Speciation of Iron in Breast Milk and Infant Formulas Whey

8/9/2019 Speciation of Iron in Breast Milk and Infant Formulas Whey

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Talanta 50 (2000) 1211–1222

Speciation of iron in breast milk and infant formulas wheyby size exclusion chromatography-high performance liquid

chromatography and electrothermal atomic absorptionspectrometry

P. Bermejo a, *, E. Peña a, R. Domı ´nguez a, A. Bermejo a , J.M. Fraga b ,J.A. Cocho c

a Department of Analytical Chemistry , Nutrition and Bromatology , Faculty of Chemistry , Uni ersity of Santiago de Compostela ,

A da de las Ciencias , 15705 Galeras SN , Spainb Department of Pediatrics , Faculty of Medicine , Uni ersity of Santiago de Compostela , Galeras SN ,15705 Santiago de Compostella , Spain

c Laboratory of Metabolic Disorders , Complejo Uni ersitario , Uni ersity of Santiago de Compostela , Galeras SN ,15705 Santiago de Compostela , Spain

Received 5 January 1999; received in revised form 1 June 1999; accepted 23 July 1999

Abstract

Speciation of iron in milk was carried out by high performance liquid chromatography (HPLC) and electrothermal

atomic absorption spectrometry (ETAAS). Milk whey was obtained and low molecular weight protein separation wasperformed by size exclusion chromatography (SEC) with a TSK Gel SW glass guard (Waters) pre-column and aTSK-Gel G2000 glass (Toso Haas) column. After studying water as a possible mobile phase, this mobile phase wascarefully selected in order to avoid alterations of the sample and to make subsequent iron determination in the proteinfractions easier by ETAAS. The proposed method is sensitive (limit of detection [LOD] and LOQ 1.4 and 4.7 g l− 1 ,respectively) and precise (relative standard deviation [RSD] 10%). Iron is principally found in the proteins of 3 and76 kDa in breast milk, and it is irregularly distributed in infant formulas. © 2000 Elsevier Science B.V. All rightsreserved.

Keywords : Iron speciation; Milk; SEC-HPLC; ETAAS

www.elsevier.com /locate /talanta

1. Introduction

Iron deciency anemia (IDA) is a priority nutri-tional problem in industrialized as well as devel-oping countries. Besides anemia per se, tissue irondeciency may lead to a defect in learning capac-

* Corresponding author. Tel.: + 34-981-591-079; fax: + 34-981-595-012.

E -mail address : [email protected] (P. Bermejo)

0039-9140 /00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.PII: S 0 0 3 9 - 9 1 4 0 ( 9 9 ) 0 0 2 3 3 - 7


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P . Bermejo et al . / Talanta 50 (2000) 1211–1222 1212

ity, cognitive performance and abnormalities inpsychomotor development in infants and pre-schoolers. This deciency also effects work perfor-mance in adults and an increase in the frequencyof low birth weight, prematurity, and prenatalmortality in pregnancy can be found [1]. Becauseof the risk of cancer and heart disease in individu-als with high iron stores [2,3] it is not recom-mended to supply iron to individuals who do notrequire it. A high priority must be assigned to theprevention of iron depletion among infants [4],and so, iron fortication has been widely used inthe industrialized world.

Breast milk and /or milk formulas are the mainnutrient uids of newborn infants. Trace elementsare usually added to infant formulas as inorganicsalts, whereas in milk, these elements are boundto different compounds, which affect bioavailabil-ity. In order to carry out a rational supplementa-tion, breast milk is used as a reference to evaluatethe nutritional content of alternative formulas,assuming that the composition of breast milk maysatisfy the growing demands of healthy infantsduring the early months of life [5]. It is interestingto know how the highest breast milk bioavailabil-ity might depend on the distribution among thedifferent milk proteins and how inorganic saltsare found in infant formulas after being added.

