Determination of metals in cow s milk by flame atomic absorption by benbenzhou

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Determination of metals in cow s milk by flame atomic absorption

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									Czech J. Anim. Sci., 48, 2003 (11): 481–486                                                       Original Paper




Determination of metals in cow’s milk by flame
atomic absorption spectrophotometry
M. SIKIRIĆ1, N. BRAJENOVIĆ1, I. PAVLOVIĆ2, J. L. HAVRANEK2, N. PLAVLJANIĆ2
1
    Ruđer Bošković Institute, Zagreb, Croatia
2
    Faculty 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 flame
atomic absorption spectrophotometry (FA-AAS) in raw cow’s milk. Milk was sampled four times in March 2000
on fifteen farms near Križ in the Zagreb region from bulk milk directly into plastic bottles (to avoid contamina-
tion). e raw cow’s milk samples were digested in an MLS-1200 Mega Microwave Digestion System with MDR
Technology, and the metals were determined directly by FA-AAS in the solutions of digested samples. Statistical
analyses 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. e
level of toxic metals (Pb and Cd) was lower than the tolerance level defined by Croatian regulations (Pb < 100 µg/l
and Cd < 10 µg/l).

Keywords: lead; cadmium; copper; iron; manganese; magnesium; calcium; raw cow’s milk; flame atomic absorption
spectrophotometry




  Increased awareness of the influence of diets on         1995a,b). erefore the essential metals are inter-
human health has prompted producers to produce             esting for determination of their adequate daily
food of higher quality, rich in nutrients and nu-          intake by the organism. On the other hand, due
traceuticals, in environmentally friendly manner.          to the growing environmental pollution it is also
On the other hand, raw materials for food pro-             necessary to determine and monitor the levels of
duction are getting poor in essential minerals and         toxic metals in milk, such as lead and cadmium,
vitamins at the same time.                                 because they can significantly influence the human
  Milk and dairy products have been recognized             and animal health (Steijns, 2001).
all over the world for a long time as good for their         e most frequently used technique to deter-
sensory properties, but also for their beneficial in-      mine metals in food and biological materials is
fluence on human health (Steijns, 2001). On the            atomic absorption spectrophotometry (AAS). If
other hand, milk is an expensive raw material pro-         the concentrations of metals are high enough, AAS
duced by animals (Boland et al., 2001). To produce         determinations are usually done by flame atomic
milk or to make its products of good quality and to        absorption spectrophotometry (FA-AAS). When
reduce their price, a good raw material is needed.         the concentrations of metals are low, electrothermal
erefore constant control of raw milk bought               atomic absorption spectrophotometry (EA-AAS) is
from farms is necessary. Milk should be controlled         applied. Both techniques should give the same
not only for fat and proteins as it is usual, but also     results if the concentration of metals is above the
for mineral content whenever possible.                     detection limit of FA-AAS (Jorhem, 1993).
  Milk is known as an excellent source of Ca, and            Because of low industrial production Croatia has
it can supply moderate amounts of Mg, smaller              a unique opportunity to produce milk rich in nu-
amounts of Zn and very small amounts of Fe                 tritional elements and with very low levels of toxic
and Cu (Levy et al., 1985; Pennigton et al., 1987,         substances at the same time. In order to determine

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Original Paper                                                 Czech J. Anim. Sci., 48, 2003 (11): 481–486


