Method validation and determination of heavy metals in cocoa beans and cocoa products by microwave assisted digestion technique with inductively coupled plasma mass spectrometry

Food Chemistry 303 (2020) 125392

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Method validation and determination of heavy metals in cocoa beans and cocoa products by microwave assisted digestion technique with inductively coupled plasma mass spectrometry

T

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Rahmat Mohamed, Badrul Hisyam Zainudin , Abdul Syukor Yaakob Analytical Services Laboratory, Cocoa Downstream Technology Division, Malaysian Cocoa Board, Cocoa Innovation and Technology Centre, Lot 12621 Kawasan Perindustrian Nilai, 71800 Nilai, Negeri Sembilan, Malaysia

A R T I C LE I N FO

A B S T R A C T

Keywords: Cocoa Measurement uncertainty Heavy metals Microwave acid-digestion ICP-MS

In this article, an easy and quick method based on microwave assisted acid digestion technique prior to quantification using inductively coupled plasma mass spectrometry for the analysis of heavy metals in cocoa beans, cocoa powder and chocolate was established and validated for arsenic (As), cadmium (Cd), lead (Pb), and antimony (Sb). Limit of quantification for all elements were product dependent and varies from 7.84 to 194.52 µg/kg. The recoveries of the heavy metals at 250 and 1000 µg/kg spiking levels were ranged between 96.27–108.75%, 90.43–101.97% and 89.72–106.26% for cocoa beans, cocoa powder, and chocolate, respectively. Relative standard deviation values obtained were all below 20% and the expanded uncertainty measurements for the elements were less than 25%. The analysis of real samples found that the concentration level is far from the national alarming level except for cadmium in cocoa beans.

1. Introduction Heavy metals and trace elements are essential for human metabolism and play an important role in normal growth and development. However, they can be toxic when taken in excess. Many of the heavy metals, such as zinc, copper, chromium, iron and manganese, are essential to body function in very small amounts. However, if these metals accumulate in the body in high concentrations, they can cause poisoning and may lead to a number of human health disorders, which can induce systemic health problems, such as decreased immunological defenses, impaired psycho-social behavior, disabilities associated with malnutrition and a high prevalence of upper gastrointestinal cancer (Järup, 2003; Türkdoǧan, Kilicel, Kara, Tuncer, & Uygan, 2002). On the other hand, heavy metals such as arsenic, lead, cadmium, and mercury are hazardous and can be classified as non-essential to metabolic and other biological functions (Rai, Lee, Zhang, Tsang, & Kim, 2019). Ingestion of crops contaminated with these metals may cause adverse effect to human health. Cadmium, which is highly enriched in women, can enter the embryo through the placenta and destroy the placenta’s morphological structure, which result in fetal growth restriction (Geng & Wang, 2019). Prenatal exposure to mercury during pregnancy may also affect neurodevelopment in children (Barbone et al., 2019). Cadmium and lead also linked to smoking-related respiratory diseases such

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as chronic obstructive pulmonary disease and lung cancer (Taylor, Isley, & Glover, 2019; Xiong et al., 2019). Elevated cadmium levels in humans have been significantly correlated with adverse effects such as pre-term labor or a low birthweight for gestation age. On the other hand, cadmium blood concentrations have been correlated with adverse effects on several sperm parameters (California Environmental Protection Agency, 1996). Cocoa beans are derived from the fermented and dried seeds of cocoa tree (Theobroma cacao). These beans are an important global agricultural commodity used as a raw ingredient in the chocolate industry and other cocoa-related products such as beverages, cakes, biscuits, and ice cream (Hashimoto et al., 2018). Food contaminants especially metals played important role in chocolate industry where children are vulnerable to metals such as cadmium and lead. The source of heavy metals in cocoa products can be contributed from industrial agricultural activities in which the industrial discharge can accumulate in water or soil and later transferred into cocoa beans during vegetation through the roots system (Bell, 1992). These metallic contaminants might withstand the post-harvest process and find their occurrence in the semi-finished products such as cocoa liquor, cocoa butter and cocoa powder, as well as chocolate. In response, global legislative bodies especially CODEX Alimentarius Commission and EU Commission enforced strict

Corresponding author. E-mail addresses: [email protected] (R. Mohamed), [email protected] (B.H. Zainudin), [email protected] (A.S. Yaakob).

https://doi.org/10.1016/j.foodchem.2019.125392 Received 13 February 2019; Received in revised form 1 August 2019; Accepted 18 August 2019 Available online 20 August 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.

