Toxic metal levels in cocoa powder and chocolate by ICP-MS method after microwave-assisted digestion

Accepted Manuscript Toxic metal levels in cocoa powder and chocolate by ICP-MS method after microwave-assisted digestion Gianluigi Maria Lo Dico, Fabio Galvano, Giacomo Dugo, Carlo D'ascenzi, Andrea Macaluso, Antonio Vella, Giuseppe Giangrosso, Gaetano Cammileri, Vincenzo Ferrantelli PII: DOI: Reference:

S0308-8146(17)31872-1 https://doi.org/10.1016/j.foodchem.2017.11.052 FOCH 22029

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

23 May 2016 8 August 2017 13 November 2017

Please cite this article as: Maria Lo Dico, G., Galvano, F., Dugo, G., D'ascenzi, C., Macaluso, A., Vella, A., Giangrosso, G., Cammileri, G., Ferrantelli, V., Toxic metal levels in cocoa powder and chocolate by ICP-MS method after microwave-assisted digestion, Food Chemistry (2017), doi: https://doi.org/10.1016/j.foodchem.2017.11.052

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Toxic metal levels in cocoa powder and chocolate by ICP-MS method after microwave-assisted digestion

GIANLUIGI MARIA LO DICO1*, FABIO GALVANO2, GIACOMO DUGO3, CARLO D’ASCENZI4, ANDREA

MACALUSO1,

ANTONIO

VELLA1,

GIUSEPPE

GIANGROSSO 1,

GAETANO

CAMMILERI1, VINCENZO FERRANTELLI1 1

Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri”, Via Gino Marinuzzi 3, 90129 Palermo (Italy)

2

Department of Biological Chemistry, Università degli studi di Catania, Città Universitaria - Via Santa So-

fia, 64, Catania. 3

Department of Organic and Biological Chemistry and Department of Animal Biology and Marine Ecolo-

gy, Università degli Studi di Messina, Vill. S. Agata, 98166 Messina. 4

Department of Veterinary science, Università degli Studi di Pisa, viale delle Piagge, Pisa.

*+390916565258; [email protected]

ABSTRACT The Commission Regulation (EC) Regulation N. 488/2014, established the concentration limits for cadmium in specific products based on cocoa and chocolate products as from January 2019. Based on this information there is a need to determine ultratrace levels of elements that might be presents in cocoa and chocolate products. In this work, the concentrations of Arsenic, Antimony, Cadmium, Chromium, Lead, Selenium and Vanadium were evaluated in cocoa powder and chocolate by the validation of an ICP-MS method. Good selectivity/specificity, recovery, repeatability and within-laboratory reproducibility, LOD, LOQ, range of linearity, standard measurement uncertainty parameters for method validation were achieved, in accordance with Commission Regulation. The cocoa powder revealed the maximum metal concentrations of 0.303 ± 0.035 mg / Kg for cadmium, 1.228 ± 0.146 mg / Kg for lead and 0.094 ± 0.013 mg / Kg for arsenic. A significant difference was found between cocoa powder and chocolate samples (p < 0.05).

Chemical compounds studied in this article: Arsenic (PubChem CID: 5359596), Antimony (PubChem CID: 5354495), Cadmium (PubChem CID: 23973), Chromium (PubChem CID: 23976), Lead (PubChem CID: 5352425), Selenium (PubChem CID: 6326970), Vanadium (PubChem CID: 23990).

1

Keywords: ICP-MS, cocoa, chocolate, toxic metals.

