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Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Lead and cadmium in functional health foods and Korean herbal medicines a
Wooseok Kim & Kwang-Geun Lee
a
a
Department of Food Science and Biotechnology , Dongguk University-Seoul , Seoul , Korea Accepted author version posted online: 25 Jan 2013.Published online: 26 Feb 2013.
To cite this article: Wooseok Kim & Kwang-Geun Lee (2013) Lead and cadmium in functional health foods and Korean herbal medicines, Food Additives & Contaminants: Part B: Surveillance, 6:2, 146-149, DOI: 10.1080/19393210.2013.769026 To link to this article: http://dx.doi.org/10.1080/19393210.2013.769026
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives & Contaminants: Part B, 2013 Vol. 6, No. 2, 146–149, http://dx.doi.org/10.1080/19393210.2013.769026
Lead and cadmium in functional health foods and Korean herbal medicines Wooseok Kim and Kwang-Geun Lee* Department of Food Science and Biotechnology, Dongguk University-Seoul, Seoul, Korea
Downloaded by [Australian National University] at 06:03 24 January 2015
(Received 18 April 2012; final version received 18 January 2013) Lead (Pb) and cadmium (Cd) in functional health foods (FHF) and Korean herbal medicines (KHM) were analysed by the standard addition method with inductively coupled plasma–mass spectrometry. A total of 672 samples were collected from 2347 people (1015 adults, 557 students and 775 infants and children) who lived in Korea. Pb and Cd concentrations were analysed in the samples (FHF, n = 535; KHM, n = 50). Method validation was carried out using standard reference material (SRM), recovery rate and limits of detection and quantification. Recovery rates for Pb and Cd using three SRMs were 94.9%–101.6% and 96.7%–115.2%, respectively. Mean Pb values in FHF and KHM were 0.146 and 0.349 mg kg–1, respectively. Mean Cd levels in FHF and KHM were 0.035 and 0.056 mg kg–1, respectively. Mean values in Spirulina and yeast products were the highest in the FHF samples (0.940 mg kg–1 for Pb in Spirulina products and 0.115 mg kg–1 for Cd in yeast products). Keywords: lead; cadmium; functional health food; Korean herbal medicine; standard addition method; ICP-MS
Introduction Many Koreans are eating functional health food (FHF) and Korean herbal medicine (KHM) for their health benefit. In Korea, FHF is defined as a processed food used with the intention to enhance and preserve human health through physiologically functional ingredients or components in the form of a tablet, capsule or powder (Ministry of Health and Welfare 2004). FHF is divided into six groups by functional ingredients such as phenols, fatty acids and lipids, sugars and carbohydrates, nutrients, terpenes and fermentation microorganisms (KFDA 2010). KHM is defined as oriental medicine manufactured with the principles of medicine following the pharmaceutical affairs law of Korea (Ministry of Health and Welfare 2010). However, these medicines can be contaminated with heavy metals such as lead (Pb) and cadmium (Cd) because of environmental pollution, which has negatively affected the ecosystem. Likewise, foodstuffs are exposed to environmental pollutants. Heavy metals are a serious problem, because they remain in the environment for a long time and accumulate in the food chain (Szyczewski et al. 2009). Pb and Cd are toxic heavy metals that have a longer half-life than that of other organic contaminants. They are also classified as toxic substances by the International Agency for Research on Cancer. Pb affects the central nervous system–related cognitive development and behaviour in infants and children (Center for Disease Control 1991). It also causes illnesses such as peripheral artery disease, hypertension, neurodegenerative disease, kidney disease and cognitive impairment (Ahamed & Siddiqui 2007). Prolonged exposure to Cd can lead to nephrotoxicity, immunotoxicity, osteotoxicity and cancer (Liu et al. 2009). *Corresponding author. Email: [email protected] © 2013 Taylor & Francis
