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Speciation Analysis of Arsenics in Commercial Hijiki by High Performance Liquid Chromatography-Tandem-Mass Spectrometry and High

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Speciation Analysis of Arsenics in Commercial Hijiki by High Performance Liquid Chromatography-Tandem-Mass Spectrometry and High Journal of Health Science, 56(1) 47–56 (2010) 47 Speciation Analysis of Arsenics in Commercial Hijiki by High Performance Liquid Chromatography-tandem-mass Spectrometry and High Performance Liquid Chromatography-inductively Coupled Plasma Mass Spectrometry Yasuyo Shimoda,a Yoshihiro Suzuki,b Yoko Endo,b Koichi Kato,a Mariko Tachikawa,a Ginji Endo,c and Kenzo Yamanaka∗, a aEnvironmental Toxicology and Carcinogenesis Research Unit, Nihon University College of Pharmacy, 7–7–1 Narashinodai, Fun- abashi, Chiba 274–8555, Japan, bResearch Center for Occupational Poisoning, Tokyo Rosai Hospital, 4–13–21 Ohmori-minami, Ohta-ku, Tokyo 143–0013, Japan and cDepartment of Preventive Medicine and Environmental Health, Osaka City University Medical School, 1–4–3, Asahimachi, Abeno, Osaka 545–8585, Japan (Received August 4, 2009; Accepted December 3, 2009; Published online December 4, 2009) Edible brown alga (hijiki in Japanese, Hijikia fusiforme) contains not only a high content of inorganic arsenic (iAs) but also various arsenosugars (AsSugs) which are metabolized to dimethylarsinic acid (DMA) in mammals. Since DMA is considered to be carcinogenic in rodents, it is necessary to accurately measure the contents of AsSugs as well as iAs for the risk assessment of seaweed consumption. Seven commercially available dried-hijiki products and two raw hijiki products were analyzed. Total-As was measured by inductively coupled plasma mass spectrometry (ICP-MS) with the Dynamic Reaction Cell (DRC) mode after acid-digestion. After water-extraction, AsSugs were detected by HPLC-MS/MS with multiple reaction monitoring in the positive ion mode and speciation analysis of arsenics was performed by HPLC-ICP-MS. The ranges of total-As obtained by acid digestion (A-TAs) in nine hijiki samples were 37.1–118.6 µg As/g dry weight (dw), and those of water extracted total-As (W-TAs) were 18.4–81.0 µg As/g dw. The ratios of water extracted iAs (W-iAs) to A-TAs ranged from 24.5 to 60.1%. The major compound detected was arsenate in all samples (8.9–70.5 µg As/g dw). Dimethylarsenosugar sulfate, AsSug 408, showed the highest peak among AsSugs detected. The content ratio of water extracted AsSugs (W-AsS) to A-TAs was estimated to be from 3.7 to 27.6%. The contents of A-TAs, W-iAs and W-AsS varied depending on the hijiki product. HPLC-MS/MS detected AsSugs more sensitively than HPLC-ICP-MS. Since iAs could not be detected by HPLC-MS/MS, combined analysis consisting of HPLC-MS/MS and HPLC-ICP-MS is necessary for accurate determination of arsenic species in seaweed products and also for the toxicological evaluation of AsSugs. Key words ——hijiki, arsenosugar, HPLC-MS/MS, inorganic arsenic, dimethylarsinic acid INTRODUCTION advised consumers to avoid eating hijiki.5) The iAs concentration in hijiki depends on the producing The consumption of seaweed as a diet food has district and manufacturing method.6, 7) Five oxo- been increasing in recent years in Western coun- arsenosugars (oxo-AsSugs) have been detected in 1–3) tries. The edible brown alga Hijikia fusiforme, hijiki as well as other seaweeds.8–10) In humans, called hijiki in Japanese, has a high content of in- oxo-AsSug is metabolized and excreted into urine organic arsenic (iAs). Therefore, in 2004, the Food as dimethylarsinic acid (DMA) and several oxo- Standards Agency (FSA) of the United Kingdom4) or thio-dimethylarsenic compounds.11, 12) In 2004, ∗ the International Agency for Research on Cancer To whom correspondence should be addressed: Environmen- (IARC)13) declared that there is sufficient evidence tal Toxicology and Carcinogenesis Research Unit, Nihon Uni- indicating that DMA, a major metabolite of iAs in versity College of Pharmacy, 7–7–1 Narashinodai, Funabashi, Chiba 274–8555, Japan. Tel. & Fax: +81-47-465-6077; E- mammals, is carcinogenic in animal experimental mail: [email protected] models. Therefore, in order to assess the risk from C 2010 The Pharmaceutical Society of Japan 48 Vol. 56 (2010) hijiki ingestion, both AsSugs and iAs should be de- and packed by a Japanese provider. Two raw hi- termined. Arsenic speciation analysis in biological jiki samples were harvested on the seashores of Am- samples is generally performed using HPLC induc- atsu and Kominato, respectively, which are located tively coupled plasma mass spectrometry (HPLC- on the Boso peninsula of Chiba prefecture. Each ICP-MS).14–16) Recent publications have reported raw sample was immediately washed with fresh wa- the usefulness of HPLC with tandem mass spec- ter, sealed in an airtight plastic bag, and then stored trometry (HPLC-MS/MS) for the identification and by freezing at −80◦C until analysis. The manufac- determination of organoarsenic compounds in bio- turing methods for commercial dried-hijiki can be logical samples.17–19) These analytical