Speciation analysis for iodine in milk by size-exclusion chromatography with inductively coupled plasma mass spectrometric detection (SEC-ICP MS) Luiza Fernandez Sanchez{ and Joanna Szpunar* CNRS EP132, He�lioparc, 2, av. Pr. Angot 64053 Pau-Pyre�ne�es, France. E-mail: joanna.szpunar@univ-pau.fr Received 9th July 1999, Accepted 25th August 1999 A method allowing the determination of iodine species in milk and infant formulas was developed. It was based on the coupling of size-exclusion chromatography (SEC) with on-line selective detection of iodine by ICP MS.Iodine species were quantitatively eluted with 30 mM Tris buffer within 40 min and detected by ICP MS with a detection limit of 1 mg l21 (as I). A systematic study of iodine speciation in milk samples of different animals (cow, goat) and humans, of different geographic origin (several European countries) and in infant formulas from different manufacturers was carried out. Whey obtained after centrifugation of fresh milk or reconstituted milk powders contained more than 95% of the iodine initially present in milk in all the samples investigated with the exception of the infant formulas in which only 15±50% of the total iodine was found in the milk whey.An addition of sodium dodecyl sulfonate (SDS) improved considerably the recovery of iodine from these samples into the milk whey. Iodine was found to be principally present as iodide in all the samples except infant formulas.In the latter, more than half of the iodine was bound to a high molecular (w1000 kDa) species. The sum of all the species recovered from a size-exclusion column accounted for more than 95% of the iodine present in a milk sample. For the determination of total iodine in milk a rapid method based on microwave-assisted digestion of milk with ammonia followed by ICP MS was optimized and validated using CRM 151 Skim Milk Powder. Introduction Iodine is an essential micronutrient to animals and man. It is a constituent of the thyroid hormones, the lack of which causes poor mental and physical development in children, and goiter (enlargement of the thyroid gland) in adults.1 Supplementation with iodine is a common practice.2 The addition of iodine to cow feed in order to enhance production of milk and meat makes milk an important source of iodine.2 In infant nutrition the iodine level in breast milk is known to be affected by the maternal diet whereas infant formula needs to be supplemented with iodine.3 Since excessive intake of iodine can cause toxic goiter (thyrotoxicosis) the supplementary iodine should be strictly limited and controlled by manufacturers and government institutions; a number of methods for precise and accurate quantiÆcation of iodine in food have therefore been developed.4 To date, milk has been analysed for total iodine only without looking into the nature of the species present.Inductively coupled plasma mass spectrometry (ICP MS) has been the usual analytical technique applied,4±10 replacing the classical Sandell and Kolthoff kinetic-catalytic method,11 cumbersome radiochemical neutron activation analysis,12 and tedious GC with electron capture detection (ECD), which requires the conversion of iodine to a 2-iodopentan-3-one derivative.13,14 Despite some reports of the direct analysis of milk diluted with ammonia by ICP MS,10 digestion has always been an integral step of the sample preparation procedure.Tetramethylammonium hydroxide (TMAH),4±6 a TMAH±KOH mixture,9 and diluted ammonia8 have been the most widely used approaches for milk digestion, an alternative being the combustion of a milk sample in an oxygen stream.7 These procedures lead to the conversion of iodine into iodide, which is then determined by ICP MS without any concern for the speciation of the iodine in the original sample. It is well known that the absorption of an endogenous trace element in food by man can be different from that of a supplemented one and, in particular, the absorption of this element by formula-fed children is usually different than that of breast-milk-fed children.This fact renders trace element speciation analysis of milk necessary.15 Most iodine in biological matrices is said to be covalently bound but Bra» tter et al.16 reported