Determination of total sulfite in shrimp, potatoes, dried pineapple, and white wine by flow injection analysis: collaborative study

A method for the determination of total sulfite in shrimp, potatoes, dried pineapple, and white wine by flow injection analysis (FIA) was collaboratively studied by 8 laboratories. In the method, the sample solution is reacted with sodium hydroxide to liberate aldehyde-bound sulfite. The sample stream is acidified to produce SO2 gas, which diffuses across a Teflon membrane in the gas diffusion cell into a flowing stream of malachite green. The degree of discoloration of the malachite green is proportional to the amount of sulfite in the sample solution. Red wine was included in the study but interlaboratory precision for these samples was not satisfactory and correlation with Monier-Williams results was poor. The present method is not recommended for use with these samples. For shrimp, potatoes, dried pineapple, and white wine, average reproducibility (RSDR) of results was 25% for samples at 10 ppm SO2 and 10% for samples at greater than 50 ppm. Overall average reproducibility was 14%. Recoveries of sulfite added to samples averaged 80%. Comparison of FIA with the Monier-Williams method indicated comparable results by the 2 methods. The FIA method has been adopted official first action for determination of greater than or equal to 5 ppm total sulfite in shrimp, potatoes, dried pineapple, and white wine.     

Differential detection of shrimp and crab for food labeling using polymerase chain reaction

Shrimp and crab are well-known as allergenic ingredients. According to Japanese food allergy labeling regulations, shrimp species (including prawns, crayfishes, and lobsters) and crab species must be differentially declared when ≥10 ppm (total protein) of an allergenic ingredient is present. However, the commercial ELISA tests for the detection of crustacean proteins cannot differentiate between shrimp and crab. Therefore, two methods were developed to discriminate shrimp and crab: a shrimp-PCR method with postamplification digestion and a crab-PCR method that specifically amplifies a fragment of the 16S rRNA gene. The sensitivity and specificity of both PCR methods were verified by experiments using DNA extracted from 15 shrimp species, 13 crab species, krill, mysid, mantis shrimp, other food samples (cephalopod, shellfish, and fish), incurred foods, and commercial food products. Both PCR methods could detect 5 pg of DNA extracted from target species and 50 ng of genomic DNA extracted from incurred foods containing 10 ppm (μg/g) total protein of shrimp or crab. The two PCR methods were considered to be specific enough to separately detect species belonging to shrimp and crab. Although false-positive and false-negative results were obtained from some nontarget crustacean species, the proposed PCR methods, when used in conjunction with ELISA tests, would be a useful tool for confirmation of the validity of food allergy labeling and management of processed food safety for allergic patients.     

Determination and confirmation of melamine residues in catfish, trout, tilapia, salmon, and shrimp by liquid chromatography with tandem mass spectrometry

Pet and food animal (hogs, chicken, and fish) feeds were recently found to be contaminated with melamine (MEL). A quantitative and confirmatory method is presented to determine MEL residues in edible tissues from fish fed this contaminant. Edible tissues were extracted with acidic acetonitrile, defatted with dichloromethane, and cleaned up using mixed-mode cation exchange solid-phase extraction cartridges. Extracts were analyzed by liquid chromatography with tandem mass spectrometry with hydrophilic interaction chromatography and electrospray ionization in positive ion mode. Fish and shrimp tissues were fortified with 10-500 microg/kg (ppb) of MEL with an average recovery of 63.8% (21.5% relative standard deviation, n = 121). Incurred fish tissues were generated by feeding fish up to 400 mg/kg of MEL or a combination of MEL and the related triazine cyanuric acid (CYA). MEL and CYA are known to form an insoluble complex in the kidneys, which may lead to renal failure. Fifty-five treated catfish, trout, tilapia, and salmon were analyzed after withdrawal times of 1-14 days. MEL residues were found in edible tissues from all of the fish with concentrations ranging from 0.011 to 210 mg/kg (ppm). Incurred shrimp and a survey of market seafood products were also analyzed as part of this study.     

