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Simple Analysis of Total Mercury and Methylmercury in Seafood Using Heating Vaporization Atomic Absorption Spectrometry

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Simple Analysis of Total Mercury and Methylmercury in Seafood Using Heating Vaporization Atomic Absorption Spectrometry The Journal of Toxicological Sciences (J. Toxicol. Sci.) 489 Vol.41, No.4, 489-500, 2016 Original Article Simple analysis of total mercury and methylmercury in seafood using heating vaporization atomic absorption spectrometry Keisuke Yoshimoto1,2, Hoang Thi Van Anh1,2, Atsushi Yamamoto3, Chihaya Koriyama4, Yasuhiro Ishibashi2, Masaaki Tabata5, Atsuhiro Nakano1 and Megumi Yamamoto1,2 1Department of Basic Medical Science, National Institute for Minamata Disease, 4058-18 Hama, Minamata, Kumamoto 867-0008, Japan 2Graduate School of Environmental and Symbiotic Science, Prefectural University of Kumamoto, 3-1-100 Tsukide, Higashi-ku, Kumamoto 862-8502, Japan 3Department of Aquaculture, Graduate School of Fisheries, Kagoshima University, 4-50-20 Shimoarata, Kagoshima 890-0056, Japan 4Department of Epidemiology and Preventive Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan 5Graduate School of Science and Engineering, Saga University, 1 Honjo-machi, Saga 840-8502, Japan (Received March 24, 2016; Accepted May 12, 2016) ABSTRACT — This study aimed to develop a simpler method for determining total mercury (T-Hg) and methylmercury (MeHg) in biological samples by using methyl isobutyl ketone (MIBK) in the degreasing step. The fat in the samples was extracted by MIBK to the upper phase. T-Hg transferred into the water phase. This was followed by the extraction of MeHg from the water phase using HBr, CuCl2 and toluene. The MeHg fraction was reverse-extracted into L-cysteine-sodium acetate solution from toluene. The con- centrations of T-Hg and MeHg were determined by heating vaporization atomic absorption spectrome- try. Certified reference materials for T-Hg and MeHg in hair and fish were accurately measured using this method. This method was then applied to determine T-Hg and MeHg concentrations in the muscle, liv- er and gonads of seafood for the risk assessment of MeHg exposure. The mean T-Hg and MeHg concen- trations in squid eggs were 0.023 and 0.022 μg/g, and in squid nidamental glands 0.052 and 0.049 μg/g, respectively. The MeHg/T-Hg ratios in the eggs and nidamental glands of squid were 94.4% and 96.5%, respectively. The mean T-Hg and MeHg concentrations in the gonads of sea urchins were 0.043 and 0.001 μg/g, respectively, with a MeHg/T-Hg ratio of 3.5%. We developed an efficient analytical meth- od for T-Hg and MeHg using MIBK in the degreasing step. The new information on MeHg concentration and MeHg/T-Hg ratios in the egg or nidamental glands of squid and gonads of sea urchin will also be use- ful for risk assessment of mercury in seafood. Key words: Methylmercury, Heating vaporization atomic absorption spectrometry, Degreasing, Methyl isobutyl ketone, Seafood INTRODUCTION crossing the blood-brain barrier and/or the blood-pla- cental barrier (World Health Organization, 2010). Mer- Human beings are exposed to methylmercury (MeHg) curic toxicity is known to vary according to its chemical mainly through the consumption of seafood; there- form (World Health Organization, 2008). Determining the fore, determining the concentration of MeHg in sea- chemical form and the ratio of mercury compounds in tis- food is important for assessing the risks of MeHg expo- sues is, thus, also important for understanding the behav- sure (World Health Organization, 2008). MeHg is readily ior of MeHg and its toxicity in vivo. absorbed from the gastrointestinal tract and capable of In many studies, the total mercury (T-Hg) content of Correspondence: Megumi Yamamoto (E-mail: [email protected]) Vol. 41 No. 4 490 K. Yoshimoto et al. biological samples has been commonly analyzed using the upper organic solvent layer containing fat can be eliminat- atomic absorption method and MeHg by methods such as ed by an aspirator during the degreasing step, the analyti- gas chromatography-electron capture detector (GC-ECD). cal procedure can be expected to be simpler and faster for In this case, it is necessary to prepare two sets of appara- many routine sample treatments. Therefore, in the present tus and two samples for T-Hg and MeHg analysis. One study we aim to investigate replacing chloroform with method developed for the selective analysis of inorgan- methyl isobutyl ketone (MIBK) in the degreasing step. ic mercury (InHg) and MeHg used reductive vaporization MIBK may make it easier to remove fat using an aspira- atomic absorption spectrometry (Magos and Clarkson, tor because its specific gravity is lower than that of aque- 1972). This was under the premise that MeHg was the ous solutions. It also solubilizes fat components enough predominant chemical form of organic mercury in most to allow the upper phase to separate from the water phase. biological samples such as seafood and animal tissues. Furthermore, MIBK has already been used for atom- This experimental procedure was simpler than using gas ic absorption spectrometry owing to its