Inductively coupled plasma optical emission spectrometry (ICP-OES) is widely used to determine heavy metal concentrations, although it can have low sensitivity and some interference problems caused by complex matrices such as FHF and KHM. As an alternative technique, ICP– mass spectrometry (MS) is becoming more and more attractive, because it has high sensitivity and specificity (Wang et al. 1995; Nardi et al. 2009). In studies of GarcíaRico et al. (2007) and Avula et al. (2010), Pb and Cd were analysed in dietary supplements by ICP-MS. Yim et al. (2009) and Kim et al. (2010) reported analysis of Pb and Cd in limited numbers of KHM. Matrix and interference effects can be overcome by applying the standard addition method (Saxberg & Kowalski 1979), which requires some materials that have to be added to the standard solution in the samples (Zagatto et al. 1983). The objective of this study was to analyse Pb and Cd using ICP-MS in FHF and KHM samples collected directly from 2347 Koreans. The results will provide data for an integrated exposure assessment of heavy metals in FHF and KHM. Materials and methods Reagents All reagents were analytical grade. Calibration solutions were prepared daily. A multi-element calibration standard (PerkinElmer Life and Analytical Sciences, Shelton, CT, USA) was used for Pb and Cd calibration. HNO3 (70%) and H2O2 (31%) (Dong Woo Fine-Chem, Iksan, Korea) were used for digestion of samples and standard reference materials (SRMs). Ultrapure deionised water (Milli-Q;
Food Additives & Contaminants: Part B
Downloaded by [Australian National University] at 06:03 24 January 2015
Millipore, Bedford, MA, USA) was used in all analyses. SRMs of oyster tissue (SRM 1566b), corn bran (SRM 8433) and Ginkgo biloba (SRM 3246) were purchased from the National Institute of Standards and Technology (Gaithersburg, MD, USA). Sampling FHF (n = 622) and KHM (n = 50) were collected from 2347 Koreans (1015 adults, 557 students, 547 infants and 228 parents of infants) who lived in all regions of Korea except Jeju Island, using amber-coloured bottles. The 547 infants were classified into three groups: 12, 12–36 and >36 months of age. If samples were not collected directly from the examinees, they were purchased from local pharmacies or online websites. The number of samples bought was 94 (only FHF, no KHM). The amber-coloured sampling bottles were cleaned in H2SO4 (Dong Woo FineChem, Iksan, Korea) for 24 hours, and then they were rewashed in ultrapure deionised water and air dried. After collecting the samples, they were accurately sorted into FHF and KHM by KFDA’s Functional Health Food database and protocol (KFDA 2010; Ministry of Health and Welfare 2010). Sample digestion After sorting, samples were classified as solid, semi-solid or liquid according to the sample matrix. A microwave closed system (model START D, Milestone, Bergamo, Italy) was used to digest the samples with HNO3-H2O2. Homogenised samples (solid or liquid) of 0.5 g were weighed in Teflon vessels cleaned with 5% HNO3 for 24 hours. Then, 7 mL of 70% HNO3 and 1 mL of 31% H2O2 were added to the Teflon vessel. Microwave digestion involved four steps, each using 1000 W, with phased temperatures being gradual at 80°C, 50°C, 190°C and 190°C. Digested samples and SRMs were transferred to 25-mL volumetric flasks after cooling, diluted to 25 mL with ultrapure deionised water and filtered with a 0.45-μm syringe filter. However, as semi-solid samples tended to explode because of organic matter in the Teflon vessels, they were digested after being preheated on a hot block (ED16, Labtech, Beijing, China). Determination of Pb and Cd Pb and Cd concentrations in the FHF and KHM were determined by the standard addition method to be able to correct for matrix effects. Analysis was done in two steps. Step 1 was a preliminary analysis to verify the presence of Pb and Cd in FHF and KHM. Step 2 was the actual analysis using the standard addition method, making use of the known presence of Pb and Cd from step 1. External calibration included 0.01, 0.1, 1, 5, 10, 20 and 100 ng mL–1
Table 1. Inductively operating conditions.