Few reports can be found in the literature.Previous results have been obtained by means of size exclusion chromatography-high performanceliquid chromatography (SEC-HPLC) and induc-tive coupled plasma-atomic emission spectrometry(ICP-AES) [5– 7]. Negretti de Brätter et al. [8]used instrumental neutron activation analysis(INAA) as the reference method for the qualitycontrol of the shape of the element proles (Se,Fe, Zn) obtained with ICP-AES after chromato-graphic separation. When the sensitivity of ICP-AES is not sufcient ICP-MS may be used.However, instruments for these techniques areexpensive and not available in many laboratories.

Based on a cost-benet analysis, atomic absorp-tion spectrometry (AAS) seems to be the preferredmethod. The detection limits obtained by AASmethods, requiring a small sample volume, arelow enough to allow the determination of most of the trace elements in speciation. Consequently,

the use of AAS, in particular electrothermalatomic absorption spectrometry (ETAAS) needsto be considered [9,10]. Thus, in an earlier studyFransson and Lönnerdal [11] determined the dis-tribution of iron among various fractions of breast milk by HPLC, ultraltration and AAS.The samples were freeze dried, ashed at 600°C for5 h, and dissolved in 1:1:1 HCl /HNO 3 /H 2O beforeanalysis.

In this work, iron speciation in milk was carriedout by SEC-HPLC and ETAAS. Milk fat andcasein micelles were removed and the milk wheyobtained was chromatographied. Special attentionwas paid to the column and mobile phase selec-tion, when looking for the best separation in thelowest range of molecular weights, where ironpresence had been reported [5–8,11].

Water was studied as mobile phase [12,13].Then, the mobile phase was carefully selected,salinity was decreased, in order to avoid contami-nations, stability problems of the organometalcomplex, undesired interactions with the sampleto a great extent and interferences of our mobilephase in the direct determination of iron inprotein fractions by ETAAS.

2. Experimental

2 .1. Apparatus

Milk whey was obtained using an ultracen -trifuge L8-Beckmann with a SW-40 rotor.

A Crison pH-meter equipped with a combinedelectrode gas-calomel INGOLD U455-Ag 7.0DIN pH 0–14 was utilized to determine pH of mobile phases.

For the chromatographic separation of wheyproteins, a High Performance Liquid Chro-matograph 625-LC System (Waters, USA)equipped with a TSK gel SW glass guard pre-column, 4 cm × 8 mm, and a TSK gel G 2000

glass, 30 cm × 8 mm (Toso Haas, Japan) columnwas employed. The column has a selected silica-based packing, derivatized using the glycol etherfunction containing spherical 10 m particles withpore sizes of 13 nm. The Waters 625 LC system isa non-metallic HPLC solvent delivery system for


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liquid chromatography applications where wettedsurface material is made of inert, polymeric mate-rials and exible PEEK tubing. Peaks were de-tected by an UV detector model 486 (Waters,USA) at 254 nm, and acquisition and processingof data were performed by the Millennium Chro-matography Manager System, version 2.15 (Wa-ters, USA) programme.

Iron measurement in milk and milk whey wascarried out using an Atomic Absorption Spec-trophotometer Perkin Elmer model 5000 (PerkinElmer, Germany) at 248.3 nm, a hollow cathodelamp operating at 30 mA, a slit of 0.2 nm, and anacetylene air-ame.

Iron measurement in protein fractions wascarried out using an Atomic AbsorptionSpectrophotometer 1100 B (Perkin Elmer, Ger-many) with a hollow cathode lamp and aHGA-700 graphite furnace (Perkin Elmer,Germany). The instrument was tted withan AS-70 autosampler. Pyrolitic graphite tubeswith L’vov platforms were used throughoutthe course of this study. Instrument settingsfor Fe determination are summarized in Table1.

2 .2 . Reagents

All solutions were performed with ultrapurewater, specic resistivity 18 M cm, from a Milli-

Q purication system (Millipore).