lower level of chromatography. Organic mercury in natural biological contamination by metals (Cabrera-Vique et al., 2012; samples can be assumed to be MeHg, because only MeHg El-Mufleh et al., 2013; List et al., 1971). has been detected in natural biological samples, with the We will apply this modified method to determine the exception of human specimens treated with vaccines con- levels of T-Hg and MeHg in tissues of five species of taining sodium ethylmercuric thiosalicylate. The meth- seafood. Although many studies have investigated the od of Magos and Clarkson (1972) has, thus, been modi- concentrations of mercury in edible muscle (Ministry fied to become an analytical method for T-Hg and MeHg of Health, Labour and Welfare, 2010), little informa- in biological materials using heating vaporization atom- tion is available concerning mercury levels, especial- ic absorption spectrometry (Miyamoto et al., 2010). In ly MeHg, in seafood such as gonads, despite being com- contrast to the reducing-vaporization method, the heat- monly consumed (Atta et al., 2012; Donald and Sardella, ing vaporization atomic absorption spectrometry meth- 2010; Hammerschmidt et al., 1999; Joiris et al., 1999; od does not need complex pretreatment of samples, and Seixas et al., 2005; Watanabe et al., 2012; Webb et al., large volumes of waste liquid such as SnCl2 are not pro- 2006; Yamashita et al., 2005). In the present study, we will duced. The advantages of this method are that: (1) a sin- determine the concentration of T-Hg and MeHg in the mus- gle apparatus (heating vaporization atomic absorption cle, liver and gonads of several commercial fish, squid and spectrometry) is used for both T-Hg and MeHg determi- sea urchins using the currently modified method to obtain nation; (2) both T-Hg and MeHg can be measured using information for assessing the risk of MeHg exposure. the same biological sample in two consecutive steps; (3) only one standard solution needs to be prepared for T-Hg MATERIALS AND METHODS and MeHg in each experiment. Both T-Hg and MeHg can be analyzed using a commercial mercury standard solu- Reagents and fish samples tion (1,000-ppm HgCl2), which has the stability required Analytical grade reagents were used in this study. for a calibration curve; and (4) the protocol is easy and Five M sodium hydroxide (NaOH) solution, hydro- cost-effective compared with other methods such as gas gen bromide (HBr), copper (II) chloride dihydrate chromatography-inductively coupled plasma/mass spec- (CuCl2), nitric acid (HNO3), methyl isobutyl ketone trometry or liquid chromatography-inductively coupled (MIBK: atomic absorption spectrometry grade), hex- plasma/mass spectrometry. These methods require expen- ane (HPLC analysis grade), toluene (HPLC analy- sive apparatus and conditions (e.g. carrier gas) and high- sis grade) and L-cysteine (Cys) were purchased from er levels of experimental skills (Clémens et al., 2012; the Kanto Chemical Co. (Tokyo, Japan). Methylmercu- Rodrigues et al., 2010). Thus, there are many limitations ry chloride (MeHgCl; > 98% purity) was obtained from to conducting assessments of Hg exposure using these Tokyo Chemical Industry (Tokyo, Japan). Sodium ace- assay systems, especially in developing countries. tate trihydrate (NaOAc: Emsure grade) was purchased In our previous method, chloroform was used to elimi- from Merck (Darmstadt, Germany). The Hg standard nate the fat that inhibited the extraction of the MeHg frac- solution (Hg 1,000 mg/L, HgCl2 in 0.1-mol/L HNO3) tion, because it has a high degreasing ability (Miyamoto was purchased from Wako Pure Chemical Industries et al., 2010). The separated lower chloroform layer was (Osaka, Japan). Certified reference materials for MeHg removed using a micro-syringe. However, the bounda- were obtained: hair (NIES CRM No.13) from the ry between the aqueous layer containing mercury and National Institute of Environmental Studies (Tsukuba, the chloroform layer containing lipid was not clear. If the Japan), cod fish tissue (NMIJ CRM 7402-a No.250) and Vol. 41 No. 4 491 Simple method for total mercury and methylmercury analysis in seafood swordfi sh tissue (NMIJ CRM 7403-a No.165) from the (1 mL) were added to samples (0.6-1.5 g: wet) in 15-mL National Institute of Advanced Industrial Science and PP tubes then incubated in a block incubator at 80°C for Technology (Tsukuba, Japan). Ultra-pure quality water 1-2 hr with vortexing every 15 min. The resulting degen- was prepared using a Milli-Q system (Merck Millipore, erated solutions were cooled to room temperature in a Tokyo, Japan). Cys (0.1%) solution and Cys (0.2%)- water bath and their volumes made up to 5 mL with dis- NaOAc (2%) solution were freshly prepared for each tilled water (DW). MIBK (6 mL) was added to the sol- experiment. ubilized sample solutions to degrease them, followed by Fresh marine organisms (red seabream (Pagrus major), vigorous shaking using an SR-1 reciprocal shaker set at n = 10; golden threadfi n bream (Nemipterus virgatus), n = 220 rpm for 10 min (Taitec Corp., Nishikata, Japan) and 10; cutlass fi sh (Trichiurus lepturus), n = 10; bigfi n reef centrifugation (1,750 × g, 10 min).
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