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Increased awareness of the inuence of diets onhuman health has prompted producers to producefood of higher quality, rich in nutrients and nu-traceuticals, in environmentally friendly manner.On the other hand, raw materials for food pro-duction are getting poor in essential minerals andvitamins at the same time.

Milk and dairy products have been recognizedall over the world for a long time as good for theirsensory properties, but also for their benecial in-

uence on human health (Steijns, 2001). On theother hand, milk is an expensive raw material pro-duced by animals (Boland et al., 2001). To producemilk or to make its products of good quality and toreduce their price, a good raw material is needed.erefore constant control of raw milk boughtfrom farms is necessary. Milk should be controllednot only for fat and proteins as it is usual, but alsofor mineral content wheneverpossible.

Milk is known as an excellent source of Ca, andit can supply moderate amounts of Mg, smaller

amounts of Zn and very small amounts of Feand Cu (Levyet al., 1985; Pennigton et al., 1987,

1995a,b). erefore the essential metals are inter-esting for determination of their adequate dailyintake by the organism. On the other hand, dueto the growing environmental pollution it is alsonecessary to determine and monitor the levels oftoxic metals in milk, such as lead and cadmium,because they can signicantly inuence the humanand animal health (Steijns, 2001).

e most frequently used technique to deter-mine metals in food and biological materials is

atomic absorption spectrophotometry (AAS). Ifthe concentrations of metals are high enough, AASdeterminations are usually done by ame atomicabsorption spectrophotometry (FA-AAS). Whenthe concentrations of metals are low, electrothermalatomic absorption spectrophotometry (EA-AAS) isapplied. Both techniques should give the sameresults if the concentration of metals is above thedetection limit of FA-AAS (Jorhem, 1993).

Because of low industrial production Croatia hasa unique opportunity to produce milk rich in nu-

tritional elements and with very low levels of toxicsubstances at the same time. In order to determine

Determination of metals in cows milk by ame

atomic absorption spectrophotometryM. SIKIRI1, N. BRAJENOVI1, I. PAVLOVI2, J. L. HAVRANEK2, N. PLAVLJANI2

1Ruer Bokovi Institute, Zagreb, Croatia2Faculty of Agriculture, University of Zagreb, Zagreb, Croatia

ABSTRACT: e essential (Ca, Cu, Fe, Mg, Mn, Zn) and toxic metals (Pb and Cd) were determined by ameatomic absorption spectrophotometry (FA-AAS) in raw cows milk. Milk was sampled four times in March 2000

on fteen farms near Kri in the Zagreb region from bulk milk directly into plastic bottles (to avoid contamina-

tion). e raw cows milk samples were digested in an MLS-1200 Mega Microwave Digestion System with MDRTechnology, and the metals were determined directly by FA-AAS in the solutions of digested samples. Statisticalanalyses were performed using statistical software SAS v. 8.0. Contents of essential minerals (Ca, Cu, Fe, Mg, Mn,

Zn) in milk from Croatian farms are on the normal levels in comparison with the data from other countries. elevel of toxic metals (Pb and Cd) was lower than the tolerance level dened by Croatian regulations (Pb < 100 g/l

and Cd < 10 g/l).

Keywords: lead; cadmium; copper; iron; manganese; magnesium; calcium; raw cows milk; ame atomic absorption

spectrophotometry

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Original Paper Czech J. Anim. Sci., 48, 2003 (11): 481486

the quality of raw material bought by the Croatiandairy industry one-month research was conductedon 15 family farms in the region of Ivanic grad, theregion of biggest milk producers in Croatia. Ourattention was focused on the mineral composition

of milk.

MATERIAL AND METHODS

Sampling strategy. Samples of raw cows milkwere collected four times in March 2000 fromfteen farms located near Kri in the Zagreb re-gion. e cows on each farm were fed dry hay andmineral premixes and drank water from the pump.Raw milk was sampled from the bulk milk of the

farms directly into carefully washed plastic bottlesbecause of the relatively great possibility of samplecontamination, especially by lead and cadmium.

Precautions against contamination. All chemi-cals were of the highest available purity. Deionised

water was used. e laboratory ware was cleanedwith chromosulphuric acid and rinsed with deion-ised water. e laboratory ware was dipped in10% HNO

3for 24 h, rinsed with deionised water,

dipped in 3% EDTA solution for 24 h and rinsedwith deionised water again.

