Article
Exposure to arsenic (As) in humans mainly occurs via the food chain [Ackley et al., 1999; Gallagher et al., 2002; Soros et al., 2003], especially in the form of inorganic As (iAs), such as arsenite (AsIII) and arsenate (AsV), which are classified as human carcinogens [IARC, 2004]. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has established the provisional tolerable weekly intake level of iAs as 15.0 g/kg bw per week (equivalent to 2.1 g/kg bw per day) [WHO, 1988]. Because the toxicity of As in food depends on the iAs content, and not that of total As [Benramdane et al., 1999], iAs content may be regarded as the regulatory limit of As in food. However, at present, as determined by JECFA, there is no sufficient basis to decide the maximum level of As due to the lack of suitable analytical methods. Therefore, specific data for As is urgently needed to address the large difference of toxicity to humans among various forms of As [WHO, 2009]. Rice is considered as an agricultural commodity containing higher iAs content, especially AsIII, than other cereals such as wheat and barley [Su et al., 2010]. However, speciation studies have been widely performed only on As in marine products [Hirata and Toshimitsu, 2005; Park et al., 2005; Koch et al., 2007]; thus,
*Corresponding author Phone: +82-31-290-0577; Fax: +82-31-290-0508 E-mail: mink1114@korea.kr doi:10.3839/jksabc.2010.096
speciation studies of As in rice are limited [Schoof et al., 1999; Pizarro et al., 2003; DAmato et al., 2004]. Here, we evaluated an As speciation method using the methanol-water digestion method applied to polished rice. The results allowed a more accurate assessment of human risk associated with the consumption of polished rice.
Arsenic species in rice Table 1. HPLC-ICP-MS operating conditions for the analysis of As species Descriptions RF power Nebulizer Plasma gas Auxiliary gas Nebulizer gas Selected isotopes (m/z) Anion exchange column Gradient mobile phase A Gradient mobile phase B Gradient profile Flow rate Sample injection volume Re-equilibration time Sample preparation Sample filtration Total analysis time Conditions
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1.3 kW Concentric type 10.00 L/min 0.50 L/min 0.95 L/min 75 (77, 82) Hamilton Column PRP-X100, 4.1 mm240 mm, 10 m 20 mM Ammonium bicarbonate, pH 8.2 15 mM Ammonium nitrate, Ammonium phosphate, pH 7.2 5 min at 100% A; Step to 100% B for 3 min; Step to 100% A for 7 min; 5 min at 100% A (Run time: 15 min) 1.5 mL/min 100 L 25 min 50:50 (MeOH:D.W.) Oasis Max Cartridge, 30 m 45 min
fine powder by a homogenizer (Ace Homogenizer, Nihonseiki Kaisha Ltd., Tokyo, Japan) for 1 min three times with intervals of 3 min and stored in a freezer at 20oC before analysis. Extraction procedure for samples. The modified extraction method of Pizarro et al. [2003] was used for speciation of As. One gram ground sample was mixed with 10 mL methanol-water (1:1) mixture containing 1% HNO3 in a 50-mL capped high-density polyethylene (HDPE) centrifuge tube (Becton Dickinson, NJ), and the mixture was sonicated for 30 min. The extracts were centrifuged at 6,000 rpm for 5 min, and the supernatant was filtered through a 30-m anion exchanger (Oasis Max cartridge, Waters, MA, USA). Extracts were stored at 4oC prior to analysis. Determination of As species by HPLC-ICP-MS. The HPLC apparatus (Agilent 1100 Series, Agilent Technologies, Tokyo, Japan) was equipped with a PRP-X100 anion exchange column (250 mm4.1 mm i.d., 10 m; Hamilton, Reno, NV). An Agilent 7500a ICP-MS system (Agilent Technologies) was used. The operating conditions and the elution program are given in Table 1. The gradient mobile phases were 20 mM ammonium bicarbonate adjusted to pH 8.2, 15 mM ammonium nitrate, and ammonium phosphate adjusted to pH 7.2. The flow rate was 1.5 mL/ min. Seven calibration standards of each species were prepared by serial dilutions of the starting compounds in laboratory pure water: 0.1, 0.5, 1, 5, 10, 30, and 50 ng/ mL. Determination of total Arsenic. Ground sample (0.5 g) was accurately weighed into a 50-mL polypropylene
digest vessel, and 9 mL of 70% nitric acid and 1 mL of hydrogen peroxide were added [Zavala and Duxvury, 2008]. Samples were extracted in a microwave oven (CEM Mars, CEM Corp, Matthews, NC). After digestion, extracts were cooled to room temperature, and water was added to adjust the volume to 50 mL. The total content of As was measured by an Agilent 7500a ICP-MS (Agilent Technologies). Efficiency of extraction. Efficiency of extraction (%) was evaluated as the ratio of extracted As content to the total As content. This method was checked using the National Institute of Standards and Technology (NIST; Gaithersburg, MD) certified 1568a rice flour as the standard reference material.
