Bioavailability of cadmium and lead in cocoa: comparison of

extraction procedures prior to size-exclusion fast-flow liquid

chromatography with inductively coupled plasma mass spectrometric

detection (SEC-ICP-MS){

Sandra Mounicou,*a Joanna Szpunar,a Ryszard Lobinski,a Daniel Andreyb and

Christopher-John Blakeb

aCNRS UMR 5034, Group of Bio-inorganic Analytical Chemistry, Helioparc, 2, av. Pr.Angot, 64053 Pau, France

bCentre de recherche de Nestle, NESTEC LTD., Vers chez les Blanc, 1000 Lausanne 26,Switzerland

Received 13th February 2002, Accepted 21st May 2002

First published as an Advance Article on the web 14th June 2002

Fifteen extraction methods were investigated for the recovery of different classes of Cd and Pb species in 8

different cocoa powder samples. The procedures targeted water-soluble compounds, polypeptide and

polysaccharide complexes and compounds soluble in simulated gastrointestinal conditions. The extracts were

analysed by size-exclusion fast-flow liquid chromatography with ICP-MS detection. The detection limit was

0.5 mg l21 and the RSD was less than 7.5%. Cd and Pb were very firmly bound to the insoluble matrix

components, of which the binding capacity exceeded about 1000 times the naturally present metal levels. Cocoa

powder may show possible detoxifying properties for Pb and Cd by binding them into stable complexes, which

are resistant in gastrointestinal conditions. The maximum average recovery for Cd and Pb was, respectively,

15% and 5% of the total metal present.

Introduction

Cadmium and lead are considered as toxic elements. Acutetoxic cases are rare but chronic exposure can lead to theaccumulation of Cd in kidney, leading to renal damage,1 andchronic exposure to Pb is blamed for saturnism.2 The concen-trations of Cd and Pb in edible plants and food products aretherefore a matter of public attention. The provisional tolera-ble weekly intakes proposed by the WHO are 7.5 mg and 50 mg(25 mg for children) per week per kg body weight for Cd and Pb,respectively.3 The maximum permissible levels of Cd inchocolate and in cocoa powder have been set at 0.4 mg kg21

in Germany, 0.5 mg kg21 in Finland and Central Europeancountries4 and 1.0 mg kg21 in Malaysia. For Pb the corres-ponding values are 2 mg kg21 and 3 mg kg21 proposed byCodex Alimentarius5 and by the legislation of some Europeancountries, respectively.4

Cocoa bean is a farm product. Properly fermented and dried,it is used for the production of cocoa and cocoa-based products(beverages, chocolate, sweets and butter). The accumulation oftoxic metals in beans of the cocoa tree (Theobroma cacao L) hasbeen an issue of increased interest for many years.3,4,6–9

Cadmium is known to be accumulated by foliar and rootabsorption.1 The metal is mobilized by the action of earth-worms on a thick layer of Cd-rich cocoa leaves, usuallycovering the soil. The level of Cd in the beans is affected by theold and current mining activities in the area, and by the appli-cation of phosphate fertilizers and municipal sewage sludge toagricultural soil.Information on metal concentrations in cocoa beans is rela-

tively scarce. The available data show highly variable concen-trations of Cd and Pb, often exceeding the permissible levels,depending on sample origin. About 80% of cocoa comes from

West Africa and Brazil, where low Cd values have beenmeasured (0.3 ¡ 0.1 mg kg21).10,11 A survey of the Swissmarket showed elevated concentrations in Venezuelan (0.86–1.22 mg kg21) and Ecuadorian (0.56–0.77 mg kg21) cocoabeans.12 Levels above 1 mg kg21 have been measured indepen-dently in Ecuadorian and Venezuelan varieties,10 whereas thehighest reported values of 3–6.5 mg kg21 have been reportedfor those of Colombian origin.6 The varieties of cocoa farmedin Asia and Oceania show varying concentrations, from withintolerable limits to exceeding them by far (0.07–1.83 mg kg21).11

A series of analyses carried out by Knezevic in the years 1978–1986 demonstrated that Cd content is a function of the cocoacontent in chocolate and diminishes in the order: bitterchocolate (used for baking), dark chocolate (bittersweet andsemi-sweet), milk chocolate and white chocolate.7,8,13 In con-trast to Cd, Pb concentrations were reported to be within per-missible levels, in the range 0.09–0.143 or 0.18–1.2 mg kg21.6