In order to prepare mobile phases, differentreactives (ammonium nitrate, NH 4NO 3, Suprapur(Merck, Germany), ammonia solution, NH 3,Suprapur (Merck, Germany), sodium azide, NaN 3(Sigma, St. Louis, MO) were used. The calibra-tion column was performed using protein stan-dards ribonuclease A, ovalbumine, albumine,aldolase, coming from HMW Gel Filtration cali-bration Kit and LMW Gel Filtration CalibrationKit (Pharmacia Biotech, USA), and cianocobal-amine (Sigma).

A previously diluted stock solution of iron 1 gl− 1 (Merck, Germany), was employed to optimizeETAAS temperature programme and to obtainthe calibration graphs. Finally, magnesium ni-trate, MgNO 3, Suprapur (Merck, Germany), wasassayed as a possible modier.

A Reference Material A-11 non-fat milk of theInternational Atomic Energy Agency (IAEA) witha certied iron content was used.

2 .3 . Procedure

2 .3 .1. Sampling Breast milk samples were collected following

strict precautions in order to minimize contamina-tion and avoid alterations. The samples were col-lected by hoc trained personnel in polyethyleneasks using a motorized pump. Care was paid toavoid touching the inner wall of the device or

ask.Table 1Instrumental conditions and furnace programme for iron determination in protein fractions

Step Signal read outGas ow (ml min − 1)t (s)T (°C)

HoldRamp

3003015100Dry −10700 −Pyrolysis 1 25 300

20 3005 −Pyrolysis 2 13500 032400Atomization +

3002600 −1 3Clean

248.3 Purge gasWavelength (nm) ArgonPyroliticBackground corrector Graphite tubesDeuterium20Lamp (mA) Injection volume ( l)30

Peak areaSignal processing 0.2 nmSlit width20 lSample volume


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Fig. 1. Total procedure.

the retention time of the different peaks. A secondinjection of 100 l was performed afterwards andthe eluent emerging from the UVA-VIS detectorwas collected in different fractions according tothe number of different chromatographic peaksobtained for each sample.

2 .3 .4 .2 . Iron determination . Iron was directly de-termined in the collected fractions by ETAAS,without addition of a chemical modier. Calibra-tion was performed using standards of iron di-luted with the mobile phase at concentrations of 0, 10, 20, 30 g l− 1. The volume of sampleinjected was 20 l. As the volumes of fractions areknown (0.5–1.5 ml), the iron concentration wasgiven as ng fraction − 1.

3. Results and discussion

3 .1. Water as a mobile phase

Water was assayed to nd a simple mobilephase which avoids interferences in iron measure-ment and undesirable interactions with the sam-ple. Chromatograms of an infant formula werecompared, using only water as the mobile phaseand using a solution of the bactericide sodiumazide 0.05% m V − 1 [6], these results are shown inFig. 2. Negative peaks and signal distortions ap-peared indicating that the aforementioned com-pound must be taken out of the mobile phase.Nevertheless to avoid the rapid deterioration of the column the use of the sodium azide wasproposed only in the washing programme of thecolumn.

3 .2 . Chromatographic conditions

The term ‘non size effects’ in SEC includesattractive interactions, such as ion exchange andhydrophobic binding, which tend to increase the

elution volumes of solutes, and increase forces of electrostatic repulsion with the opposite effect. Abalance must be made between the need to in-crease ionic strength to reduce ionic electrostaticinteractions and to decrease ionic strength to limithydrophobic interaction [17].

With regard to infant formulas, commerciallyavailable, solutions were prepared by dissolvingmilk powder using ultrapure water, following themanufacturer’s instructions.

Containers and covers (polyethylene) were keptin nitric acid for at least 48 h, rinsed three times

with ultrapure water and maintained dried untilused. Samples were stored at − 20°C until treat-ments were performed.