Gas chromatographic determination of pentachlorophenol in gelatin: collaborative study

Eleven collaborators participated in this study of a gas chromatographic method for the determination of pentachlorophenol (PCP) in gelatin. Following acid hydrolysis of a 2 g sample, PCP is extracted with hexane and partitioned into KOH solution. After reacidification, PCP is again extracted with hexane for determination by electron capture gas chromatography on a 1% SP-1240DA column. Three duplicate practice samples (0.0, 0.5, and 1.5 ppm) and 5 blind duplicate collaborative samples (0.0, 0.02, 0.1, 0.5, and 2.0 ppm) were analyzed by each collaborator. Mean recoveries of PCP in the collaborative samples ranged from 88% at the 0.02 ppm fortification level to 102% at the 0.1 ppm level; the overall mean recovery was 96%. Interlaboratory coefficients of variation ranged from 16.4% for the 0.1 ppm fortification level to 22.9% for the 0.5 ppm level; the overall interlaboratory coefficient of variation was 19.5%. The method has been adopted official first action.     

Rapid determination of methyl mercury in fish and shellfish: method development

The AOAC official first action method for methyl mercury in fish and shellfish was modified to provide more rapid determination. Methyl mercury is isolated from homogenized, acetone-washed tissue by addition of HCl and extraction by toluene of the methyl mercuric chloride produced. The extract is analyzed by electron capture gas chromatography (GC) on 5% DEGS-PS treated with mercuric chloride solution. The quantitation limit of the method is 0.25 micrograms Hg/g. Swordfish, shark, tuna, shrimp, clams, oysters, and NBS Research Material-50 (tuna) were analyzed for methyl mercury by the AOAC official first action method. All products also were analyzed by the modified method and the AOAC official method for total Hg. In addition, selected extracts obtained with the modified method were analyzed by GC with Hg-selective, microwave-induced helium plasma detection. There was no significant difference between the results for the various methods. Essentially all the Hg present (determined as total Hg) was in the organic form. Coefficients of variation from analyses by the modified method ranged from 1 to 7% for fish and shellfish containing methyl mercury at levels of 0.50-2.30 micrograms Hg/g. The overall average recovery was 100.5%.     

Rapid and sensitive determination of sulfonamide residues in milk and chicken muscle by microfluidic chip electrophoresis

A new, rapid, and sensitive method was proposed for the determination of sulfonamide residues in milk and chicken muscle samples by microchip electrophoresis with laser-induced fluorescence detection. Separation of fluorescamine-labeled sulfonamides was accomplished by using a buffer containing 5 mmol/L boric acid and 1% (w/v) polyvinyl alcohol (PVA). The pH, amount of PVA, and concentration of boric acid in the running buffer were found to have great influence on the separation. By optimizing these conditions, the separation of four sulfonamides, sulfamethazine, sulfamethoxazole, sulfaquinoxaline, and sulfanilamide, was achieved within 1 min with limits of detection (S/N = 3) of 0.2-2.3 μg/L, which are well below the maximum residue limit. The proposed method also exhibited very good repeatability; the relative standard deviations for both within-day and between-day measurements were ≤3.0%. With a simplified sample pretreatment protocol, fast determination of sulfonamides in real samples was successfully performed with standard addition recoveries of 93.3-100.8 and 82.9-92.3%, respectively.     

Determination of total N-nitroso compounds and their precursors in frankfurters, fresh meat, dried salted fish, sauces, tobacco, and tobacco smoke particulates.

Total N-nitroso compounds (NOC) and NOC precursors (NOCP) were determined in extracts of food and tobacco products. Following Walters' method, NOC were decomposed to NO with refluxing HBr/HCl/HOAc/EtOAc and NO was measured by chemiluminescence. NOC were determined after sulfamic acid treatment to destroy nitrite, and NOCP were determined after treatment with 110 mM nitrite and then sulfamic acid. Analysis without HBr gave results < or =20% of those with HBr. This NOC method was efficient for nitrosamines but not nitrosoureas. The standard nitrosation for determining NOCP gave high yields for readily nitrosated amines, including 1-deoxy-1-fructosylvaline, but not for simple amines, dipeptides, and alkylureas. Mean NOC and NOCP results were (respectively, in micromol/kg of product) 5.5 and 2700 for frankfurters, 0.5 and 660 for fresh meat, 5.8 and 5800 for salted, dried fish, and 660 and 2900 for chewing tobacco (all for aqueous extracts) and 220 and 20000 nmol/cigarette for MeCN extracts of cigarette smoke filter pads.     