The metal bioavailability, and thus its toxicity, is known todepend on the chemical form in which it is present in the sampleand on the behavior of this form in the gastrointestinal tract.14

From the chemical point of view, cocoa powder is composed offatty acids (the total fat content is about 10–12%), lignin,polyuronides, cellulose and protein, which also containsphytate. All these components have a marked ability to bindmetal cations but further experiments have shown that Cd andPb are not accumulated in fat (data not shown). Therefore,strong binding of Cd in cocoa is expected, which can affectconclusions as to the toxicity of trace elements made on thebasis of the total element content.Examining the speciation and proving the non-bioavail-

ability of Cd (should this be the case) is an alternative methodto research into genetic modification of the cocoa plant in orderto decrease the level of Cd assimilation. The coupling of size-exclusion fast-flow LC to ICP-MS is a widely accepted tool forthe screening and quantification of trace element species inplant tissues and the products of their processing.15,16 The

{Presented at the 2002 Winter Conference on Plasma Spectro-chemistry, Scottsdale, AZ, USA, January 6–12, 2002.

880 J. Anal. At. Spectrom., 2002, 17, 880–886 DOI: 10.1039/b201639g

This journal is # The Royal Society of Chemistry 2002

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prerequisite of its successful application is the transfer of themetal species in the sample into the aqueous phase whilepreserving their original form, or while using a controlleddegradation procedure. The literature on water-insoluble metalspecies in biological samples of plant origin is very scarce,according to a recent review.17

The goals of this research were (i) an investigation ofdifferent extraction procedures using aqueous buffers, proteinsolubilizing agents (SDS, CHAPS), and pectino- and proteo-lytic enzymes for the recovery of metal species from cocoa and(ii) the evaluation of the coupling of SE HPLC-ICP-MS for thefractionation of Cd–bioligand complexes in cocoa.

Experimental

Instrumentation

Chromatographic separations were carried out using a Series410 BIO HPLC pump (PerkinElmer, Palo Alto, CA, USA) asthe sample delivery system. Injections were made using aModel 7725 injection valve with a 100 ml injection loop(Rheodyne, CA, USA). All the connections were made ofPEEK tubing (id 0.17 mm). Analyte species were separated ona 10 6 300 mm 6 13 mm SuperdexTM-75 HR 10/30 SEC(Pharmacia Biotech, Uppsala, Sweden) with an exclusion limitof 100 kDa and an effective separation range between 0.5 and50 KDa (pullulans).Element-specific detection was realized with an Elan 6000

ICP mass spectrometer (PE-SCIEX, Concord, Canada). Thesample introduction system included a RytonTM spray cham-ber fitted with a cross-flow nebulizer. For total analyses thesamples were fed by means of a Minipuls 3 peristaltic pump(Gilson, France) that also served for draining the spraychamber. Chromatographic data were processed using theTurbochrom4TM software (PerkinElmer). All signal quantifi-cations were done in peak area mode.

Reagents, standards and samples

Analytical-reagent grade chemicals purchased from Sigma–Aldrich (St. Quentin Fallavier, France) were used throughoutunless otherwise specified. 18 MV cm Milli-Q water (Millipore,Bedford, MA, USA) was used throughout.

Rapidase Liq1TM (Gist Brocades, Seclin, France) andPectinex Ultra-SPLTM (Novo Nordisk, Copenhagen, Den-mark) commercial enzymatic preparations containing pecti-nases, hemicellulases and cellulases were used.The enzymes lipase (type VII from Candida cylindacea),

protease (type XIV from Streptomyces griseus), driselase (fromBasidiomycetes), amyloglucosidase (from Aspergillus miger),a-amylase and pepsin (from porcine stomach mucosa) werepurchased from Sigma–Aldrich, St Quentin Fallavier, France.The fraction reported earlier as RG-II3 (predominantly (87%)as dimer) was used as the dRG-II-B standard.18

Eight cocoa powder samples originating from differentSouth American countries were analysed.