that ca. 80% of iodine was present in the breast milk in the form of iodide, in addition to various organic compounds.Studies of speciation of iodine in milk and body Øuids have been practically non-existent because of the lack of a suitable, species-selective methodology, as demonstrated by the recent review papers.17,18 In terms of potential methodology available for speciation analysis for iodine in milk, approaches described for speciation of essential elements (Zn, Cu, Fe, Se),15,16,19,20 toxic elements (Cd)21 and radioactive elements (90Sr, 99Tc, 137Cs and 152Eu)22 in this matrix should be considered.Size-exclusion chromatography has been the most widely used fractionation technique for trace element species present in milk, whereas ICP AES,15,16 ICP MS,16 scintillation radiodetection22 and stripping voltammetry 21 were the most commonly used detection techniques. Since milk is a slurry that cannot be introduced on to the column directly, milk whey was actually analysed. Care should be taken to assure that the composition (in terms of trace elements) of the whey obtained by the centrifugation of the milk matches that present in the original milk sample.The objective of this research was to develop a method able to distinguish and to quantify the different iodine species potentially present in milk. The approach was based on the coupling of SEC and ICP MS following a sample preparation procedure, assuring the complete transfer of iodine-containing {On leave from: Department of Analytical Chemistry, Universidad de Santiago de Compostela, 15706 Santiago, Spain.J. Anal. At. Spectrom., 1999, 14, 1697±1702 1697 This Journal is # The Royal Society of Chemistry 1999species present in milk to the milk-whey fraction. Since problems were encountered during the direct determination of iodine in milk by ICP MS, a rapid open-vessel focussed microwave-assisted digestion was developed for this purpose. Experimental Instrumentation Chromatographic separations were carried out using an HP Model 1100 HPLC pump (Hewlett-Packard, Wilmington, DE, USA) as the sample delivery system.Injections were performed using a Model 7725 injection valve with a 100 ml injection loop (Rheodyne, Cotati, CA, USA). All the connections were made of polyether ether ketone (PEEK) tubing (id 0.17 mm). Analyte species were separated on 106300 mm613 mm Superdex-75 and Superdex-200 columns (Pharmacia Biotech, Uppsala, Sweden) with an exclusion limit of 100 kDa (an effective separation range of 0.5 kDa and 80 kDa) and an exclusion limit of 1300 kDa (an effective separation range of 10 kDa and 600 kDa), respectively. A guard column, TSK PWXL (40 mm63 mm id) (Tosoh Corp., Stuttgart, Germany) was always used.The columns were calibrated (UV detection was used) with the following standards: glutathione (Mr 307), PC2 (Mr 539), PC3 (Mr 771), rabbit liver metallothionein±Cd complex (Mr 6918), cytochrome c (Mr 12 384), bovine albumin (Mr 66 000), and thyroglobulin (Mr 660 000).An ELAN 6000 ICP mass spectrometer (PE-SCIEX, Thornhill, Ontario, Canada) was used as the element-speciÆc detector in HPLC. The column eluate was introduced into the ICP via a cross-Øow nebulizer Ætted in a Ryton spray chamber. For total analyses the samples were fed by means of a Minipuls 3 peristaltic pump (Gilson, Villiers-le-bel, France) that also served for draining the spray chamber. Chromatographic data was processed using the Turbochrom4 software (Perkin-Elmer, Norwalk, CT, USA).All signal quantiÆcations were performed in the peak area mode. A Hitachi Model Himac CS 120GX refrigerated ultracentrifuge (Jouan, Saint Herblain, France) was used for the separation of the milk whey. Lyophilization was carried out using a Model LP3 lyophilizer (Jouan). For the total iodine analysis, samples were digested in a 22 ml open vessel of borosilicate glass Ætted with a 10 censer using a Synthewave S402 microwave digester (2.45 GHz, maximum power 300 W) (Prolabo, Fontenay-sous-Bois, France).Reagents, standards and samples Analytical-grade reagents purchased from Sigma±Aldrich (St. Quentin Fallavier, France) were used throughout unless speciÆed otherwise. 