Analytical Procedures for the Determination of Aflatoxin B 1 in Eggs of Laying Hens Using Immunoaffinity Columns and Liq
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Analytical Procedures for the Determination of Aflatoxin B1 in Eggs of Laying Hens Using Immunoaffinity Columns and Liquid Chromatography with Post-Column Derivatisation and Fluorescence Detection Katarina Pavšič-Vrtač & Suvi Ojanperä & Juha Apajalahti & Karin Šrimpf & Gabrijela Tavčar-Kalcher
Received: 16 October 2013 / Accepted: 2 March 2014 # Springer Science+Business Media New York 2014
Abstract An analytical procedure for the determination of aflatoxin B1 in eggs was introduced and validated in laboratory 1. The method consisted of the extraction of aflatoxin B1 from a sample, purification of the extract with solvents, immunoaffinity column cleanup and the determination by liquid chromatography with post-column bromination and fluorescence detection at λex =362 nm and λem =425 nm. The method was transferred to laboratory 2, where it was modified and validated. The limit of detection (LOD) and limit of quantification (LOQ) obtained in laboratory 1 were 2 and 6 ng/kg, respectively, and 2 and 5 ng/kg in laboratory 2, respectively. The repeatability of measurements in laboratory 1, represented by differences between results of duplicate measurements, was 10 ng/kg at the contamination level of 50 ng/kg. At the same concentration level, the standard deviation (sR) and the relative standard deviation (RSDR) for the within-laboratory reproducibility were 5.5 ng/kg and 11 %, respectively, and the measurement uncertainty was ±10 ng/kg. The mean recovery was 70 %. In laboratory 2, the repeatability of measurements at the contamination level of 20 ng/kg, represented by the standard deviation (sR), repeatability (r) and relative standard deviation (RSDR) was 4 ng/kg, 11 ng/kg and 20 %, respectively, and the recovery was 67 %. The results indicate that the procedures are suitable for the determination of aflatoxin B1 in eggs and can be implemented for the routine analysis. Using the procedure validated in laboratory 1, 25 samples from farms in Slovenia were analysed. In none of the analysed samples, aflatoxin B1 was detected. K. Pavšič-Vrtač : K. Šrimpf : G. Tavčar-Kalcher (*) National Veterinary Institute, University of Ljubljana, Veterinary Faculty, Gerbičeva 60, 1115 Ljubljana, Slovenia e-mail: [email protected] S. Ojanperä : J. Apajalahti Alimetrics Ltd, Koskelontie 19 B, 02920 Espoo, Finland
Keywords Aflatoxin B1 . Eggs . Immunoaffinity cleanup . Liquid chromatography . Post-column derivatisation . In-house validation
Introduction Aflatoxins are secondary metabolites produced by moulds Aspergillus flavus and Aspergillus parasiticus under specific conditions (high moisture and temperature between 25 and 30 °C) and may contaminate plants and plant products intended for animal and human consumption (Muscarella et al. 2009). They are associated with chronic or acute aflatoxicosis and may pose a threat to humans and animals through their mutagenic, carcinogenic, teratogenic, immunosuppressive and other adverse effects (Hussein and Brasel 2001; Muscarella et al. 2009; Zain 2011; Han et al. 2013). Aflatoxins p
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