1. Introduction Cocoa powder and chocolate are products arising from the manufacture of cocoa beans (Theobroma cacao) largely consumed around the world due to its flavor, texture and eating pleasure. The main components come from fermented, crushed, and roasted cocoa beans. The cocoa trees are mostly planted in West Africa, South America and Asia. Côte d'Ivoire, Ghana, Indonesia, Nigeria, Cameroon, Brazil, Ecuador and Malaysia represent about 90 % of the world cocoa production [1]. Chocolate can be classified as white, milk and dark, depending mainly on the amounts of dry cocoa, cocoa butter and milk added during the manufacturing process. The definitions and characteristics of cocoa and chocolate products intended for human consumption are defined by the EC Directive 2000/36 [2]. It is known that cocoa contained heavy metals trace and their high concentration in chocolates is a very old problem that has evolved in time [3, 4]. The presence of heavy metals such as lead and cadmium in chocolate is a matter of health consideration because the chemical composition of cocoa allows strong binding of these elements [5]. Considering this issue, children are the biggest consumers of chocolate; the development and validation of analytical methods for the control of ultratrace metals is an important task in order to preserve their health. Trace elements and minerals are essential for biological processes and play an important role in normal growth and development but can be toxic when taken in excess [6]. Due to environmental pollution by industrialization, further studies about the heavy metals exposure are needed [7]. The heavy metals enter in the human body by inhalation and ingestion. Children are more sensitive to heavy metals such as Pb and Cd than adults [8]. Analytical measurement is essential to maintain the quality of a product. Quadrupole Inductively Coupled Plasma Mass Spectrometry (ICP-MS) has been widely employed for the detection of elements at very low concentration [9]. Gases such as H2 or He are introduced into a collision/reaction cell, which consists of an octapole, operated in the rf-only mode. The rf-only mode focuses the ions, which then collide and react with molecules of the collision/reaction gas [6]. In this work we examined the concentrations of Chromium (Cr), Selenium (Se), Cadmium (Cd), Antimony (Sb), Arsenic (As), Lead (Pb) and Vanadium (V) in cocoa powder and chocolate samples by an ICP-MS method in accordance to the conditions suggested by the Commission Decision 2002/657/EC [10], UNI CEI EN ISO/IEC 17025/2005 [11], Commission Regulation (EC) n° 333/2007 [12], Commission Regulation (EC) n° 836/2011 [13] for acceptability criteria of method and

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Commission Regulation (EC) n° 488/2014 [14] and taking into account the Commission Regulation (EC) n° 1881/2006 for the maximum acceptable levels [15].

2. Materials and Methods 2.1. Instrumentation All the elements were determined by an ICP-MS (7700x series, Agilent Technologies, Santa Monica CA, USA) equipped with octopole reaction system (ORS3). The sample solutions were pumped by a peristaltic pump from tubes arranged on an autosampler ASX-500 Series (Agilent Technologies, Santa Monica (CA), USA) and then conducted on a quartz cyclonic spray chamber. A fast and efficient sample digestion was achieved by a microwave-assisted system Multiwave 3000 (Anton-Paar, Graz, Austria) equipped with a rotor for eight MF100 PTFE-TFM (poly-tetrafluoroethylene-tetrafluoroethylene) vessels.

2.2. Reagents and gases All solutions were prepared with ultra-pure grade analytical reagent. Ultrapure deionized water, with resistivity of 18,2 MΩ cm was obtained by Milli-Q® Integral water purification system with Q-pod (Millipore, Bedford, MA, USA) Ultrapure nitric acid (60 % V/V) was purchased from Merck KgaA (Darmstadt, Germany). Standard solutions: the multielement calibration solutions were prepared at different concentration levels (0.05 - 10 µg/L) from 1000 mg/L of single element ICP-MS grade standard, traceable to NIST [16] were purchased from VWR International LTD. (Randon, Pennsylvania, USA). To optimize the performance of ICP-MS a Tuning Solution for ICP-MS, capable of covering a wide range of masses (Ce, Co, Li,Mg, Tl and Y 1 µg/l) was purchased from Agilent Technologies, Santa Monica, CA, USA). Internal Standard solutions: 100 mg/L standard stock solution of scandium (Sc), yttrium (Y), indium (In), terbium (Tb), rhodium (Rh), lutetium (Lu), Lithium6 (Li6), indium (In), germanium (Ge) and bismuth (Bi) were purchased from Agilent Technologies (Santa Monica, CA, USA). Ultrapure grade carrier gas (argon (Ar), 99.9995 % pure) was purchased from SOL S.p.a. (Monza, Mi, Italy). Ultrapure grade diluition gas (helium (He), 99.9995 % pure) was purchased from SOL S.p.a. (Monza, Mi, Italy). Ultrapure grade diluition gas (hydrogen (H2), 99.9995 % pure) was purchased from SOL S.p.a. (Monza, Mi, Italy).

