The methylmercury to total mercury ratio in selected marine,freshwater, and terrestrial organisms

Total and methylmercury concentrations were determined in muscle and organ tissue from a wide variety of marine and terrestrial organisms spanning several trophic levels. Sediment and water samples from many of the tissue sampling sites were also analyzed to assess the degree of mercury contamination to which the animals were exposed. The methylmercury to total mercury ratios were examined to determine whether this ratio is indicative of elevated exposure to organic or inorganic mercury and how it varies relative to tissue type and position in the food chain. As an ancillary study, a subset of these tissues was analyzed as 1) wet tissue, and 2) freeze-dried, ball-milled tissue to determine whether the form of sample preparation can adversely affect mercury analysis. Results indicate that the methylmercury to total mercury ratios generally approach unity only in muscle tissue of higher food chain carnivorous fish residing in waters that are relatively uncontaminated with respect to inorganic mercury species. Herbivorous terrestrial mammals and low food chain marine organisms tend to have very low methylmercury to total mercury ratios. Marine animals placed higher on the food chain, such as crabs and lobsters, exhibit somewhat higher methylmercury to total mercury ratios and can exhibit a large variation in this ratio between, organ tissue and muscle tissue of the same animal. The samples analyzed as both wet and freeze-dried, ball-milled tissue indicate that freezedrying and ball-milling in no way result in mercury loss or contamination and, in fact, result in better replicate analyses and create a sample sufficiently stable to be archived for several years without refrigeration.     

Mercury and Methylmercury in Freshwater Fish and Sediments in South Korea Using Newly Adopted Purge and Trap GC-MS Detection Method

The use of purge and trap gas chromatography–mass spectrometry (GC-MS) technique for the determination of methylmercury in biological and sediment samples was described. The GC-MS detection system was combined with the dithizone extraction method for biological samples and the distillation method for sediment samples to alleviate matrix interference problems. The method was validated by analysis of CRMs such as SRM 966 (human blood), BCR 463 (tuna fish), IAEA 407 (fish), ERM CC580 (estuarine sediment), and IAEA 405 (sediment). The performance of the purge and trap GC-MS method was also tested on field samples of freshwater fish and sediment. The results were compared with those of the GC-ECD and the GC-CVAFS, which were used widely for methylmercury analysis. Additionally, total mercury and methylmercury levels in freshwater fish and sediments from various reservoirs and streams in Korea were measured to understand mercury contamination status in Korean peninsula. Methylmercury concentrations in freshwater fish were found to correlate with body weight, diet habit, and food availability. In sediment, total mercury concentrations correlated with methylmercury concentrations and organic matter such as %C and %S. However, no significant relationships between methylmercury and sediment organic matter have been found.     

Determination of Organic Mercury in Biota, Plants and Contaminated Sediments Using a Thermal Atomic Absorption Spectrometry Technique

A simple, rapid procedure for the determination of organic mercury in sediments, plants and fish tissues has been developed and validated. Extraction and separation of organic mercury compounds from the sample matrix was achieved by an established procedure based on an acid leaching of the sample (H₂SO₄/KBr/CuSO₄), followed by extraction of the organic mercury halide with toluene and back-extraction with an aqueous solution of thiosulphate. Detection and quantification of mercury, in the liquid extracts, was made by atomic absorption spectrometry (AAS), following thermal decomposition of the sample. The method was evaluated using Certified Reference Material (CRM) BCR 463 (tuna fish), BCR 580 (estuarine sediment), IAEA-140TM (sea plant homogenate) and NRCC TORT-2 (lobster hepathopancreas). The recovery factors for organic mercury in all tested CRM were between 81–107%. The precision of the method has relative standard deviations of less than 10% for sediments and fish tissues and of less than 16% for plant material. The method was successfully applied to natural samples of sediments, plants, macroalgae and fish tissues collected from an estuarine ecosystem and could, therefore, be used for routine analyses.     