coupled
Descriptions RF generator RF power Coolant gas flow rate Auxiliary gas flow rate Nebuliser gas flow rate Sample uptake flow Nebuliser Spray chamber Torch Interface cones Quadrupole chamber Dwell time Pb/Mass Cd/Mass
plasma–mass
147
spectrometry
Conditions Free-running type, 40 MHz 1400 W 17.0 L/min 2.00 L/min 1.05 L/min 1.00 mL/min Concentric type Cychronic type Demountable Platinum 1 × 10–6 torr 600 ms 208, 206 mu 114, 112 mu
with mixed standard solutions of Pb and Cd. The standard addition method used three points of calibration: high, low and an untreated sample addition. The highest concentration of the standard addition calibrant was set at three times the concentration obtained from the preliminary analysis. The lowest concentration was determined to be half of the highest concentration obtained from the preliminary analysis. The three digested 3 mL sample solutions used for standard addition were respectively diluted to 5 mL with a suitable amount of standard solution and ultrapure deionised water. Then the three 5 mL sample solutions were analysed with an ELAN 6100 DRC plus ICP-MS (Perkin Elmer SCIEX, Concord, Ontario, Canada), which was calibrated using a smart-tune solution (10 µg/L Ba, 1 µg/L Be, Ce, Co, Fe, In, Mg, Pb, Th and U). The operating conditions are specified in Table 1. Pb and Cd concentrations were determined as the absolute value of the x-axis intercept. Method validation The analytical method was validated in terms of linearity, recovery, limit of detection (LOD) and limit of quantification (LOQ). LOD and LOQ were calculated using the equations LOD = 3.3 × σ/S and LOQ = 10 × σ/S, where σ is the residual standard deviation and S the slope of the calibration curve (KFDA 2004). The LODs of Pb and Cd were measured with a blank and a 5 ng g–1 standard solution, respectively, in seven replicates. Recovery rate was evaluated by SRM using two samples spiked with 0.100 mg kg–1 standard solution.
Results and discussion Method validation LODs for Pb and Cd were 0.06 and 0.09 ng g–1, respectively. Recovery was evaluated as the accuracy of the
148
W. Kim and K.-G. Lee
Downloaded by [Australian National University] at 06:03 24 January 2015
analytical method in semi-solid and liquid forms, with two samples that were spiked with standard solution. Recoveries of Pb and Cd from the semi-solid were 101.8% and 104.0% and from liquid 105.3% and 105.6%, respectively. Recovery rate from solids was determined by SRM 1566b, SRM 8433 and SRM 3249 (Table 2). The z score was used to assess laboratory performance. It was calculated as z = (x – xa)/σp, where x is the certified value of the SRM, xa is the measured value of the SRM and σp is the standard deviation of the measured value. Values of z score in the ranges |z| ≤ 2, 2 < |z| < 3 and |z| ≥ 3 were defined as acceptable, questionable and unacceptable, respectively (Dolan & Capar 2002). Further, z score results for the SRMs were evaluated as acceptable (Table 2). Determination of Pb and Cd Analytical results are listed in Table 3. The arithmetical means for Pb and Cd in KHM were higher than those in FHF. When arithmetic means were calculated from positive values only, the values for Pb and Cd were 0.200 and 0.085 mg kg–1 in FHF (n = 451 for Pb and n = 257 for Cd) and 0.425 and 0.220 mg kg–1 for KHM (n = 41 for Pb and n = 12 for Cd), respectively. Maximum values in KHM were higher than those in FHF, suggesting that ingredients for FHF were cleaned during the manufacturing process (Food and Drug Administration 2003). Individual data can be found in the database. According to product specifications and FHF Code (KFDA 2010), FHF ingredients were divided into docosahexaenoic acid (DHA), N-931, Garcinia cambogia extract, γ-linolenic acid, cereal enzyme, glucosamine, protein, gigantic angelica extract, soy bean and dimethylsulfone. Pb concentration for Spirulina products was the highest in the FHF ingredients (0.940 mg kg–1), wherever it was still safe as the standard regulation is
Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Lead and cadmium in functional health foods and Korean herbal medicines a
Wooseok Kim & Kwang-Geun Lee
a
a
Department of Food Science and Biotechnology , Dongguk University-Seoul , Seoul , Korea Accepted author version posted online: 25 Jan 2013.Published online: 26 Feb 2013.
