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1 Korean Journal of Environmental Agriculture Korean J Environ Agric. 2017;36(2): Korean Online ISSN: Published online 2017 June Print ISSN: Research Article Open Access HPLC-ICP-MS 를활용한잡곡의비소화학종및위해분석 * 안재민, 홍경숙, 김성연, 김대중, 이호진, 신희창 Arsenic Speciation and Risk Assessment of Miscellaneous Cereals by HPLC-ICP-MS Jae-Min An, Kyong-Suk Hong, Sung-Youn Kim, Dae-Jung Kim, Ho-Jin Lee and Hee-Chang Shin * (Division of Safety Analysis, Experiment & Research Institute, National Agriculture Products Quality Management Service, Gimcheon 39660, Korea) Received: 16 June 2017 / Revised: 20 June 2017 / Accepted: 23 June 2017 ORCID Copyright c 2017 The Korean Society of Environmental Agriculture This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. JAE MIN AN Hee-Chang Shin Abstract BACKGROUND: Miscellaneous cereal have been largely consumed in Korea as due to their physiological functions beneficial to human health. The cereals are currently a social concern because they have been found to contain heavy metals. Thus, monitoring heavy metals in the cereals is an important requirement for food safety analysis. In this study, we determined arsenic concentration in the cereals randomly harvested from different markets. METHODS AND RESULTS: Inorganic arsenic was determined by ICP-MS coupled with HPLC system. The HPLC-ICP-MS analysis was optimized based on the limit of detection and recover test to reach μg/kg and %, respectively. The concentrations of inorganic arsenic equivalent to daily exposure were levels of μg/day in mixed grain, 1.07 μg/day in glutinous rice, 0.77 μg/day in black brown rice, 0.13 μg/day in barley and 0.11 μg/day in soybeans. CONCLUSION: The levels of arsenic in miscellaneous cereals were found lower than the recommended The Joint FAO/WHO Expert Committee on Food Additives (JECFA) *Corresponding author: Hee-Chang Shin Phone: ; Fax: ; cafestol@korea.kr levels, suggesting that the cereals marketed in Korea are not potential concern in risk assessment. Key words: Arsenic species, Miscellaneous cereals, PTWI, Risk assessment 서,,, 4,. (arsenic, As) 15 (metalloid) 33, mg/kg, 0.01 mg/l, mg/l (The Joint FAO/WHO Expert Committee on Food Additives (JECFA), 1988).,. 2 (inorganic arsenic) arsenite (As( Ⅲ)), arsenate (As( Ⅴ)), (organic arsenic) DMA(dimethylarsinic acid), MMA (monomethylarsonic acid), AsB (arsenobetaine), AsC (arsenocholine) (Devesa, 2001). Kim (2000) 론 119

2 120 An et al. (arsenic species) As( Ⅲ)>As( Ⅴ)>MMA> DMA>AsC>AsB. (International Agency for Research on Cancer, IARC),, Group 1 (carcinogenic to humans) (IARC, 2004). JECFA, (PTWI, Provisional Tolerable Weekly Intake) mg/kg b.w./week1988 (JECFA, 1988) JECFA PTWI PTWI, PTWI (JECFA, 2010; JECFA/72/SC, 2010).,,, ( ). (Munoz et al., 2002).,, CODEX (CODEX, 2012).,.,. 재료및방법 실험재료 ,,. ( ) 1-3 kg, Table 1. Sampling items of miscellaneous cereals in Korean markets Table 1. 시약및초자 Sample Total black rice 14 oat 14 millet 14 mung bean 13 barley 16 sorghum 18 adlay 14 foxtail millet 16 glutinous rice 12 soy bean 16 adzuki bean 16 mixed grain 25 Total 188, 70% (Dong woo Fine-Chem, Korea) 30% (Dong woo Fine-Chem, Korea). Milli-Q ultrapure water purification system (Millipore Co., Massachusetts, USA) 18.2 MΩ 3. (P.E.), 5% ,000 mg/l (Merck, Germany) 5%, (CRM, Certified Reference Materials) National Institute of Standards and Technology (NIST) 1568b (Rice Flour, USA) NMIJ 7503-a (Rice Flour, Japan). arsenobetaine (Fluka, Switzerland), dimethylarsinic acid (Chem Service, USA), disodium methyl arsenate (Chem Service, USA), arsenite (High- Purity Standards, USA), arsenate (High-Purity Standards, USA). 1,000 mg/l stock solution, 10 mg/l 1% working solution, standard solution. HPLC ammonium carbonate(fluka, Switzerland), ammonium phosphate(fluka, Switzerland).

