2-tetradecylcyclobutanone and 2-dodecylcyclobutanone

The DNA-damaging and tumour-promoting effects of two 2-alkylcyclobutanones (2-ACBs), which are found in irradiated fat-containing foods, were investigated by use of the comet assay and in an azoxymethane (AOM)-induced colon-carcinogenesis study in rats, respectively. We conducted genotoxicity tests of 2-dodecylcyclobutanone (2-dDCB) and 2-tetradecylcyclobutanone (2-tDCB) according to the test guidelines for chemicals or drugs. In addition, a cell-transformation assay with Bhas 42 cells was performed to investigate their promoting potential in vitro. The Salmonella typhimurium mutagenicity assay (Ames test), conducted with five tester strains, revealed that neither 2-dDCB nor 2-tDCB possessed mutagenic activity. Moreover, both in the in vitro chromosomal aberration test on CHL/IU cells and the in vivo bone-marrow micronucleus test where mice were given 2-dDCB and 2-tDCB (orally, up to 2000 mg/kg bw/day), we did not detect any clastogenic effects. Furthermore, DNA strand-breaks were not detected in the in vitro comet assay with CHL/IU cells, and DNA adducts derived from 2-dDCB and 2-tDCB were not detected in the colon tissues of the mice used for the micronucleus tests, in rats from a repeated dose 90-day oral toxicity test (0.03% 2-tDCB in the diet), or in rats from the AOM-induced carcinogenesis study (0.025% 2-tDCB in the diet). An in vitro tumour-promotion assay with Bhas 42 cells revealed that the number of transformed foci increased significantly following treatment of cells in the stationary phase with 2-dDCB or 2-tDCB for 10 days. Our results indicate that neither 2-dDCB nor 2-tDCB were genotoxic chemicals. However, they exhibited promoting activity, at least in vitro, when Bhas 42 cells were continuously exposed to these chemicals at toxic doses.

Topics: Animals; Azoxymethane; Carcinogens; Cell Line; Chromosome Aberrations; Colon; Comet Assay; Cricetinae; Cyclobutanes; DNA Damage; Dose-Response Relationship, Drug; Fatty Acids; Female; Food Irradiation; Male; Mice; Mice, Inbred ICR; Micronucleus Tests; Mutagenicity Tests; Mutagens; Neoplasms; Rats; Rats, Sprague-Dawley; Salmonella typhimurium

Food irradiation is a common preservation method that is used in many countries. The ability to identify irradiated food is important for assuring compliance with regulatory policies, such as food labeling requirements, and for informed consumer choice. There is thus a significant demand for analytical methods of high sensitivity and selectivity to identify irradiated food, especially for foods subjected to low-dose irradiation and for processed or composite foods that contain small quantities of irradiated ingredients. 2-Alkylcyclobutanones (2-ACBs) are uniquely formed during food irradiation and have been adopted by the European Committee for Standardization as signature biomarkers for the identification of irradiated foods. We now report the development of a novel assay for quantification of 2-ACBs in γ-irradiated food by liquid extraction of fat content followed by precolumn derivatization and liquid chromatography-tandem mass spectrometric (LC-MS/MS) detection. Precolumn derivatization with hydroxylamine introduced a polar functional group into the otherwise nonpolar 2-ACBs, which greatly enhanced ESI-MS response. The method was validated for extraction efficiency, precision, accuracy, and detection limit. In comparison with the current GC-MS based European official method (EN1785:2003) for 2-ACBs determination, our new LC-MS/MS method offers a more efficient sample processing protocol with reduced solvent consumption. More importantly, the combination of chemical derivatization and LC-MS/MS detection significantly enhanced the analytical sensitivity of the method, which allows confident identification of food irradiated with as little as 10 Gy. To the best of our knowledge, this is the first report of 2-ACB determination by LC-MS/MS and the first analytical method allowing confident identification of irradiated food at dosage of down to 10 Gy.

