Validation of an HPLC Methodology for the Identification and Quantification of Biogenic Amines in Chicken Meat
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Validation of an HPLC Methodology for the Identification and Quantification of Biogenic Amines in Chicken Meat César A. Lázaro & Carlos A. Conte-Júnior & Fernanda L. Cunha & Eliane T. Mársico & Sérgio B. Mano & Robson M. Franco
Received: 23 October 2012 / Accepted: 9 January 2013 # Springer Science+Business Media New York 2013
Abstract This study validated a high performance liquid chromatography (HPLC) method to determine biogenic amines in chicken meat. For the identification of biogenic amines, an isocratic elution system coupled with a UV detector (254 nm) was used after a perchloric acid (5 %) extraction and benzoyl chloride derivatization of the samples. The standards of tyramine, putrescine, cadaverine, spermidine, and spermine were used for the following validation parameters: selectivity, linearity, accuracy, recovery, limit of detection and quantification, and robustness. Finally, chicken meat commercialized in two types of packaging was evaluated. The results showed an excellent selectivity and separation of all amines, r2 >0.99, relative standard deviation 12 was reached. Samples were derivatized using benzoyl chloride (40 μL), homogenized (vortex, 15 s), and kept at room temperature for 20 min. The mixture was extracted two times with 1,000 μL of diethyl ether. The ether layer was aspirated and evaporated to dryness under a stream of nitrogen (Sample Concentrator Techne®, Cambridge, UK). Finally, the residue was dissolved in 1,000 μL of the mobile phase and stored at 4±1 °C.
Sample Preparation Two commercial packages (an expanded polystyrene tray and a low-density polyethylene bag) of frozen chicken breasts (Gallus gallus domesticus) were purchased (n=10 for each package) at markets in Rio de Janeiro, Brazil, and transported to the laboratory in insulated polystyrene boxes on ice. For biogenic amine extraction, 5 g of minced chicken meat was homogenized with 5 mL of 5 % perchloric acid. The homogenates were kept under refrigeration (4±2 °C) for 1 h and shaken continuously (Certomat® MV, B. Braun Biotech International, Melsungen, Germany); then, the mixture was centrifuged at 503×g for 10 min at 4±1 °C (Hermle Z 360 K) and filtered through Whatman no. 1 filter paper. The filtrates were neutralized (pH>6) with 2 N NaOH and kept in an ice bath (0 ± 2 °C) for approximately 20 min, followed by a second filtration, and addition of NaOH (pH>12) under the same conditions. The derivatization procedure was carried out in the same way as for the standards. Chromatographic Conditions The chromatographic system consisted of a LC/10AS pump coupled to a SPD/10AV UV–Vis detector and a C-R6A chromatopack integrator (Shimadzu, Kyoto, Japan). Biogenic amine separations were performed on a Teknokroma Tracer Extrasil ODS2 (15 × 0.46 cm id., 5 μm) column equipped with a Supelco Ascentis C18 (2 × 0.40 cm id., 5 μm) guard column, under isocratic conditions. The mobile phase was prepared by mixing acetonitrile (Tedia®) and Milli-Q water, 42:58 (v/v); the mixture was degassed in an ultrasonic bath (Cleaner USC 2800 A). The f
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