Rapid digestion and flameless atomic absorption spectroscopy of mercury in fish: collaborative study

A collaborative study of the determination of mercury in fish has been completed in which wet oxidation of fish tissue in nitric acid, using vanadium as a catalyst, is compared with the AOAC official final action digestion technique, 25.103-25.105, involving a nitric-perchloric acid mixture. The study used tuna fish samples of known mercury content and included spike recovery studies in which methyl mercury solutions of known composition were provided to each laboratory. The study was designed to provide recovery information that bracketed the regulatory level of mercury in fish. The results indicate that the proposed digestion method is at least as precise and accurate as the AOAC method. The proposed method is also more rapid and less hazardous. It has been adopted as official first action.     

Determination of chlorinated methylthiobenzenes and their sulfoxides and sulfones in fish

A procedure was developed to determine chlorinated methylthiobenzenes and their respective sulfur oxidation products in fish. Perch samples fortified at the 0.1 ppm level with 2,4,5-trichloromethylthiobenzene, pentachloromethylthiobenzene, and their sulfoxides and sulfones were extracted and cleaned up using an adaptation of the official AOAC method for multiple residues of organochlorine pesticides. The Florisil column cleanup was modified; 200 mL 6% petroleum etherethyl ether eluted the methylthiobenzenes, 200 mL 50% PE-EE eluted the sulfones, and 200 mL EE eluted the sulfoxides. Recoveries determined by electron capture (ECD) gas chromatography (GC) were 75-101% for the methylthiobenzenes and their sulfones and 63-93% for the sulfoxides. Co-extracted materials in the Florisil eluates that interfered with the ECD/GC quantitation were removed by partitioning the sulfoxides and sulfones into sulfuric acid and by thin layer chromatography on silica gel, using methylene chloride-hexane (50 + 50) as the developing solvent. Seven fish samples containing residues of chlorinated benzenes or polychlorinated biphenyls (PCBs) were examined for chlorinated methylthiobenzenes, methylthio-PCBs, and their oxidation products by matching GC retention times obtained with the EC detector and a flame photometric detector operated in the sulfur mode. These analytes were not found in the fish samples above a detection level equivalent to 0.02 ppm 2,4,5-trichloromethylthiobenzene.     

Rapid, Micro-Methods to Estimate Plant Silicon Content by Dilute Hydrofluoric Acid Extraction and Spectrometric Molybdenum Method

By treating 0.5 g DW of a plant sample directly with 10 ml of a dilute hydrofluoric acid solution (HF solution, 1.5 M HF—0.6 M HCl), all the silica in plant (as much as 150 mg SiO₂) was dissolved within 1 h. After dilution of the extract with 40 mL of distilled water, the silica in the extract was measured by the spectrometric molybdenum yellow method. The molybdenum yellow method, in which silica in 0.1 mL of the diluted extract can be determined in 8 min, is well suited to rapid, micro-estimations of the silica content in plants. In the micro-modification, the size of the plant sample was reduced to 100 mg DW. The analytical procedure was simple, and the analytical time was less than 2 h. The method can save much labor and time, compared with the gravimetric analysis. The dissolution with HF solution and the molybdenum yellow method were also applied to the measurement of the content of silica separated by acid digestion of rice plants. Excellent agreement in the silica measurement of rice plants was confirmed among the direct extraction method, the gravimetric method and the digestion-separation-dissolution method. In the molybdenum yellow method, the addition of boric acid enabled to mask the interference of hydrofluoric acid, and the least amount of citric acid required for the elimination of phosphorus interference was proposed. In conclusion in this report, recommended methods for the rapid estimation of the silica content in rice plants were presented.     