Topics: Alkylation; Analytic Sample Preparation Methods; Animals; Biomarkers; Chickens; Chromatography, High Pressure Liquid; Cyclobutanes; Food Inspection; Food Irradiation; Food, Preserved; Gamma Rays; Hydroxylamine; Indicators and Reagents; Limit of Detection; Meat; Molecular Structure; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

2-Alkylcyclobutanones (2-ACBs) are recognized as a marker of irradiation in lipid-containing food products. Here, a rapid method for the analysis of 2-dodecylcyclobutanone (2-DCB) and 2-tetradecylcyclobutanone (2-TCB) in irradiated food products using direct solvent extraction (DSE) was developed, and the extraction efficiency was compared to that of the Soxhlet extraction method (EN 1785), which is the official method for this analysis. Briefly, 2-ACBs were extracted either by using a Soxhlet apparatus or by DSE with n-hexane. The lipid extract was purified by GPC followed by a silica gel cartridge column. Finally, 2-DCB and 2-TCB were measured using GC-MS/MS. The extraction efficiency of 2-ACBs by the DSE method was similar to that of the Soxhlet extraction method. The trueness of 2-DCB and 2-TCB spiked at 50 ng/g in lipid extracts of beef, pork, chicken, cheese and salmon with the proposed method were 76.6% to 91.6% and 81.3% to 109.0%, respectively. The limits of detection for 2-DCB and 2-TCB in lipid extracts were 15 and 20 ng/g, respectively.

Topics: Animals; Cattle; Cheese; Chickens; Cyclobutanes; Fish Products; Food Analysis; Food Irradiation; Gas Chromatography-Mass Spectrometry; Hexanes; Lipids; Meat; Salmon; Silica Gel; Swine; Tandem Mass Spectrometry

2-Alkylcyclobutanones (2-ACBs) have long been considered as unique radiolytic products that can be used as indicators for irradiated food identification. A recent report on the natural existence of 2-ACB in non-irradiated nutmeg and cashew nut samples aroused worldwide concern because it contradicts the general belief that 2-ACBs are specific to irradiated food. The goal of this study is to test the natural existence of 2-ACBs in nut samples using our newly developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with enhanced analytical sensitivity and selectivity ( Ye , Y. ; Liu , H. ; Horvatovich , P. ; Chan , W. Liquid chromatography-electrospray ionization tandem mass spectrometric analysis of 2-alkylcyclobutanones in irradiated chicken by precolumn derivatization with hydroxylamine . J. Agric. Food Chem. 2013 , 61 , 5758 - 5763 ). The validated method was applied to identify 2-dodecylcyclobutanone (2-DCB) and 2-tetradecylcyclobutanone (2-TCB) in nutmeg, cashew nut, pine nut, and apricot kernel samples (n = 22) of different origins. Our study reveals that 2-DCB and 2-TCB either do not exist naturally or exist at concentrations below the detection limit of the existing method. Thus, 2-DCB and 2-TCB are still valid to be used as biomarkers for identifying irradiated food.

Topics: Anacardium; Chromatography, Liquid; Cyclobutanes; Food Irradiation; Myristica; Nuts; Pinus; Prunus; Tandem Mass Spectrometry

Raw and cooked beef and pork loins were irradiated at 0 or 5 kGy. The radiation-induced marker compounds, such as hydrocarbons, 2-alkylcyclobutanones (2-ACBs), and sulfur volatiles, were determined after 0 and 6 mo of frozen storage. Two hydrocarbons (8-heptadecene [C(17:1)] and 6,9-heptadecadiene [C(17:2)]) and two 2-ACBs (2-dodecylcyclobutanone [2-DCB] and 2-tetradecylcyclobutanone [2-TCB]) were detected only in irradiated raw and cooked meats. Although precooked irradiated meats produced more hydrocarbons and 2-ACBs than the irradiated cooked ones, the amounts of individual hydrocarbons and 2-ACBs, such as 8-heptadecene, 6,9-heptadecadiene, 2-DCB, and 2-TCB, were sufficient enough to detect whether the meat was irradiated or not. Dimethyl disulfide and dimethyl trisulfide were also determined only in irradiated meats but dimethyl trisulfide disappeared after 6 mo of frozen storage under oxygen-permeable packaging conditions. The results indicated that 8-heptadecene, 6,9-heptadecadiene, 2-DCB, 2-TCB, and dimethyl disulfide, even though they were decreased with storage, could be used as marker compounds for the detection of irradiated beef and pork regardless of cooking under the frozen conditions for 6 mo.. Radiation-induced chemical changes such as specific hydrocarbons, 2-ACBs, and sulfur volatiles may be used as potential identification markers by regulatory authorities to confirm irradiation history of frozen stored raw or cooked beef and pork.