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2.3. Quality assurance The accuracy of the analysis was verified by comparison of the results with the interlaboratory evaluation test 2015/1 “Determination of total concentrations of arsenic, cadmium, mercury and lead in chocolate” organized by the National Reference Laboratory of Italy – National Institute of Health (ISS) where all the concentrations found, in November 2015, were satisfactory, with a Z-score value less than 2, demonstrating the above method. Certified Reference Material DORM-4 (fish protein) was analyzed for analytical batch. The obtained values demonstrated the accuracy of analytical determination, as showed in the table 1.

2.4. Samples Collection and preparation One hundred and forty-five samples of cocoa powder and chocolate produced in Italy were collected from local supermarkets all over Italy in 2015. All the samples represented five popular brands available in the market and were categorized into four different types, according to the Commission Regulation (EC) Reg. 488/2014 [14]. Therefore, 35 samples of chocolate with < 30 % total dry cocoa solids, 35 samples of chocolate with total dry cocoa solids contents between 30 % and 50 %; 35 samples of chocolate with ≥ 50 % total dry cocoa solids, 35 samples of cocoa powder sold to the final consumer and 5 samples cocoa powder for chocolate shake. All the samples were transferred and stored at +4 °C and then processed for analysis before the expiry date. A sample of chocolate with dry cocoa solids contents less than 30 % was chosen for the ICP-MS validation. The digestion of the samples was performed according to the UNI EN 13805:2002 [17]. Approximately 1 g of the samples were transferred into previously decontaminated vessels with 3 mL of 60 % (V/V) ultrapure nitric acid and 5 mL of deionized water [17, 18]. The microwaves digestion for ten vessels followed a power ramp to 600 W in 10 min. The same power was maintained for 40 min. Finally, the vessels were cooled for 15 minutes.

2.5. ICP-MS analysis The instrumental conditions were set as follows following: RF-Power 1550W, reflect power < 30, carrier gas flow 1.0 mL / min, plasma gas flow 15 mL / min, auxiliary gas flow 0.9 mL / min, spray chamber temperature 2 °C, lens voltage 6.25 V, mass range 6 – 220 a.m.u., mass resolution 0.7, integration time 3 point / ms, 3 point of peak, 4 replicates. Torch axis, electron multiplier, plasma correction, standard lens tune, axis, P/A factor, 7 – 89 – 205 mass resolution, ratio (oxide mass 156/140) and ratio (doubly-charged ions 70/140) in NoGas, He and H2 mode. These parameters were optimized daily with the tuning solution in or-

4

der to maximize the signal and to minimize interference effects from polyatomic ions and doubly-charged ions. RSD values lower than 3 % were accepted for the resolution. The concentration was determined by the sum of the isotopes, the signal intensity was attenuated by matrix effect, and the correction of the matrix effect was made by on-line determination of Internal Standard (I.S.) associated to all elements, as showed in table 2. The analyses were conducted by using He and H2 gas into Dynamic Reaction Cell, in order to reduce interferences (ArO, ArCl…). The mathematical equations correct the signal of the analytes when the level of interfering elements produce a signal less than 20 % of the total signals.

2.6. Method validation 2.6.1. Instrumental/method detection and quantification limits The limits of detection and quantification (LoDs and LoQs), were determined by the 3σ and 10σ approach [9, 19, 20]. A pool of 15 blank samples spiked with 0.05 µg / L of all elements were analyzed.

2.6.2. Range of the linearity and calibration curve The calibration curve was constructed with 8 standard additions (BlankCal – 0.01 – 0.05 – 0.1 – 0.2 – 0.5 – 1 – 2 – 5 – 10 – 50 µg/L) checked by the r2. The linearity range was acceptable when r2 was greater than 0.999.

2.6.3. Recovery study The recovery of the method was evaluated at three different concentration levels (50 – 100 – 250 µg / Kg). An acceptance limits between 90 and 110 % was selected according to the scheme of table 2.