Methylmercury accumulation in rice (Oryza sativa L.) grown at abandoned mercury mines in Guizhou, China

Mercury is a global pollutant that can transform into methylmercury, a highly toxic and bioaccumulative organic form. Previous surveys have shown that fish is the main source of human methylmercury exposure, whereas most other food products have an average value below 20 microg/kg and primarily in the inorganic form. This paper reports that methylmercury in rice (Oryza sativa L.) grown at abandoned mercury mining areas contained levels >100 microg/kg in its edible portion and proved to be 10-100 times higher than other crop plants. The daily adult intake of methylmercury through rice consumption causes abnormally high methylmercury exposure to humans. The results demonstrate that rice is a methylmercury bioaccumulative plant and the main methylmercury source for human exposure in the areas studied.     

Factors affecting the bioaccessibility of methylmercury in several marine fish species

Bioaccessibility refers to the maximum bioavailability of pollutant ingested with food, and its measurements can lead to a more accurate risk assessment as compared to the measurements of total concentrations of pollutant in food. This study examined the factors affecting the bioaccessibility of methylmercury (MeHg) in nine species of marine fish with an aim to identify ways of reducing MeHg bioaccessibility. MeHg bioaccessibility without any treatment in the nine species of marine fish ranged from 16.0 to 67.7%. Steaming, grilling, and frying reduced MeHg bioaccessibility by 29.4-77.4% for rabbitfish and 74.6-95.8% for grouper. Co-consumption of phytochemical-rich foods such as green tea decreased the bioaccessibility of MeHg by 72.2% for rabbitfish and 74.0% for grouper, whereas meso-2,3-dimercaptosuccinic acid increased it by 39.2-108% for rabbitfish and 45.3-75.7% for grouper. The bioaccessibilities of both MeHg and inorganic mercury were independent of the total Hg concentration and the exposure route (dietary vs dissolved). In eight of the nine species studied, bioaccessibility was negatively correlated with the extent to which MeHg was partitioned into the metal-rich granule fraction and the trophically available fraction. It was positively correlated with partitioning into the cellular debris fraction. This study demonstrated the important control of subcellular distribution in MeHg bioaccessibility.     

Determination of taurine in infant formulas using ultrafiltration and cation-exchange chromatography

A fast and simple method for determination of taurine in infant formulas has been developed. The sample preparation uses disposable ultrafiltration cartridges to remove protein and clarify the sample. Hydrolysis is avoided, simplifying the procedure and increasing efficiency. One mL sample is centrifuged in a cartridge for 45 min. The filtrate is diluted with pH 2.2 citrate buffer and injected into a high performance amino acid analyzer. A cation-exchange column (sodium phase) is used with a single buffer eluant and an isocratic chromatographic program. Colorimetric detection is performed following post-column ninhydrin reaction. Chromatographic resolution from other ninhydrin-positive compounds is excellent. Average recoveries for 3 levels of spike for various products were 100-102%. Precision is 1-3% RSD, depending on product. Linearity, specificity, and ruggedness are excellent. The method is applicable to quality control testing of milk-based, soy-based, and prehydrolyzed protein-based infant formulas in the ready-to-use, concentrate, and powder forms. A variety of commercially available infant formulas from different manufacturers were analyzed and all were found to contain taurine levels comparable to human milk. Some human milk and cow's milk samples were also analyzed and results compare well with literature values.     

Polybrominated diphenyl ethers in retail fish and shellfish samples purchased from Canadian markets

Fish and shellfish retail samples (n = 122) were purchased from three Canadian cities in the winter of 2002 and analyzed for a total of 18 polybrominated diphenyl ether (PBDE) congeners. The samples (salmon, trout, tilapia, Arctic char, mussels, oysters, shrimp, and crab) represented the range of fish and shellfish commercially available to Canadian consumers at the time of purchase. Trout and salmon (geometric mean SigmaPBDE = 1600 and 1500 pg/g, wet weight, respectively) were found to contain significantly higher amounts of PBDEs than the mussel, tilapia, and shrimp groups (geometric mean SigmaPBDE = 260, 180, and 48 pg/g, wet weight, respectively). These differences in SigmaPBDE concentrations among fish and shellfish products were partly driven by differences in lipid content among the samples. Mean SigmaPBDE concentrations in domestic samples were also significantly greater than in imported samples, possibly reflecting global environmental distribution of PBDEs. These concentration differences will contribute to variations in dietary exposure to PBDEs when assorted fish and shellfish items from various origins are consumed.     