Procedures

Determination of total Cd and Pb. 0.2 g of powder wasdigested with concentrated HNO3 in a closed system (highpressure asher) according to a German official mineralizationmethod prior to determination of Cd and Pb in foods.6 Afterdigestion, the sample was diluted to 10 ml with water andanalysed by ICP-MS. The digestion procedure was carried outin duplicate, and a blank was run in parallel. A certifiedreference material, NIST SRM 8433 (Corn Bran) as similar aspossible to cocoa powder was also analysed in duplicateaccording to this procedure. Pb and Cd were quantified using acalibration curve by means of standard solutions from 5 to50 mg l21 and the internal standard was In at 50 mg l21.

Defatting. 4 g of cocoa powder sample were shaken with30 ml of petroleum ether for 5 min. The mixture was centri-fuged at 3000 rev min21. The supernatant was discarded andthe procedure was repeated twice. The residue was dried over-night at ambient temperature.

Single step extractions. 1 g of cocoa powder was extractedwith 5 ml of an extractant solution for a given time at a giventemperature. The compositions of the extracting solutions aswell as the time and temperature conditions are given inTable 1. In the case of amylase and amyloglucosidase extrac-tions, the cocoa powder was suspended in water for 2 h atambient temperature prior to the addition of enzyme solution

Table 1 Details of extraction procedures investigated in this work

Procedure number Extractant Extraction time Extraction temperature

Water soluble speciesA-1 Cold water 4 h 20 uCA-2 Hot water 4 h 80 uCA-3 Water–methanol (4 1 1) 2 h 20 uCA-4 0.5% HNO3 (pH 1.2) 2 h 20 uCA-5 10 mM HCOONH4 (pH 5.5) 2 h 20 uCProtein fractionB-1 4% SDS in 30 mM Tris-HCl (pH ~ 8) 4 h 80 uCB-2 2% CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-

1-propanosulfonate), 1% dithiothreitholand 4 M urea in water

4 h 25 uC

B-3 0.1% protease in 30 mM Tris-HCl (pH 8) 4 h 37 uCB-4 Lipase (0.4%) and protease (0.2%)

in 30 mM Tris-HCl (pH 8)4 h 37 uC

Polysaccharide fractionC-1 2% Driselase (mixture of laminarase, xylenalase and cellulase)

in 30 mM Tris-HCl (pH 8.0)24 h 37 uC

C-2 0.1% solution Rapidase LIQ and pectinex Ultra-SPL enzymes(pectinase, hemicellulase, cellulase) in 30 mM Tris-HCl (pH 8)

4 h 37 uC

C-3 4% amyloglucosidase in 10 mM ammonium formate buffer(pH 5.2)

4 h 60 uC

C-4 1% a-amylase in 20 mM Tris buffer (pH 8.0) was used 4 h 80 uCSimulated gastrointestinal conditionsD-1 1% pepsin in 150 mM NaCl solution (pH 2) 24 h 37 uCD-2 Simulated gastrointestinal juice: 1.5% pancreatin, 0.15% bile

salts, 50 ml amylase in 150 mM NaCl4 h 37 uC

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and an increase in temperature. After extraction the suspensionwas centrifuged for 15 min at 3000 rev min21. The supernatantwas analysed by ICP-MS (extraction yield) and size-exclusionLC-ICP-MS (speciation).

Determination of the extraction yield. A 400 ml aliquot of thesupernatant was spiked with an internal standard (Rh) andmade up with nitric acid in order to obtain the concentration ofthe latter of 2% (v/v). Cd and Pb were quantified by use of acalibration curve established by analysing standard solutions at1, 2, 5 10, 20 mg l21. The calibration solutions contained thesame concentration of Rh and HNO3 as the analysed samples.A blank was run in parallel and analyses were carried out induplicate. The ICP-MS measurement conditions (nebulizer gasflow, rf power and lens voltage) were optimized daily using astandard built-in software procedure.

Size-exclusion fast-flow LC-ICP-MS conditions. An aliquotof 100 ml was injected. The mobile phase was 30 mM Tris-HClbuffer, pH 7.2, at a flow rate of 0.6 ml min21. The eluate fromthe column was fed directly into the ICP. The column wascalibrated with narrow pullulan molecular weight standards(P-5, Mr ~ 5800; P-10, Mr ~ 12200; P-20, Mr ~ 23700; P- 50,Mr ~ 48000, Showa Denko, Tokyo, Japan) using refractometricdetection as described elsewhere.19,20 Note that pullulans arelinear polysaccharides and the use of this calibration may leadto some underestimation of the molecular masses of highlyramified polysaccharides. The dwell time for each isotope was200 ms and the number of replicates allowing for continuousscanning for the duration of the chromatogram was applied.