3 Arsenic Speciation and Risk Assessment of Miscellaneous Cereals by HPLC-ICP-MS 121 Table 2. Operating condition of microwave instrument Step Power (Watt) Time a) (min) Temp b) ( ) Hold c) (min) : : : : : :00 a) Ramping time, b) Target temperature, c) Stay time in target temperature Table 3. ICP-MS Condition for total arsenic analysis Parameter RF Power Plasma gas flow Nebulizer gas flow Auxillary gas flow Lens voltage Ion monitored Operating conditions 1,300 W 15.0 L/min 0.91 L/min 1.45 L/min 6.50 V As m/z 75, DRC mode Fig. 1. Flow chart for total arsenic analysis. 1%. 시료의전처리 , (, 2014). (Hadde VCM- 41, Sweden) 200 g, ICP-MS 를이용한총비소분석 g microwave (vessel) 70% 10 ml heating block (ED 16, LabTech, USA) ml microwave digestion system , , ( Table 2). heating block ml μm (Fig. 1). (Merck, Germany) 1,000 mg/l 5% 1.0 mg/kg working solution 0.1, 1.0, 5.0, 10.0, 25.0, 50.0 μg/kg. (Wang et al, 1991). (Inductiely Coupled Plasma- Mass Spectrometer, ICP/MS), ICP-MS(ELAN DRC, PerkinElemer, USA) DRC mode(dynamic reaction gas cell), Table 3. HPLC-ICP-MS 를이용한비소화학종분석 HPLC ICP-MS HPLC-ICP-MS.,5mM,1% (Choi et al., 2016; Chen et al., 2016; Rintala et al., 2014). Ronkart (2007)

122 An et al. Table 4. HPLC-ICP-MS condition for arsenic species analysis Instrument parameters HPLC As speciation AsB a), As( Ⅲ), DMA, MMA, As( Ⅴ) Column Anion exchange, Hamilton PRP X-100(4.

4 122 An et al. Table 4. HPLC-ICP-MS condition for arsenic species analysis Instrument parameters HPLC As speciation AsB a), As( Ⅲ), DMA, MMA, As( Ⅴ) Column Anion exchange, Hamilton PRP X-100( mm, 10 μm) Mobile phase A 12 mm ammonium carbonate Mobile phase B 20 mm ammonium phosphate Gradientprofile Run1:1min/A100%,Run2:7min/B100% Flow rate 2.0 ml/min Injection volume 50 μl ICP/MS RF Power 1,300 W Nebulizer gas flow 0.95 L/min Auxillary gas flow 1.14 L/min Plasma gas flow 16.0 L/min Lens voltage 7.2 V DRC mode AsO 91, gas flow 0.5 ml/min a) AsB: Arsenobetaine, As( Ⅲ): Arsenite, DMA: Dimethylarsonic acid, MMA: Monomethylarsinic acid, As( Ⅴ): Arsenate ph 8 anion exchange column, 12 mm ammonium carbonate, 20 mm ammonium phosphate gradient Table g centrifuge tube 1% 10 ml (TOMY MV-307, Hanil, Korea) 105,000 G 0.45 μm (Fig. 2). 5 1,000 mg/l 1% 10.0 mg/kg stock solution 1.0, 5.0, 10.0, 20.0 μg/kg. 시험법검증 (Limit of Detection, LOD) (Limit of Quantitation, LOQ), LOQ 2 LOQ 7 y. LOD=3.14 σ/s LOQ=10 σ/s ( σ :, S : ) 5 Fig. 2. Flow chart for arsenic species analysis.. (NIST) (Certified Reference Materials) 3, C.V. (Coefficient of Variation, %). 위해도평가