2 .3 .2 . Iron determination in milk and milk wheyTotal values of iron concentration in milk and

milk whey were directly determined by an AASame [14] using a high performance nebulizer.The addition procedures were always used.

2 .3 .3 . Sample preparation : ultracentrifugation

Milk samples were ultracentrifuged [14–16] at31 000 rpm (160 000 × g ) for 60 min, with 1 minacceleration and 1 min deceleration times. Milkwhey was taken out with a micropipette after fatseparation. The lower phase (casein micelles) re-mained in the bottom of the tube.

2 .3 .4 . Iron speciation

2 .3 .4 .1. Chromatographic separation . Milk wheyswere ltered using Millex GV 13 0.22 m sterile

units (Millipore, France), and 100 l were injectedin the chromatographic system, with a ow rateof 1 ml min − 1 . Measurement wavelength was 254nm (Fig. 1). As each milk can present a differentprotein prole it was always necessary to performa rst injection to know the protein prole and


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Early in the development of electrothermal at-omization L’vov [18] found that molecular ab-sorption by alkali halides was one of the majorcauses of interferences, when these halides arepresent in a concentration four or ve orders of magnitude higher than the analyte element. Dueto the very high sensitivity of the graphite furnacetechnique, this relationship is very quickly

reached. For this reason the utilization of sodiumchloride at concentrations of 0.05–0.3 M as oneof the components of the mobile phase, as otherauthors using ICP-AES [5–8] had reported, wasnot possible here.

Ammonium nitrate concentration was opti-mized in the range of 0.1–0.4 M using a proteinmixture (0.02 mg of cianocobalamine, 0.19 mgribonuclease A, 0.21 mg of ovalbumine, 0.19 mgof albumin, 0.77 mg of aldolase). Using ammo-nium nitrate 0.2 M the best separation at higher

retention times (lower molecular weights) was ob-tained. This fact was observed because of thepeak appearance corresponding to the aggregatewhich elutes slightly before the true peak of ri-bonuclease A (retention time = 15 min, Fig. 3).Different pHs around neutrality were assayed (pH6.1, 6.7, 7.3) without observing signicant changesin the elution. Ammonia solution was added toobtain pH 6.7, reported as milk pH [19]. The nalcomposition of mobile phase was 0.2 M NH 4NO 3and 3.24 × 10 − 4 M NH 3.

Using this mobile phase the best peak denitionwas obtained, as can be seen in the chro-

matograms shown in Fig. 4, for a breast milksample and the same infant formula used in thechromatograms of Fig. 2.

3 .3 . Column calibration

In order to know the molecular weight of theproteins found in the milk whey, the column was

calibrated using a series of standard proteins withcertied molecular weight. These proteins werechromatographied and the retention times ob-tained as well as the molecular weight are shownin Table 2.

The equation obtained to column calibrationwas:

Log MW (kDa) = − 0.276 + 7.591 tR (min)

with a correlation coefcient r = 0.974.

3 .4 . Precision in the protein separation

The nal aim of this work is to determine ironin the different proteins of the milk whey, and forthis reason it is necessary to know the repeatibilityof the protein retention times in order to be ableto separate the protein fractions. To perform thisstudy ten replicates of a breast milk sample and12 replicates of an infant formula sample werechromatographed; the results obtained for thedifferent proteins found in both types of milk are

shown in Table 3. The retention times for allproteins were constant, and the relative standard

Fig. 2. Chromatograms demonstrating the negative effect of sodium azide addition (used as bactericide) in the elution. Mobile phase:(1) water; (2) sodium azide 0.05% (w /v).


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Fig. 3. Effect of different concentrations of ammonium nitrate in the elution of a standard protein mixture.

Fig. 4. Chromatograms: (1) breast milk sample; (2) infant formula sample.

deviation (RSD) for the absorbance expressed inheight or area peak mode can be considered goodin all cases. An R.S.D. of 12.3% was obtained forthe peak corresponding to 2 kDa, this value couldbe produced because of being the nearest peak tothe separation limit of the column.