Analysis of Flavan-3-ols and procyanidins in food samples by reversed phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (RP-HPLC-ESI-MS/MS)

Concentrations of the main dimeric and trimeric procyanidins (PC) and their monomeric constitutive units catechin (CT) and epicatechin (EC) were determined in food samples by using reversed phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (RP-HPLC-ESI-MS/MS). In a first step, 12 PCs (PC B1, B2, B3, B4, B5, B6, B7, B8, C1, C2, and A2 and cinnamtannin B1), of which most are not commercially available, were isolated from plant materials or synthesized and purified by a combination of column chromatographic separation techniques with different stationary phases. These PCs in combination with CT and EC were used as standard substances for identification and quantification during the following screening of food samples by RP-HPLC-ESI-MS/MS analysis. The main focus of the newly developed RP-HPLC-ESI-MS/MS method is the compensation of matrix effects by using the echo-peak technique simulating internal standard injection. The suitability of this new method was demonstrated by the determination of recovery rates being 90% or higher. Use of this method allowed the determination of patterns and concentrations of PCs in 55 food samples.     

Simultaneous determination of eight water-soluble vitamins in supplemented foods by liquid chromatography

A fast, simple, and reliable method for the isolation and determination of the vitamins thiamin, riboflavin, niacin, pantothenic acid, pyridoxine, folic acid, cyanocobalamin, and ascorbic acid in food samples is proposed. The most relevant advantages of the proposed method are the simultaneous determination of the eight more common vitamins in enriched food products and a reduction of the time required for quantitative extraction, because the method consists merely of the addition of a precipitation solution and centrifugation of the sample. Furthermore, this method saves a substantial amount of reagents as compared with official methods, and minimal sample manipulation is achieved due to the few steps required. The chromatographic separation is carried out on a reverse phase C18 column, and the vitamins are detected at different wavelengths by either fluorescence or UV-visible detection. The proposed method was applied to the determination of water-soluble vitamins in supplemented milk, infant nutrition products, and milk powder certified reference material (CRM 421, BCR) with recoveries ranging from 90 to 100%.     

Determinations of residual furazolidone and its metabolite, 3-amino-2-oxazolidinone (AOZ), in fish feeds by HPLC-UV and LC-MS/MS, respectively

The antibacterial drug furazolidone belonging to the group of nitrofuran antibacterial agents has been widely used as an antibacterial and antiprotozoal feed additive for poultry, cattle, and farmed fish in China. During application a large proportion of the administered drug may reach the environment directly or via feces. Although the use of furazolidone is prohibited in numerous countries, there are indications of its illegal use. It is known that furazolidone can be rapidly metabolized to 3-amino-2-oxazolidinone (AOZ) in the body of the target organism. In this study, a total of 21 fish feed samples, including 17 commercial fish feeds from local markets in China (representing 15 different formulations) and 4 fish feeds obtained from Germany and Turkey, respectively, are analyzed to determine whether the drug is still illegally used or commercially available feeds are contaminated by this drug. High-performance liquid chromatography (HPLC) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methods have been implemented to determine furazolidone and its metabolite AOZ in fish feeds containing animal protein, respectively. An efficient and convenient cleanup method for the determination of furazolidone in fish feeds is developed, and a simple cleanup method for the determination of AOZ is used. Method recoveries for samples used were determined as 87.7-98.3% for furazolidone at two spike levels of 2.0 and 5.0 ng g-1 and as 95.6-102.8% for AOZ at spike levels of 0.4 and 0.8 ng g-1. Limits of detections were 0.4 ng g-1 for furazolidone and 0.05 ng g-1 for AOZ. The established methods are therefore suitable for the determination of furazolidone and its metabolite AOZ in fish feeds at trace contamination levels. Using the established methods, all fish feed samples have been proved to be furazolidone negative; however, AOZ is tested in 16 of 17 fish feeds obtained from local markets in the Hubei province of China, with a positive rate as high as 94.1%.     