Anakytical procedure. e fat and protein con-tent (%) was determined on Bently 150 Infraredmilk analyser.

To determine the concentrations of metalschemicals of the highest available purity wereused. HNO

3(Riedel) was used for the dilution

and mineralization of raw milk samples, H2O

2(9.791 mol/dm3, Riedel) was used for digestionof all milk samples. Lead nitrate (3.019 103mol/dm3, Merck) and cadmium nitrate (4.230 103 mol/dm3, Merck) solutions were used ascalibration standards. All solutions were prepared

with deionised water.e metals (calcium, copper, iron, magnesium,

manganese, zinc, lead and cadmium) were ana-lysed by FA-AAS in a total of 60 milk samples. Toeliminate the organic part of milk, 0.500 g of rawcows milk was treated with 6 ml of 65% HNO3and 1 ml 30% H

2O

2, mixed and digested in an

MLS-1200 Mega Microwave Digestion Systemwith MDR Technology. Lead and cadmium weredetermined with ammonium salts pyrrolidine-1-dithiocarboxylic acid (APDC) into methyl-isobutyl

ketone by FA-AAS. e other investigated metalswere determined directly by FA-AAS.

Instrumentation. PU 9100X FA-AAS equippedwith Epson FX-850 printer was used. e instru-ment was controlled by Philips P 3120 PC. e air/acetylene ame was used for the analysis of metals.Instrumental conditions for the determinations of

metals in raw cows milk are given in Table 1.Statistics. Data were analysed by General Linear

Models procedure. Mean dierences were sepa-rated by Duncans Multiple Range Test. e levelof signicance was set at P< 0.05. All analyses wereperformed using statistical software SAS v. 8.0.

RESULTS

Average mineral concentration of raw cows milk is

shown in Figure 1. e results of ANOVA are sum-marized in Table 2. Signicant correlations betweenthe investigated parameters are shown in Figure 2.

Fat and protein

In the period of investigations fat content in rawcows milk from 15 dierent farms was signicantlydierent, ranging from 2.55 to 4.62%. Similar re-sults were obtained for protein content that rangedfrom 2.81 to 4.1%. e correlation between fat andprotein content was not statistically signicant.

Calcium and magnesium

Dierences between farms were signicantfor calcium content, ranging from 1 125.76 to2 019.04 mg/kg, but not for magnesium content136.02196.67 mg/kg. Calcium content correlated

with fat content (R= 0.647).

Iron, zinc, copper and manganese

Iron, zinc, copper and manganese contents inmilk from dierent farms were statistically dif-ferent. Relatively high coecients of correlationof manganese and copper with the other threeminerals were obtained (Mn Fe: R= 0.641, Mn Zn: R= 0.731, Mn Cu: R= 0.618, Cu Fe:R= 0.613, Cu Zn: R= 0.629) while the contentsof iron and zinc were not so highly correlated. In

addition, correlations existed between copper andcalcium content (R = 0.629) as well as between

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Table1.Instrumentalconditionsforlead

,cadmium,calcium,copper,iron,m

anganese,magnesiumandzincdeterminationinrawcowsmilkbyFA-AAS

Lead

Cadmium

Calcium

Copper

Iron

Manganese

Magnesium

Zinc

Prim

arywavelength(nm)

217.0

228.8

422.7

324.8

248.3

279.5

285.2

213.9

Band

-pass(nm)

0.5

0.5

0.5

0.5

0.2

0.5

0.5

0.5

Injec

tionvolume(ml)

1

1

1

1

1

1

1

1

Flam

etype

air/acetylene

Fuel

owrate(l/min)

0.91.2

1.01.3

0.91.2

0.81.1

0.81.0

0.91.2

0.91.2

0.91.2

Sensitivity(mg/l)

0.10

0.032

0.09

0.041

0.060

0.029

0.003

0.013

Table2.ANOVAsummaryofthefarme

ectonminerallevelsinrawcowsmilk.Meanswiththesameletterare

notsignicantlydierent

Protein

Fat

Ca

Mg

Fe

Zn

Cu

Mn

Pb

Cd

(%)

P

Mn. is sequence is similar tothat obtained by Rodrguez et al. (1999), the onlydierence being the sequence of Cu and Fe.