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Fig. 1. HPLC-ICP-MS chromatograms of four standard As compounds. Concentrations of standard As compounds, 30 ng As/mL, (b) Polished rice sample treated by methanol-water digestion method
shows the results of validation method in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision. As shown in, four As species were quantified via a linear calibration plot (0.965 -0.999) established by using each standard solution having As concentrations of 0.1, 0.5, 1, 5, 10, 30, and 50 ng/mL (Table 3). The LOD and the LOQ values of AsIII, AsV, MMA and DMA were 0.1, 0.1, 0.5, and 0.1 g/kg, and 0.5, 0.5, 1.0, and 0.5 g/kg, respectively. Accuracy was evaluated by determining the recovery of arsenic species from five replicated analyses. Four As species were added to the polished rice at a final concentration of 25.0 g/kg on dry weight basis. The recoveries of AsIII, AsV, MMA, and DMA were 103.1, 112.4, 88.6, and 80.3% (data not shown), respectively, suggesting that this method can be applied to polished rice. Precision was evaluated by calculating the coefficient of variance (CV) based on ten replicated analyses of a sample with a total As concentration of 50.0 g/kg on dry
weight basis. CVs obtained for AsIII, AsV, MMA, and DMA were 2.79, 4.53, 6.25, and 10.27%, respectively, suggesting a good reproducibility. The efficiency of extraction using a methanol-water mix was evaluated by using Certified Reference Material (CRM). However, no certified CRM was commercially available for As speciation. Thus, NIST rice flour has been used for As speciation in a number of studies, which were reviewed by Zhu et al. [2008]. The recovery of total As from NIST rice flour CRM was 309.24 g/kg (Table 2), showing 106.5% recovery of its certified value of 290 3 g/kg. Furthermore, the characterization of this CRM for the present study had much higher extraction efficiency than the previously reported As speciation methods, in which this CRM was extracted by using a trifluoracetic acid extraction method (80.0%) or ultrasonic and enzymatic hydrolysis method (79.7%) [Sanz et al., 2005; Williams et al., 2005; Zhu et al., 2005]. Application of CRM to polished rice samples. CRM was applied to eleven polished rice samples cultivated
Table 2. The results of validation in this analytical method for As species Validation items Linearity Precision LODa LOQb NIST SRM 1568ac
a
Inorganic arsenic As
III
Organic arsenic
V
As
LOD (Limit of Detection) was determined as the elemental concentration giving a signal three times the standard deviation (n=7) of the blank b LOQ (Limit of Quantification) was determined as the elemental concentration giving a signal ten times the standard deviation (n=7) of the blank c Certified value of NIST 1568a rice flour (309.24.0 g/kg), and efficiency of extraction is 106.5%
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Table 3. The concentrations of four As species and the ratio of iAs content to the total As in polished rice samples Concentration (g/kg on dry weight basis) Sample 1 2 3 4 5 6 7 8 9 10 11 Average AsIII 19.60.10 31.10.90 15.10.50 8.60.20 11.90.30 24.20.80 5.80.1 3.60.2 5.80.2 6.50.3 7.50.2 12.70.30 AsV N.D. N.D. N.D. N.D. 8.10.4 11.40.50 13.80.60 54.02.40 22.71.00 30.71.40 N.D. 12.80.60 MMA 6.30.4 12.80.80 5.20.3 2.10.1 4.70.3 18.01.10 12.30.80 54.03.30 19.91.20 51.93.20 3.00.2 17.31.10 DMA 3.30.4 1.20.1 1.80.2 2.00.3 3.20.4 3.60.3 4.10.4 19.52.10 1.90.3 3.30.4 2.40.2 04.20.50 iAs (AsIII+AsV) 19.60.1 31.10.9 15.10.5 08.60.2 20.00.3 35.60.4 19.60.3 57.62.6 28.50.7 37.21.1 07.50.2 25.50.4 Total As 29.04.2 48.05.0 20.63.1 13.31.1 28.03.0 57.37.1 44.03.9 150.018.6 48.05.3 105.09.80 15.02.1 50.74.7 % iAs/Total As 67.60.3 64.71.4 73.30.7 64.40.3 71.30.4 62.20.6 44.50.7 38.46.7 59.31.2 35.43.1 50.00.4 57.40.7
near the mining areas in Korea. The measured concentrations of AsIII, AsV, MMA, and DMA are given in Table 3. Total As concentrations were in the range of 13.3 1.1 to 150.018.6 g/kg on dry weight basis. The mean concentration of iAs (sum of AsIII and AsV) detected in polished rice samples was approximately 25.50.4 g/kg on dry weight basis. MMA and DMA were detected in all polished rice samples. The mean percentage of iAs based on total As in polished rice was approximately 57%, ranging from 35 to 71%. Acknowledgment. This study was supported by 2009 Post Doctoral Course Program of National Academy of Agricultural Science, Rural Development Administration, Republic of Korea
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