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the data quality (Azevedo et al., 2009). The aim of this study is to validate a method for the simultaneous determination of heavy metals such Arsenic (As), Cadmium (Cd), Lead (Pb) and Antimony (Sb) in cocoa beans and cocoa derived products such as cocoa powder (semi-finished product) and chocolate (finished product). These metals were selected based on the MPLs contained in Schedule 14th Malaysian Food Act (1983), and Regulations 1985 (Food Regulations, 2010). In this case, it is important to study the metal levels in both the raw materials, intermediate products and final products since the sources of metals contamination were varied between these three matrices. Previous study found that lead and cadmium concentrations were higher in cocoa powder and butter than in the raw cocoa beans (Yanus et al., 2014). Earlier research by Rankin et al. indicated that atmospheric emissions of leaded gasoline is one of the probable sources of contamination of the finished products (Rankin et al., 2005). The argument whether the metal contaminations are naturally present in soil or man-made during cocoa processing are still ongoing. This method validation took into consideration the importance method validation criteria namely limit of detection (LOD), limit of quantification (LOQ), linearity, and measurement of uncertainty. The validated method was then applied to cocoa beans and cocoa products samples collected from smallholders and manufacturers respectively.

regulations to minimise exposure of certain heavy metals through establishment of Maximum Levels (MLs). In addition to CODEX, other International and National Standards for heavy metals in cocoa have been established. Australia New Zealand Food Standards Code established a maximum level of 0.5 mg/kg for chocolate and cocoa products for cadmium (Code, 2016). On the other hand, China has introduced maximum levels for cocoa products, chocolate and chocolate products and candies for lead and arsenic at 0.5 mg/kg (GB 2762, 2014). The Food and Drug Administration of the United States has no provision for maximum level for cocoa beans. However, they recommended that lead levels in candy products including chocolates likely to be consumed frequently by small children, should not exceeded 0.1 mg/kg (US Food and Drug Administration, 2006). Malaysia has established a Maximum Permissible Limit (MPL) for cocoa and cocoa products as described in Schedule 14th Malaysian Food Act (1983), and Regulations 1985 (Food Regulations, 2010). From the Malaysian perspective, EU countries are major consumers of semi-finished cocoa products. Malaysia export value keeps increasing every year and in 2017 alone, the export value of those products was about USD 1.02 billion (Malaysian Cocoa Board, 2018). Currently the European Commission has introduced the EC 1881/2006 regulation, setting maximum levels (wet weight) for certain heavy metals contaminants in cocoa products that will come into effect in 2019 (European Commission, 2006). The Commission proposed the level of cadmium in dark chocolate with more than 50% total dry cocoa solids should be 0.8 mg/kg. For chocolate with less than 50% solids a 0.30 mg/kg level of cadmium is proposed and for milk chocolate (less than 30% solids) a limit of 0.1 mg/kg is proposed. Finally, a limit of 0.60 mg/kg would apply for cocoa powder sold to the final consumer (chocolate drink). This EU’s proposal would not really affect the cocoa producing countries of raw cocoa beans. However, it will certainly affect some of the countries which produce semi-finished cocoa products and chocolate manufacturers. As a result, products containing cadmium above permitted levels will not be allowed in the EU’s market. Thus, producing countries of semi-finished products including Malaysia will suffer losses in export value. Consequently, there is a need to develop reliable method for the determination of heavy metals in cocoa products which conforms to both validation criteria and uncertainty measurements. Cocoa samples were prominent for their complexity and difficulty because of their high fat content and the presence of other chemical constituent such as fatty acid, fatty acid esters, phytosterols, tocopherols, sugars, polyphenols, theobromine, and caffeine (Zainudin, Salleh, Mohamed, Yap, & Muhamad, 2015). Heavy metals analysis can be divided into two techniques, the open-system dry ashing technique and the close-system microwave digestion technique. Nowadays, the time consuming dry ashing technique had been replaced by more efficient and fast microwave digestion technique. While dry ashing technique could take one to two days to finish, the microwave technique can digest the samples in merely 1 h. Many analytical methods had been proposed and used as routine analysis for cocoa and cocoa products either using dry ashing or microwave digestion technique (Aikpokpodion, Atewolara- Odule, Osobamiro, Oduwole, & Ademola, 2013; Arévalo-Gardini, ArévaloHernández, Baligar, & He, 2017; Assa, Noor, Yunus, Misnawi, & Djide, 2018; Barraza et al., 2017; Bertoldi, Barbero, Camin, Caligiani, & Larcher, 2016; Chavez et al., 2015; Dahiya, Karpe, Hegde, & Sharma, 2005; Lewis, Lennon, Eudoxie, & Umaharan, 2018; Ramtahal et al., 2015; Salama, 2018; Shittu & Badmus, 2009; Vītola & Ciproviča, 2016; Yanus et al., 2014). However, the information related to method validation is very limited (Kruszewski, Obiedziński, & Kowalska, 2018; Lo Dico et al., 2018). Method validation is an essential component of the measures that a laboratory should implement in order to produce reliable analytical data (Magnusson & Örnemark, 2014). For consistent interpretation of the measurement results, it is necessary to evaluate the confidence that can be placed on the method. Therefore, the presentation of an analytical result must be accompanied by indication of

