ARTICLE pubs.acs.org/JAFC
Determination of Water-Soluble Arsenic Compounds in Commercial Edible Seaweed by LC-ICPMS Toni Llorente-Mirandes,† Maria Jose Ruiz-Chancho,†,§ Mercedes Barbero,‡ Roser Rubio,*,†,# and Jose Fermín Lopez-Sanchez†,# †
Department of Analytical Chemistry, Universitat de Barcelona, Martí i Franques 1-11, Barcelona E-08028, Spain Department of Plant Biology, Universitat de Barcelona, Avinguda Diagonal 643, Barcelona E-08028, Spain # Water Research Institute, Universitat de Barcelona, Avinguda Diagonal 684, Barcelona E-08034, Spain ‡
bS Supporting Information ABSTRACT: This paper reports arsenic speciation in edible seaweed (from the Galician coast, northwestern Spain) produced for human consumption. Chondrus crispus, Porphyra purpurea, Ulva rigida, Laminaria ochroleuca, Laminaria saccharina, and Undaria pinnatifida were analyzed. The study focused on arsenosugars, the most frequently occurring arsenic species in algae. As(III) and As(V) were also determined in aqueous extracts. Total arsenic in the samples was determined by microwave digestion and inductively coupled plasma mass spectrometry (ICPMS). For arsenic speciation, a water extraction especially suitable for arsenosugars was used, and the arsenic species were analyzed by liquid chromatography with both anionic and cationic exchange and ICPMS detection (LC-ICPMS). The total arsenic content of the alga samples ranged from 5.8 to 56.8 mg As kg1. The mass budgets obtained in the extracts (column recovery extraction efficiency) ranged from 38 to 92% except for U. pinnatifida (4%). The following compounds were detected in the extracts: arsenite (As(III)), arsenate (As(V)), methylarsonate (MA), dimethylarsinate (DMA), sulfonate sugar (SO3-sug), phosphate sugar (PO4-sug), arsenobetaine (AB), and glycerol sugar (Gly-sug). The highest concentrations corresponded to the arsenosugars. KEYWORDS: arsenic speciation, LC-ICPMS, edible seaweed, inorganic arsenic, arsenosugars
’ INTRODUCTION Marine algae have high contents of iodine, minerals, and vitamins and form part of the human diet, especially in Asian countries, due to their recognized therapeutic properties.1 Several countries cultivate seaweed for industrial purposes because they are used as a source of agar, carrageenans, and alginates; China, Korea, Japan, and the Philippines are the leaders in such production. Each algal species requires different farming methods as several factors, such as the morphology and regeneration capacity of the thallus, as well as the complex interactions between irradiance, temperature, nutrients, and water movements, are responsible for the success of large-scale seaweed production.2 In the European Union (EU), seaweeds are considered novel foods, defined as food that does not have a significant history of consumption within the EU before May 15, 1997,3 although it is a subject of controversy in some EU countries.4 Nowadays different types of edible seaweed are increasingly consumed in many European countries. In Spain, seaweed is not a widespread constituent of the diet, although the number of consumers has increased considerably in recent years. The edible seaweed sold in Spain is mainly cultivated in the northern area of the country, although it is also imported from Asian countries. Many types of seaweed are consumed raw or after only light cooking, for example, Porphyra spp. (red algae, commercialized as “nori”), which is frequently consumed and rich in proteins and vitamins B and C. However, Chondrus spp. (red algae, commercialized as “Irish moss”) is an industrial source of carrageenan, which is commonly used as a thickener and r 2011 American Chemical Society