2.6.4. Repeatability and within-laboratory reproducibility The repeatability was calculated with metrological approach and with the formula HORRATr where: observed RSDr divided by the RSDr value estimated from the modified Horwitz equation, using the assumption r = 0.66 R; within-laboratory reproducibility is calculated as HORRATR where: the observed RSD R divided by the RSD R value estimated from the modified Horwitz equation percentage variation coefficient (CV %); all the values must be smaller than 2.

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2.6.5. Measurement uncertainty The validation of the analytical method allowed us to identify all the uncertainty contributions in order to calculate the expanded uncertainty [9, 19, 20].

2.7. Statistical analysis All the data obtained were subjected to statistical analysis performed with the statistical software R (3.2.4). A Shapiro-Wilk test was carried out in order to verify the normality of distribution. A Kruskal-Wallis test was carried out in order to verify significant differences between the sample groups (cocoa powder, chocolate with dry cocoa solid contents less than 30 %, chocolate with dry cocoa solid contents between 30 and 50 % and chocolate with dry cocoa solid contents more than 50 %).

3. Results and discussion The instrumentation has achieved all the objectives required by the Commission Regulation n° 836/2011 [13] for the determination of cadmium concentrations in cocoa powder and chocolate for official food control. The linearity test gave a correlation coefficient (r2) was greater than 0,999 for each element and the equation of the straight line demonstrated a low instrumental variability, achieving limits of quantification (LoQ) between 5.22 µg / Kg (LoD Antimony) or 5.74 µg / kg (LoQ Cadmium) and 9.12 µg / kg (LoQ Chromium) or 9.55 µg / kg (LoQ Selenium). ICP-MS is well known to give a linear broad dynamic range; the proposed method revealed acceptable linear regression models for all the analytes examined in the defined range. All the LOQ values obtained in this study were in compliance with the Commission Regulation (EC) n. 333/2007 [12], establishing the methods of analysis for the official control of the levels of Pb and Cd in foodstuff. Furthermore, the instrumentation allowed us to reach LOQ values for Pb, Cd and Cr more than 10 times lower than the method based on Atomic Absorption proposed by Chukwujindu 2011 [21] (0.40, 0.05 and 0.2 mg / kg, respectively) and the same times lower than Pb LOQ value obtained by Jalbani et al. 2009 [4] with another Atomic Absorption method. Slight differences were found in As, Cd and Sb LOQ values between our method and another based on ICP-MS proposed by Millour et al. 2011 [20] (0.01, 0.001 and 0.001 respectively), while the same LOQ values were achieved for Pb and V. The recovery values were found between 93 % and 105 % for all the elements examined, greater than other methods proposed in literature based on ICP-AES and Atomic Absorption applied on the same matrices [21, 22, 23]. The repeatability limit was calculated by adding to 15 digested samples spiked 1.00 - 2.00 to 5.00 µg /

