Doctor of Philosophy / Department of Animal Sciences and Industry / J. Scott Smith / The effect of fat level and antioxidant Origanox on the formation of 2-dodecylcylobutanone (2-DCB) was investigated in fresh irradiated ground beef patties. Patties containing 15% and 25% fat were irradiated by electron beam at 1, 2, 3, and 4.5 kGy. Ground beef patties with 0.08% Origanox were gamma irradiated at 3.0 kGy. Commercially available irradiated ground beef with different fat levels was analyzed in order to estimate dose absorbed by these samples. The 2-DCB was extracted by Supercritical fluid extraction (SFE) and analyzed by gas chromatography-mass spectrometry (GC-MS). The concentration of 2-DCB increased linearly with dose with no significant difference in 2-DCB concentrations between the two fat levels. The estimated doses applied to the commercial samples ranged between 1.38 kGy and 1.55 kGy. Origanox did not affect the concentration of 2-DCB. Mutagenicity of 2-DCB was evaluated by the Ames assay using five standard Salmonella tester strains with S9 enzyme activation. The Ames assay did not show a mutagenic effect of 2-DCB, including samples incubated with S9. Acute toxicity of 2-DCB was evaluated by the Microtox acute toxicity system and compared with cyclohexanone and 2-nonenal (both GRAS additives). The toxicity of 2-DCB was between that of cyclohexanone and 2-nonenal while the maximum toxic effect elicited by 2-DCB was the least of the three compounds. Metabolism of 2-DCB was investigated in Female Sprague-Dawley rats. Hexane extracts of feces and fat were analyzed by GC-MS. Urine with and without added β-glucuronidase, was monitored for glucuronide complexes by hexane extraction GC-MS. The total amount of 2-DCB recovered in feces was 1.78 ± 0.63 mg over five days, about 3-11% of the total 2-DCB administered. The total amount recovered in fat was 0.08 ± 0.01 mg which was approximately 0.33% of the total 2-DCB administered. No metabolites were recovered in any of the urine extracts.

Master of Science / Food Science Institute / J. Scott Smith / Food irradiation is one of the most important food processing methods utilized to reduce microbial load and extend shelf life. In 1995 the World Health Organization (WHO) declared irradiated foods to be safe from a nutritional and toxicological point of view. Various methods have been applied to detect irradiated foods. Detection of 2-alkylcyclobutanones has been found to be a useful method in identifying irradiated foods. The solvent extraction method utilizes a Soxhlet apparatus for lipid extraction followed by clean up with Florisil. However, this method is very time consuming. The other methods available to detect 2-alkylcyclobutanone include supercritical fluid extraction (SFE), and accelerated solvent extraction method using a Dionex ASE 200 instrument. The SFE is a fast method to detected 2-alkylcyclobutanone. All the above mentioned methods involve costly equipment. The aim of this study was to eliminate the requirement of costly extraction equipment for lipid extraction before clean up or direct isolation of 2-alkylcyclobutanone as in case of SFE instrument using Florisil cartridges. In this study, the manual solvent extraction method was applied to isolate alkylcylcobutanone followed by clean up with 2 g silica cartridge. The clean up extract was injected to gas chromatography-flame ionization detector (GC-FID) for detection of 2-dodecylcyclobutanone (2-DCB). Gas chromatography-mass spectrometry (GC-MS) was used to confirm that the compound detected was 2-dodecylcyclobutanone. The ions m/z 98 and 112 were selected for 2-DCB for monitoring in selected ion monitoring (SIM) mode of GC-MS. The results showed that this method was able to detect 2-DCB from irradiated ground beef. The manual method does not require costly equipment such as supercritical fluid extractor, Dionex, or Soxlet apparatus for extraction process.

Doctor of Philosophy / Food Science Institute -- Animal Science & Industry / J. Scott Smith / A rapid direct solvent extraction method for the extraction of 2-dodecylcyclobutanone (2-DCB) in irradiated beef using acetonirile was developed and evaluated. The 2-DCB in commercially irradiated ground beef patties was extracted with n-hexane by using a Soxhlet apparatus or with acetonitrile via direct solvent extraction. The hexane and the acetonitrile extracts were evaporated to dryness. Then, the fat in the hexane extract was removed with filtration by standing at -20 °C after the addition of a mixture of ethylacetate and acetonitrile. The defatted extract as well as the acetonitrile extract were purified with a 1 g silica cartridge and was injected into a gas chromatography-mass spectrometry (GC-MS). The 2-DCB concentration in irradiated patties was 0.031 ± 0.0026 ppm (n = 5) for the Soxhlet method and 0.031 ± 0.0025 ppm (n = 10) for direct solvent extraction. The effect of low-energy X-rays on the formation of 2-DCB was investigated in frozen ground beef. Beef patties (85/15) were irradiated by low-energy X-rays and gamma rays at 3 targeted doses of 1.5, 3.0, and 5.0 kGy. The 2-DCB was extracted with n-hexane by using a Soxhlet apparatus and analyzed by GC-MS. There was no significant difference in 2-DCB concentration between gamma-ray- and low-energy X-ray-irradiated patties at all targeted doses. The metabolism of 2-DCB was investigated in vitro and in vivo. The incubation mixture from the in vitro study was extracted with n-hexane by using a Soxhlet apparatus and injected into a GC-MS). The hexane extract from the in vitro study and of rat feces was also derivatized with a silylation reagent and injected into a GC-MS. The average percentage of 2-DCB recovered from the test incubations (2-DCB with S9 and NADPH) was 23%, compared with 50% from the controls (2-DCB in buffer). The GC-MS chromatograms of the derivatized samples showed that 2-DCB was metabolized to 2-dodecylcyclobutanol.