It is known that the metal concentration in milkcan vary due the factors inuencing its secretionfrom the mammary gland, such as breed of the ani-mal, season of the year, feeding and factors related

Table 3. Average mineral composition of raw cows milk from dierent countries

Country Ca (mg/kg) Mg (mg/kg) Fe (mg/kg) Cu (mg/kg) Zn (mg/kg) References

Italy 0.65 0.21 3.82 Del Petere, DiStanislao (1984)

Spain 1 251 48 116 3 0.46 0.10 0.16 0.02 3.70 0.22 Zuerera-Cosanoet al. (1994)

Pakistan 0.60 0.45 4.20 Bano et al.(1985)

Croatia 1 403.94 322.33 165.01 37.97 0.13 0.02 0.38 0.12 0.51 0.16 this paper

Figure 1. Composition of raw cows milk from dierent farms: a) protein and fat; b) Ca and Mg; c) Fe, Zn, Cuand Mn; d) Pb and Cd

I II III IV V VI VIIVIIIIX X XI XIIXIIIXIVXV0

1

2

3

4

5

6

(%)

Farm

Protein

Fat

I II III IV V VI VIIVIIIIX X XI XIIXIIIXIVXV0

500

1000

1500

2000

(mg/kg)

Farm

Ca

Mg

I II III IV V VI VIIVIIIIX X XI XIIXIIIXIVXV0.0

0.2

0.4

0.6

0.8

(mg/kg)

Farm

Fe

Zn

Cu

Mn

I II III IV V VI VIIVIIIIX X XI XIIXIIIXIVXV0

20

40

60

(g/kg)

Farm

Pb

Cd

c) d)

a) b)

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to animal handling by humans (Moreno-Rojas etal., 1993; Zurera-Cosano et al., 1994). In this studythe animals on the farms were of the same breed,samples were taken in a relatively short period, cows

were fed the same concentrate but dierent dry hay.erefore the statistically signicant dierences inthe content of essential minerals between the farmscan be ascribed to dierent dry hays. Lead and cad-mium are toxic minerals often associated with trac

pollution, therefore variations of their levels in milkfrom dierent farms are likely due to the locationof meadows in relation to roads. Tolerance limitsofthese two toxic metals according to Croatian legis-lation are: Pb < 100 g/l, Cd < 10 g/l (NarodneNovine, 1994). Lead and cadmium levels in milkfrom all investigated farms were below these limits.

In comparison with the average mineral composi-tion of raw milk obtained by investigations in Italy(Del Petre and Di Stanislao, 1984), Spain (Zuerera-Cosano et al., 1994) and Pakistan (Bano et al.,

1985), milk from Croatian farms has more Ca andMg, similar content of Cu and lower content of Fe

2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4

0.00

0.02

0.04

0.06

0.2

0.4

0.6

0.8

1.0Zn

Cu

Mn

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

0.0

0.2

0.4

0.6

0.8

1.0

1000

1500

2000

2500Ca

Fe

Zn

Figure 2. Correlations between: a) Zn, Cu, Mn and protein; b) Ca and fat; c) Ca, Fe, Zn and Cu; d) Fe, Zn, Cuand Mn in raw cows milk from dierent farms

1 2 3 4 5 6 7

0

500

1000

1500

2000

2500 Ca

0.02 0.03 0.04 0.05 0.06 0.07

0.0

0.2

0.4

0.6

0.8

1.0 Fe

Zn

Cu

and Zn (Table 3). However, it should be noted thatdeviations are much larger in our investigation.

Relatively low correlation coecients are in ac-cordance with previous studies (Rodrguez et al.,1999). is was explained by the relatively narrowrange of metal concentrations found in certaintypes of milk.

CONCLUSIONS

Contents of essential minerals (Ca, Mg, Fe, Zn.Cu, Mn) in milk from Croatian farms are on thenormal levels in comparison with the data fromother countries. e statistically signicant dif-ference in their contents in milk obtained fromdierent farms implies the necessity of control-ling mineral levels in milk by the dairy industry.Lead and cadmium levels in milk from all inves-tigated farms are below the limits as dened by

the Croatian legislation, indicating a possibility ofproducing organic products.