To cite this article: Wooseok Kim & Kwang-Geun Lee (2013) Lead and cadmium in functional health foods and Korean herbal medicines, Food Additives & Contaminants: Part B: Surveillance, 6:2, 146-149, DOI: 10.1080/19393210.2013.769026 To link to this article: http://dx.doi.org/10.1080/19393210.2013.769026
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives & Contaminants: Part B, 2013 Vol. 6, No. 2, 146–149, http://dx.doi.org/10.1080/19393210.2013.769026
Lead and cadmium in functional health foods and Korean herbal medicines Wooseok Kim and Kwang-Geun Lee* Department of Food Science and Biotechnology, Dongguk University-Seoul, Seoul, Korea
Downloaded by [Australian National University] at 06:03 24 January 2015
(Received 18 April 2012; final version received 18 January 2013) Lead (Pb) and cadmium (Cd) in functional health foods (FHF) and Korean herbal medicines (KHM) were analysed by the standard addition method with inductively coupled plasma–mass spectrometry. A total of 672 samples were collected from 2347 people (1015 adults, 557 students and 775 infants and children) who lived in Korea. Pb and Cd concentrations were analysed in the samples (FHF, n = 535; KHM, n = 50). Method validation was carried out using standard reference material (SRM), recovery rate and limits of detection and quantification. Recovery rates for Pb and Cd using three SRMs were 94.9%–101.6% and 96.7%–115.2%, respectively. Mean Pb values in FHF and KHM were 0.146 and 0.349 mg kg–1, respectively. Mean Cd levels in FHF and KHM were 0.035 and 0.056 mg kg–1, respectively. Mean values in Spirulina and yeast products were the highest in the FHF samples (0.940 mg kg–1 for Pb in Spirulina products and 0.115 mg kg–1 for Cd in yeast products). Keywords: lead; cadmium; functional health food; Korean herbal medicine; standard addition method; ICP-MS
Introduction Many Koreans are eating functional health food (FHF) and Korean herbal medicine (KHM) for their health benefit. In Korea, FHF is defined as a processed food used with the intention to enhance and preserve human health through physiologically functional ingredients or components in the form of a tablet, capsule or powder (Ministry of Health and Welfare 2004). FHF is divided into six groups by functional ingredients such as phenols, fatty acids and lipids, sugars and carbohydrates, nutrients, terpenes and fermentation microorganisms (KFDA 2010). KHM is defined as oriental medicine manufactured with the principles of medicine following the pharmaceutical affairs law of Korea (Ministry of Health and Welfare 2010). However, these medicines can be contaminated with heavy metals such as lead (Pb) and cadmium (Cd) because of environmental pollution, which has negatively affected the ecosystem. Likewise, foodstuffs are exposed to environmental pollutants. Heavy metals are a serious problem, because they remain in the environment for a long time and accumulate in the food chain (Szyczewski et al. 2009). Pb and Cd are toxic heavy metals that have a longer half-life than that of other organic contaminants. They are also classified as toxic substances by the International Agency for Research on Cancer. Pb affects the central nervous system–related cognitive development and behaviour in infants and children (Center for Disease Control 1991). It also causes illnesses such as peripheral artery disease, hypertension, neurodegenerative disease, kidney disease and cognitive impairment (Ahamed & Siddiqui 2007). Prolonged exposure to Cd can lead to nephrotoxicity, immunotoxicity, osteotoxicity and cancer (Liu et al. 2009). *Corresponding author. Email: [email protected] © 2013 Taylor & Francis