Dry rehydratable film for enumeration of total aerobic bacteria in foods: collaborative study

A rehydratable dry-film plating procedure for aerobic plate counts has been compared to the standard agar plate method (966.23B and C, 15th ed.; 46.014-46.015, 14th ed.) in a collaborative study by 12 laboratories. Each laboratory analyzed the normal microflora of 3 samples in duplicate for 6 products. The aerobic plate counts ranged from 1.0 x 10(3) to 1.0 x 10(8) cfu/g. The products were flour, nuts, frozen raw shrimp, spice, frozen raw ground turkey, and frozen and refrigerated vegetables. Repeatability standard deviations of the 2 methods did not differ significantly for 13 of 18 test samples. For 1 shrimp and 2 turkey samples, the dry-film method had lower repeatability variances (P less than 0.05) and for 1 spice sample the agar method had lower repeatability variances (P less than 0.05). Relative standard deviations of repeatability were between 1.7 and 15.5% for the dry-film method and 1.2 and 16.0% for the agar method. Relative standard deviations of reproducibility ranged from 2.4 to 23.4% for the dry-film method and 2.3 to 18.8% for the agar method. The dry rehydratable film method has been adopted official first action for determination of the aerobic plate count.     

Collaborative study of the determination of available lysine in proteins and feeds.

In the proposed method 1-fluoro-2,4-dinitrobenzene (DNFB) is reacted with the free epsilon-amino groups in protein of form DNFB-epsilon-amino lysine which is stable to acid hydrolysis. The sample is acid hydrolyzed and unavailable lysine is determined with an amino acid analyzer; total lysine is determined on the untreated sample. The available lysine, which was bound by DNFB, is determined by difference. The available lysine has been determined in 3 samples of 44% protein soybean meal by 5 collaborators, following the method outlined. The range for available lysine in reference standard 1 was 2.02-2.14%, in reference standard 2, 2.59-2.73% and in reference standard 3, 0.55-0.91%. The method has been adopted as official first action.     

Bioaccumulation of chlorinated paraffin residues in fish fed chlorowax 500C.

Fingerling rainbow trout were fed a diet fortified with 10 ppm Chlorowax 500C, a chlorinated paraffin, for up to 82 days. Chlorinated paraffin residues as high as 1.1 ppm (tissue basis) were found. Recoveries from fortified fish tissue were greater than 92%, and the method sensitivity (tissue basis) is at least 0.5 ppm. Ultraviolet irradiation of the sample extracts and microcoulometric gas chromatography minimized interferences. No gross toxicological effects were noted in the experimental fish, although their weight gain was less than that of the controls.     

Sonication-assisted extraction of chitin from North Atlantic shrimps (Pandalus borealis)

The influence of sonication during extraction of chitin from North Atlantic shrimp (NAS) shells (Pandalus borealis) on chitin yield, purity, and crystallinity was investigated. Shells were peeled, washed, lyophilized, ground, and suspended for 4 h in 0.25 M HCl (1:40) at 40 degrees C followed by ultrasonication at 41 W/cm(2) for 0, 1, and 4 h, respectively. Demineralized shells were lyophilized, resuspended in 0.25 M NaOH (1:40), and ultrasonicated at 41 W/cm(2) for 0, 1, and 4 h to remove proteins. The yield and mineral and protein contents were determined after each processing step. The purity of extracted chitin was determined from the total amount of glucosamine. The crystallinity index and size of crystals were calculated from wide-angle X-ray scattering measurements. Scanning electron microscope images were recorded to evaluate morphological changes in samples. The yield of chitin from NAS decreased from 16.5 to 11.4% for 0 and 1 h sonicated samples, respectively, which was attributed to increased concentrations of depolymerized materials in the wash water. Sonication did not enhance the removal of minerals. The application of ultrasound enhanced the removal of proteins from 39.8 to 10.6, 8.3, and 7.3% after 0, 1, and 4 h of sonication treatments. The crystallinity index of chitin decreased from 87.6 to 79.1 and 78.5% after 1 and 4 h of sonication, yielding chitosans with crystallinity indices of 76.7, 79.5, and 74.8% after deacetylation, respectively. Fourier transform infrared spectroscopy scans indicated that the degree of acetylation of chitins was unaffected by sonication. Comparison of the extraction results of NAS with that from freshwater prawns indicated that more impurities were left in NAS chitin, suggesting that composition and structural arrangement of chitin in shells influence the efficiency of ultrasound-assisted extraction.     