2. Materials and methods 2.1. Reagents and materials Water was purified through an Elga Purelab Option-Q system (High Wycombe, UK). Ultrapure nitric acid (HNO3, 65%) and hydrogen peroxide (H2O2, 30%) were obtained from Merck (Darmstadt, Germany). Certified Reference Standard solution: 1000 mg/L single element of arsenic, cadmium, lead and antimony were purchased from Ultra Scientific (North Kingstown, USA). Mixed standard solutions for calibration curve ranging from 0.1 µg/L to 50 µg/L were prepared by successive dilution using 8% nitric acid. Internal standard solution containing Bi, Ge, Li, Lu, Rh, Sc and Tb for instrument control and optimization was purchased from Agilent Technologies (Santa Clara, USA). 2.2. Cocoa beans and cocoa products samples for fortification 1 g of dried cocoa beans and cocoa products were weighed and transferred into PTFE vessels and spiked with 0.25 mL and 1 mL from the 1 mg/L intermediate mixed standard solution, prior to digestion to give final spiking concentration levels of 250 and 1000 µg/kg. Digested samples were then filtered, diluted and transferred into 50 mL polypropylene volumetric flask. 2.3. Samples digestion Digestion was performed using microwave digestion system Milestone Ethos Up (Milestone, Sorisole, Italy). 1 g samples were weighed into dry and clean PTFE vessels. Then, 6 mL of HNO3 and 2 mL of H2O2 were added. The vessels were capped and introduced to the microwave digestion system and digested using a two-step digestion program. Temperature of the vessels was increased to 200 °C in 15 min and stayed another 15 min for digestion to complete. The resulting solutions were cooled, diluted with 50 mL ultrapure water and filtered using filter paper. 2.4. Instruments optimization Heavy metals quantification was carried out by inductively coupled plasma quadrupole mass spectrometry (ICP-MS) Agilent 8800 ICP-QQQ equipped with a quartz spray chamber, glass concentric nebulizer, on2

Food Chemistry 303 (2020) 125392

3.2 0.1 5.7 0.8 12.3 13.0 11.5 7.9

10.6 0.2 19.0 2.5

LOD RSD (%) Rec. (%)

99.5 95.3 89.7 92.5

RSD (%)

5.0 4.9 5.6 5.8

2.5. Method validation criteria The validation parameters included in this study were linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ) and measurement of uncertainty (MU). The method validation followed the protocol from EURACHEM guidelines (Magnusson & Örnemark, 2014).

30.7 185.1 50.3 194.5 9.2 55.5 15.1 58.4

106.3 101.0 94.1 94.5

LOQ

Rec. (%)

2.6. Real samples monitoring

LOD

1000 µg/kg 250 µg/kg

Chocolate

line internal standard (ISTD) addition kit, and Ni interface cones (Agilent Technologies, Tokyo, Japan). The ICP-MS was allowed to stabilize for 20–30 min and the performance was optimized based on radio frequency (RF) power, sampling depth, argon flow rate, collision cell gas flow rate, lens voltage and sample uptake rate. For quantification, the following analytes isotopes were used; 75As, 111Cd, 121Sb and 208Pb.

