Determination of aflatoxins B1, B2, G1, and G2 in olive oil, peanut oil, and sesame oil using immunoaffinity column cleanup, postcolumn derivatization, and liquid chromatography with fluorescence detection: first action 2013.05.

A collaborative study of a method for determination of aflatoxins (AFs) B1, B2, G1, and G2 in olive oil, peanut oil, and sesame oil using immunoaffinity column cleanup, postcolumn derivatization, and LC with fluorescence detection, previously published in J. AOAC Int. 95, 1689-1700 (2012), was approved as First Action 2013.05 on March 29, 2013 by the Method-Centric Committee for Aflatoxins in Edible Oils. The method uses methanol for extraction followed by filtration. The extract is applied to an immunoaffinity column with antibodies specific for AFs, which are then eluted from the column with a methanol solution. Determination and quantification occur using RP-LC with fluorescence detection after postcolumn derivatization. The average recovery of AFs in olive, peanut, and sesame oils in spiked samples (levels between 2.0 and 20.0 microg/kg) ranged from 84 to 92%. The recoveries for AFs B1, B2, G1, and G2 were 86-93, 89-95, 85-97, and 76-85%, respectively. Within-laboratory RSD (RSDr) values for AFs ranged from 3.4 to 10.2%. RSDr values forAF B1, B2, G1, and G2 were 3.5-10.9, 3.2-9.5, 6.5-14.9, and 4.8-14.2%, respectively. Between-laboratory RSD (RSDR) values for AFs were 6.1-14.5%. RSD, values for AFs B1, B2, G1, and G2 were 7.5-15.4, 7.1-14.6, 10.8-18.1, and 7.6-23.7%, respectively. Horwitz ratio values were < or =2 for the analytes in the three matrixes.

Determination of aflatoxins B1, B2, G1, and G2 in olive oil, peanut oil, and sesame oil using immunoaffinity column cleanup, postcolumn derivatization, and liquid chromatography/fluorescence detection: collaborative study.

The accuracy, repeatability, and reproducibility characteristics of a method using immunoaffinity column (IAC) cleanup with postcolumn derivatization and LC with a fluorescence detector (FLD) for determination of aflatoxins (AFs; sum of AFs B1, B2, G1, and G2) in olive oil, peanut oil, and sesame oil have been established in a collaborative study involving 15 laboratories from six countries. Blind duplicate samples of blank, spiked at levels ranging from 0.25 to 20.0 microg/kg for AF, were analyzed. A naturally contaminated peanut oil sample was also included. Test samples were extracted with methanol-water (55 + 45, v/v). After shaking and centrifuging, the lower layer was filtered, diluted with water, and filtered through glass microfiber filter paper. The filtrate was then passed through an IAC, and the toxins were eluted with methanol. The toxins were then subjected to RPLC-FLD analysis after postcolumn derivatization. Average recoveries of AFs from olive oil, peanut oil, and sesame oil ranged from 84 to 92% (at spiking levels ranging from 2.0 to 20.0 microg/kg); of AFB1 from 86 to 93% (at spiking levels ranging from 1.0 to 10.0 microg/kg); of AFB2 from 89 to 95% (at spiking levels ranging from 0.25 to 2.5 microg/kg); of AFG1 from 85 to 97% (at spiking levels ranging from 0.5 to 5.0 microg/kg); and of AFG2 from 76 to 85% (at spiking levels ranging from 0.25 to 2.5 microg/kg). RSDs for within-laboratory repeatability (RSD(r)) ranged from 3.4 to 10.2% for AF, from 3.5 to 10.9% for AFB1, from 3.2 to 9.5% for AFB2, from 6.5 to 14.9% for AFG1, and from 4.8 to 14.2% for AFG2. RSDs for between-laboratory reproducibility (RSDR) ranged from 6.1 to 14.5% for AF, from 7.5 to 15.4% for AFB1, from 7.1 to 14.6% for AFB2, from 10.8 to 18.1% for AFG1, and from 7.6 to 23.7% for AFG2. Horwitz ratio values were < or = 2 for the analytes in the three matrixes.

Use of fatty acid profiles to identify food-borne bacterial pathogens and aerobic endospore-forming bacilli.

Capillary gas chromatography (GC) with flame ionization detection was used to determine the cellular fatty acid profiles of various food-borne microbial pathogens and to compare the fatty acid profiles of spores and vegetative cells of the same endospore-forming bacilli. Fifteen bacteria, representing eight genera (Staphylococcus, Listeria, Bacillus, Yersinia, Salmonella, Shigella, Escherichia, and Vibrio) and 11 species were used to compare the extracted fatty acid methyl esters (FAMEs). Endospore-forming bacilli were processed to obtain pure spores and whole cell FAMEs for GC analysis. A data set for each bacterial agent was prepared using fatty acid profiles from five replicates prepared on different days. The results showed that these fatty acid intensity profiles were unique for each of the 11 species and that they could be used as a fingerprint for the organisms. The cellular fatty acid profiles for Bacillus anthracis and Bacillus cereus show that there are two branched chain fatty acids, iso 17:1 omega10c and 17:1 anteiso, which are unique in these species. Iso 17:1 omega10c is present in B. cereus vegetative cells and spores but is not observed in B. anthracis. The 17:1 anteiso fatty acid is present in B. anthracis cells but not in B. cereus cells. Fatty acids 16:0 2OH and 17:0 iso 3OH are present in B. anthracis and B. cereus spores but not in the vegetative cells. In summary, analysis of FAMEs from bacteria and spores can provide a sensitive procedure for the identification of food-borne pathogens.

Accelerating bacterial identification by infrared spectroscopy by employing microarray deposition of microorganisms.

A microarray method for the deposition of bacteria onto an agar slide was developed to accelerate the formation of microcolonies. Representative microarrays each consisting of 40 micro-spots of five replicates of eight foodborne bacteria (Yersinia enterocolitica, Staphylococcus aureus, Salmonella typhimurium, Listeria monocytogenes, Enterobacter cloacae, Citrobacter freundii, Klebsiella pneumoniae, and Escherichia coli) were printed on a Brain Heart Infusion (BHI) agar slide using a contact micro-spotting robotic system. Within 3 h, sufficient bacterial cells were obtained to allow accurate identification of the microorganism by infrared spectroscopy. This approach allows a "complete-in-a-single-day" analysis of a large array of samples.