Determination of carotenoids in spear shrimp shells (Parapenaeopsis hardwickii) by liquid chromatography

The objectives of this study were to develop a high-performance liquid chromatography method for analysis of carotenoids in spear shrimp shells (Parapenaeopsis hardwickii) and to compare the extraction efficiency of carotenoids by supercritical carbon dioxide (SCD) and solvents. Results showed that the most appropriate HPLC method was accomplished by employing a Cosmosil 5C18-AR-II column and a mobile phase of methanol-dichloromethane-acetonitrile (90:5:5, v/v/v) (A) and water (100%) (B) with the following gradient elution: 92% A and 8% B in the beginning, decreased to 4% B in 9.5 min, 1% B in 26 min, 0% B in 35 min, maintained for 25 min, and returned to 92% A and 8% B in 61 min. All-trans-astaxanthin and its two cis isomers, as well as five astaxanthin monoesters and 11 diesters were resolved within 60 min with a flow rate at 2 mL/min and detection at 480 nm. Astaxanthin diesters were found to contain 12 fatty acids, of which palmitic acid and stearic acid constituted a large portion, whereas astaxanthin monoesters were found to contain 10 fatty acids with arachidonic acid dominating. Solvent extraction could generate a higher content of trans-astaxanthin and astaxanthin esters, while SCD extraction could produce greater levels of 9-cis-astaxanthin and 13-cis-astaxanthin.     

基于改进主成分分析和AdaBoost算法的运动虾苗识别方法

针对虾行为量化过程中运动虾苗较难检测与识别的问题,该文以南美白对虾虾苗为例,提出了一种基于改进主成分分析(principal component analysis,PCA)+AdaBoost算法的运动虾苗自动识别方法。在室内自然光条件下,利用工业相机采集承装容器中虾苗的灰度图像。提取图像中大小为100×100像素的不同运动状态的虾苗图像,首先使用改进PCA算法进行主成分分析,并进行特征提取。根据特征参数的分布情况,对其进行归一化处理,利用归一化的特征构建多个弱分类器,利用Adaboost方法将弱分类器构建成强分类器。最后,利用强分类器对运动虾苗进行识别。试验结果表明,在150幅不同运动状态虾苗测试样本中,基于改进PCA+Adaboost方法的识别正确率98%,平均每个样本识别时间为0.027 898 s,满足行为量化中的自动识别要求。     

Uniformly (14)C-ring-labeled 2,4-dichlorophenoxyacetic acid: a metabolism study in bluegill sunfish, Lepomis macrochirus.

The fate of 2,4-dichlorophenoxyacetic acid (2,4-D), a mixture of [phenyl(U)-(14)C]-2,4-D and unlabeled 2,4-D, in bluegill sunfish was investigated after exposure to approximately 11 ppm under static conditions for 4 days. Total radioactive residues (TRR) in whole fish increased from 0.41 ppm on day 1 to 0.60 ppm on day 3. TRR levels in fillet (edible) and viscera (nonedible) of treated fish on day 4 were 0.41 and 1.9 ppm, respectively. Most residues in both matrices were acetonitrile soluble; small amounts were hexane soluble or unextractable with solvents. Acid and base hydrolyses with ethyl acetate partitioning were used to release the fillet unextractable residues. The identification of 2,4-D and 2,4-dichlorophenol (2,4-DCP) in the fillet was conclusively confirmed by GC-MS analysis. On the basis of the experimental data from this study, a metabolic pathway for 2,4-D in bluegill sunfish in which the 2,4-D is metabolized to 2,4-DCP and conjugates of 2,4-D and 2,4-DCP is proposed.     