the quality of raw material bought by the Croatian        Instrumentation. PU 9100X FA-AAS equipped
dairy industry one-month research was conducted         with Epson FX-850 printer was used. e instru-
on 15 family farms in the region of Ivanic grad, the    ment was controlled by Philips P 3120 PC. e air/
region of biggest milk producers in Croatia. Our        acetylene flame was used for the analysis of metals.
attention was focused on the mineral composition        Instrumental conditions for the determinations of
of milk.                                                metals in raw cow’s milk are given in Table 1.
                                                          Statistics. Data were analysed by General Linear
                                                        Models procedure. Mean differences were sepa-
MATERIAL AND METHODS                                    rated by Duncan’s Multiple Range Test. e level
                                                        of significance was set at P < 0.05. All analyses were
   Sampling strategy. Samples of raw cow’s milk         performed using statistical software SAS v. 8.0.
were collected four times in March 2000 from
fifteen farms located near Križ in the Zagreb re-
gion. e cows on each farm were fed dry hay and         RESULTS
mineral premixes and drank water from the pump.
Raw milk was sampled from the bulk milk of the            Average mineral concentration of raw cow’s milk is
farms directly into carefully washed plastic bottles    shown in Figure 1. e results of ANOVA are sum-
because of the relatively great possibility of sample   marized in Table 2. Significant correlations between
contamination, especially by lead and cadmium.          the investigated parameters are shown in Figure 2.
   Precautions against contamination. All chemi-
cals were of the highest available purity. Deionised
water was used. e laboratory ware was cleaned          Fat and protein
with chromosulphuric acid and rinsed with deion-
ised water. e laboratory ware was dipped in              In the period of investigations fat content in raw
10% HNO3 for 24 h, rinsed with deionised water,         cow’s milk from 15 different farms was significantly
dipped in 3% EDTA solution for 24 h and rinsed          different, ranging from 2.55 to 4.62%. Similar re-
with deionised water again.                             sults were obtained for protein content that ranged
   Anakytical procedure. e fat and protein con-        from 2.81 to 4.1%. e correlation between fat and
tent (%) was determined on Bently 150 Infrared          protein content was not statistically significant.
milk analyser.
   To determine the concentrations of metals
chemicals of the highest available purity were          Calcium and magnesium
used. HNO3 (Riedel) was used for the dilution
and mineralization of raw milk samples, H 2O2             Differences between farms were significant
(9.791 mol/dm3, Riedel) was used for digestion          for calcium content, ranging from 1 125.76 to
of all milk samples. Lead nitrate (3.019 × 10 –3        2 019.04 mg/kg, but not for magnesium content
mol/dm3, Merck) and cadmium nitrate (4.230              136.02–196.67 mg/kg. Calcium content correlated
× 10–3 mol/dm3, Merck) solutions were used as           with fat content (R = 0.647).
calibration standards. All solutions were prepared
with deionised water.
   e metals (calcium, copper, iron, magnesium,         Iron, zinc, copper and manganese
manganese, zinc, lead and cadmium) were ana-
lysed by FA-AAS in a total of 60 milk samples. To         Iron, zinc, copper and manganese contents in
eliminate the organic part of milk, 0.500 g of raw      milk from different farms were statistically dif-
cow’s milk was treated with 6 ml of 65% HNO3            ferent. Relatively high coefficients of correlation
and 1 ml 30% H2O2, mixed and digested in an             of manganese and copper with the other three
MLS-1200 Mega Microwave Digestion System                minerals were obtained (Mn – Fe: R = 0.641, Mn
with MDR Technology. Lead and cadmium were              – Zn: R = 0.731, Mn – Cu: R = 0.618, Cu – Fe:
determined with ammonium salts pyrrolidine-1-           R = 0.613, Cu – Zn: R = 0.629) while the contents
dithiocarboxylic acid (APDC) into methyl-isobutyl       of iron and zinc were not so highly correlated. In
ketone by FA-AAS. e other investigated metals          addition, correlations existed between copper and
were determined directly by FA-AAS.                     calcium content (R = 0.629) as well as between

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      Table 1. Instrumental conditions for lead, cadmium, calcium, copper, iron, manganese, magnesium and zinc determination in raw cow’s milk by FA-AAS

                                                Lead            Cadmium            Calcium             Copper            Iron            Manganese      Magnesium     Zinc
      Primary wavelength (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
      Injection volume (ml)                      1                  1                 1                   1                 1                1              1          1
      Flame type                                                                                             air/acetylene
      Fuel flow rate (l/min)                   0.9–1.2          1.0–1.3            0.9–1.2             0.8–1.1          0.8–1.0           0.9–1.2        0.9–1.2     0.9–1.2
      Sensitivity (mg/l)                         0.10            0.032               0.09               0.041            0.060             0.029          0.003       0.013


      Table 2. ANOVA summary of the farm effect on mineral levels in raw cow’s milk. Means with the same letter are not significantly different

                 Protein            Fat                    Ca              Mg                 Fe                Zn              Cu             Mn            Pb         Cd
                                                                                                                                                                                 Czech J. Anim. Sci., 48, 2003 (11): 481–486