3 .5 . Graphite furnace programme

The chemical modier proposed for the irondetermination in ETAAS is magnesium nitrate.

Table 2Column calibration

Retention time (min)Standard protein MW (kDa)

1.355 15.5Cianocobalamine13.700Ribonuclease A 13.643.000Ovalbumine 10.5

9.667.000Albumine158.000 8.9Aldolase


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Table 3Precision in protein separation

Infant Formula ( n = 12)Breast milk ( n = 10)

RSD (%)MW (kDa) MW (kDa) RSD (%)

Peak height Peak areaRetention time Retention time Peak height Peak area

2.3 2.9 250.0 0.0101 2.2 1.6

0.078 0.8 2.4 15 0.0 1.4 0.90.053 2.0 5.0 4 0.0 3.7 3.71.0 0.6 3.50.0 0.014 2.7 4.09.1 5.6 23 0.00.0 3.8 12.39.0 8.30.02

To optimize the pyrolysis and atomization tem-peratures an aqueous iron standard solution of 10

g l− 1 with Mg(NO 3)2 and without Mg(NO 3)2were used. No important improvement in the ironstabilization was observed. For this reason and to

avoid risk of contamination and to shorten theanalytical procedure we propose the eliminationof the use of the Mg(NO 3)2 and thus the directintroduction of the protein fractions become pos-sible. The optimum pyrolisis and atomizationtemperatures were 1350 and 2400°C, respectively.An intermediate pyrolisis step at 700°C was neces-sary to follow a complete mineralization of thesample. Finally a cleaning step at 2600°C wasintroduced to avoid possible memory effects. Theinstrumental conditions and the furnace pro-gramme are summarized in Table 1.

3 .6 . Calibration

Solutions prepared in mobile phase with ironstandard concentrations between 0 and 30 g l− 1

were used to determine a calibration curve. Theequation obtained was:

A = 4.48 × 10 − 3 [Fe]–5.0 × 10 − 3 r = 0.999

where A is the absorbance (peak area) and [Fe] is

the iron concentration expressed in g l− 1

.

3 .7 . Sensiti ity

The limit of detection (LOD) is dened as 3S.D. /m and the limit of quantication is given by

10 S.D. /m (m = slope of the calibration graph andS.D. = the within-run standard deviation of theblank signals). The values based on ten replicatesof the blank were 1.4 and 4.7 g l− 1, respectively.

The characteristic mass ( m0) dened as the mass

of analyte that provides an integral absorbance of 0.0044 for an aliquot sample of 20 l was 20.4 pg.

3 .8 . Iron – protein complex stability

To know the stability of the Fe–protein com-plexes a study about the time effects on the chro-matographic protein separation was performed. Amilk whey sample (infant formula) was chro-matographed at different times after preparation1.0–2.0–3.0–4.0 and 24 h later. The results areshown in Fig. 5(1)). It can be seen that only theabsorbance of the protein corresponding to 2 kDachanges with the time and this absorbance wasconstant after 24 h. For this reason, the perfor-mance of chromatographic separation 24 h afterthe milk preparation was proposed.

On the other hand to know the effect of thesample freezing, the same sample was chro-matographed before and after freezing, both chro-matograms are shown in Fig. 5(2). It can be seenthat the freezing did not affect to the protein

separation.In the same way the iron determination in the

different protein fractions was performed beforeand after freezing. The levels of iron obtained arein Table 4 and it can be seen that there are nosignicant differences due to the sample freezing.


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3 .9 . Precision in the iron determination

The within-run precision (RSD) of the method(instrumental and matrix factors) was studied us-

ing both types of milk. The iron content of thefractions obtained for a breast milk sample (sixreplicates) and for infant formula milk (nine repli-cates) was determined using the proposed proce-

Fig. 5. (1) Chromatograms of a sample after ultracentrifugation: (a) 1 h later; (b) 2 h; (c) 3 h; (d) 4 h; (e) 24 h. 2) Chromatogramsof a sample stabilized: (a) after ultracentrifugation; (b) after defreezing.