Liquid chromatographic determination of benzo[a]pyrene in total particulate matter of cigarette smoke

The benzo[a]pyrene (BaP) delivery of reference and commercially available tobacco cigarettes, as well as reference and placebo marijuana cigarettes, is determined using a sequential liquid chromatographic/liquid chromatographic procedure. The total particulate matter of sample cigarette smoke is collected using a Cambridge filter pad, which is ultrasonically extracted with acetone. The resulting extract is filtered, then fractionated using semipreparative-scale normal phase liquid chromatography (LC). Quantitative determination is achieved using analytical-scale reverse phase LC equipped with a fluorescence detector. The method is precise (+/- 10-15% relative standard deviation) and yields 85% or better BaP recovery at the ng/cig. level. A single pad may be analyzed in 8 person-hours, while a more typical lot of 12 pads (6 pads each for 2 cigarette brands) may be analyzed in 10 person-days.     

Use of solid phase Florisil cartridges to separate fat from semivolatile organic compounds in adipose tissue

A quick method for separation of semivolatile organic compounds from fat in adipose tissue has been developed. This method uses commercially available solid phase cartridges for sample cleanup. The results indicate that the recoveries, from hexane-extracted fat, of 4 representative classes of organic compounds range from 86.2 to 116%. The solid phase cartridges provide excellent separations of the fat from the analytes; no extraneous interference peaks were detected in the gas chromatograms. The method requires only 0.1 g sample and is quick and simple to use. Although results are reported for samples containing 1-14 ppm, the final extract can be concentrated to a volume allowing detection between 10 and 100 ppb.     

Fish species identification in surimi-based products

Whole fish morphologically identified as belonging to Theragra chalcogramma, Merluccius merluccius, Merluccius hubbsi, and Merluccius capensis and 19 fish products commercialized as surimi with different commercial brands and labeled as T. chalcogramma were analyzed by direct sequence analysis of the cytochrome b gene. A phylogenetic analysis of surimi products was performed as well. Results demonstrated that mislabeling is a large-scale phenomenon, since 84.2% of surimi-based fish products sold as T. chalcogramma (16/19) were prepared with species different from the one declared. In fact, only three samples (samples 15-17) were found to belong to T. chalcogramma. In the remaining samples, Merluccidae (samples 4-14), Gadidae (samples 18 and 19), Sparidae (sample 1), and Pomacentridae (samples 2 and 3) families were detected. A phylogenetic tree was constructed, and the bootstrap value was calculated. According to this methodology, 11 samples were grouped in the same clade as Merluccius spp.     

Determination of polybrominated diphenyl ethers and polybrominated dibenzo-p-dioxins/dibenzofurans in marine products

Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants in plastics and textile coatings, and these compounds have been recognized as ubiquitous environmental contaminants. Furthermore, it is considered a serious problem that polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/DFs), having toxicities similar to those of chlorinated dioxins, are generated by the manufacture of brominated flame retardants (BFRs) such as PBDEs, and formed by the combustion of substances containing BFRs. Several congeners of PBDD/DFs and PBDEs have been detected in the adipose tissue of the Japanese. Although food is suspected as an exposure source, little information is available regarding the levels of these brominated compounds in food, as compared with information regarding dioxin or polychlorinated biphenyls. It is necessary to investigate the levels of these brominated organic compounds in various foods and to estimate their influence in the case of human exposure. We developed an efficient method of analyzing PBDEs and PBDD/DFs contents in food samples using accelerated solvent extraction and determined the concentrations in several marine products such as raw fish, processed foods, and seaweed purchased in Japan. A recovery test (n = 5) using the method and involving dried fish showed acceptable recoveries of 57.7-78.5% (RSD 5.4-15.9%) for PBDEs and 50.0-56.4% (RSD 1.5-7.9%) for PBDD/DFs. In the analysis of marine product samples, several congeners of PBDEs were detected in raw fish, processed fish, and seaweed; the highest concentration of sigmaPBDEs was detected in yellowtail (1161 pg/g whole basis), followed by mackerel (553.5 pg/g whole basis). The most dominant congener present in these marine samples was 2,2',4,4'-tetraBDE (#47).     