18 MV Milli-Q water (Millipore, Bedford, MA, USA) was used throughout. The Tris±HCl buffer was prepared by dissolving 30 mM of Tris [tris(hydroxymethyl)- aminomethane] in water and adjusting the pH to 7.0 by the addition of hydrochloric acid (1 : 10, v/v).The buffer solution was degassed in an ultrasonic bath before use. Standards containing iodine: thyroglobulin (iodine content approx. 1%), 3,3',5-triiodothyronine sodium salt and potassium iodide were used. The standards were dissolved in water with the exception of triiodothyronine which was dissolved in diluted ammonia and diluted with the chromatographic mobile phase prior to injection. A certiÆed reference material (CRM) 151 Skim Milk Powder (BCR, Brussels, Belgium) with a certiÆed iodine concentration of 5.35°0.14 mg g21 was used to control the accuracy of the total iodine determination.The human milk samples were collected from mothers having delivered at the Hospital Xeral in Santiago, Spain. The samples were collected in polypropylene containers cleaned with 10% HNO3 and immediately frozen (220 �C). Infant formula samples and milk samples of different geographical origin (France, Nactalia Brick; UK, Safeway Long Life Milk; Poland, Mleko Ëaciate; Germany, Haltbare Alpen Milch; Spain, Leche Pascual) were purchased in supermarkets in the appropriate countries.Analytical procedures Sample preparation. Infant formulas and the CRM powder were reconstituted with water according to manufacturers' recommendations. Other milk samples were analysed as received. In order to obtain milk whey, a sample aliquot of a size sufÆcient for the subsequent chromatographic analysis (typically 500 ml) was centrifuged at 50 000 rpm at 4 �C for 15 min (infant formulas and breast milk) or for 60 min (cow and goat milk).The fat (upper layer) and the insoluble residue at the bottom were discarded. The medium fraction was Æltered through a 0.45 mm syringe Ælter prior to analysis for total iodine or prior to chromatography. Freeze-drying was occasionally used for preconcentration of iodine species in milk whey samples; the powder obtained was redissolved in the chromatographic mobile phase. The preconcentration factor obtained was 2- to 9-fold.For experiments with the use of sodium dodecyl sulfonate (SDS), a 500 ml portion of a 4% aqueous solution of the reagent was mixed with a 500 ml aliquot of milk sample in an ultrasonic bath and incubated for 2 h at 37 �C. Enzymolysis was carried out with a mixture (1z2 w/w) of lipase and pronase. An amount of 30 mg of this mixture was added to 5 ml of milk whey diluted twice with the chromatographic mobile phase and incubated for 16 h at 37 �C.Determination of total iodine by ICP MS. For an analysis for the total iodine, a 2 ml aliquot of a milk sample was placed in a reaction tube together with 5 ml of 0.5% v/v ammonia solution and digested in the focussed microwave system at 45 W for 2.5 min. The resulting solution was diluted to 10 ml and fed directly into the ICP. The method of standard additions (at two levels: 100 and 200 mg l21) was applied for the quantiÆcation of the iodine content. Rh was used as the internal standard.The BCR CRM 151 Skim Milk Powder was analysed at least once every day to check the accuracy of the total iodine determination. ICP MS measurement conditions (nebulizer gas Øow, RF power and lens voltages) were optimized daily using a standard built-in software procedure. An aqueous solution of potassium iodide 10 mg l21 was used for sensitivity optimization. Typical examples of the optimum measurement conditions are a nebulizer gas Øow of 1.05 l min21, ICP RF power of 1.1 kW and a lens voltage of 9 V.The same instrumental conditions were employed when ICP MS was used as the chromatographic detector. Speciation analysis of iodine by SEC-ICP MS. The chromatographic mobile phase was the 30 mM Tris±HCl buffer at pH 7.0. The Øow rate was 0.75 ml min21. A sample aliquot of 100 ml was injected. The eluate from the column was fed directly into the ICP. 127I isotope was monitored together with 57Fe, 114Cd, 63Cu, 64Zn and 208Pb. The particular set of elements included the typical toxic and essential metals present in milk.The detailed discussion of speciation of the other elements is beyond the scope of this paper. The dwell time for each isotope was 100 ms and a