Korean Journal of Environmental Agriculture Korean J Environ Agric. 2017;36(2):119-128. Korean Online ISSN: 2233-4173 Published online 2017 June 30. https://doi.org/10.5338/kjea.2017.36.2.20 Print ISSN: 1225-3537 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 (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. JAE MIN AN http://orcid.org/0000-0002-9964-2438 Hee-Chang Shin http://orcid.org/0000-0002-0600-4972 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 0.13-1.24 μg/kg and 94.3 102.1%, respectively. The concentrations of inorganic arsenic equivalent to daily exposure were levels of 19.91 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: +82-54-429-7770; Fax: +82-54-429-7779; E-mail: 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, 74.9 2 mg/kg, 0.01 mg/l, 0.001-0.008 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
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) 0.015 mg/kg b.w./week1988 (JECFA, 1988). 2010 JECFA PTWI PTWI, PTWI (JECFA, 2010; JECFA/72/SC, 2010).,,, ( ). (Munoz et al., 2002).,, CODEX (CODEX, 2012).,.,. 재료및방법 실험재료 2015-2016 188.,,. ( 2015-52 ) 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% 243. 1,000 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).
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) 1 1600 5:00 90 10:00 2 1600 5:00 150 10:00 3 1600 5:00 190 30:00 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 7. 7.1. 7.1.1., (, 2014). (Hadde VCM- 41, Sweden) 200 g, ICP-MS 를이용한총비소분석 1. 1.0 g microwave (vessel) 70% 10 ml heating block (ED 16, LabTech, USA) 85 30. 1 ml microwave digestion system 5 90 10, 5 150 10, 5 190 30 ( Table 2). heating block 12 0 4 1-2 ml 20 3 0.45 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(4.1 250 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 4. 1.0 g centrifuge tube 1% 10 ml5 85 90. (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 12188 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±3.7 43.8±6.5 68.2±7.1 63.8±8.8 52.7±8.3 5 mm malonic acid 77.1±8.0 76.6±8.2 82.5±4.9 79.6±6.8 93.4±10.1 1% nitric acid 97.8±8.2 106.4±6.9 95.6±10.1 97.1±8.4 102.9±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 2010 15 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
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 0.13 0.87 0.73 0.76 0.98 1.24 LOQ 0.43 2.87 2.41 2.51 3.23 4.09 R 2 1.0000 0.9984 0.9994 0.9991 0.9999 0.9986 Table 7. Recovery and Coefficient of varidation Item Species Total As AsB As ( Ⅲ) DMA MMA As ( Ⅴ) Recovery (%) 98.6±0.7 94.5±1.3 102.1±1.8 98.4±2.3 94.3±2.0 96.2±2.4 C.V. (%) 1.35 2.11 1.86 1.47 0.98 3.44 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 188 0.241±0.137 b) (0.029-0.449) c) 0.136±0.075 (0.017-0.278) 0.031±0.019 (0.009-0.078) 0.022±0.011 (0.008-0.044) 0.037±0.023 (0.011-0.084) 0.037±0.014 (0.007-0.063) 0.038±0.025 (0.007-0.104) 0.033±0.013 (0.008-0.064) 0.045±0.019 (0.017-0.080) 0.267±0.090 (0.126-0.442) 0.044±0.025 (0.014-0.109) 0.028±0.011 (0.014-0.053) 0.183±0.045 (0.086-0.262) 0.085±0.098 (0.007-0.449) 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 (0.004-0.044) 0.009±0.009 (N.D.-0.024) 0.018±0.014 (N.D.-0.041) 0.021±0.011 (N.D.-0.042) 0.021±0.017 (N.D.-0.058) 0.019±0.009 (N.D.-0.041) 0.026±0.014 (N.D.-0.051) 0.149±0.056 (0.054-0.254) 0.024±0.015 (N.D.-0.053) 0.014±0.008 (N.D.-0.028) 0.110±0.035 (0.041-0.162) 0.048±0.057 (N.D.-0.278) phosphate gradient (Fig. 3). 시험법검증 (Limit of Detection, LOD) (Limit of Quantitation, LOQ) LOQ LOQ 2 7. 0.1, 1.0, 5.0, 10.0, 25.0, 50.0 μg/kg 1.0, 5.0, 10.0, 20.0 μg/kg,r 2 0.998 ( Table 6). 0.43 μg/kg, 2.41-4.09 μ g/kg, arsenite(as( Ⅲ)) arsenate(as( Ⅴ)). (NIST 1568b, USA ; NMIJ 7503-a, Japan), AsB MMA 50.0 μg/l 3. 94.3-102.1%, C.V.% (Coefficient of Varidation) 5% AOAC (AOAC International, 2002) U.S. FDA (Patrick et al., 2015) (Table 7). 잡곡중의총비소농도분석 ICP-MS, 12 188 0.085±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). 0.01 mg/kg 8 8
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 0.136 5.63 7.66 10-1 1.39 10-2 0.65 oat 0.017 0.01 2.00 10-4 3.09 10-6 - millet 0.009 0.27 2.40 10-3 4.42 10-5 - mung bean 0.018 0.10 1.80 10-3 3.27 10-5 - barley 0.021 6.20 1.30 10-1 2.37 10-3 0.11 sorghum 0.021 0.42 8.80 10-3 1.60 10-4 0.01 adlay 0.019 0.15 2.90 10-3 5.18 10-5 - foxtail millet 0.026 1.00 2.60 10-2 4.73 10-1 0.02 glutinous rice 0.149 7.17 1.07 10 0 1.94 10-2 0.91 soy bean 0.024 4.61 1.11 10-1 2.01 10-3 0.09 adzuki bean 0.014 0.47 6.60 10-3 1.19 10-4 0.01 mixed grain 0.110 181.0 a) 19.90 10 0 3.62 10-1 16.89 a) Food intake of mixed grain was based on polished rice, 0.01-0.05 mg/kg,0.3mg/kg 2 10. 잡곡중의무기비소농도분석 HPLC-ICP/MS, 12 188 0.048±0.057 mg/kg, 56.5%. 0.149±0.056 mg/kg, 0.136± 0.075 mg/kg, 0.110±0.035 mg/kg, 0.009±0.009 mg/kg (Table 8, Fig. 4). Kim (2013) 0.06-0.08 mg/kg, 53-88%. 총비소농도와무기비소농도와의상관분석, 56.5%. 60.1% 40.9%. 12 (r >0.57), (Fig. 5). 잡곡류섭취로인한무기비소위해도평가 12 188 (2012). (55 kg),
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, 0.13 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,. 12 188, 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) 19.91 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, 1-38. Horwitz, W. (2002). AOAC guidelines for single laboratory validation of chemical methods for dietary supplements and botanicals. AOAC International,
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