5 Arsenic Speciation and Risk Assessment of Miscellaneous Cereals by HPLC-ICP-MS 123 Table 5. Extraction efficiency by extract solution Species Extract AsB (%) As ( Ⅲ)(%) DMA (%) MMA (%) As ( Ⅴ)(%) Distilled water 39.6± ± ± ± ±8.3 5 mm malonic acid 77.1± ± ± ± ±10.1 1% nitric acid 97.8± ± ± ± ±6.5 Fig. 3. HPLC-ICP-MS chromatogram for a standard mixture of 5 arsenic species. [AsB: Arsenobetaine, As( ): Arsenite, DMA: Dimethylarsonic acid, MMA: Monomethylarsinic acid, As( ): Arsenate] (Korea Centers for Disease Control & Prevention, 2012). SPSS(ver 14.0, SPSS Inc., USA) 3, 55 kg. JECFA (Provisional Tolerable Weekly Intake, PTWI). PTWI μg/kg b.w./week PTWI., (determination approach). (%) = (mg/kg b.w./week) 100 JECFA (mg/kg b.w./week) 결과및고찰 비소화학종분리시험법확립 2 3. Huang (2010), 90%, ICP- MS ArCl tetramethyl ammonium hydroxide phosphoric acid swelling0.28 M.,5mM,1% 1% 95% (Table 5). ph HPLC, Hamilton PRP-X100 (anion exchange column). Raber (2012) HPLC sodium sodium, malonic acid ammonium. 5 mm isocratic arsenobetaine (AsB) peakarsenite (As( Ⅲ)) peak (AsB), 12 mm ammonium carbonate, 20 mm ammonium

6 124 An et al. Table 6. Limit of detection (LOD) and limit of quantitation (LOQ) Item Species Total As AsB As ( Ⅲ) DMA MMA As ( Ⅴ) LOD LOQ R Table 7. Recovery and Coefficient of varidation Item Species Total As AsB As ( Ⅲ) DMA MMA As ( Ⅴ) Recovery (%) 98.6± ± ± ± ± ±2.4 C.V. (%) Table 8. Total arsenic and inorganic arsenic concentration of each agricultural products Concentration (mg/kg) Sample N a) Total Arsenic Inorganic Arsenic black rice 14 oat 14 millet 14 mung bean 13 barley 16 sorghum 18 adlay 14 foxtail millet 16 glutinous rice 12 soy bean 16 adzuki bean 16 mixed grain 25 Total ±0.137 b) ( ) c) 0.136±0.075 ( ) 0.031±0.019 ( ) 0.022±0.011 ( ) 0.037±0.023 ( ) 0.037±0.014 ( ) 0.038±0.025 ( ) 0.033±0.013 ( ) 0.045±0.019 ( ) 0.267±0.090 ( ) 0.044±0.025 ( ) 0.028±0.011 ( ) 0.183±0.045 ( ) 0.085±0.098 ( ) N.D. : not detected a) N, Number of sample b) Mean value±sd (standard of deviation) c) Concentration range (minimum maximium) 0.017±0.011 ( ) 0.009±0.009 (N.D ) 0.018±0.014 (N.D ) 0.021±0.011 (N.D ) 0.021±0.017 (N.D ) 0.019±0.009 (N.D ) 0.026±0.014 (N.D ) 0.149±0.056 ( ) 0.024±0.015 (N.D ) 0.014±0.008 (N.D ) 0.110±0.035 ( ) 0.048±0.057 (N.D ) phosphate gradient (Fig. 3). 시험법검증 (Limit of Detection, LOD) (Limit of Quantitation, LOQ) LOQ LOQ , 1.0, 5.0, 10.0, 25.0, 50.0 μg/kg 1.0, 5.0, 10.0, 20.0 μg/kg,r ( Table 6) μg/kg, μ g/kg, arsenite(as( Ⅲ)) arsenate(as( Ⅴ)). (NIST 1568b, USA ; NMIJ 7503-a, Japan), AsB MMA 50.0 μg/l %, C.V.% (Coefficient of Varidation) 5% AOAC (AOAC International, 2002) U.S. FDA (Patrick et al., 2015) (Table 7). 잡곡중의총비소농도분석 ICP-MS, ±0.098 mg/kg, 0.267±0.090 mg/kg, 0.241±0.137 mg/kg, 0.183±0.045 mg/kg, 0.022±0.011 mg/kg (Table 8, Fig. 4). Choi (2010) mg/kg 8 8