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L of all analytes. The results were satisfactory for the limit of repeatability (metrological approach), less than the double value of the expanded uncertainty [11, 19]. All concentration levels showed Horrat values of repeatability and reproducibility lower than 2 as specified by the Commission Regulation n° 836/2011 [13]. The method proposed confirmed ICP-MS as the preferred technique for rapid multi-elemental analysis of food matrix, offering multi-elemental capability, extreme sensitivity and selectivity in a short timeframe (only 2 minutes for each sample with the method validated in this study). More than 94 % of cocoa powder and chocolate with a dry cocoa content major than 50 % samples revealed toxic metals concentrations over the limit of quantification; results below 0.001 mg / Kg are indeterminable. The results of analysis showed an increasing trend of toxic metals levels depending on the amount of dry cocoa used in the formulation. In fact, cocoa powder samples revealed the highest metals concentrations, with maximum Pb, Cd, As, V, Cr, Sb and Se of 1.228 ± 0.146, 0.303 ± 0.035, 0.094 ± 0.013, 0.627 ± 0.078, 5.864 ± 0.601, 1.564 ± 0.165 and 0.146 ± 0.019 mg / Kg, respectively. However, the Cd concentrations obtained were under the permissible levels established by the International Cocoa Organization [24] (0.6 mg / kg) and by Commission Regulation Reg. 488/20114 [14]. The chocolate samples with a dry cocoa content less than 30 % revealed the lowest metals values, with maximum Pb, Cd, As, V, Cr, Sb and Se of 0.545 ± 0.070, 0.050 ± 0.006, 0.013 ± 0.002, 0.094 ± 0.013, 0.952 ± 0.097, 0.027 ± 0.003 and 0.047 ± 0.007 mg / Kg, respectively (table 3). Even in the case of preparation for milk shake, with a dry cocoa content below 30 % (the toxic metals concentrations were under the limit of quantification of the method). The data obtained did not follow a log-normal distribution, therefore Kruskal-Wallis tests were carried out. Significant differences were found between cocoa powder and chocolate samples (p < 0.05) for all the toxic metals examined. The Nemeney post-hoc test revealed that the toxic metals concentrations found in cocoa powder and chocolate with dry cocoa contents more than 50 % were significantly higher than the other chocolate samples. No significant difference was found between chocolate brands (Kruskal-Wallis chi-squared = 7.2053, df = 4, p > 0.05), suggesting that the toxic metals amounts found in our study doesn’t depend on manufacturing processes such as contamination via utensils, transportation and storage, in contrast to what was reported by Rankin et al. 2005 [25] for Pb levels assessment (lead contamination in cocoa and cocoa products). The data distributions of toxic metals levels, sorted by sample types, were reported in fig 1. Our findings seem to be in compliance with what was reported by Lee and Low 1985 [22], which have not verified contamination of toxic metals, either from the processes involved or from the ingredients added during chocolate production. It is therefore probable that the artificial contamination of soil is the principal route of heavy metals uptake by cocoa plants [25], and consequently the main cause of heavy metals presence in their derived

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products. The distribution of toxic metals, such as cadmium, in cacao plant generally decreased from beans to leaves; cadmium was detected in shells when the concentration in beans was more than 1 mg/kg [24]. The results obtained in this study revealed As, Cd and Pb levels much lower than that was found by Lee et al., 1985 [20] in dark and milk chocolate samples from Malaysia (0.83 mg / Kg, 0.41 mg / Kg, 1.94 mg / Kg, respectively). However, same correlation was found between toxic metals levels and percentage of dry cocoa used for chocolate production. The levels of Pb found in the chocolate samples with a dry cocoa content more than 50 % and between 30 and 50 % were much higher than those from chocolate samples examined by Yanus et al., 2013 [6] (0.088 and 0.083 mg / Kg), on the contrary, a trend reversal, was found for Cr levels (2.913 and 0.566 mg / Kg). The levels of Cd and Pb in chocolate samples examined by Rehman and Husnain, 2012 [23] were comparable to those from our study (Cd 0.046, Pb 0.360 mg / Kg for chocolate > 50 % cocoa powder; Cd 0.033, Pb 0.088 mg / Kg) suggesting adaptation by food industry to the recent European provisions.

4. Conclusion In this study, we performed a validated method that has been proved effective in the quantification of Cd, Pb, As, Cr, Sb, Se and V extracted from cocoa powder and chocolate matrices, with efficient recovery (90 – 110 %). The proposed method obtained results in accordance to the performance criteria and the requirements set by European Union for method validation to be used in official food controls [17, 18]. Furthermore, the present method revealed satisfactory detection limits due to the technique employed. The results obtained might exclude a contamination of chocolate due to manufacturing processes. All the chocolate samples reached cadmium levels under the limits imposed by Commission Regulation (EC) Reg. 488/20114 [14]. Even in the study a correlation was confirmed between the levels of trace metals in chocolate and the dry cocoa content. Based on our findings, further studies on preparation process and selection of raw materials are needed in order to have an overview on the presence of toxic metals in these products. In cocoa powder the maximum tolerable levels for Pb and Cd have been set to 1 mg / Kg and 0.3 mg / Kg by European Legislation, so cocoa powder, if taken daily, could be harmful for health. Children are great consumers of chocolate and can be categorized as risk group because of their low body weight and higher digestive track uptake. However, the low content of Toxic metals detected in this study and the presents of molecules such as antioxidants and fatty acids make chocolate a food that can improve the human health.

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5. Conflict of Interest The authors declare that they have no conflict of interest related to the publication of this manuscript.