stabilizer in milk products such as ice cream and processed foods including luncheon meats and is also commonly eaten raw in salads and cooked in soups. Ulva rigida (green algae, commonly called “sea lettuce”) is eaten raw in salads, cooked in soups, or served as a side dish to accompany fish or seafood; it is high in protein, soluble dietary fiber, and a variety of vitamins and minerals, especially iron. Brown algae, Laminaria spp. (generic commercial name, “kombu”, with more than 12 species), and Undaria spp. (generic name “wakame”) are consumed the most worldwide. Seaweed can absorb arsenic (mainly inorganic) from seawater and can accumulate and biotransform this arsenic into less toxic organo-arsenicals. The arsenic species usually identified in seaweed are arsenosugars (derivatives of dimethylarsinoylribosides and trimethylarsonioribosides).5,6 The structures of the four arsenosugars most reportedly found in algae are presented in Figure 1. As well as these compounds, other organo-arsenicals such as methylarsonate (MA), dimethylarsinate (DMA), trimethylarsine oxide (TMAO), the tetramethylarsonium ion (TETRA), arsenobetaine (AB), arsenocholine (AC), and inorganic arsenic (As(III) and As(V)) can also be found in some seaweed. Although arsenosugars are the most abundant arsenic compounds found in algae, some researchers have detected high contents of inorganic arsenic in some seaweed,7 mainly in Hizikia fusiforme.811 Speciation of arsenic in food analysis is Received: May 12, 2011 Accepted: November 14, 2011 Published: November 14, 2011 12963
dx.doi.org/10.1021/jf2040466 | J. Agric. Food Chem. 2011, 59, 12963–12968
Journal of Agricultural and Food Chemistry
ARTICLE
determined using microwave digestion and ICPMS. The certified reference material (CRM) NIES 9 Sargassum fulvellum was used throughout the study to assess the accuracy and the reliability of the analytical results.
’ MATERIALS AND METHODS Figure 1. Structures of the four arsenosugars commonly found in algae.
necessary to evaluate the toxicological risk, which is strongly related to the specific chemical molecule.12 The International Agency for Research on Cancer (IARC) considers inorganic arsenic compounds (arsenite and arsenate) highly toxic and classifies them as group I (human carcinogens).13 The same considerations are reported by the Joint FAO/WHO Expert Committee on Food and Additives14 and by the European Food Safety Authority.15,16 Organic forms of arsenic, such as arsenobetaine and arsenosugars, are considered to be nontoxic, although there are no reliable data on arsenosugars.17 Recent publications suggest that arsenosugars should be reported as potentially toxic arsenic compounds due to the fact that they are metabolized by humans and so far there are no conclusive results about their toxicity.11,18 Specific regulations for toxic elements in edible seaweed have been established in the United States with 3 mg kg1 (dw) as the maximum permitted inorganic arsenic.19 Other countries such as Australia and New Zealand have established different limits for inorganic As in seaweed: 1 mg As kg1 (dw).20 In the EU the Commission Regulations do not establish maximum levels for arsenic in food.21 France was the first European country to regulate the human consumption of seaweed as a nontraditional food substance, and the French limit for inorganic As in edible seaweed is 3 mg As kg1 (dw). Currently, 12 macroalgae (6 brown algae, 4 red algae, 2 green algae) and 2 microalgae are licensed in France as vegetables and condiments.19,22 There is no specific legislation regarding seaweed in Spain, and the only Spanish legislation concerns seaweed for animal consumption; it establishes a maximum level of 2 mg As kg1 (dw) for inorganic As and warns of the possible risk of H. fusiforme.23 The aim of the present study is to determine the total arsenic content, inorganic arsenic as well as organoarsenicals (some of them potentially toxic), in commercially available edible seaweed and to evaluate the safety and assess the risk associated with its consumption. This may contribute to increase the availability of reliable results, which will be necessary for establishing and implementing future EU directives on inorganic and organic arsenic compounds in edible seaweed and for further studies on risk assessment. This study also focuses on the speciation of arsenic in different types of edible seaweed. To carry out the study, six seaweed (Chondrus crispus (Irish moss), Porphyra purpurea (nori), Ulva rigida (sea lettuce) Laminaria ochroleuca (kombu), Laminaria saccharina (kombu), and Undaria pinnatifida (wakame)) were selected and purchased in retail stores in Barcelona (Spain). Water was used as the extracting reagent, and a coupled technique, LC-ICPMS, was applied to quantify the arsenic species detected using both anionic and cationic chromatographic systems. We used an extract from the seaweed Fucus serratus, in which four dimethylarsinoylribosides were previously identified and quantified,24 to identify the arsenosugars present in our seaweed samples. Total arsenic in the samples was also