Samples for monitoring purposes consisted of cocoa beans collected from local farmers. Cocoa powder samples were obtained from local cocoa manufacturers and traders. Chocolate samples were collected from local markets. In total, 86 cocoa beans samples, 97 cocoa powder samples and 35 chocolate samples were collected for this study in 2016 and 2017. Samples were analyzed in duplicate.

3.4 9.1 16.0 9.3 102.0 95.5 90.4 97.0

4.2 4.5 18.7 7.1

3.1. Optimization of ICP-MS The optimization of ICP-MS is important because the nebulizer gas and make-up gas flows had to be adjusted to ensure plasma stability. This was achieved by adjusting the torch position and tuning for reduced oxide and doubly charged ion formation with a standard tuning solution containing 10 ng/g of 7Li, 89Y, 140Ce and 205Tl in 2% HNO3. The optimized parameters were as follows; Nebulizer Gas flow rates: 0.86 L/min; Auxiliary Gas Flow: 1.2 L/min; Plasma Gas Flow: 15 L/min; Lens Voltage: 7.25 V; ICP RF Power: 1100 W; CeO/Ce = 0.031; Ba++/ Ba+ = 0.016.

100.0 96.2 89.6 97.1

Rec. (%) RSD (%) Rec. (%)

3.2. Method validation

Rec. (%) RSD (%)

4.4 8.3 8.1 4.6

Rec. (%)

108.7 100.7 96.3 102.4

3.6 3.8 6.7 2.4

2.4 5.6 4.4 20.8

7.8 18.5 14.7 69.3

Selectivity and specificity depend on the selected element and possible interferences. It is always relating to “the extent to which the method can be used to determine particular analytes in mixtures or matrices without interferences from other components of similar behavior” (Vessman et al., 2001). The selectivity of the method in this work was investigated by studying its ability to measure the analyte of interest by the use of specific isotopes and in this case the primary isotopes for each element were used; 75As, 111Cd, 121Sb and 208Pb. In order to evaluate linearity of the method, seven calibration standards of each analyte with concentrations of 0.1, 0.5, 1.0, 5.0, 10.0 and 20.0 and 50.0 µg/L were analyzed by ICP-MS and the responses recorded. The calibration curves were found to be linear in the range of 5–2500 µg/kg with correlation coefficient values better than 0.990 for all elements. This result demonstrated linearity of this method over the specified range. The LOD indicates the lowest concentration that can be distinguished from noise, but not necessarily quantified, while LOQ is the lowest concentration of the analyte that can be determined with an acceptable level of repeatability, precision, and trueness. LOD was estimated from the calibration function using Eq. (1) while LOQ was estimated from the calibration function using Eq. (2) (Lo Dico et al., 2015).

3·sdblank × Cspiked ⎛ ⎞ LOD = ⎜ Signalspiked − Signalblank ⎟ ⎝ ⎠

n=6

As Cd Sb Pb

108.5 99.5 97.2 98.8

1000 µg/kg 250 µg/kg

Cocoa beans

RSD (%)

LOD

LOQ

1000 µg/kg 250 µg/kg

Cocoa powder

RSD (%)

3. Results and discussion

Element

Table 1 Average recovery (%), RSD (%), LOD (µg/kg) and LOQ (µg/kg) obtained by microwave digestion of cocoa beans, cocoa powder and chocolate, spiked at 250 and 1000 µg/kg and analyzed by ICP-MS.

LOQ

R. Mohamed, et al.

3

(1)

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⎛ 10·sdblank × Cspiked ⎞ LOQ = ⎜ Signalspiked − Signalblank ⎟ ⎝ ⎠

percentage recovery. Samples were spiked at two different concentrations (250 µg/kg and 1000 µg/kg) in six replicates for each concentration. The recovery values obtained were further used in the calculation of standard uncertainty for recovery, u(Rec) and relative standard uncertainty, (RSU) using Eqs. (5) and (6) respectively (Supplementary 2). Additionally, a Student’s t test was used to determine whether the mean recovery is significantly different from 1. From the results obtained, it was found that the critical value tcrit were greater than the tcal values, hence recoveries obtained in the validation data were not significantly different from 100%, and hence no corrections needed to the test results. Another source of uncertainty that contributes to the uncertainty in bias is the purity of the reference standards from which the spiked solutions are prepared. The purity of each reference standards is given by the manufacturer and the standard uncertainty, u(purity) was calculated using rectangular distribution. Finally, the Bias uncertainty is obtained by combining the RSU of each uncertainty contributors using Eq. (7) as 0.016, 0.018, 0.016, and 0.028 for As, Cd, Pb and Sb respectively.