Table 4Iron distribution in fractions of an infant formula sample before and after freezing

MW (kDa) After freezingBefore freezing

[Fe] (ng fraction − 1) %%[Fe] (ng fraction − 1 )

321 14.086.5 1.812.074.7 0.617.8110.3 1.659 106.7 5.4 17.3

211.2 1.6 34.2207.0 0.038 33.415 24.3150.4 0.0162.4 3.5 26.2

7.4 39.6 0.8 6.43 46.1 0.83.823.4 0.13.219.6 0.12

Table 5Precision in iron determination by electrothermal atomic absorption spectrometry (ETAAS)

Infant formula ( n = 9)Breast milk ( n = 6)

Fe (ng fraction − 1)MW (kDa) S.D. RSD (%) MW (kDa) Fe (ng fraction − 1) S.D. RSD (%)

1.526.5101 1.6 0.1 6.3 25 5.7

9.0 15 19.81.0 1.478 7.111.21.0 7.0 453 109.214.4 8.8 8.1

14 ND a – – 3.5 144.3 9.2 6.40.43 11.83.4 2 22.8 1.6 6.9 – ND a – 2

a ND, not detected.


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Table 6Mass balance study

After freezingBefore freezing[Fe][Fe]

(ng fraction − 1) (ng fraction − 1)

[Fe] fractions 619.9 617.6(ng 100 l− 1)

[Fe]milk (ng 1000 l− 1) 1150.0 0.0

720.0 20.0[Fe]whey ( g ml− 1

) 86.1[Fe] fractions[Fe]whey

× 100 (%) 86.1

Table 6. It can be seen that the iron mass balancesalways approached to 100% (86.1%), and it can beconcluded that there is no contamination or lossproblems.

3 .12 . Applications

The proposed method has been applied to thestudy of the iron distribution in the milk wheyproteins of ten infant formulas and ten breastmilk samples. The infant formula samples of different brands were prepared at the concentra-tions suggested by the manufacturer, whereasbreast milk was individual samples from womenliving in Galicia, North West of Spain with mostof them corresponding to the rst stage of lacta-tion.

The protein identication of the milk whey isusually performed by comparison of the retentiontimes of standards and samples, but in case of anincomplete separation metals can not be clearlyattributed to proteins [13], or may be attributed tothe wrong one. To avoid this problem the ironfound in a protein fraction was only associatedwith the molecular weight of the protein obtainedin the calibration column.

The iron in the protein fractions was deter-mined by ETAAS using the established conditionsat least twice. The iron concentrations in milk andmilk whey are reported in Table 7.

Previous researchers concluded that a morecareful speciation should be performed in therange of low molecular weight compounds. First,Fransson and Lönnerdal [11] demonstrated usingultraltration (membrane molecular weight cut-off of 15 kDa) and SEC that a considerablefraction of the iron was bound to LMW com-pounds in breast milk. Brätter and al. [5] foundiron in a fraction of HMW ( 600 kDa) and inthe fraction of LMW (9–10 kDa) eluting togetherwith citrate. Suzuki et al. [6] studied daily changesin components of breast milk with number of

lactation days. They found iron in one or twopeaks, whose elutions times coincided with thetransferrin peak and citrate peak time. However,their chromatographic separation seems to beworse. Negretti de Brätter [8] studied a wide rangeof MWs protein obtaining a complicate chro-

dure. The results obtained are shown in Table 5.The values of RSD (%) obtained are acceptable inall cases because this precision includes all theanalytical procedure: the chromatographic separa-tion, the fraction collection and the irondetermination.