number of replicates that allowed continuous data acquisition in the peak hopping mode for the duration of the chromatographic run was applied. Typically, 1000 replicates were applied to give a scan duration of 50 min. 1698 J. Anal. At. Spectrom., 1999, 14, 1697±1702Results and discussion Milk cannot be analysed directly by size-exclusion chromatography because it is an emulsion containing solid particles that would clog the inlet Ælter of the column.A prerequisite of a successful speciation analysis is therefore the development of a sample preparation procedure that will allow the quantitative transfer of iodine-containing species present in an original milk sample into a solution that would pass through a 0.2 mm inlet Ælter.For this purpose an approach based on the extraction of iodine-containing species into an aqueous phase (whey) that could be separated from the solid particles (caseine) and fat by (ultra)centrifugation was investigated. The evaluation of the extraction efÆciency was based on the comparison of the iodine concentration in the milk whey with that in the whole milk. Therefore a reliable method for the determination of iodine in these matrices (whey and whole milk) was required. Attempts to determine iodine in milk by direct introduction of samples diluted with ammonia into an ICP MS10 failed.The values obtained for the analysis of the BCR CRM 151 Skim Milk Powder were ca. 60±80% of the certiÆed value. Because the existing digestion methods were judged to be too cumbersome it was decided to develop a rapid open-vessel focussed microwave-assisted digestion method for the determination of the total iodine in milk and milk whey. Optimization of the total iodine determination Three factors were frequently cited in the literature concerning the determination of total iodine in milk by ICP MS: the unsuitability of the commonly used internal standards (In and Rh) in alkaline media because of their hydrolysis and the precipitation of the hydroxides,8 the risk of oxidation of iodide to iodine giving rise to an erroneous ICP MS signal,8 and the need for harsh (prolonged heating at elevated temperatures) conditions for the extraction of iodine from milk.A 3 h extraction at 90 �C with TMAH,4,6 microwave-assisted digestion with ammonia in a pressurized vessel at 700 W (three times),8 or sample combustion in a stream of oxygen7,13 were judged inappropriate for routine analyses in this work. Preliminary experiments indicated that the digestion of a milk sample with diluted ammonia in an open vessel using a focussed microwave Æeld could be the basis of a simple method yielding accurate results.Under the optimized working conditions (0.5% ammonia solution, microwave power of 45 W) a transparent solution that could be fed directly into an ICP MS could be obtained within 2±3 min. Rhodium used as an internal standard did not create any inconvenience, the signal was stable, probably because of the formation of Rh complexes with ammonia. The method of standard additions was evaluated to correct for matrix effects but since the slope of the standard additions curve was similar to that for an external calibration graph it was considered to be rather a preventive measure.The method developed was validated by analysing the BCRCRM151 Skim Milk Powder. The typical precision of Æve measurements realised during a day was ca. 3±4%. The mean of the resultsfferent days was 5.43°0.06 mg g21, in comparison with the certiÆed value of 5.35°0.14 mg g21. Determination of iodine in the whole milk and in the Æltrable milk fraction Three series of samples were investigated: (i) milk samples from different mammals including cow, goat and human breast milk, (ii) cow milk from different European countries including France, Spain, England, Germany and Poland, and (iii) different infant formulas.Such a choice was judged sufÆciently representative to develop a valid methodology for speciation of iodine in milk. The milk samples (natural milk or reconstituted infant formula powder) were subjected to ultracentrifugation and the total iodine concentration in the milk whey [referred to as a Æltrable (0.2 mm) fraction] was compared with the iodine concentration in the initial milk sample.Results