(2)

In order to determine these values, sample blank and spiked samples with expected concentration of 250 µg/kg were analyzed and the results were summarized in Table 1. Results showed that the LOQ for all elements were product dependent and varies from 7.84 to 194.52 µg/kg. All these values are lower than national MPLs (Food Regulations, 2010) and hence concluded that the method is sensitive enough to quantify these elements in cocoa beans, cocoa powder and chocolate. As demonstrated in Table 1, the recoveries of the heavy metals at 250 and 1000 µg/kg were ranged between 96.27–108.75 %, 90.43–101.97% and 89.72–106.26% for cocoa beans, cocoa powder, and chocolate, respectively. On the other hand, the RSD values obtained were all below 20%. Both the recoveries and RSD values met the method performance criteria and indicate the good precision and accuracy of the method. 3.3. Estimation of measurement uncertainty The uncertainty is a quantification of the doubt about the result and determined whether the measurement result is fitted for the intended purpose. There are various approaches related to measurement uncertainty, but the most common are known as Top-Down and Bottomup approaches. Then, the overall uncertainty is obtained by identifying, quantifying and combining all individual contributions to uncertainty. “Bottom-up’’ approach was used in this study using the data obtained during method validation and found that uncertainty of extraction belongs to precision and bias were shown to represent the main source of combined standard uncertainty. The relative expanded uncertainty was then calculated by using the coverage factor (k) of 2 at 95% confidence level. Coverage factor is a numerical factor used as a multiplier of the combined standard uncertainty in order to obtain an expanded uncertainty. A typical coverage factor ranges from 2 to 3 depending on the degree of freedom (95–99%) and can be obtained from the Student’s T table.

u (P ) =

(RSD1) + (RSD2 ) (n1 − 1) + (n2 − 1)

(n1 − 1)(RS D12) + (n2 − 1)(RS D22) + ⋯⋯⋯ (n1 − 1) + (n2 − 1) + ⋯⋯

(Csd )2 n × (Cmean )2

(5)

Relative standard uncertainty (RSU) =

μ (Rec )2 (Recovery )2

(6) 2

RSUpooled =

2 2 μ ⎛ (n1 − 1)(μ (Rec1) ) + (n2 − 1)(μ (Rec 2) ) ⎞ + ⎜⎛ (purity) ⎟⎞ (n1 − 1) + (n2 − 1) ⎝ ⎠ ⎝ Cpurity ⎠ ⎜

⎟

(7) Other sources of uncertainty are adequately covered by the precision and recovery data. Since analytical balances, volumetric devices and environmental conditions were under regular control, and the verification were carried out over a longer period of time with variations in analyst, laboratory tools, and calibrations, it can be assumed, that the influences of the variability of most sources on the measurement uncertainty are covered by the within-laboratory precision.

3.3.1. Precision study (repeatability) The overall run to run variation (precision) of analytical procedure was performed during method validation studies with two different batches of calibration solutions, two different batches of reagents, two different analysts, at two concentration levels, for three different types of samples; cocoa beans, cocoa powder and chocolate. The precision assessments were done by determining relative standard deviation (RSD) of six blank sample solutions spiked at 250 µg/kg and 1000 µg/ kg. Results showed that the RSD observed for the different concentrations were all of a similar order of magnitude for all heavy metals between different samples (Supplementary 1). This indicates that the RSD is approximately proportional to analyte concentration. In such cases, it is appropriate to pool the RSD of each analyte using Eq. (3). Finally, the RSDpooled of each analyte and from different samples were pooled using Eq. (4), to obtain a representative or single estimation of precision uncertainty, u(P) as 0.063, 0.079, 0.066, and 0.117 for As, Cd, Pb and Sb respectively.

RSDpooled =

Standard uncertainty, μ (Rec ) = Rec ×

3.3.3. Combined standard uncertainty During the in-house validation study of the analytical procedure, the precision and the bias uncertainty sources had been thoroughly investigated. Both uncertainties were combined using Eq. (8). Finally, the expanded uncertainty U(Cmetal) is calculated by multiplying the combined standard uncertainty with a coverage factor of 2 with a confidence level of 95% and the values are summarized in Table 2.