Inductively coupled plasma optical emission spectrometry (ICP-OES) is widely used to determine heavy metal concentrations, although it can have low sensitivity and some interference problems caused by complex matrices such as FHF and KHM. As an alternative technique, ICP– mass spectrometry (MS) is becoming more and more attractive, because it has high sensitivity and specificity (Wang et al. 1995; Nardi et al. 2009). In studies of GarcíaRico et al. (2007) and Avula et al. (2010), Pb and Cd were analysed in dietary supplements by ICP-MS. Yim et al. (2009) and Kim et al. (2010) reported analysis of Pb and Cd in limited numbers of KHM. Matrix and interference effects can be overcome by applying the standard addition method (Saxberg & Kowalski 1979), which requires some materials that have to be added to the standard solution in the samples (Zagatto et al. 1983). The objective of this study was to analyse Pb and Cd using ICP-MS in FHF and KHM samples collected directly from 2347 Koreans. The results will provide data for an integrated exposure assessment of heavy metals in FHF and KHM. Materials and methods Reagents All reagents were analytical grade. Calibration solutions were prepared daily. A multi-element calibration standard (PerkinElmer Life and Analytical Sciences, Shelton, CT, USA) was used for Pb and Cd calibration. HNO3 (70%) and H2O2 (31%) (Dong Woo Fine-Chem, Iksan, Korea) were used for digestion of samples and standard reference materials (SRMs). Ultrapure deionised water (Milli-Q;
Food Additives & Contaminants: Part B
Downloaded by [Australian National University] at 06:03 24 January 2015
Millipore, Bedford, MA, USA) was used in all analyses. SRMs of oyster tissue (SRM 1566b), corn bran (SRM 8433) and Ginkgo biloba (SRM 3246) were purchased from the National Institute of Standards and Technology (Gaithersburg, MD, USA). Sampling FHF (n = 622) and KHM (n = 50) were collected from 2347 Koreans (1015 adults, 557 students, 547 infants and 228 parents of infants) who lived in all regions of Korea except Jeju Island, using amber-coloured bottles. The 547 infants were classified into three groups: 12, 12–36 and >36 months of age. If samples were not collected directly from the examinees, they were purchased from local pharmacies or online websites. The number of samples bought was 94 (only FHF, no KHM). The amber-coloured sampling bottles were cleaned in H2SO4 (Dong Woo FineChem, Iksan, Korea) for 24 hours, and then they were rewashed in ultrapure deionised water and air dried. After collecting the samples, they were accurately sorted into FHF and KHM by KFDA’s Functional Health Food database and protocol (KFDA 2010; Ministry of Health and Welfare 2010). Sample digestion After sorting, samples were classified as solid, semi-solid or liquid according to the sample matrix. A microwave closed system (model START D, Milestone, Bergamo, Italy) was used to digest the samples with HNO3-H2O2. Homogenised samples (solid or liquid) of 0.5 g were weighed in Teflon vessels cleaned with 5% HNO3 for 24 hours. Then, 7 mL of 70% HNO3 and 1 mL of 31% H2O2 were added to the Teflon vessel. Microwave digestion involved four steps, each using 1000 W, with phased temperatures being gradual at 80°C, 50°C, 190°C and 190°C. Digested samples and SRMs were transferred to 25-mL volumetric flasks after cooling, diluted to 25 mL with ultrapure deionised water and filtered with a 0.45-μm syringe filter. However, as semi-solid samples tended to explode because of organic matter in the Teflon vessels, they were digested after being preheated on a hot block (ED16, Labtech, Beijing, China). Determination of Pb and Cd Pb and Cd concentrations in the FHF and KHM were determined by the standard addition method to be able to correct for matrix effects. Analysis was done in two steps. Step 1 was a preliminary analysis to verify the presence of Pb and Cd in FHF and KHM. Step 2 was the actual analysis using the standard addition method, making use of the known presence of Pb and Cd from step 1. External calibration included 0.01, 0.1, 1, 5, 10, 20 and 100 ng mL–1
Table 1. Inductively operating conditions.