are shown in Table 1. The concentrations of iodine in the whole milk are generally between 100 and 200 mg l21, which is a typical level reported earlier in the literature for Denmark9 and Turkey.11 The clearly elevated level in the British sample can be attributed to iodinesupplemented feed or disinfection of teats with iodophores9 or to supplementation with iodide salt.The low level of iodine in the Polish milk sample indicates no supplementation. The concentrations in infant formulas were distinctly lower, with an average value of ca. 50 mg l21. Recovery of iodine into the aqueous (Æltrable) phase. The prerequisite of speciation analysis by SEC-ICP MS, is the presence of all the iodine species potentially present in milk in a 0.2 mm Æltrable solution (whey fraction). The results shown in Table 1 indicate that in all the `natural' milk samples iodine is mostly present in the milk whey with the exception of one (British) sample.On the other hand, the average concentrations of the iodine present in the Æltrable phase in infant formulas are well below 50% of the total iodine present in the whole milk. One preparation showed the recovery as low as 15%. This iodine was found to be present in the solid residue after ultracentrifugation and attempts were made to enhance its recovery in view of investigating the speciation of this iodine by SEC-ICP MS.The approach investigated assumed that the non-extractable iodine was bound to (incorporated in) high-molecular weight compounds abundant in milk. It was based on the incubation of milk with sodium dodecyl sulfonate (SDS), which is a surfactant reagent used in protein chromatography to disrupt the aggregated proteins and to solubilize the proteins (by forming ion pairs) enabling their separation by liquid chromatography. This reagent has been recently successfully employed to improve the recovery of macromolecule bound selenium in yeast samples.23 It was found that the addition of SDS to milk improves the recovery of iodine from the supernatant (whey fraction).In the case of the natural milk samples, this increase was ca. 10±20% but for infant formula samples the amount of iodine recovered in the supernatant was more than twice that in the samples not incubated with SDS.Irrespective of the sample origin, more than 85% of the iodine initially present in milk could thus be recovered in the supernatant after ultracentrifugation, which was then subject to speciation analysis by SEC-ICP MS. Table 1 Total iodine concentration in milk samples of different origin Sample Total iodine concentration/mg l21 Iodide/mg l21 Whole milk Milk whey Milk whey Commercial cow's milk of different geographical origin– France (I) 167°14.6 148.7°13.6 145 France (II) 185.0 163.5 160.0 Spain 190.0 122.5 102.9 Germany 191.5 149.5 137.5 England 625 254 239 Poland 58 51.5 37.1 Milk from other species– Goat milk 326°11.4 293°9.5 279.1 Human milk 108.5°4.0 104.6°6.8 45.8 Infant formulas of different origin– Nidal 47.4°3.3 24.4°5.1 19.8 Nestle� 48.5 18.5 2.6 Milupa 51.2 20.0 3.8 Ordesa 53.1 30.0 1.5 Mead Johnson 21.5 3.76 0.12 Sandoz 32.0 16.5 4.9 J. Anal.At. Spectrom., 1999, 14, 1697±1702 1699Optimization of the SEC-ICP MS conditions for speciation of iodine in milk There is little information on iodine species in milk, which makes the choice of standards to be used for the optimization of chromatographic conditions difÆcult.Iodide should deÆnitely be included since it is often assumed to be the only iodine species in milk; indeed, the iodide concentration in milk is sometimes considered to be a measure of the total iodine present.14,24 Other potentially present species include the thyroid hormones (tetraiodothyronine and triiiodothyronine), reported in milk at concentration levels of between 2 and 12 ng ml21.25,26 Since infant formulas are often produced on the basis of hydrolysed cow's milk proteins, an iodinecontaining protein (thyroglobulin) was included in the array of standards used for the optimization of the chromatographic separation conditions.It should be noted that the presence of this particular protein in milk is unlikely but this was the only iodine-containing protein standard available commercially. In terms of chromatographic