氨基酸配合硼喷施对油麦菜硼营养及生长、品质的影响

为探究有机营养类物质氨基酸与硼酸配合喷施对油麦菜生长的应用效果,本研究采用盆栽试验,设置单独喷施1%硼酸处理,以及谷氨酸、丙氨酸和天冬氨酸3种氨基酸各设3个喷施浓度(5、10、20 mmol·L^-1) 与1%硼酸配合喷施处理,并以喷施清水为对照(CK),共11个处理,收获后测定植株的生长指标和硼含量。结果表明,单独喷施硼酸对油麦菜生物量无明显影响,低浓度氨基酸与硼配合喷施均能显著提高油麦菜生物量,其中丙氨酸-硼喷施处理油麦菜生物量较CK平均增加了9.4%。与CK相比,3种氨基酸与硼配合喷施均能明显提高油麦菜叶片总蛋白含量,同时降低硝酸盐积累,从而改善品质。与单独喷施硼酸相比,适宜浓度氨基酸与硼配合喷施均可显著提高油麦菜氮、钾积累量,另外谷氨酸-硼和天冬氨酸-硼喷施对油麦菜磷积累量也有明显促进作用。与单独喷施硼酸相比,氨基酸与硼配合喷施均可显著提高油麦菜地上部硼含量,其中丙氨酸-硼喷施处理油麦菜地上部硼含量平均增幅为41.0%;此外,丙氨酸-硼喷施对油麦菜地下部硼含量也有明显提升作用,平均增幅为15.6%;喷施丙氨酸浓度与油麦菜地上部和地下部硼含量均呈显著正相关关系。综合比较不同氨基酸与硼酸配合喷施对油麦菜的影响,可考虑将丙氨酸作为增效剂与硼肥配合施用,提高硼肥的应用效应。本研究为开发新型硼肥提供了理论依据。     

Determination of chlorate and chlorite and mutagenicity of seafood treated with aqueous chlorine dioxide.

The use of chlorine dioxide (ClO(2)) as a potential substitute for aqueous chlorine to improve the quality of seafood products has not been approved by regulatory agencies due to health concerns related to the production of chlorite (ClO(2)(-)) and chlorate (ClO(3)(-)) as well as possible mutagenic/carcinogenic reaction products. Cubes of Atlantic salmon (Salmo salar) and red grouper (Epinephelus morio) were treated with 20 or 200 ppm aqueous chlorine or ClO(2) solutions for 5 min, and extracts of the treated fish cubes and test solutions were checked for mutagenicity using the Ames Salmonella/microsome assay. No mutagenic activity was detected in the treated fish samples or test solutions with ClO(2). Only the sample treated with 200 ppm chlorine showed weak mutagenic activity toward S. typhimurium TA 100. No chlorite residue was detected in sea scallops, mahi-mahi, or shrimp treated with ClO(2) at 3.9-34.9 ppm. However, low levels of chlorate residues were detected in some of the treated samples. In most cases, the increase in chlorate in treated seafood was time- and dose-related.     

Liquid chromatographic determination of seven antioxidants in dry food

The liquid chromatographic determinative step of the official method for propyl gallate, trihydroxybutyrophenone, tert-butylhydroquinone, nordihydroguaiaretic acid, butylated hydroxyanisole (BHA), 3,5-di-tert-butyl-4-hydroxymethylphenol, and butylated hydroxytoluene (BHT) in fats and oils has been applied to their determination in a number of dry foods. A representative sample (10 g) is homogenized first with hexane (25 mL), then with 5 mL added water, and finally with 75 mL added acetonitrile. The hexane and acetonitrile are decanted, filtered, and separated; the hexane and rehydrated food are reextracted with 2 additional portions of acetonitrile, and the combined acetonitrile extracts are concentrated and diluted to 10 mL. An aliquot is analyzed as described in the official method, using a 150 x 4.6 mm 5 microns C-18 column. The need for rehydration to maximize the recovery of BHA and other antioxidants from marketplace dry food samples such as potato flakes, dry coffee whiteners, and dessert topping mixes was demonstrated. Rehydration was not required for cheese snacks, breakfast cereals, cake mixes, and some other foods. The need for rehydration should be determined by analyzing other foods with and without the addition of water. Potato and corn chips, popcorn and cheese snacks, breakfast cereals, dry beverage mixes, rice, potato flakes, french fried potatoes, and cake mixes were spiked with the above antioxidants at 10-50 ppm. Overall recoveries ranged from 64.3 to 105.6% and repeatabilities ranged from 0.7 to 10.8%. A total of 109 samples of the above foods were analyzed, and 64% contained detectable (greater than 1-2 ppm) antioxidants, mainly BHA and BHT.