of 1 ng µl -1 of 10 kinds of mixed standard solution heterocyclic amines 40, 100 and 200µL, adding levels were 10, 25 and 50 ng g -1, and then the pre-treatment and HPLC analysis (n = 6), calculate the recovery and precision. Results and Discussion Evaluation of Method Performance. Accuracy was estimated by spiking blank samples in recovery experiments. The spiked concentrations of HAAs were valued at 10, 25 and 50 ng g - 1 for each standard, and the results of average recovery and precision at 6 parallel tests are shown in Table 3. Table 3 Recoveries and precision of method (n = 6 ) Spiked levels( ng g - 1 ) HAAs Recovery/% RSD/% Recovery/% RSD/% Recovery/% RSD/% IQ MeIQ MeIQx PhIP Trp-P Trp-P Glu-P Glu-P AαC MeAαC Application to Samples. The samples after pretreatment of the injection of HPLC analysis, the calibration curve method were used to determine the contents of 10 kinds of heterocyclic amines in 5 samples, each sample in 2 parallel determinations. The results are shown in Table 4. Table 4 Content of heterocyclic aromatic amines (HAAs ) in test samples ng g - 1 HAAs Roast chicken Roast mutton Roast beef Roast pork Roast fish IQ ND* MeIQ ND ND ND ND ND MeIQx ND PhIP Trp-P ND Trp-P ND Glu-P-1 ND ND ND ND ND Glu-P-2 ND 2.1 ND ND ND AαC MeAαC ND ND 1.5 * ND, not detected. Table 4 shows: (1) Heterocyclic amines could be detected in five kinds of barbecue, but the measured content is not same in different types of barbecue products, which in roast mutton, beef and pork are more than roasted chicken and fish. Many kinds of HAAs were detected in roast mutton, except MeIQ and Glu-P-1. However, only PhIP, AαC and MeAαC were detected in grilled fish. This may be that beef, mutton and pork are red meat, and chicken, fish belonging to white meat, both fat and free amino acids are different, so the final determination will affect the results.

4 372 Environmental Protection and Resources Exploitation (2) In 5 kinds of meat products are capable of detecting PhIP and AαC, respectively its content is 0.92 ~ 10.5 ng g -1 and 0.21 ~ 2.5 ng g -1. MeIQ and Glu-P-1 were not detected. Glu-P-2 only detected in roast mutton. (3) The types of heterocyclic amine are numerous, different content of heterocyclic amines in meat are different. IQ, PhIP, Trp-P-1, AαC content in the roast mutton is generally more than beef, pork, fish. However, other heterocyclic amines, like Trp-P-2 in the roast mutton usually less than in the pork and beef. (4) The formation of PhIP in polarity heterocyclic amines was the highest, followed by MeIQx. In roast mutton PhIP generation is the largest, and in roast beef MeIQx generation is the largest. Non-polar heterocyclic amines, AαC formation is the largest, while Trp-P-1, Glu-P-2 generates the least amount. Conclusions This experiment using solid phase extraction and high performance liquid chromatography - UV detection method for the determination of 10 kinds of HAAs which are classified into suspected or potential carcinogens by IARC in roasted chicken, mutton, beef, pork and fish. Results show that the detection limits (S/N=3) were in the range of 0.13 ~ 0.42ng g -1, and the recoveries were ~ % with relative standard deviations (RSD, n=6) of 1.96 ~ 8.77%. All can meet the detection requirements. This method is simple and rapid with a good accuracy and reliability, which can be easily popularized to application. These results are similar to values reported previously [8]. References [1] Nagao M, Honda M, Seino Y, Yahagi T, Sugimura T. Cancer Lett. 1977, 2(9): [2] Robert J. Toxicol Lett, 2007, 168( 3) : 219. [3] Busquets R, Bordas M, Toribio F, Puignou L, Galceran M T. J. Chromatogr. B, 2004, 802(4): [4] Skog K. Food Chem Toxicol, 2002, 40(8): [5] F.Toribio,M.T. Galceran, L. Puignou. Separation of hetero aromatic amines in food products. Journal of Chromatography B, 747(2000): [6] Skog K I, Johansson M A E, Jagerstad M I. Carcinogenic heterocyclic amines in model systems and cooked foods: a review on formation, occurrence and intake [J]. Food and Chemical Toxicology, 1998, 36(10): [7] Gross GA, Grüter A. Quantitation of mutagenic / carcinogenic heterocyclic amines in food products [J]. Journal of Chromatography, 1992, 592: [8] Solyakov A, Skog K. Screening for heterocyclic amines in chicken cooked in various ways [J]. Food and Chemical Toxicology, 2002, 40(1):

5 Environmental Protection and Resources Exploitation / Determination of 10 Heterocyclic Aromatic Amines in Roast Meat by HPLC / DOI References [6] Skog K I, Johansson M A E, Jagerstad M I. Carcinogenic heterocyclic amines in model systems and cooked foods: a review on formation, occurrence and intake [J]. Food and Chemical Toxicology, 1998, 36(10): /S (98)