3 .10 . Accuracy for the Fe determination in total milk and milk whey

The accuracy of the total iron concentration inmilk was performed using a Certied ReferenceMaterial, non-fat milk A-11 of the IAEA with acertied content of 3.65 0.76 g Fe g − 1 . Thisreference material was prepared at 15% (W /V)and the Fe content was determined using theaddition procedure. The results obtained for vereplicates were 3.04 0.17 g F e g − 1. On the

other hand the recovery of the method was stud-ied measuring iron added to whole milk, due tothe fact that this sample has a more complexmatrix than milk whey. Different amounts of Feadded to the milk sample were studied, the resultsobtained were 100.0, 96.0 and 102.0% for 0.50,1.00 and 1.5 g ml − 1 of Fe added.

3 .11 . Mass balance study

To check the accuracy in the iron determination

in the protein fraction a study about the massbalance in a milk sample was performed. Thesample was studied after its collection and afterfreezing and each sample was chromatographedby duplicate. The iron levels for the total milk, themilk whey and for different fractions are shown in


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Fig. 6. Iron distribution among protein fractions: (1) breast milk; (2) infant formula. Concentration is expressed as ng Fe fraction − 1 .


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Table 7Iron concentration in milk and milk whey

Breast milk Infant formula

Sample Milk wheyMilk Sample Milk Milk whey

[Fe] ( g ml− 1) [Fe] ( g ml− 1)

0.22 0.001 10.28 0.01 3.6 0.30 1.8 0.00

0.12 0.00 20.23 0.00 5.8 0.102 1.6 0.000.60 0.013 0.41 0.00 3 5.7 0.10 1.6 0.100.24 0.00 44 4.2 0.100.33 0.00 1.1 0.100.17 0.00 50.19 0.00 3.3 0.105 2.0 0.20

0.23 0.006 0.17 0.00 6 3.1 0.30 1.8 0.000.13 0.00 77 5.7 0.100.22 0.00 1.8 0.10 – a 8 – a 6.3 0.308 1.4 0.100.17 0.00 99 4.4 0.600.28 0.01 1.5 0.000.22 0.00 100.24 0.00 2.9 0.1010 1.5 0.00

a Results not available.

matographic pattern and concluded that columnswith a separation range 250 kDa should beused for a detailed human milk speciation. Thechromatographic patterns are similar to that ob-tained by Coni et al.[7]. They found a homogene-ity of iron distribution in protein fractions of infant formulas, whereas there is an increase inthe amounts of elements in those intermediatefractions of mature breast milk that are related tosubstances with molecular weights ranging be-tween 10 and 100 kDa. Underlying this distribu-tion, an important role is played by thedifferences in percentage of these proteins in thecomposition of cow, cow-based formulas and hu-man milk. Colostrum shows a different elementcomposition from that of mature milk.

It was found that the behaviour of the infantformulas is very different and the iron distributionin the proteins is not regular. This can be ex-plained by the different chemical composition of the formulas, which are prepared in different pro-

portions of whey, proteins and minerals accordingto the needs of the infants. For the studied breastmilks, the iron was bound principally to theproteins corresponding to molecular weights of 3and 76 kDa (Fig. 6), these results agree with theresults obtained by other authors.

4. Conclusions

A speciation method for iron in milk byHPLC and ETAAS has been developed in orderto compare the iron distribution among lowmolecular weight proteins in breast milk andinfant formulas. With the use of a simplemobile phase a good protein separation wasobtained, and moreover its use does not pre-sent problems in the iron determination byETAAS.

The results obtained showed a differentiron distribution in the proteins of the milkwhey of infant formulas and human milk,and this can be important in the iron bioavail-ability of both types of milk. For this reasonit is necessary to continue these studies in or-der to prepare new infant formulas wherethe iron distribution is more similar to humanmilk.

Acknowledgements

This work was partially supported by the re-search project 1997, CEO12 Laboratorios Ordesa,Barcelona, Spain.


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Speciation of Iron in Breast Milk and Infant Formulas Whey

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