techniques size-exclusion was the separation mechanism investigated.The SEC-ICP MS coupling was a method of choice for the determination of iodine speciation in human serum.27,28 Despite the fact that, in theory, the separation should be based on the analyte molecular weight, secondary adsorption and ion-exchange effects make SEC a universal separation technique, as was demonstrated recently for organoselenium16 and organoarsenic compounds.29 SEC has the advantage over other HPLC techniques in terms of a high tolerance to the matrix and the compatibility of the mobile phase with ICP MS.A typical SEC-ICP MS chromatogram of a mixture of standards is shown in Fig. 1. The recovery of thyroglobulin and iodide exceeds 90% whereas the response of the triiodothyronine standard is poor (v10%), probably because of its sorption on the column stationary phase. Note that the elution of iodide is markedly delayed in comparison with the time predicted on the basis of the calibration of the column with the molecular weight standards.Iodide elutes well after the total volume of the column, which means the occurrence of strong non-exclusion interactions. The precision of the SEC-ICP MS analyses was ca. 5% (4.9% for peak area, 5.1% for peak height based on 5 consecutive injections). Quantitative determination of iodide in milk whey by SEC-ICP MS is possible using external calibration with aqueous iodide standards, a good agreement with values obtained by the method of standard additions was obtained.The limit of species-selective determination of iodide (10 times the standard deviation of the blank) was about 1 mg L21. The intensity of the blank was calculated as the average of the intensities (peak height mode) for the replicate measurements within the elution volume of iodide. The standard deviation of the blank is understood as the standard deviation of these intensities.Speciation of iodine in milk SEC-ICP MS chromatographic proÆles of natural milk whey samples and infant formulas of different origin, for which the results of the total iodine determination are given in Table 1, are shown in Fig. 2. Prior to injection on a chromatographic column the samples were preconcentrated by freeze-drying. The preconcentration factor depended on the possibility of the redissolution of the lyophilisate and varied from 2 (cow milk) Fig. 1 SEC-ICP MS chromatogram of iodine-containing standards (10 ng ml21 as I): 1, thyroglobuline; 2, triiodothyrosine; 3, iodide. Column: Superdex-75. Fig. 2 SEC-ICP MS chromatographic proÆles of milk whey samples of different origins. (a) Animal milk: A, goat milk; B, cow milk (Germany); C, cow milk (UK); D, cow milk (France); E, cow milk (Poland); F, cow milk (Spain). (b) Breast milk. (c) Infant formulas: A, Mead Johnson; B, Ordesa; C, Milupa; D, Nestle�.The chromatograms in (a) and (c) were off-set from each other by 56104 cps for the sake of the clarity of presentation. Column: Superdex-75. Peak identiÆcat 1, excluded species (w100 kDa); 2, iodide. 1700 J. Anal. At. Spectrom., 1999, 14, 1697±1702to 9-fold (breast milk). Three basic types of chromatograms can be distinguished. The basic SEC-ICP MS patterns obtained for commercial cow and goat milk samples (Fig. 2a) show one major signal at the elution volume matching that of iodide.The identity of this signal was conÆrmed by spiking the milk sample with a solution of iodide. In addition, a small signal corresponding to a compound excluded from the column can sometimes be seen. This pattern is characteristic for all cow milk samples investigated irrespective of their geographic origin. Human breast milk (Fig. 2b) shows two additional signals: one excluded from the column and one in the middle of the chromatogram; iodide accounts only for ca. 50% of the total iodine present. A completely different pattern is observed for iodine speciation in all but one of the samples of milk formula. Chromatograms of all but one of the samples investigated show a major signal corresponding to an iodine-containing compound excluded from the column. This signal represents 87z6% of the total iodine for Milupa, 73°5% for Ordesa, 65°5% for Mead Johnson, and 84°6% for the Nestle� formula powders. One sample (Nidal) shows a