6. Acknowledgement We want to thank Rea Lo Dico, MD of the INSERM U965 CART, Paris Diderot University, Sorbonne Paris Cite, for the technical support.

7. References [1] UNCTAD. (2008). Cocoa Study: Industry Structures and Competition. In United Nations Conference on Trade and Development 2008 ed., (pp. 62).

[2] European Commission. (2000). Directive 2000/36/EC of the European Parliament and of the Council of 23 June 2000 relating to cocoa and chocolate products intended for human consumption. In, vol. 197 (pp. 1925). Official Journal of the European Communities.

[3] Hou, S., Yuan, L., Jin, P., Ding, B., Qin, N., Li, L., Liu, X., Wu, Z., Zhao, G., & Deng, Y. (2013). A clinical study of the effects of lead poisoning on the intelligence and neurobehavioral abilities of children. Theor Biol Med Model, 10, 13; doi: 10.1186/1742-4682-10-13.

[4] Nusrat Jalbani, T. G. K., Hassan I. Afridi and Mohammad Bilal Arain. (2009). Determination of Toxic Metals in Different Brand of Chocolates and Candies, Marketed in Pakistan. Pak. J. Anal. Environ. Chem., 10(1 & 2), 48-52.

[5] Valiente, C., Molla, E., MartinCabrejas, M. M., LopezAndreu, F. J., & Esteban, R. M. (1996). Cadmium binding capacity of cocoa and isolated total dietary fibre under physiological pH conditions. Journal of the Science of Food and Agriculture, 72(4), 476-482.

[6] Yanus, R. L., Sela, H., Borojovich, E. J., Zakon, Y., Saphier, M., Nikolski, A., Gutflais, E., Lorber, A., & Karpas, Z. (2014). Trace elements in cocoa solids and chocolate: an ICPMS study. Talanta, 119, 1-4; //dx.doi.org/10.1016/j.talanta.2013.10.048.

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[7] Orun, E., Yalcin, S. S., Aykut, O., Orhan, G., Morgil, G. K., Yurdakok, K., & Uzun, R. (2011). Breast milk lead and cadmium levels from suburban areas of Ankara. Sci Total Environ, 409(13), 2467-2472; doi: 10.1016/j.scitotenv.2011.02.035.

[8] Tripathi, R. M., Raghunath, R., Sastry, V. N., & Krishnamoorthy, T. M. (1999). Daily intake of heavy metals by infants through milk and milk products. Science of the Total Environment, 227(2-3), 229-235; doi: 10.1016/S0048-9697(99)00018-2.

[9] Gianluigi Maria Lo Dico, G. C., Andrea Macaluso, Antonio Vella, Giuseppe Giangrosso, Mirella Vazzana and Vincenzo Ferrantelli. (2015). Simultaneous Determination of As, Cu, Cr, Se, Sn, Cd, Sb and Pb Levels in Infant Formulas by ICP-MS after Microwave-Assisted Digestion: Method Validation. J Environmental & Analytical Toxicology, 5, 328; doi:10.4172/2161-0525.1000328.

[10] European Commission (2002). Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. In, vol. 221 (pp. 8-36). Official Journal of the European Communities.

[11] ISO. (2005). EN ISO/IEC 17025:2005 - General requirements for the competence of testing and calibration laboratories. 28.

[12] European Commission (2007) Commission Regulation (EC), No 333/2007 of 28 March 2007 laying down the methods of sampling and analysis for the official control of the levels of lead, cadmium, mercury, inorganic tin, 3-MCPD and benzo(a)pyrene in foodstuffs. Official Journal of the European Union, 88, 29-38.

[13] European Commission (2011) Commission Regulation (EU) No 836/2011 of 19 August 2011 amending Regulation (EC) No 333/2007 laying down the methods of sampling and analysis for the official control of the levels of lead, cadmium, mercury, inorganic tin, 3-MCPD and benzo(a)pyrene in foodstuffs. Official Journal of the European Union, 215, 9-16.

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[14] European Commission (2014) Commission Regulation (EU) No 488/2014 of 12 May 2014 amending Regulation (EC) No 1881/2006 as regards maximum levels of cadmium in foodstuffs. Official Journal of the European Union, 138, 75-79.