Determination of the complexing capacity of the cocoapowder. A 1 g sample of cocoa powder was suspended in10 ml, and spiked with a known amount of Cd and Pb. Uponequilibration (48 h) the solution was centrifuged and Cd and Pbwere determined in the supernatant.

Results and discussion

Determination of the total Cd and Pb concentrations

Wet sample digestion with HNO3 and elevated temperatureand pressure in closed systems (high pressure asher), which wasadopted in Germany as an official method of mineralizationprior to determination of Pb and Cd in foods,6 was chosen forthe total Cd and Pb determination.Table 2 shows that all the samples analysed contained Cd

concentrations higher than the permissible limit of 400 mg kg21.In contrast, all the concentrations of lead measured were about10 times lower than the legally permissible limit. An insight intohow these metals are bound can be obtained by investigatingthe metal recoveries by means of extraction reagents specific tothe different classes of metal-binding ligands potentially pre-sent in cocoa powder. More information regarding the identity

of the different metal forms extracted (or ligands only) can beobtained by examining the extracts by SEC-ICP-MS.

Cd and Pb binding forms in cocoa—rationale for a strategy

The different constituents of cocoa have a marked ability tobind Cd and Pb, which can affect the toxicity assessment madeon the basis of the total element content. Components posi-tively affecting the bioavailability of minerals and traceelements are, for example, citric acid, ascorbic acid, lactoseand some amino acids, while polysaccharides, phytic acid,dietary fibre and polyphenolic compounds may have a negativeeffect.The applied analytical strategy examined the extraction

recovery with reagents having the potential to select the differ-ent classes of Cd and Pb complexes. The investigatedextractants (summarized in Table 1) that were targeted are:(i) Water-soluble Cd and Pb (present as free Cd21 and Pb21

ions, halide complexes as well as polysaccharides, amino acids,carboxylic hydroxy acids and polypeptides). The extraction ofthese species was attempted with water and aqueous buffers atdifferent pH, concentrations of organic modifier (methanol),ionic force and temperature.(ii) Water-insoluble protein complexes. They can be either

solubilized or degraded by solutions of surfactants (SDS,CHAPS) and of proteolytic enzymes, the latter not onlydenaturing the complex but also digesting the protein.(iii) Water-insoluble polysaccharide complexes. They can be

solubilized by means of hydrolysis of bonds between the carbo-hydrate monomer units in polysaccharides and between themonomer units and a functional pendant, such as the phenolicgroup, using a glycohydrolase type enzyme. The latter includeda number of enzymes divided according to their specific func-tions, e.g., pectinases, cellulases, hemicellulases and amylases.(iv) Bioavailable metal complexes, defined as the part of the

metals liberated in simulated gastrointestinal conditions.

Recovery of Cd and Pb from cocoa powder using differentextraction procedures

The recoveries obtained by extraction/leaching procedures (cf.,Table 1) are summarized in Table 3. The results show that onlya small fraction of Cd and Pb was water-soluble. With coldwater, only ca. 7% of Cd and ca. 5% Pb was extracted. Equallylow yields were obtained with a water-methanol mixture. Thechange in the acidity of the extractant, by using 0.5% HNO3 ora formate buffer at pH 5.5, did not affect the recovery (resultsnot shown). The increase in the water temperature did notaffect the extraction of Pb, however, the average extractionyield of Cd was increased by ca. 50%.The highest recoveries were obtained with the protein-

solubilizing chemicals, such as CHAPS and SDS. Both for Cdand Pb, ca. 30% of the metal was extracted. This is in sharpcontrast to the poor recoveries observed for extraction withproteolytic enzymes. Indeed, except for one case, the pro-teolytic extraction yield for Pb hardly exceeded that obtainedwith water. In the case of Cd, the recovery was twice thatobtained with water, but only half of that was obtained withsolutions of surfactants.Pectinolysis, using the mixture of pectinases (degrading the

principal chain of pectins, chains of rhamnogalacturonans, andside-chain of pectins), cellulases (degrading amorphous cellu-lose and cellobioses) and hemicellulases (degrading xyloglu-cans, arabinoxylans and mannans present in plant cell walls)did not improve the recovery, either for Cd or for Pb. Thiswould suggest that complexes with pectic polysaccharides, ifpresent, are water-soluble.On the other hand, the mixture of laminarase and xylenase