μ (Cmetal ) = Cmetal

(precision)2 + (bias )2

(8)

3.4. Real samples monitoring The validated method was used in the analysis of Cd, As, Pb, and Sb in cocoa beans, cocoa powder, and chocolate samples collected in 2016–2017. In total, 86 cocoa bean samples were collected from small holders, 97 cocoa powder samples were collected from local grinders Table 2 Uncertainty components, combined standard uncertainty and expanded uncertainty for the analysis of heavy metals in cocoa products.

(3)

Elements

Precision

Bias

Combined Standard Uncertainty

Expanded Uncertainty

Arsenic Cadmium Antimony Lead

0.063 0.079 0.117 0.066

0.016 0.018 0.028 0.016

0.06 0.08 0.12 0.07

U(CAs) = CAs × 0.12 U(CCd) = CCd × 0.16 U(CSb) = CSb × 0.24 U(CPb) = CPb × 0.14

(4)

3.3.2. Bias study (recovery) The bias of the analytical procedure was investigated using spiked samples of cocoa beans, cocoa powder and chocolate, and expressed as 4

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good agreement with the data stated by Yanus et al. (2014) (0.04 mg/ kg) and Lo Dico et al. (2018) (0.026 mg/kg). However, the results obtained were higher compared to the values reported by Salama (2018) (0.018 mg/kg). As shown in Table 3, more than 90% of the samples analyzed were detected for Cd and Pb. However, based on the median values, most of the concentration’s levels are below national MPL (Regulations, 2010). The Cd content in this study was more than double the amount when compared to the data reported by Yanus et al. (2014) (0.125 mg/kg), Kruszewski et al. (2018) (0.15–0.17 mg/kg), Salama (2018) (0.041 mg/kg) and Lo Dico et al. (2018) (0.159 mg/kg), but similar to the data compiled by CODEX Alimentarius Commission (CODEX, 2017), which reported that the Cd from different continents (Latin America, North America, Africa, Asia and Europa) varies between 0.04 and 1.30 mg/kg which below the proposed CODEX MPL at 1.50 mg/kg for Cd in cocoa powder. The Pb content considerably below the national MPL set at 2.0 mg/kg, and the median concentration recorded at 0.20 mg/kg was slightly higher compared to data reported by Yanus et al. (2014) (0.10 mg/kg) and Salama (2018) (0.098 mg/kg). However, the results reported in this study were lower compared to data stated by Lo Dico et al. (2018) (0.417 mg/kg). Chocolate samples were collected from different brand of milk chocolate and dark chocolate containing more than 50% cocoa solids. The concentration of As, Cd, and Pb in chocolates samples were found in the range of 0.01–0.02 mg/kg, 0.01–0.20 mg/kg, 0.01–0.20 mg/kg while the concentration of Sb was less than 0.01 mg/kg. The median concentration of Cd content in this study is in good agreement with the data described by Sager (2012) (0.03–0.51 mg/kg), Lo Dico et al. (2018) (0.116 mg/kg), and Salama (2018) (0.011 mg/kg). Additionally, CODEX Alimentarius Commission (CODEX, 2017), reported that the Cd from 552 samples collected were below 0.6 mg/kg and conform to the proposed EU MPL set at 0.8 mg/kg for chocolate containing cocoa solids more than 50%. The median concentration of Pb in this study also in good agreement to the figures reported by Yanus et al. (2014) (0.09–0.15 mg/kg), Sager (2012) (0.01–0.11 mg/kg), Lo Dico et al. (2018) (0.133 mg/kg), and Salama (2018) (0.061 mg/kg). Meanwhile, the median concentration of As and Sb in this study are detected at very low level. Both metals do not pose any issue, because there are no MPL that has been regulated internationally so far.

Table 3 The concentration (mg/kg) of As, Cd, Sb and Pb in cocoa beans, cocoa powder, and chocolate samples. Element

Cocoa beans

MPL* (mg/kg)

Mean (mg/kg)

Min (mg/ kg)

Max (mg/ kg)

Median (mg/kg)

Positive results (%)

As Cd Sb Pb

0.05 0.25 0.00 0.50

0.01 0.01 0.01 0.01

0.27 1.27 0.05 1.6

0.01 0.21 0 0.43

40 92 14 91

Element

Cocoa powder

± ± ± ±

0.07 0.22 0.01 0.42

MPL* (mg/kg)

Mean (mg/kg)

Min (mg/ kg)

Max (mg/ kg)