                                                                                                       (%)
      P          <0.0001          <0.0001                0.0011           0.5022            0.0050           <0.0001       <0.0001           0.0069        0.0007    <0.0001
      Farm                  (%)                                                         (mg/kg)                                                            (µg/kg)
                      a                   ab                      a                                a                 a               a              a
          I       2.81             3.15                  1 151.62         141.53            0.10              0.31          0.20             0.026         23.06a      3.02a
         II       3.97e            5.90c                 2 019.04b        196.67            0.14b             0.83b         0.69d            0.055b        70.56b      5.47bcd
       III        3.22abc          4.23b                 1 463.57ab       162.34            0.12ab            0.49a         0.40bc           0.043ab       41.36ab     6.09cd
        IV        3.32bc           3.81ab                1 314.40a        161.21            0.13ab            0.41a         0.32a            0.035ab       29.61ab     6.47d
         V        3.23abc          4.08b                 1 384.77ab       157.59            0.14b             0.54a         0.41bc           0.040ab       40.36ab     5.65bcd
        VI        3.86de           4.62bc                1 588.46ab       180.52            0.13ab            0.64b         0.39bc           0.048ab       43.50ab     5.65bcd
       VII        3.23abc          3.69ab                1 401.71ab       159.35            0.13ab            0.50a         0.43bc           0.040ab       37.53ab     4.06ab
      VIII        3.17abc          3.61ab                1 094.49a        153.61            0.12ab            0.47a         0.37bc           0.040ab       47.22ab     6.12cd
        IX        3.41cde          4.45b                 1 478.34ab       196.18            0.13ab            0.48a         0.41bc           0.044ab       58.59ab     5.63bcd
         X        3.06abc          4.01b                 1 309.12a        159.70            0.12ab            0.39a         0.34bc           0.034ab       32.51ab     5.80cd
        XI        2.97ab           3.89abc               1 279.91a        150.44            0.12ab            0.38a         0.31a            0.033ab       39.98ab     5.46bcd
       XII        3.14abc          3.54ab                1 364.10ab       162.88            0.12ab            0.52a         0.33bc           0.039ab       36.32a      5.16bcd
      XIII        3.45bcd          3.79ab                1 389.10ab       163.55            0.13ab            0.63b         0.31a            0.043ab       36.08a      4.49abc
      XIV         4.10e            4.38ab                1 694.72ab       193.60            0.16b             0.63b         0.48c            0.052b        56.43b      4.91bcd
       XV         3.50bcd          2.55a                 1 125.76a        136.02            0.13ab            0.49a         0.32a            0.039ab       38.58ab     5.67bcd




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                                                                                                                                                                                 Original Paper
Original Paper                                                                   Czech J. Anim. Sci., 48, 2003 (11): 481–486


 a)                                                    Protein          b)                                                     Ca
         (%)                                           Fat                   (mg/kg)                                           Mg
          6                                                                    2000
           5
                                                                               1500
           4
           3                                                                   1000
           2
                                                                                500
           1
           0                                                                      0
                I II III IV V VI VIIVIIIIX X XI XIIXIII IVXV
                                                      X                                                                       X
                                                                                        I II III IV V VI VIIVIIIIX X XI XIIXIII IVXV
                                  Farm                                                                    Farm


 c)                                                                     d)
                                                       Fe                                                                         Pb
      (mg/kg)                                          Zn                    (µg/kg)                                              Cd
         0.8                                           Cu
                                                       Mn                        60
         0.6
                                                                                 40
         0.4

         0.2                                                                     20


         0.0                                                                      0
                                                      X
                I II III IV V VI VIIVIIIIX X XI XIIXIII IVXV                                                                  X
                                                                                        I II III IV V VI VIIVIIIIX X XI XIIXIII IVXV
                                  Farm                                                                    Farm

Figure 1. Composition of raw cow’s milk from different farms: a) protein and fat; b) Ca and Mg; c) Fe, Zn, Cu
and Mn; d) Pb and Cd


protein content and zinc (R = 0.669), copper (R =                       DISSCUSION
0.656) and manganese (R = 0.630).
                                                                          e concentrations of different essential minerals
                                                                        in milk from 15 farms included in this investiga-
Lead and cadmium                                                        tion can be arranged in the sequence Ca > Mg >
                                                                        Zn > Cu > Fe > Mn. is sequence is similar to
  Both lead and cadmium contents in milk from                           that obtained by Rodríguez et al. (1999), the only
different farms were statistically different. No cor-                   difference being the sequence of Cu and Fe.
relation was obtained between these minerals and                          It is known that the metal concentration in milk
other investigated parameters.                                          can vary due the factors influencing its secretion
                                                                        from 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 cow’s milk from different 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, Di
                                                                                                                     Stanislao (1984)
Spain             1 251 ± 48              116 ± 3           0.46 ± 0.10         0.16 ± 0.02        3.70 ± 0.22       Zuerera-Cosano
                                                                                                                     et 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

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Czech J. Anim. Sci., 48, 2003 (11): 481–486                                                                                                 Original Paper


   a)                                                                  b)
               1.0        Zn                                                       2500                Ca
                          Cu
               0.8        Mn                                                       2000
               0.6




                                                                       mg(mg/kg)
                                                                                   1500
  (mg/kg)




                                                                          kg-1
    mg kg-1




               0.4
                                                                                   1000
               0.2
              0.06                                                                  500
              0.04
              0.02                                                                     0
              0.00
                  2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4                                   1          2          3          4          5          6          7

                                Protein (%)                                                                           Fat (%)

  c)                                                                    d)
                          Ca
              2500                                                                                     Fe
                          Fe                                                          1.0
              2000        Zn                                                                           Zn
                                                                                      0.8              Cu
              1500
 mg (mg/kg)