(driselase) liberated Cd species from many samples as effi-ciently as surfactant solutions. In contrast to the latter,

Table 2 Total concentrations of Cd and Pb found in cocoa powders.Certified reference material NIST 8433 measured values (mg kg21): Cd10¡ 1; Pb126¡ 3 and certified values (mg kg21):Cd12¡ 5; Pb140¡ 34

Sample

Concentration/mg kg21

Cadmium Lead

A 677 ¡ 4 290 ¡ 2B 676 ¡ 3 295 ¡ 5C 660 ¡ 0 297 ¡ 30D 642 ¡ 0 248 ¡ 4E 535 ¡ 2 140 ¡ 3F 524 ¡ 1 156 ¡ 8G 521 ¡ 1 181 ¡ 6

882 J. Anal. At. Spectrom., 2002, 17, 880–886

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however, there were some cocoa samples from which therecoveries were significantly lower. Driselase did not affect theextraction yield of Pb at all; the recoveries were identical tothose obtained with water. Extraction of Cd was unaffected byamyloglucosidase or amylase (note that the latter was carriedout at 80 uC so the yields should be compared with hot water).The extraction recovery of Pb was slightly improved byamylase but not by amyloglucosidase. The poor recoveries withglycohydrolase type enzymes may be due to their being blockedby polyphenols. Polyphenols are an abundant constituent ofcocoa, therefore this is a strong possibility. Polyphenols wereremoved by triple extraction with acetone,21,22 and the samplewas then extracted with enzymic mixtures. The recovery wasnot improved, and no Cd or Pb could be detected in the acetoneextract.The solubility of minerals and trace elements under

simulated conditions of the stomach (pH 1–2, 37 uC) orstomach and small intestine (pH 6.5–8, 37 uC) was consideredas an index of bioavailability.23 The use of an extractantsimulating gastrointestinal conditions did not improve therecovery in the case of Pb but twice as much Cd was liberated.The average recovery of Cd reached ca. 15% of the total Cdpresent.The results indicate a strong binding of Cd and Pb in cocoa.

About 70% of these elements is bound in very stable species andcannot be released by any of the extraction methods used. Thissuggests that the elements stick to the total dietary fibreconstituents, such as microfibres of crystalline cellulose that aredifficult to destroy. On the other hand, up to 30% of the com-plexes present can be solubilized, usually by the destruction ofthe complex. An insight into the identity of extractable speciescan be gained by SEC-ICP-MS.

Optimization of size-exclusion LC-ICP-MS conditions

The Cd and Pb concentrations of concern in this work areamong the lowest ever investigated by SEC-ICP-MS in biolo-gical tissue extracts. Indeed, considering a 10% recovery, valuesof ca. 10 mg L21 and lower need to be measured, which resultsin a severe risk of contamination. Cadmium and, to a lesserdegree, lead are known to be retained by the stationary phaseand can be released by injection of any ligand showing a com-plexing affinity if present in a subsequently analysed sample.24

The most promising results were obtained by conditioningthe column with a 0.1% (w/v each) solution of 2-mercaptoetha-nol and EDTA in 30 mM Tris buffer at pH 7.5 for 30 min. Theremoval of the chelating agent prior to next injection wasaccomplished by conditioning the column with the mobilephase for another 30 min. Care was taken to avoid traces ofmetals in the conditioning solutions by using a Chelex-100sorption column at the exit of the pump. Otherwise trace metalswill accumulate at the head of the SEC during the conditioningphase. The state of the size-exclusion column was controlledprior to introduction of a sample by injection of 0.1% EDTA–2-mercaptoethanol solution. Typical peak height values werelower than 200 counts s21 for Cd and Pb.The concentrations of interest were hardly above the detec-