Median (mg/kg)

Positive results (%)

As Cd Sb Pb

0.05 0.33 0.03 0.27

0.01 0.01 0.01 0.01

0.14 0.83 0.08 1.1

0.05 0.31 0.02 0.2

73 96 65 91

Element

Chocolate

As Cd Sb Pb

± ± ± ±

0.04 0.26 0.02 0.22

Min (mg/ kg)

Max (mg/ kg)

Median (mg/kg)

Positive results (%)

0.02 0.09 0.01 0.10

0.01 0.01 0.01 0.01

0.02 0.19 0.01 0.2

0.02 0.04 0.01 0.09

43 100 11 100

0.01 0.07 0.00 0.07

1 1 1 2 MPL* (mg/kg)

Mean (mg/kg)

± ± ± ±

1 1 1 2

1 1 1 2

* Food Regulations (2010).

and 35 chocolate samples were purchased from local markets. The levels of As, Cd, Sb and Pb in cocoa beans, cocoa powder and chocolates are shown in Table 3. The concentrations of As, Cd, Sb, and Pb for cocoa beans from different regions across Malaysia were found in the range of 0.01–0.27 mg/kg, 0.01–1.27 mg/kg, 0.01–0.05 mg/kg, and 0.01–1.60 mg/kg respectively. From Table 3, almost 90% of samples analyzed were detected for Cd and Pb. However, based on the median values, most of the concentration’s levels are below national MPL except for one sample which was detected for cadmium at 1.27 mg/kg. The As content is in good agreement with the data reported by Yanus et al. (2014) and Vītola and Ciproviča (2016), but much lower compared to figures reported by Lee and Low (1985) (2.52–3.19 mg/kg) on Malaysia cocoa beans. The Cd content in this study was comparable to data reported by Chavez et al. (2015) and Lee and Low (1985), but was slightly higher compared to data reported by Yanus et al. (2014), Vītola and Ciproviča (2016), Aikpokpodion et al. (2013) and Assa et al. (2018). However, the results obtained were definitely lower compared to what was found by Lewis et al. (2018), Ramtahal et al. (2015) and Arévalo-Gardini et al. (2017). On the other hand, Cd content in Malaysian beans were higher than Dominican Republic but lower than Ecuador, which were 0.13 and 0.63 mg/kg respectively (Kruszewski et al., 2018). The Pb content was considerably below the National or Draft CODEX Maximum Limit, and the median concentration was slightly higher compared to data reported by Yanus et al. (2014) (0.04 mg/kg), Kruszewski et al. (2018) (0.13–0.16 mg/kg) and Assa et al. (2018) (< 0.1 mg/kg), but much lower compared to data reported by Lee and Low (1985) (3.54–4.25 mg/kg) and Arévalo-Gardini et al. (2017) (1.00–3.78 mg/kg). The concentrations of As, Cd, Sb, and Pb in cocoa powder from different manufacturers were found in the range of 0.01–0.14 mg/kg, 0.01–0.83 mg/kg, 0.01–0.08 mg/kg, and 0.01–1.10 mg/kg respectively. The median concentrations of As and Sb found in this study were considerably low, and do not pose any alarming issue since there are no MPL at the moment internationally. The As content in this study is in

4. Conclusion In this study, the quantification of heavy metals in cocoa beans and cocoa products via a microwave assisted acid digestion technique and analysed by ICP-MS were successfully validated. It is considered to be excellent, reliable and fast technique for trace metal analysis of cocoa products. The method showed good selectivity, linearity, limit of detection/quantification, recovery and precision which acceptable under the validation criteria of EURACHEM guidelines (Magnusson & Örnemark, 2014). The expanded uncertainty measurements (using the coverage factor (k) of 2 at 95% confidence level) for cocoa beans, cocoa powder, and chocolate were less than 25% in which the uncertainty associated to precision and repeatability strongly contributes to the total uncertainty. The proposed method was effectively applied for the routine analysis of heavy metals in cocoa beans, cocoa powder and chocolates where some of them were found positive for As, Cd, Pb, and Sb. However, overall results showed that the concentration level is far from the national alarming level except for Cd in cocoa beans. Finally, the analysis technique presented here can be considered as time- and cost-efficient, suitable for a routine analysis in determining the concentration of heavy metals contaminants in cocoa beans and cocoa products. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to 5

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influence the work reported in this paper.

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