apparatuses broadly divided into two systems, one that involves have made it possible to detect known arsenic com- a boiling system in a large vessel, and the other pounds in samples without standard compounds and a closed or open steam-system.22) In Japan, major to estimate As concentration using appropriate stan- manufacturing methods for commercial dried-hijiki dards.20, 21) basically use a steam system.22) A system involv- In this study, we determined total arsenic and ing boiling has been used traditionally in South Ko- water-extracted iAs, and estimated the AsSugs con- rea and China.22) Moreover, there are two types of tents of 7 commercially available dried-hijiki prod- commercial dried-hijiki, naga-hijiki, which consists ucts and two raw hijiki products using HPLC-ICP- of the entire seaweed plant, and me-hijiki, which is MS and HPLC-MS/MS for the speciation analysis the sprouts of the seaweed.22) of arsenics in hijiki products from various regions Preparation of the Hijiki Samples —— The com- of Japan and elsewhere in east Asia produced using mercial dried-hijiki samples and the lyophilized raw several different production systems. hijiki samples were finely ground to powder using a food processor. For total-As analysis, twenty mil- ligrams of the powdered hijiki samples was digested MATERIALS AND METHODS by adding 0.5 ml of HNO3 and 0.5 ml of H2SO4 and then kept at 330◦C for 1 hr using a dry block Chemicals —— Sodium arsenite (AsIII), sodium bath. After cooling, the solutions were adjusted to arsenate (AsV), methanearsonic acid (MMA), and 50 ml with 0.1 M HNO3 and their total arsenic con- arsenobetaine (AsBe) were purchased from Wako centrations were determined by ICP-MS using the Pure Chemical (Osaka, Japan). DMA, trimethy- Dynamic Reaction Cell (DRC) mode. larsine oxide (TMAO), and arsenocholine (AsCho) For speciation analysis, powdered hijiki sam- were obtained from Tri Chemical Laboratory (Ya- ples (0.5 g) swollen by the addition of 20 ml of ultra- manashi, Japan). Germanium standard solution pure water were treated with an ultrasonic cell dis- (Kanto Chemical, Tokyo, Japan) was used as an ruptor (Misonix, Inc., Farmingdale, NY, U.S.A.) internal standard for HPLC-ICP-MS analysis. Ni- for 1 min, and centrifuged at 3000 rpm for 15 min. tric acid (HNO3, TAMAPURE AA-100, 68%, Tama The supernatant collected was adjusted to 50 ml by Chemicals, Kawasaki, Japan) and H2SO4 [for Ultra- adding ultra-pure water. The supernatant was ap- trace Analysis (Ultratrace Anal.), 97%, Wako Pure plied to speciation analysis of arsenic compounds Chemical] were used for sample treatment. Ammo- by HPLC-ICP-MS and by HPLC-MS/MS. nium bicarbonate (NH4HCO3, Wako Pure Chemi- Determination of Total Arsenic Concentra- cal) was used for the HPLC mobile phase. Ultra- tion —— The acid digested solution was diluted pure water was purified by Milli-Q Element A-10 to 50 times with buffer containing 0.5% aqueous with Quantum ICP cartridges (Millipore, Tokyo, ammonia, 2 mM EDTA, and 0.8 mM TritonX-100. Japan). A certified reference material, CRM No. 18 The determination was performed by the standard (human urine), obtained from the National Institute addition method. The diluted solution was intro- for Environmental Studies (NIES), Tsukuba, Japan, duced into an Elan DRCII ICP-MS (PerkinElmer was used to validate the procedure used for analysis. SCIEX, Concord, Ontario, Canada) using the DRC Samples of Commercial Dried-hijiki and Raw- mode. The instrument settings were as follows: hijiki —— As shown in Table 1, nine hijiki sam- radio-frequency (RF) power 1300 W, argon plasma ples derived from six different producing districts gas flow 15 l/min, auxiliary flow 1.2 l/min, and were collected from six stores in each producing nebulizer flow 1.0 l/min. A coaxial-type nebu- district. Two of the commercial samples were im- lizer was used; skimmer and sample cones were ported from China (Hong Kong) and South Korea platinum, and elemental As was measured at m/z No. 1 49 Table 1. Hijiki Producing Areas and Production Systems Used for Their Dried-products Sample Producing area Production system 1 me-hijiki (sprouts of weed) China (Hong Kong) unknown 2 me-hijiki (sprouts of weed) South Korea boiling systema) 3 me-hijiki (sprouts of weed) Mie prefecture (Japan) boiling systema) 4 naga-hijiki (whole of weed) Ohita prefecture (Japan) closed-steam system 5 me-hijiki (sprouts of weed) Ohita prefecture (Japan) closed-steam system 6 naga-hijiki (whole of weed) Amatsu (Japan) open-steam system 7 naga-hijiki (whole of weed) Kominato (Japan) open-steam system 8 raw hijiki (whole of weed) Amatsu (Japan) 9 raw hijiki (whole of weed) Kominato (Japan) a) The production systems used as printed on their packages. Table 2. Fragmentation Parameters of MRM for Arsenic Compounds24) compounds Precursor ion Product ion Cone voltage Collision (m/z)(m/z) (V) energy (eV) DMA 138.9 90.8 30 24 MMA 140.9 90.8 30 30 AsBe 179.0 119.9 30 24 AsSug 254 255.0 96.9 25 30 AsSug 328 329.0 96.9 25 32 AsSug 391 392.0 96.9 25 30 AsSug 392 393.0 96.9 25 32 AsSug 408 409.0 96.9 20 38 AsSug 482 483.0 96.9 30 36 of 75 and 77.
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