7 Arsenic Speciation and Risk Assessment of Miscellaneous Cereals by HPLC-ICP-MS 125 Fig. 4. Histogram of total arsenic and inorganic arsenic. Table 9. Daily mean dietary exposure and risk of inorganic arsenic Sample Inorganic As content (mg/kg) Food intake (g/day) μg/day InorganicAsexposure μg/kg b.w./day %PTWI black rice oat millet mung bean barley sorghum adlay foxtail millet glutinous rice soy bean adzuki bean mixed grain a) a) Food intake of mixed grain was based on polished rice, mg/kg,0.3mg/kg 잡곡중의무기비소농도분석 HPLC-ICP/MS, ±0.057 mg/kg, 56.5% ±0.056 mg/kg, 0.136± mg/kg, 0.110±0.035 mg/kg, 0.009±0.009 mg/kg (Table 8, Fig. 4). Kim (2013) mg/kg, 53-88%. 총비소농도와무기비소농도와의상관분석, 56.5%. 60.1% 40.9%. 12 (r >0.57), (Fig. 5). 잡곡류섭취로인한무기비소위해도평가 (2012). (55 kg),

8 126 An et al. Fig. 5. Histogram of total arsenic and inorganic arsenic.

Arsenic Speciation and Risk Assessment of Miscellaneous Cereals by HPLC-ICP-MS 127 Fig. 6. Contribution rate of inorganic arsenic by agricultural products. 19.91 μg/day, 1.07 μ g/day, 0.77 μg/day, 0.

9 Arsenic Speciation and Risk Assessment of Miscellaneous Cereals by HPLC-ICP-MS 127 Fig. 6. Contribution rate of inorganic arsenic by agricultural products μg/day, 1.07 μ g/day, 0.77 μg/day, 0.13 μg/day, 0.11 μg/day. PTWI 16.89%, 0.91%, 0.65%, 0.11%, 0.09% (Table 9)., 51%, 36%, 6%, 5% (Fig. 6)., 12 PTWI 18.69%. 요, , microwave ICP-MS. HPLC ICP-MS HPLC-ICP-MS.,, 1% 1%, ph ammonium carbonate, ammonium phosphate gradient.,, AOAC. 약 0.267±0.090 mg/kg, 0.241±0.137 mg/kg, 0.183±0.045 mg/kg, 0.149±0.056 mg/kg, 0.136±0.075 mg/kg, 0.110±0.035 mg/kg.. (2012) μg/day, 1.07 μg/day, 0.77 μg/day, 0.13 μg/day, 0.11 μg/day. PTWI 16.89%, 0.91%, 0.65%, 0.11%, 0.09%. 12 PTWI 18.69%,. Acknowledgement This study was carried out with the support of Research & Technology Development (Project year, 2016), National Agricultural Products Quality Management Service, Republic of Korea. References AOAC. (2002). AOAC Guidelines for single laboratory validation of chemical methods for dietary supplements and botanicals, AOAC, Horwitz, W. (2002). AOAC guidelines for single laboratory validation of chemical methods for dietary supplements and botanicals. AOAC International,