tion limit estimated for ca. 0.5 ppb. Therefore, an attempt wasmade to preconcentrate the extracts by lyophilization. How-ever, it turned out to be impossible to redissolve the residue.Attempts of evaporation were unsuccessful, either because ofthe increasing viscosity of the sample and/or difficulties withthe chromatography.Each SEC-ICP-MS experiment included the analysis of three

samples: a blank that was the extractant solution only, asample and a sample spiked with a significant (ca. 100 ppb)concentration of Cd21 and Pb21. The latter analysis was foundto be useful to obtain an insight into the presence of the differ-ent ligands potentially binding these elements in the sample.T

able

3TherecoveryofCdandPbfrom

cocoapowder

bymeansofthedifferentextractionprocedures

Extraction

procedure

Recovery(%

)

Meanvalue

AB

CD

EF

G

Cd

Pb

Cd

Pb

Cd

Pb

Cd

Pb

Cd

Pb

Cd

Pb

Cd

Pb

Cd

Pb

A-1

8¡

03¡

07¡

14¡

17¡

03¡

07¡

15¡

16¡

05¡

27¡

02¡

07¡

09¡

47¡

15¡

2A-2

7¡

06¡

19¡

16¡

110¡

15¡

110¡

09¡

113¡

25¡

020¡

14¡

016¡

17¡

012¡

56¡

2A-3

7¡

04¡

06¡

06¡

16¡

04¡

14¡

18¡

16¡

05¡

06¡

01¡

26¡

04¡

11

6¡

14¡

2B-1

31¡

226¡

534¡

117¡

232¡

333¡

321¡

319¡

127¡

210¡

325¡

216¡

332¡

423¡

428¡

519¡

8B-2

33¡

329¡

036¡

023¡

034¡

027¡

031¡

232¡

129¡

129¡

333¡

137¡

239¡

049¡

11

34¡

332¡

8B-3

18¡

010¡

015¡

413¡

318¡

18¡

118¡

211¡

08¡

04¡

015¡

14¡

120¡

211¡

116¡

410¡

4B-4

14¡

07¡

216¡

38¡

014¡

09¡

113¡

27¡

08¡

03¡

19¡

04¡

210¡

08¡

112¡

36¡

2C-1

27¡

16¡

132¡

06¡

222¡

45¡

010¡

17¡

013¡

27¡

235¡

18¡

131¡

28¡

325¡

10

6¡

1C-2

9¡

05¡

111¡

210¡

38¡

06¡

18¡

28¡

17¡

04¡

28¡

18¡

38¡

06¡

19¡

17¡

2C-3

8¡

04¡

011¡

33¡

07¡

03¡

07¡

24¡

19¡

03¡

010¡

18¡

28¡

06¡

310¡

44¡

2C-4

12¡

012¡

418¡

310¡

418¡

17¡

215¡

414¡

318¡

26¡

229¡

213¡

025¡

018¡

319¡

512¡

4D-1

13¡

13¡

019¡

26¡

011¡

22¡

010¡

13¡

20¡

00¡

00¡

00¡

00¡

00¡

08¡

72¡

2D-2

15¡

05¡

120¡

35¡

116¡

04¡

114¡

27¡

210¡

03¡

111¡

06¡

011¡

05¡

114¡

35¡

2

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Size-exclusion LC-ICP-MS of water extracts

Fig. 1a and b shows chromatograms obtained for the aqueousextracts of cocoa powder for Cd and Pb species, respectively.One peak (ca. 6 kDa) at the detection limit was observed in theCd chromatogram and the Pb chromatogram showed threepeaks: one close to the void, one matching the 12.2 kDastandard and one of ca. 6 kDa. The analysis of a spiked samplegave an intense peak in the void of the Cd chromatogram and anumber of peaks in the Pb chromatogram. The 6 kDa peak didnot change in intensity upon spiking, which suggests it is due toa plasma instability rather than due to the presence of a Cdspecies.The intensity of the Cd peak in the spiked sample was

distinctly lower (ca. 20%) than expected from the concentrationspiked. The remaining Cd was retained on the column. Thisobservation, together with the absence of a Cd peak in thechromatogram of the unspiked sample, leads to the conclusionthat the water-extractable Cd is in the Cd21 form, able to bindloosely to large-molecular weight ligands.An insight into the identity of the peaks in the Pb chro-

matogram can be obtained by comparing the Pb chromato-gram with those of other elements (Fig. 1c). The elutionpatterns of B and Sr, which overlap with Pb (Fig. 1b, line 1) interms of the first two peaks (void and 48 kDa peak) may herehelp to reveal the identity of the species. Pb and Sr have similarionic radii and are known to enter into a cavity formed by theproduct of the dimerization of Rhamnogalacturonan II byesterification with boric acid.25