Reagents and Standards. All solutions were prepared with doubly deionized water obtained from Millipore water purification systems (Elix & Rios) (18.2 MΩ cm1 resistivity and total organic carbon < 30 μg L1). Nitric acid (69%) (Panreac, Hiperpur), 98% formic acid (Panreac, p.a.), ammonium dihydrogen phosphate (Panreac, p.a.), 25% aqueous ammonia solution (Panreac, p.a.), pyridine (Scharlau, p.a.), and 31% hydrogen peroxide (Merck, Selectipur) were used. Stock standard solutions (1000 mg L1) were prepared as follows: arsenite, from As2O3 (NIST, USA, Oxidimetric Primary Standard 83d, 99.99%) dissolved in 4 g L1 NaOH (Merck, Suprapure); arsenate, from Na2HAsO4 3 7H2O (Carlo Erba) dissolved in water; MA, prepared from (CH3)AsO(ONa)2 3 6H2O (Carlo Erba) dissolved in water; and DMA, prepared from (CH3)2AsNaO2 3 3H2O (Fluka) dissolved in water. AC from (CH3)3As+(CH2)CH2OHBr was supplied by the “Service Central d0 Analyse” (CNRS Vernaison, France); arsenobetaine (AB) from (CH3)3 As+CH2COO was supplied by BCR, as CRM 626 standard solution; and TMAO was prepared from (CH3)3AsO (Argus Chemicals srl) dissolved in water. All of the stock solutions were kept at 4 °C, and further diluted solutions for the analysis were prepared daily. Arsenate, arsenite, DMA, MA, AC, TMAO, and AB were standardized against As2O3 (NIST Oxidimetric Primary Standard 83d) as our internal control. Arsenic standard solution from NIST High-Purity Standards with a certified concentration of 1000 ( 2 mg As L1 was used as the calibrant in the determination of total arsenic content using ICPMS. CRM NIES 9 Sargasso (S. fulvellum) seaweed, supplied by the National Institute for Environmental Studies (Japan), had a certified total arsenic content of 115 ( 9.2 mg As kg1. An aliquot of freeze-dried extract of F. serratus, containing the four common arsenosugars, that is, phosphate (PO4-sug), sulfate (SO4-sug), sulfonate (SO3-sug), and glycerol (Gly-sug),24 was used to identify the arsenosugar peaks in the chromatograms. Instruments. A microwave digestion system, Milestone Ethos Touch Control, with a microwave power of 1000 W and temperature control, was used for digestion. An Agilent 7500ce ICPMS with a microflow nebulizer (Agilent, Germany) was used to measure total arsenic content. For arsenic speciation, LC-ICPMS was used with an Agilent 1200 LC quaternary pump, equipped with an autosampler. The analytical columns, a Hamilton PRP-X100 (250 4.1 mm, 10 μm, Hamilton, USA) and Zorbax-SCX300 (250 4.6 mm, 5 μm, Agilent), were protected by guard columns filled with the corresponding stationary phases. Chromatographic conditions are reported.25 The outlet of the LC column was connected via PEEK capillary tubing to the nebulizer (BURGENER Ari Mist HP type) of the ICPMS system (Agilent 7500ce), which was the arsenic-selective detector. The ion intensity at m/z 75 (75As) was monitored using time-resolved analysis software. Additionally, the ion intensities at m/z 77 (40Ar37Cl and 77Se) were monitored to detect possible argon chloride (40Ar35Cl) interference at m/z 75. Samples. Dry algae (from the Galician coast) were purchased in a food market in Barcelona, Spain. Six seaweed samples were analyzed in this study: C. crispus, P. purpurea, U. rigida, L. ochroleuca, L. saccharina, and U. pinnatifida. Details of algal taxonomy can be found in the Supporting Information (Table SI-1). The samples were dried in an oven at 40 °C for 24 h and then ground to a fine powder in a tungsten carbide disk mill. 12964
dx.doi.org/10.1021/jf2040466 |J. Agric. Food Chem. 2011, 59, 12963–12968
0.017
0.030 0.058 0.031 0.025
0.009 0.007
0.016 0.024 LOQ (mg As kg 1)
0.005 0.007 LOD (mg As kg 1)