[15] European Commission (2006) Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union, 364, 5-24.

[16] NIST; 2012; National Institute of Standards and Technology, Certificate of analysis, standard reference material, USA.

[17] ISO. (2002). UNI EN 13805:2002 - Foodstuff, Determination of trace elements, Pressure Digestion.

[18] ISO. (2010). UNI EN 15763:2010. Foodstuff, Determination of trace elements, Determination of arsenic, cadmium, mercury and lead in foodstuff by inductively coupled plasma mass spectrometry (ICP-MS) after pressure digestion.

[19] Örnemark, B. M. a. U. (2014). The Fitness for Purpose of Analytical Methods: A Laboratory Guide to Method Validation and Related Topics: Second edition.

[20] Millour, S., Noel, L., Kadar, A., Chekri, R., Vastel, C., & Guerin, T. (2011). Simultaneous analysis of 21 elements in foodstuffs by ICP-MS after closed-vessel microwave digestion: Method validation. Journal of Food Composition and Analysis, 24(1), 111-120; doi:10.1016/j.jfca.2010.04.002.

[21] Iwegbue, C. M. (2011). Concentrations of selected metals in candies and chocolates consumed in southern Nigeria. Food Addit Contam Part B Surveill, 4(1), 22-27; doi: 10.1080/19393210.2011.551943

[22] C.K. Lee and K.S. Low (1985). Determination of Cadmium, Lead, Copper and Arsenic in Raw Cocoa, Semifinished and Finished Chocolate Products. Pertanlka 8 (2). pp. 243-248. ISSN 0126-6128

[23] Husnain, S. R. a. S. M. (2012). Assessment of trace metal contents in chocolate samples by Atomic Absorption Spectrometry. Journal of Trace Element Analysis, 1(1), 1-11; doi:10.7726/jtea.2012.1001.

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[24] Chavez, E., He, Z. L., Stoffella, P. J., Mylavarapu, R. S., Li, Y. C., Moyano, B., & Baligar, V. C. (2015). Concentration of cadmium in cacao beans and its relationship with soil cadmium in southern Ecuador. Sci Total Environ, 533, 205-214.

[25] Rankin, C. W., Nriagu, J. O., Aggarwal, J. K., Arowolo, T. A., Adebayo, K., & Flegal, A. R. (2005). Lead contamination in cocoa and cocoa products: isotopic evidence of global contamination. Environ Health Perspect, 113(10), 1344-1348.

[26] Lucho-Constantino, C. A., Alvarez-Suarez, M., Beltran-Hernandez, R. I., Prieto-Garcia, F., & PoggiVaraldo, H. M. (2005). A multivariate analysis of the accumulation and fractionation of major and trace elements in agricultural soils in Hidalgo State, Mexico irrigated with raw wastewater. Environ Int, 31(3), 313-323.

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Highlight

• • •

145 chocolate and cocoa powder samples were examined for heavy metals detection; An ICP-MS method was improved and validated for the intended purpose; The method has proved fast and reliable according to the EC Regulations;

•

Significant differences were found between cocoa powder and chocolate samples (p<0.05);

•

A correlation between heavy metal levels and dry cocoa amount of the samples was found.

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14

0.301 ± 0.020

Certified Proficiency Test 0.303 ± 0.022

Measured Proficiency Test 0.281 ± 0.030

0.416 ± 0.053

0.411 ± 0.048

0.027 ± 0.007

0.038 ± 0.004

Arsenic

6.80 ± 0.64

6.22 ± 0.51

0.016 ± 0.002

0.016 ± 0.004

Copper

15,9 ± 0.9

16.4 ± 1.86

Selenium

3.56 ± 0.34

3.47 ± 0.39

Element (mg/Kg)

Certified DORM-4

Cadmium

0.306 ± 0.015

Lead

Measured DORM-4

Table 1. Results of elemental analyses for CRM DORM-4 and interlaboratory evaluation test (Proficiency Test).