Food as the Dominant Pathway of Methylmercury Uptake by Fish

A field experiment was conducted to determine the degree to which fish accumulated methylmercury (MeHg) via their food or via passive uptake from water through the gills. Finescale dace (Phoxinus neogaeus) were held in 2000 L enclosed pens floating in an undisturbed, oligotrophic lake in northwestern Ontario. Fish were exposed to water containing either low (0.10–0.40 ng L^-1), intermediate (0.45–1.30 ng L^-1), or high (0.80–2.1 ng L^-1) concentrations of MeHg. Zooplankton with either low (0.16–0.18 µg g^-1 d.w.) or high (0.28–0.76 µg g^-1 d.w.) concentrations of MeHg were added daily to each pen. Fish fed zooplankton with high concentrations of MeHg had significantly higher concentrations of mercury in muscle after 32 days than fish fed zooplankton with low concentrations of MeHg (ANCOVA, P<0.0001). Fish feeding on zooplankton with low concentrations of MeHg had the same amount of Hg in their tissues as fish at the start of the experiment. Uptake from water was at most 15%. This is the first experiment to confirm that food is the dominant pathway of MeHg bioaccumulation in fish at natural levels of MeHg.     

Cloud point extraction preconcentration prior to high-performance liquid chromatography coupled with cold vapor generation atomic fluorescence spectrometry for speciation analysis of mercury in fish samples

A cloud point extraction methodology was developed for simultaneous preconcentration of Hg(II), methylmercury (MeHg), ethylmercury (EtHg), and phenylmercury (PhHg) prior to reversed-phase high-performance liquid chromatography (HPLC) on-line coupled with cold vapor atomic fluorescence spectrometry for speciation analysis of mercury in fish. The four mercury species were taken into complexes with ammonium pyrrolidine dithiocarbamate (APDC) in aqueous nonionic surfactant Triton X-114 medium and concentrated in the surfactant-rich phase by bringing the solution to the temperature of 40 degrees C. Baseline separation of the enriched complexes was achieved on an RP-C(18) column with a mixture of methanol, acetonitrile, and water (65:15:20, v/v) containing 200 mmol L(-1) HAc (pH 3.5) as the mobile phase. An on-line postcolumn oxidation of the effluent from HPLC, in the presence of K2S2O8 in HCl, was applied in the system followed by an optimal cold vapor generation of mercury species. The variables affecting the complexation and extraction steps were examined. The preconcentration of 10 mL of solution with 0.08% w/v Triton X-114 and 0.04% w/v APDC at pH 3.5 gave enrichment factors of 29, 43, 80, and 98 for MeHg, EtHg, PhHg, and Hg(II), respectively. Low detection limits (S/N = 3) were obtained, ranging from 2 to 9 ng L(-1) (as Hg) for all species. The developed method was successfully applied to the speciation of mercury in real fish samples.     

鱼油微胶囊射流空化制备工艺优化及理化性质研究

研究目的是以大豆分离蛋白为壁材,对射流空化制备鱼油微胶囊的工艺进行优化。采用喷雾干燥法,研究壁材添加量、乳化剂添加量、芯材添加量及射流空化处理时间对乳液稳定性和鱼油微胶囊包埋率的影响,通过响应面试验分析各因素,得到最优微胶囊制备工艺,并对制备的微胶囊产品同市售产品的结构、理化特性及稳定性进行对比分析。结果表明:在壁材添加量3.21%、乳化剂添加量0.21%、芯材添加量19.70%、射流空化处理时间11.25 min工艺条件下得到的乳液稳定性较好,鱼油微胶囊的包埋率达到94.14%。微胶囊产品微观结构呈球形颗粒,结构致密,颗粒形态完整,粒径小,表面含油率较低,包埋效果好;水分含量为3.07%,溶解度为96.30%,休止角为40.39?,溶解性较好;差示扫描量热分析结果显示,微胶囊热溶解温度较高,可用于常温贮藏。包埋后的鱼油经加速贮藏试验表明微胶囊化可以提高鱼油的氧化稳定性,延长鱼油贮藏期。     