The presence of the dimer of Rhamnogalacturonan complex-ing Pb is confirmed by the overlap of another peak in the SEC-CP-MS chromatogram of the spiked sample with that of adRG-II standard (Fig. 1c). However, the degradation of thelarge molecular weight compounds to pure dRG-II, as demon-strated elsewhere for fruit and vegetable samples20 with pecti-nolytic enzymes, turned out to be less straightforward, withyields hardly exceeding 10%.

Size-exclusion LC-ICP-MS of the proteinaceous fraction

Cadmium has a strong affinity for sulfhydryl groups of severalcompounds, but also for proteins and phosphate groups.Consequently, Cd is known to be concentrated in the proteinfraction of plants.1 Research on proteins in cocoa has mainlybeen concerned with their nutritive values and proteolyticgeneration of cocoa-specific aroma precursors.26,27 Cocoa pro-teins undergo constant changes (usually by proteolysis) duringripening of the fruit, fermentation and further processing ofcocoa beans,28 which results in a number of polypeptides ofdifferent sizes being present in the sample.In order to study the Cd and Pb binding to proteins in cocoa,

extracts obtained with solutions of SDS and CHAPS as well asthose obtained with solutions of proteolytic enzymes werestudied. Fig. 2a and b shows the chromatograms obtained forextracts with SDS and CHAPS for Cd and Pb, respectively.The chromatograms of an SDS extract and of the blank wereidentical despite an elevated extraction yield. This is likely to becaused by the denaturation of extracted proteins and by thesorption of the released Cd21 and Pb21 on the column. Indeed,the post-chromatography injection of an EDTA–2-mercapto-ethanol mixture led to a release of considerable amounts of Cdand Pb. The chromatography of extracts obtained withCHAPS showed a broad peak covering the 1–20 kDa elutionrange.It is interesting to note that the molar excess of the Cd- and

Pb-binding ligands with respect to the metal available in thecocoa extract is very high. Indeed, Fig. 2c and d shows that anincrease in the spiked concentration of up to 1000 in compari-son with the original level results in a proportional increase ofthe peak intensity. This proves the availability of a large

Fig. 1 SEC-ICP-MS chromatograms of water extracts of cocoapowder. (a) 114Cd: 1—extract, 2—extract spiked with 100 mg ml21

Cd21. (b) 208Pb: 1—extract, 2—extract spiked with 100 mg ml21 Pb21.(c) 1—10B, 2—89Sr, 3—dRG-II.

884 J. Anal. At. Spectrom., 2002, 17, 880–886

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amount of Cd- and Pb-binding ligands in cocoa and indicatesthe lack of the biosynthesis of specific ligands to explain theaccumulation of Cd in cocoa. This is in sharp contrast to manyplants which biosynthesize specific ligands, e.g., phytochelatins,as a response to exposure to Cd.29 This would suggest theabsence of a genetic mechanism and, consequently, theinefficiency of genetic approaches to decrease Cd bioaccumula-tion in genetic plants.Fig. 2e and f shows SEC-ICP-MS chromatograms of extracts

obtained by enzymic extraction with a mixture of protease–lipase. A sharp peak corresponding to a 10 kDa compoundcould be seen, indicating that the enzymes degrade all the Cd-binding proteins down to one Cd-binding compound. This isconfirmed by analysing the spiked sample; the intensity of thispeak increased distinctly. It is interesting to note that the sameligand also binds Pb21, provided the latter is available in asufficiently high excess (Fig. 2d). The overall recovery of metalsfrom a cocoa powder by proteolytic enzyme extraction was 2times lower than in the case of extraction with CHAPS. Thismay mean that some CHAPS-extracted species are not Cd-bindingproteins, butothermacromolecular compounds. Indeed,for Pb, the dominating ligands seem to be pectic polysacchar-ides, and their release from cocoa is largely enhanced byextraction with CHAPS.