Topics: Alkenes; Animals; Cattle; Cooking; Cyclobutanes; Disulfides; Food Irradiation; Food Storage; Freezing; Hydrocarbons; Meat; Odorants; Swine; Taste; Volatile Organic Compounds

Thermoluminescence (TL) and 2-alkylcyclobutanone (2-ACB) analyses were performed to identify irradiated prawns ( Penaeus monodon ). With the TL method, minerals were extracted from prawns using acid hydrolysis. The experimental results satisfied the evaluation criteria of European Norm (EN) 1788, even after low-dose irradiation (0.5 kGy) and a 60 day storage at -20 °C. With the 2-ACB method, 2-dodecylcyclobutanone (2-DCB) and 2-tetradecylcyclobutanone (2-TCB) were successfully extracted from prawns by direct solvent extraction with purification using a conventional silica column and a sulfoxide column, which was used for 2-ACB for the first time. Both 2-ACB derivatives were absent from the non-irradiated samples but were identified in all irradiated samples by gas chromatography-mass spectrometry. Moreover, 2-DCB and 2-TCB production correlated with the applied dose (2.5-10 kGy), and the correlation did not diminish after 60 days of storage at -20 °C for any dose. Therefore, these two techniques provide rapid, simple, and promising methods for routine investigation of frozen prawns.

Topics: Animals; Cyclobutanes; Food Handling; Food Irradiation; Gas Chromatography-Mass Spectrometry; Luminescence; Penaeidae; Seafood; Thermoluminescent Dosimetry

The 2-Alkylcyclobutanones (2-ACBs) content was determined in three Italian cured pork products (salame Milano, coppa, and pancetta) irradiated at different targeted irradiation doses (2, 5, and 8 kGy) during vacuum-packed storage. Among 2-ACBs, three different compounds were investigated, namely, 2-dodecylcyclobutanone, 2-tetradecylcyclobutanone, and 2-(tetradec-5'-enyl)cyclobutanone. 2-ACBs were absent from the nonirradiated samples, whereas their content increased with irradiation dose. Their presence was recorded occasionally at 2 kGy and constantly at higher irradiation doses (5 and 8 kGy). The plot of 2-ACBs content against targeted irradiation doses showed an exponential relationship. The effect of vacuum-packed storage time on the 2-ACBs content was dependent on the irradiation dose. During vacuum-packed storage for up to 60 days, the 2-ACBs content remained unchanged in the cured pork products irradiated at 2 and 5 kGy, whereas a significant increase was observed in the pork products irradiated at 8 kGy.

Topics: Animals; Cyclobutanes; Food Irradiation; Food Preservation; Meat Products; Swine; Vacuum

A new analytical procedure has been developed to analyze 2-alkylcyclobutanones to detect gamma-ray-irradiated fat-containing foodstuffs. Samples were extracted with an accelerated solvent extraction system via hot and pressurized ethyl acetate in cells. A large amount of fat in the extract was precipitated and removed with filtration by standing at -20 degrees C after the addition of acetonitrile. The extract was further cleaned with a 1 g silica gel mini column, and the radiolytic compounds of 2-docecylcyclobutanone (2-DCB) and 2-tetradecylcyclobutanone (2-TCB) were determined with gas chromatography with mass spectrometry (GC/MS). Sample preparation time before GC/MS was 7-8 h. At first, the procedure was evaluated with a recovery test in eight samples spiked with 2-DCB and 2-TCB at 20 ng/g, resulting in 70-105% recoveries with mostly less than 10% relative standard deviations. The procedure was further evaluated with beef, pork, chicken, and salmon samples irradiated with gamma-rays from 0.7 to 7.0 kGy at -19 degrees C. Both 2-DCB and 2-TCB in most samples were detected with good dose-response relations at all doses, while salmon was detected more than 2 kGy irradiation. The amounts of 2-alkylcyclobutanones produced reflected precursor fatty acids levels in samples, especially for the combination of 2-TCB and stearic acid. The results indicated that the production rate of 2-TCB to stearic acid was more obvious than that of 2-DCB to palmitic acid in frozen samples with gamma-ray irradiation.

Topics: Animals; Cattle; Chickens; Cyclobutanes; Fishes; Food Irradiation; Gamma Rays; Gas Chromatography-Mass Spectrometry; Meat; Salmon; Swine