A mass spectrometric validated high-performance liquid chromatography procedure for the determination of folates in foods

A series of five food reference materials (RM) that had certified values of folate concentrations and five frozen food samples were analyzed for 5-methyltetrahydrofolic acid (5-MTHFA) and folic acid (FA) using a high-performance liquid chromatography (HPLC) method with fluorescence detection that was validated using an HPLC mass spectrometry (MS) method with electrospray ionization. Identical sample specimens were extracted and analyzed in triplicate using both instrumental methods, and a comparison was made of the mean values of 5-MTHFA and FA resulting from these determinations. The analytes were isolated on either a high capacity strong anion exchange solid phase extraction column (HPLC method) or a phenyl Bond Elut column (MS method) prior to analyses. For quantification of the analytes by MS, (13)C-labeled 5-MTHFA and FA were added to samples as internal standards prior to enzymatic digestion and conversion of the polyglutamate forms of 5-MTHFA to the monoglutamic acid. Quantification of FA and 5-MTHFA using the HPLC analysis was carried out using external standards. With the exception of one RM (pig liver), the values established for 5-MTHFA using these methods were highly comparable. In determining the variance associated with these two procedures, it was observed that the mean relative standard error for 5-MTHFA was 12 (range, 2-27%) and 11% (range, 5-25%) for the HPLC and MS methods, respectively. FA was detected in only three of the samples, and the values obtained for it by either method were similar. This is the first paper that describes a mass spectrometric method used in the validation of an HPLC determination of food folates across a wide range of sample matrixes. The comparable values for 5-MTHFA and FA suggest that HPLC analysis with fluorescent detection may be used to accurately quantify folates present in a variety of food matrixes.     

Determination of phylloquinone and menaquinones in animal products with fluorescence detection after postcolumn reduction with metallic zinc

A high-performance liquid chromatographic (HPLC) method for the determination of phylloquinone and menaquinones in foods of animal origin is described. The K vitamers were quantified with a fluorescence detector after postcolumn reduction with metallic zinc using K1(25) as an internal standard. Extraction was done either with 2-propanol-hexane (meat and fish products) or with acid hydrolysis method (dairy products). Prior to quantification, sample extracts were purified by semipreparative HPLC; in addition, the fats of cheese and rainbow trout samples were removed with lipase hydrolysis. By this method the phylloquinone and menaquinones (MK-4 to MK-10) present in a few representative samples of different animal food groups were determined. HPLC-MS was used to confirm the identification of K vitamers. Long-chain menaquinones were found from bovine and pig livers as well as from various cheeses. The total vitamin K contents calculated as the sum of quantified K vitamers were in general low (mean content 10-100 ng/g); the highest amount was analyzed in chicken meat (600 ng/g).     

Development of a Simple Method for Evaluation of Water Absorption Rate and Capacity of Rice Flour Samples

A simple method employing a commercially available canister was developed for the determination of mode of water absorption of rice flour samples. The samples prepared by four different grinding methods were used to analyze water absorption. The total amount of water in a flour sample was described by using an exponential model. Capacity and rate of water absorption of the samples were determined, and the relationship to baking quality of partially substituted rice bread was investigated. The water absorption was highly dependent on the method of grinding. Flours produced by wet jet‐milling of the grains, which absorbed a small amount of water at high speed, were most suitable for rice bread. The method was applicable to other food powders, provided that flour particles do not stick together or swell immediately upon contact with water.