10 128 An et al. Gaithersburg, MD, USA, Chen,H.L.,Lee,C.C.,Huang,W.J.,Huang,H.T.,Wu,Y. C., Hsu, Y. C., & Kao, Y. T. (2016). Arsenic speciation in rice and risk assessment of inorganic arsenic in Taiwan population. Environmental Science and Pollution Research International, 23(5), Choi,H.,Park,S.K.,Kim,D.S.,&Kim,M.H.(2010). Risk assessment of arsenic in agricultural products. Korean Journal of Environmental Agriculture, 29(3), Choi,J.Y.,Khan,N.,Nho,E.Y.,Choi,H.,Park,K.S., Cho,M.J.,Youn,H.J.,&Kim,K.S.(2016).Speciation of arsenic in rice by high-performance liquid chromatography inductively coupled plasma mass spectrometry. Analytical Letters, 49(12), Devesa,V.,Martinez,A.,Suner,M.A.,Benito,V.,Vélez,D., & Montoro, R. (2001). Kinetic study of transformations of arsenic species during heat treatment.journal of agricultural and food chemistry, 49(5), Huang, J. H., Ilgen, G., & Fecher, P. (2010). Quantitative chemical extraction for arsenic speciation in rice grains. Journal of Analytical Atomic Spectrometry, 25(6), International Agency for Research on Cancer (IARC), (2004). Some drinking-water disinfectants and contaminants, including arsenic,(vol. 84), International Agency for Research on Cancer, Lyon, France. Kim,S.T.,Lim,Y.R.,Park,K.S.,&Chung,J.H.(2000). Simultaneuous determination of As (III) and As (V) in disused mine tailing samples by hydride generationinductively coupled plasma-atomic emission spectrometry. Analytical Science and Technology, 13(2), Kim,J.Y.,Kim,W.I.,Kunhikrishnan,A.,Kang,D.W., Kim,D,H.,Lee,Y.J.,Kim,Y.J.,Kim,C.T.(2013). Determination of arsenic species in rice grains using HPLC-ICP-MS. Food Science and Biotechnology, 22(6), Munoz,O.,Diaz,O.P.,Leyton,I.,Nunez,N.,Devesa,V., Suner, M. A., Velez, D., & Montoro, R. (2002). Vegetables collected in the cultivated Andean area of northern Chile: total and inorganic arsenic contents in raw vegetables. Journal of Agricultural and Food Chemistry, 50(3), Patrick, J. G., William, R. M., &John, C. (2015). FDA elemental analysis manual: section 4.7 Inductively coupled plasma-mass spectrometric determination of arsenic, cadmium, chromium, lead, mercury, and other elements in food using microwave assisted digestion. US Food and Drug Administration, Washington DC, USA, Available from: fda. gov/downloads/ Food/FoodScienceResearch/LaboratoryMethods/ UCM pdf, 14. Raber,G.,Stock,N.,Hanel,P.,Murko,M.,Navratilova,J., & Francesconi, K. A. (2012). An improved HPLC ICPMS method for determining inorganic arsenic in food: application to rice, wheat and tuna fish. Food chemistry, 134(1), Ronkart,S.N.,Laurent,V.,Carbonnelle,P.,Mabon,N., Copin, A., & Barthélemy, J. P. (2007). Speciation of five arsenic species (arsenite, arsenate, MMAA V, DMAA V and AsBet) in different kind of water by HPLC- ICP-MS. Chemosphere, 66(4), Rintala, E. M., Ekholm, P., Koivisto, P., Peltonen, K., & Venalainen, E. R. (2014). The intake of inorganic arsenic from long grain rice and rice-based baby food in Finland Low safety margin warrants follow up. Food chemistry, 150, Wang, Y., Lu, C., Xiao, Z., Wang, G., Kuan, S. S., & Rigsby, E. J. (1991). Determination of aluminum in foods by stabilized temperature platform graphite furnace atomic absorption spectrometry. Journal of Agricultural and Food Chemistry, 39(4),

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개최요강 55 2009. 5. ( ) ( ) < > 1. 1 2. 2. 2. 3 1) 3 2) 3 3) GC-MS 4. 5 1) 5 2) 5 3) ICP-OES 6 3. 7. 7 1) 7 2) 10. 13 1) 1g 13 2),,, 14 3), 15 4),, 15 4. 17 19 < > [ 1] 2 [ 2] ICP-OES 6 [ 3] 13 [ 4] 1g 13 [ 5]