Protein (%) Fat (%)

Cu (mg/kg) Mn (mg/kg)

(m

g/kg)

(mg/kg)

(m

g/kg)

(mg/k

g)

a) b)

c) d)

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Original Paper Czech J. Anim. Sci., 48, 2003 (11): 481486

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studies in milk, milk products and eggs. J. Nat. Sci.Math.,25, 6774.

Boland M., MacGibbon A., Hill J. (2001): Designermilks for the newmillennium. Livest. Prod. Sci., 72,

99109.Del Petere V., Di Stanislao F. (1984): Trace elements in

cow milk sold in Marche Region. Nuovi Ann. Ig.Microbiol.,35, 313323.

Jorhem L. (1993): Determination of metals foodstusby atomic absorption spectrophotometry after dry

ashing: NMKL1 interlaboratory study of lead cad-mium, zinc, copper, iron, chromium, and nickel. J.

AOAC Inter., 76, 798813.

Levy Y., Zeharoa A., Grunebaum M., Nitzan M., Stein-herz R. (1985): Copper deciency in infants fed cowmilk. J. Pediatr., 106, 786788.

Moreno-Rojas R., Zurera-Cosano G., Amaro-Lpez M.(1993): Micronutrients in natural cow, ewe and goat

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cides, toxins, mycotoxins, metals and histamine andsimilar substances in food. 46, 1583.

Pennigton J.A.T., Wilson D.B., Young B., Johnson R.D.,

Vanderveen J.E. (1987): Mineral content of marketsamples of uid whole milk. J. Am. Diet. Assoc., 87,

10361042.Pennigton J.A.T., Schoen S.A, Salmon G.D., Young B.,

Johnson R.D., Marts R.W.J.E. (1995a): Compositionof core foods of the U.S. Food Supply, 19821991.II. Calcium, magnesium, iron and zinc. J. Food

Comp. Analysis, 8, 129169.Pennigton J.A.T., Schoen S.A., Salmon G.D., Young B.,

Johnson R.D., Marts R.W.J.E. (1995b): Compositionof core foods of the U.S. Food Supply, 19821991.

III. Copper, manganese, selenium and iodine. J. FoodComp. Analysis, 8,171217.

Rodrguez E.M., Sanz Alaejos M., Diaz Romero C.(1999): Chemometric studies of several minerals in

milks. J. Agric. Food. Chem., 47, 15201524.Steijns J.M. (2001): Milk ingredients as nutraceuticals.

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7578.

Received: 030121

Accepted after corrections:031015

ABSTRAKT

Stanoven obsahu kov v kravskm mlce pomoc plamenov atomov absorpn spektrofometrie

Obsahy zkladnch kov (Ca, Cu, Fe, Mg, Mn, Zn) a toxickch kov (Pb a Cd) v syrovm kravskm mlce bylystanoveny plamenovou atomovou absorpn spektrofotometri (FA-AAS). Vzorky mlka byly odebrny tyikrt

v beznu v roce 2000 na 15 farmch pobl Krie v zhebsk oblasti ze smsnho mlka pmo do plastovch lahv(aby se pedelo kontaminaci). Vzorky syrovho kravskho mlka byly podrobeny mineralizaci v mikrovlnnm

digesnm systmu MLS-1200 Mega pomoc technologie MDR; obsahy kov byly stanoveny pmo pomoc FA-AASv roztocch vzork, kter proly mineralizac. Ke statistickm analzm byl pouit statistick software SAS v. 8.0.

Obsahy zkladnch kov (Ca, Cu, Fe, Mg, Zn) v mlce z chorvatskch farem jsou ve srovnn s daji z ostatnch

zem na normln hladin. Hladina toxickch kov (Pb a Cd) byla ni ne povolen limity, kter denuj chor-vatsk pedpisy (Pb < 100 g/l a Cd < 10 g/l).

Klov slova: olovo; kadmium; m; elezo; mangan; hok; vpnk; syrov kravsk mlko; plamenov atomovabsorpn spektrofotometrie

Corresponding Author

Dr. Nataa Brajenovi, Ruer Bokovi Institute, P.O.Box 180, 10002 Zagreb, Croatia

Tel. +385 1 468 01 25, fax+385 1 468 02 45, e-mail: nbrajen@irb.hr