Size-exclusion LC-ICP-MS of the polysaccharide fraction

As discussed above, enzymolysis with a mixture of pectinase,hemicellulases and cellulases does not allow an efficient extrac-tion of metals. Only the extracts that allowed a higher recoveryof Cd in comparison with water, i.e., that with driselase andthat with amylase (degrading starch), were chromatographed.The chromatograms in Fig. 3a show, in both cases, a broadpeak in the low molecular mass elution region, which wouldsuggest that some of these ligands undergo degradation into arange of low-molecular weight carbohydrates able to complexCd.For lead, the size-exclusion profiles obtained (Fig. 3b) were

similar to those obtained for a water extract. The intensityincreased slightly which would suggest that either some metalbound to high-molecular mass structures is liberated or that

Fig. 3 SEC-ICP-MS chromatograms of extracts obtained with glyco-hydrolase-type enzymes. (a) Cd: 1—driselase, 2—a-amylase. (b) Pb:1—driselase, 2—a-amylase.

Fig. 2 SEC-ICP-MS chromatograms of proteinaceous extracts. (a)114Cd: 1—SDS, 2—CHAPS. (b) 208Pb: 1—SDS, 2—CHAPS. (c)Extract with CHAPS spiked with 1—20 ppb Cd21, 2—100 ppb Cd21,3—500 ppb Cd21. (d) Extract with CHAPS spiked with 1—20 ppbPb21, 2—100 ppb Pb21, 3–500 ppb Pb21. (e) Extract obtained withprotease–lipase: 114Cd:1—unspiked extract, 2—extract spiked with20 ppb Cd21. (f) Extract obtained with protease–lipase: 208Pb:1—unspiked extract, 2—extract spiked with 20 ppb Pb21.

J. Anal. At. Spectrom., 2002, 17, 880–886 885

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smaller molecular mass structures are formed, as describedelsewhere for vegetables samples.20

Size-exclusion LC-ICP-MS of gastrointestinal extracts

A technique was proposed to monitor the speciation in foodsunder the conditions in which the essential or toxic element istaken up by man.30 The in vitro model usually consists of threeparts: peptic digestion, pH adjustment and pancreatic digestionfollowed by equilibrium dialysis.23 The latter was replaced inthis work by SEC-ICP-MS. The Cd chromatogram of anextract is shown in Fig. 4. As discussed above no additional Pbwas liberated and its chromatogram resembled that obtainedfor a water extract. The Cd-specific chromatogram showed thatthe extracted Cd elutes as a mixture of low-molecular weight(up to 6000 Da) species.

In-vitro binding of Cd and Pb by cocoa powder

The poor extraction recoveries observed for Cd and Pb incocoa powder with practically all the extractants usedencouraged us to study the binding capacity for metals presentin a much higher excess than the natural concentrations.Results show that the percentage of Cd and Pb retained in theresidue (insoluble in water) was above 98% and is practicallyindependent of the metal excess, regardless of pH. The com-plexing sites were not saturated even in the presence of a 10000-fold excess of metal. This demonstrates an enormous capacityof the cocoa powder for binding both Cd and Pb and mayindicate its possible detoxifying properties (compared to metalcontaminants in food), which will, however, require furtherstudies.

Conclusions

The different extraction approaches followed by species-selective monitoring of the extracted compounds allowed usto obtain a fairly comprehensive, however still simplified, view

of the speciation of Pb and Cd in cocoa. Cadmium levels insome cocoa varieties may be above certain national legal limits.However, over 70% of Cd is bound in very stable complexesthat are not digested. The total Pb concentration in cocoa islargely below the legally permissible limits. Pb21 is stronglybound; only about 10% of this element can be released inbiologically realistic conditions. In-vitro binding experimentson cocoa powder suggest possible ways of detoxifying Pband Cd by binding them into stable complexes, resistant togastrointestinal conditions.

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Fig. 4 SEC-ICP-MS chromatogram of an extract (114Cd) of a cocoapowder with simulated gastrointestinal juice.

886 J. Anal. At. Spectrom., 2002, 17, 880–886

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