proÆle identical with that of the natural cow samples (a predominating signal from iodide). There are three minor signals in the chromatograms.One, poorly resolved from the major signal on the Superdex-75 column, corresponds to a compound with a molecular mass of ca. 60 kDa and accounts for a few percent of the total iodine present in all the samples except of the Ordesa milk, in which it accounts for ca. 18°2% of the total iodine. Another minor signal corresponds to iodide and represents from 4% (Ordesa) up to 27% (Mead Johnson) of total iodine present. A third minor peak in the middle of the chromatogram is always present but no hypothesis regarding its identity can be put forward.In one case (Nidal) this small peaks elutes at the elution volume of the triiodothyronine standard. Results of the determination of iodide in the milk samples investigated are summarized in Table 1. Attempts were made to obtain an insight into the identity of the excluded compounds by running size-exclusion chromatography on a Superdex-200 column with an exclusion limit of 1300 kDa.Fig. 3 shows that the compound is also excluded from this column. The peak of 60 kDa observed in Fig. 2 is baseline resolved from the excluded compound on the Superdex- 200 column and its molecular weight can be conÆrmed from the calibration curve of the Superdex-200 column. Note that the peak shapes in the chromatograms on a Superdex-200 column are worse than those on a Superdex-75 column and the recovery of iodine from this column was found to be poorer for some samples (especially the recovery of iodide from the Sandoz infant formula).Speciation of iodine in milk samples incubated with SDS The results of the total iodine determination (Table 1) suggested the presence of a substantial water-insoluble fraction of this element in some samples. As indicated above, this iodine can be recovered into the supernatant after incubation of a milk sample with SDS and ultracentrifugation. Fig. 4 compares the chromatograms of the supernatant fraction of the different milk samples without and with the addition of SDS. For natural milk samples (an example of a cow milk sample is shown in Fig. 4a) the patterns of the chromatograms obtained with and without sample incubation with SDS are similar. The iodide peak remains the same whereas there is a small increase in the signal intensity for the compound excluded from the column to account for more iodine extracted.In the case of the infant formula samples (Fig. 4b) the incubation of the reconstituted milk with SDS leads to a marked increase in the intensity of the species excluded from the column. In the absence of SDS, this species apparently stays in the solid residue left over after the centrifugation of the milk whey. The pattern of the chromatogram obtained after the incubation with SDS changes, the concentration of the excluded species being higher than that of iodide.The relative abundance of iodide in the samples incubated with SDS decreases approximately twice (from 100 to 56% for Nidal, from 19 to 10% for Milupa, from 29 to 18% for Mead Johnson). Speciation of iodine in milk after enzymolysis An alternative to the incubation with SDS that can be used to improve the recovery of iodine-containing species from milk Fig. 3 SEC-ICP MS chromatographic proÆles of infant formulas whey samples of different origins obtained on a Superdex-200 column.A, Mead Johnson; B, Ordesa; C, Sandoz; D, Milupa; E, Nestle�. Peak identiÆcation: 1, excluded species (w1300 kDa); 2, ca. 60 KDa compound; 3, iodide. Fig. 4 Effect of the SDS addition on the chromatographic proÆles of different milk samples. Solid line, sample without SDS. Dashed line, sample incubated with SDS as described in Analytical Procedures. (a) Cow milk, (b) infant formula 1, excluded species (w100 kDa); 2, iodide. J. Anal. At. Spectrom., 1999, 14, 1697±1702 1701and to get a deeper insight into the forms of iodine present is the destruction of macromolecular species present in milk by enzymic hydrolysis and monitoring the resulted changes in the speciation of iodine by SEC-ICP MS.A mixture of lipase and protease was investigated for this purpose. These enzymes were found to be effective for the decomposition of milk