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Parameters (µg/kg)

Recovery (%)

Limit of repeatability (µg/kg)

Expanded uncertainty (µg/kg)

50

100

3.12

± 6.01

100

100

7.21

± 13.9

250

104

8.56

± 31.2

50

100

2.71

± 6.40

100

100

5.74

± 13.8

250

96

7.59

± 28.0

50

99

3.50

± 6.90

100

99

7.50

± 13.7

250

95

9.88

± 34.5

50

99

2.48

± 5.40

100

100

4.22

± 14.0

250

96

6.46

± 32.4

50

101

7.56

± 7.58

100

100

9.87

± 14.6

250

105

15.23

± 29.5

50

96

6.41

± 7.00

100

98

10.30

± 17.6

Pb (204Pb, 206Pb, 207Pb, 208Pb) S.I. 209 Bi

Cd (111Cd, 112Cd) S.I. 115 In

As (75As) S.I. 72Ge

Sb (121 Sb) S.I. 115 In

Cr (52 Cr) S.I. 45Sc

Se (78 Se)

16

S.I. 74Ge

250

93

13.30

± 36.3

50

102

3.86

± 6.20

100

101

9.20

± 16.30

250

97

16.80

± 28.90

V (51V) S.I. 45Sc

Table 2: Method performance for Lead, Cadmium, Arsenic, Antimony, Chromium, Selenium and Vanadium.

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POWDER ELEMENT

MEDIAN

MAX

CHIOCCOLATE > 50% OF COCOA DRY MIN

MEDIAN

MAX

MIN

0.001 (N.D.)

0.116 ± 0.015

0.207 ± 0.027

0.019 ± 0.003

Cd

0.159 ± 0.020

Pb

0.417 ± 0.032

1.228 ± 0.146

0.049 ± 0.006

0.133 ± 0.013

0.616 ± 0.077

0.021 ± 0.003

As

0.026 ± 0.003

0.094 ± 0.013

0.003 (N.D.)

0.012 ± 0.002

0.037 ± 0.005

0.007 ± 0.001

V

0.196 ± 0.022

0.627± 0.078

0.005 (N.D.)

0.081 ± 0.009

0.347 ± 0.041

0.009 ± 0.001

Cr Sb

1.315 ± 0.147

5.864 ± 0.601

0.083 ± 0.010

0.798 ± 0.084

2.544 ± 0.274

0.153 ± 0.020

0.383 ± 0.041

1.564 ± 0.165

0.043 ± 0.005

0.008 ± 0.001

0.021 ± 0.004

0.001 (N.D.)

Se

0.076 ± 0.007

0.146 ± 0.019

0.020 ± 0.003

0.055 ± 0.006

0.102 ± 0.015

0.018 ± 0.002

0.303 ± 0.035

CHIOCCOLATE < 50% >30% OF COCOA CHIOCCOLATE < 30% OF COCOA DRY OR DRY WHITE ELEMENT

MEDIAN

MAX

MIN

MEDIAN

MAX

MIN

Cd

0.019 ± 0.002

0.040 ± 0.006

0.007 ± 0.001

0.012 (
0.050 ± 0.006

0.000 (N.D.)

Pb

0.170 ± 0.012

0.895 ± 0.093

0.019 ± 0.003

0.156 ± 0.016

0.545 ± 0.070

0.017 ± 0.002

As

0.007 ± 0.001

0.015 ± 0.002

0.003 (N.D.)

0.006 ± 0.001

0.013 ± 0.002

0.001 (N.D.)

V

0.020 ± 0.002

0.089 ± 0.012

0.005 ± 0.001

0.021 ± 0.002

0.094 ± 0.013

0.002 (N.D.)

Cr

0.416 ± 0.053

0.936 ± 0.096

0.085 ± 0.010

0.403 ± 0.052

0.952 ± 0.097

0.031 ± 0.004

Sb

0.008 ± 0.001

0.035 ± 0.004

0.002 (N.D.)

0.008 ± 0.001

0.027 ± 0.003

0.002 (N.D.)

Se

0.035 ± 0.004

0.070 ± 0.001

0.022 ± 0.004

0.032 ± 0.004

0.047 ± 0.007

0.013 ± 0.002

Table 3: median, max, min concentration values (mg / Kg) on 145 samples (n=35). N.D. not determinable (< LoQ).

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