coupled
Descriptions RF generator RF power Coolant gas flow rate Auxiliary gas flow rate Nebuliser gas flow rate Sample uptake flow Nebuliser Spray chamber Torch Interface cones Quadrupole chamber Dwell time Pb/Mass Cd/Mass
plasma–mass
147
spectrometry
Conditions Free-running type, 40 MHz 1400 W 17.0 L/min 2.00 L/min 1.05 L/min 1.00 mL/min Concentric type Cychronic type Demountable Platinum 1 × 10–6 torr 600 ms 208, 206 mu 114, 112 mu
with mixed standard solutions of Pb and Cd. The standard addition method used three points of calibration: high, low and an untreated sample addition. The highest concentration of the standard addition calibrant was set at three times the concentration obtained from the preliminary analysis. The lowest concentration was determined to be half of the highest concentration obtained from the preliminary analysis. The three digested 3 mL sample solutions used for standard addition were respectively diluted to 5 mL with a suitable amount of standard solution and ultrapure deionised water. Then the three 5 mL sample solutions were analysed with an ELAN 6100 DRC plus ICP-MS (Perkin Elmer SCIEX, Concord, Ontario, Canada), which was calibrated using a smart-tune solution (10 µg/L Ba, 1 µg/L Be, Ce, Co, Fe, In, Mg, Pb, Th and U). The operating conditions are specified in Table 1. Pb and Cd concentrations were determined as the absolute value of the x-axis intercept. Method validation The analytical method was validated in terms of linearity, recovery, limit of detection (LOD) and limit of quantification (LOQ). LOD and LOQ were calculated using the equations LOD = 3.3 × σ/S and LOQ = 10 × σ/S, where σ is the residual standard deviation and S the slope of the calibration curve (KFDA 2004). The LODs of Pb and Cd were measured with a blank and a 5 ng g–1 standard solution, respectively, in seven replicates. Recovery rate was evaluated by SRM using two samples spiked with 0.100 mg kg–1 standard solution.
Results and discussion Method validation LODs for Pb and Cd were 0.06 and 0.09 ng g–1, respectively. Recovery was evaluated as the accuracy of the
148
W. Kim and K.-G. Lee
Downloaded by [Australian National University] at 06:03 24 January 2015
analytical method in semi-solid and liquid forms, with two samples that were spiked with standard solution. Recoveries of Pb and Cd from the semi-solid were 101.8% and 104.0% and from liquid 105.3% and 105.6%, respectively. Recovery rate from solids was determined by SRM 1566b, SRM 8433 and SRM 3249 (Table 2). The z score was used to assess laboratory performance. It was calculated as z = (x – xa)/σp, where x is the certified value of the SRM, xa is the measured value of the SRM and σp is the standard deviation of the measured value. Values of z score in the ranges |z| ≤ 2, 2 < |z| < 3 and |z| ≥ 3 were defined as acceptable, questionable and unacceptable, respectively (Dolan & Capar 2002). Further, z score results for the SRMs were evaluated as acceptable (Table 2). Determination of Pb and Cd Analytical results are listed in Table 3. The arithmetical means for Pb and Cd in KHM were higher than those in FHF. When arithmetic means were calculated from positive values only, the values for Pb and Cd were 0.200 and 0.085 mg kg–1 in FHF (n = 451 for Pb and n = 257 for Cd) and 0.425 and 0.220 mg kg–1 for KHM (n = 41 for Pb and n = 12 for Cd), respectively. Maximum values in KHM were higher than those in FHF, suggesting that ingredients for FHF were cleaned during the manufacturing process (Food and Drug Administration 2003). Individual data can be found in the database. According to product specifications and FHF Code (KFDA 2010), FHF ingredients were divided into docosahexaenoic acid (DHA), N-931, Garcinia cambogia extract, γ-linolenic acid, cereal enzyme, glucosamine, protein, gigantic angelica extract, soy bean and dimethylsulfone. Pb concentration for Spirulina products was the highest in the FHF ingredients (0.940 mg kg–1), wherever it was still safe as the standard regulation is