proteins containing metals (Cu, Zn) for which the elution proÆle was completely changed after enzymolysis, giving rise to a number of low molecular metal-containing species.30 However, this procedure was found unsuitable for iodine species.The enzymic treatment improved the recovery of iodine into the aqueous phase, as in the case of the incubation with SDS, but the SEC-ICP MS chromatograms showed a continuum signal of iodine starting at the exclusion volume and lasting for several minutes (not shown). This signal was followed by a broad iodide signal.Such a chromatogram may suggest that iodine present in the species excluded from the column (Fig. 4) is not covalently bound and is attached to a mixture of macromolecular compounds by coordination bonds or even by less speciÆc interactions. Conclusion Whereas iodide seems to be the major, and practically the only, species of iodine in cow and goat milk, a more complex speciation of this element occurs in human milk and in infant formula, making species-selective analysis necessary to gain a deeper insight in the bioavailability of iodine in baby food.The method developed offers such an approach allowing the discrimination of iodide from a number of other (for the moment unidentiÆed iodine-containing species) and the quantitative determination of a particular species. The major difference between the breast milk and infant formula is the presence in the latter of a macromolecular compound comprising more than 50% of the iodine present in the preparation. Isolation, puriÆcation and characterization of this compound is the prerequisite for drawing conclusions regarding its bio-availability.Acknowledgements LFS acknowledges the research grant of the Caixa Galicia. We thank Profs. P. Bermejo (University of Santiago) and R. Lobinski (CNRS, Pau) for valuable discussions. References 1 E. J. Underwood, Trace Elements in Human and Animal Nutrition, Academic Press, New York, 4th edn., 1997, p. 271. 2 M. Anke, B. Groppel, M. Mu» ller, E.Scholz and K. Kramer, Fresenius' J. Anal. Chem., 1995, 352, 97. 3 M. F. Picciano, Biol. Neonate, 1998, 74, 84. 4 P. A. Fecher, I. Goldman and A. Nangengast, J. Anal. At. Spectrom., 1998, 13, 977. 5 E. Larsen, P. Knuthsen and M. Hansen, J. Anal. At. Spectrom., 1999, 14, 41. 6 G. Radlinger and K. G. Heumann, Anal. Chem., 1998, 70, 2221. 7 Y.Ge�linas, A. Krushevska and R. M. Barnes, Anal. Chem., 1998, 70, 1021. 8 H. Vanhoe, F. Van Allemeersch, J. Versieck and R. Dams, Analyst, 1993, 118, 1015. 9 S. Stu»rup and A. Bu» chert, Fresenius' J. Anal. Chem., 354, 323. 10 H. Baumann, Fresenius' J. Anal. Chem., 1990, 338, 809. 11 G. Go»kmen and G. DagÆh, Analyst, 1995, 120, 2005. 12 R. R. Rao and A. Chatt, Analyst, 1993, 118, 1247. 13 F. Gu, A. A. Marchetti and T. Straume, Analyst, 1997, 122, 535. 14 T. Mitsuhashi and Y. Kaneda, J. Assoc. Off. Anal. Chem., 1990, 73, 790. 15 V. E. Negretti de Bra» tter, S. Recknagel and D. Gawlik, Fresenius' J. Anal. Chem., 1995, 353, 137. 16 P. Bra» tter, I. Navarro Blasco, V. E. Negretti de Bra» tter and A. Raab, Analyst, 1998, 123, 821. 17 J. S. Edmonds and M. Morita, Pure Appl. Chem., 1998, 70, 1567. 18 A. K. Das, R. Chakraborty, M. L. Cervera and M. de la Guardia, Mikrochim. Acta, 1996, 122, 209. 19 Element Speciation in Bio-inorganic Chemistry, ed. S. Caroli, Wiley, Chichester, 1996, pp. 474. 20 B. Michalke, D. C. Muench and P. Schramel, Fresenius' J. Anal. Chem., 1992, 344, 306. 21 B. Michalke and P. Schramel, J. Trace Elem. Electrolytes Health Dis., 1990, 4, 163. 22 P. Gerhart, F. Macasek and P. Rajec, J. Radioanal. Nucl. Chem., 1998, 229, 83. 23 C. Casiot, J. Szpunar, R. Ëobin� ski and M. Potin-Gautier, J. Anal. At. Spectom., 1999, 14, 645. 24 D. Sertl and W. Malone, J. Assoc. Off. Anal. Chem., 1993, 76, 711. 25 B. Mo» ller, I. Bjo»rkhem, O. Falk, O. Lantto and A. Larsson, J. Clin. Endocrinol. Metab., 1983, 56, 30. 26 L. V. Oberkotter, J. Chromatogr., 1989, 487, 445. 27 A. Makarov and J. Szpunar, Analusis, 1998, 26, M44. 28 B. Michalke, P. Schramel and S. Hasse, Mikrochim. Acta, 1996, 122, 67. 29 S. McSheehy and J. Szpunar, unpublished work. 30 L. Fernandez Sanchez and J. Szpunar, unpublished work. Paper 9/05558D 1702 J. Anal. At. Spectrom., 1999, 14, 1697±17