A simple method for the determination of sucralose in various foods using liquid chromatography-electrospray ionization tandem mass spectrometry (LC/MS/MS) was developed. Sucralose was extracted with water or methanol, and the extract was cleaned up on a C18 cartridge, and diluted with water for injection into the LC/MS/MS. The LC separation was performed with a reversed-phase gradient on an ODS column, and the mass spectral acquisition was done in the negative ion mode by applying selected reaction monitoring (SRM). The recoveries of sucralose from various kinds of foods fortified at 100 μg/g and 5 μg/g were 88.1∼96.7% and 92.7∼98.5%, respectively. The lower limits of quantification were 0.5 μg/g in beverage, low-malt beer, yogurt and chocolate and 2.5 μg/g in other foods. Forty-three commercial foods containing sucralose were analyzed by this method. Sucralose was detected in all samples at levels of 3.8∼481 μg/g.
Subchronic animal feeding studies to examine the effect on the immune system of genetically modified corn CBH351, which contains the Cry9C protein derived from Bacillus thuringiensis subspecies tolworthi, were conducted in female BN rats and B10A mice. The studies were designed to compare the effect of a line of genetically modified corn CBH351 (GM corn) with that of isoline corn (non-GM corn). Heat-treated corn meal was incorporated into the diets of the rats and mice at a concentration of 50%. The study duration was 13 weeks. Growth, food intake, and organ weights of the thymus, spleen, and liver were compared between animals fed the non-GM and GM lines. The histological findings in thymus, spleen, mesenteric lymph nodes, Peyer's patches, small intestines, liver, kidney, and bone marrow, and the presence of Cry9C-specific IgE, IgG, IgG1 and IgA antibodies in serum were also compared. The results showed no significant differences in growth, feeding value, or the histological findings in immunity-related organs between the animals fed the GM and non-GM lines. Production of Cry9C-specific IgE and IgA was not detected in the serum of either group. Production of Cry9C-specific IgG and IgG1 was slightly increased in the 50% GM groups of BN rats. No Cry9C-specific IgG or IgG1 was detected in the serum of BN rats fed the diet containing 5% GM-corn In conclusion, no immunotoxic activity was detected in the GM-corn-fed rats and mice in this subchronic dietary study.
Ion-trap GC/MS/MS was evaluated for the multi-residue determination of pesticides in agricultural products. Matrices were extracted from samples (spinach, carrot, onion and brown rice) with acetone and submitted to gel permeation chromatography, followed by a clean-up step through a graphite carbon cartridge. Thirty-five pesticides were added to either matrix, and analyzed by GC/MS/MS. Detection limits of pesticides by GC/MS/MS was almost the same as those by GC/MS (SIM). Coefficients of variation of peak area in 5 measurements of each pesticide at 0.1 μg/mL or 0.05 μg/mL with or without matrices were mostly acceptable, though those of 20 pesticides out of 35 were higher than 10% at a concentration of 0.02 μg/mL. It was indicated that matrix artifacts, which interfere with GC/MS-Scan analysis, could be eliminated in some cases by using GC/MS/MS.
The development of a sensitive pre-column derivatization high-performance liquid chromatography (HPLC) method for determination of sucralose is reported. Sucralose is converted into a strongly ultraviolet (UV)-absorbing derivative, possessing strong absorption at 260 nm, by treatment with p-nitrobenzoyl chloride (PNBCl). Homogenized samples were dialyzed and washed with a Bond Elut ENV cartridge, then the eluate was evaporated to dryness and the residue was derivatized. Subsequently, the sucralose derivative was purified with hexane-ethyl acetate (9 : 1) in a silica cartridge, and then the sucralose derivative was eluted with acetone. HPLC was performed on a phenyl column, using acetonitrile-water (73 : 27) as a mobile phase with UV detection (260 nm). The calibration curve was linear in the range of 1 μg/mL to 50 μg/mL of sucralose. The recoveries of sucralose from eight kinds of foods spiked at the levels of 0.20 and 0.05 g/kg of sucralose were more than 76.2% with SD values in the range from 0.90% to 4.31%. The quantitative limit of the developed method was 0.005 g/kg for sucralose in samples.
A large volume injection head-space GC method was established for measuring low levels of residual methanol and ethanol in licorice extract used as a food additive. A vial was kept at 50°C in the oven of the head-space sampler. Injection of the head-space gas for 0.75 min into a Poraplot Q GC column with a initial oven temperature of 35°C, enabled the determination of low levels (5 μg/g) of methanol and ethanol. The standard deviations for five rounds of analysis of methanol and ethanol in licorice extracts were between 0.82 and 2.97. Methanol was found in 6 samples out of 9 collected in 1999, at concentrations exceeding 50 μg/g, the limit set by the Japanese Government, established in 1999 and coming into force on April 1, 2000. The highest concentration reached 10,000 μg/g. Methanol at a concentration exceeding 50 μg/g was found in 2 out of 9 samples collected in 2000. The highest concentration was 270 μg/g.
A detection method using the polymerase chain reaction (PCR) was developed to detect genetically modified (GM) potato (NewLeaf Y® potato; NL-Y), of which the mandatory assessment has not yet been completed in Japan. The potato sucrose synthase gene was used as an internal control. We designed a primer pair to specifically detect NL-Y without false-positive results in processed potato foods infected with the potato virus Y (PVY). The DNA introduced into NL-Y using the primer pair could be detected from potato powder samples containing 0.05% NL-Y. In addition, we designed primer pairs for recognizing the CryIIIA gene to detect the NewLeaf potato (NL), NewLeaf Plus potato (NL-P) and NL-Y and for recognizing p-FMV in order to detect NL-P and NL-Y. The proposed method was applied to the detection of NL-Y in 26 processed potato foods and NL-Y was not detected in any samples.
Little is known about the effects of residual veterinary drugs on the allergic reaction, except for the antigenicity of antibiotics and synthetic antimicrobials. Therefore, 59 kinds of veterinary drugs were investigated for their effects on the IgE receptor-mediated β-hexosaminidase release from RBL-2H3 cells as an index of immediate allergic reaction. We found that the antibiotics chlorotetracycline, doxycycline, monensin, the synthetic antimicrobial pyrimethamine and the steroid hormone testosterone inhibited β-hexosaminidase release. Most of the veterinary drugs showed no action, though the ionophores lasalocid, salinomycin and the steroid hormone hexestrol promoted β-hexosaminidase release from injured cells. Based on the residual levels of these drugs and the frequencies of detection in actual food samples, it seems unlikely that these drugs have any immediate allergic effect in practice.
Two typical cleanup methods, sulfuric acid treatment and multi-layer silica gel column chromatography, for the determination of polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (dioxin-like PCBs) in seventeen food samples were examined and compared. Vegetables, fruits, cereals, fish, meat and dairy foods were extracted by conventional methods (shaking with acetone/n-hexane or with n-hexane after alkaline treatment). The extracts were cleaned up by sulfuric acid treatment or multi-layer silica gel column chromatography, followed by several column chromatographic steps. Of the samples treated, the vegetable, fruit and cereal samples could be directly applied to the multi-layer silica gel column after extraction. However, the samples containing fats and oils such as fish, meat and dairy foods needed to be treated several times with concentrated sulfuric acid before multi-layer column chromatography, because these samples plugged the column with oily residues. Both cleanup methods gave similar values of isomeric concentrations and showed similar efficiency of purification, and the recoveries ranged from 40 to 120%. These results are considered to provide useful data for the efficient analysis of dioxins in foods which have wide-ranging compositions.