                                                                         (mg/kg)
              1000
                                                                                      0.6
    kg-1




                                                                            mg kg-1
               1.0
               0.8                                                                    0.4
               0.6
               0.4                                                                    0.2
               0.2
               0.0                                                                    0.0
                  0.1   0.2    0.3   0.4    0.5   0.6   0.7   0.8                               0.02       0.03       0.04       0.05       0.06       0.07
                               Cu (mg/kg)                                                                         Mn (mg/kg)



Figure 2. Correlations between: a) Zn, Cu, Mn and protein; b) Ca and fat; c) Ca, Fe, Zn and Cu; d) Fe, Zn, Cu
and Mn in raw cow’s milk from different farms



to animal handling by humans (Moreno-Rojas et                       and Zn (Table 3). However, it should be noted that
al., 1993; Zurera-Cosano et al., 1994). In this study               deviations are much larger in our investigation.
the animals on the farms were of the same breed,                      Relatively low correlation coefficients are in ac-
samples were taken in a relatively short period, cows               cordance with previous studies (Rodríguez et al.,
were fed the same concentrate but different dry hay.                1999). is was explained by the relatively narrow
erefore the statistically significant differences in               range of metal concentrations found in certain
the content of essential minerals between the farms                 types of milk.
can be ascribed to different dry hays. Lead and cad-
mium are toxic minerals often associated with traffic
pollution, therefore variations of their levels in milk             CONCLUSIONS
from different farms are likely due to the location
of meadows in relation to roads. Tolerance limits of                  Contents of essential minerals (Ca, Mg, Fe, Zn.
these two toxic metals according to Croatian legis-                 Cu, Mn) in milk from Croatian farms are on the
lation are: Pb < 100 µg/l, Cd < 10 µg/l (Narodne                    normal levels in comparison with the data from
Novine, 1994). Lead and cadmium levels in milk                      other countries. e statistically significant dif-
from all investigated farms were below these limits.                ference in their contents in milk obtained from
  In comparison with the average mineral composi-                   different farms implies the necessity of control-
tion of raw milk obtained by investigations in Italy                ling mineral levels in milk by the dairy industry.
(Del Petre and Di Stanislao, 1984), Spain (Zuerera-                 Lead and cadmium levels in milk from all inves-
Cosano et al., 1994) and Pakistan (Bano et al.,                     tigated farms are below the limits as defined by
1985), milk from Croatian farms has more Ca and                     the Croatian legislation, indicating a possibility of
Mg, similar content of Cu and lower content of Fe                   producing “organic” products.

                                                                                                                                                                  485
Original Paper                                                     Czech J. Anim. Sci., 48, 2003 (11): 481–486


REFERENCES                                                 Pennigton J.A.T., Wilson D.B., Young B., Johnson R.D.,
                                                             Vanderveen J.E. (1987): Mineral content of market
Bano N., Naeem M., Khan H.H. (1985): Trace metal             samples of fluid whole milk. J. Am. Diet. Assoc., 87,
  studies in milk, milk products and eggs. J. Nat. Sci.      1036–1042.
  Math., 25, 67–74.                                        Pennigton J.A.T., Schoen S.A, Salmon G.D., Young B.,
Boland M., MacGibbon A., Hill J. (2001): Designer            Johnson R.D., Marts R.W.J.E. (1995a): Composition
  milks for the new millennium. Livest. Prod. Sci., 72,      of core foods of the U.S. Food Supply, 1982–1991.
  99–109.                                                    II. Calcium, magnesium, iron and zinc. J. Food
Del Petere V., Di Stanislao F. (1984): Trace elements in     Comp. Analysis, 8, 129–169.
  cow milk sold in Marche Region. Nuovi Ann. Ig.           Pennigton J.A.T., Schoen S.A., Salmon G.D., Young B.,
  Microbiol., 35, 313–323.                                   Johnson R.D., Marts R.W.J.E. (1995b): Composition
Jorhem L. (1993): Determination of metals foodstuffs         of core foods of the U.S. Food Supply, 1982–1991.
  by atomic absorption spectrophotometry after dry           III. Copper, manganese, selenium and iodine. J. Food
  ashing: NMKL1 interlaboratory study of lead cad-           Comp. Analysis, 8, 171–217.
  mium, zinc, copper, iron, chromium, and nickel. J.       Rodríguez E.M., Sanz Alaejos M., Diaz Romero C.
  AOAC Inter., 76, 798–813.                                  (1999): Chemometric studies of several minerals in
Levy Y., Zeharoa A., Grunebaum M., Nitzan M., Stein-         milks. J. Agric. Food. Chem., 47, 1520–1524.
  herz R. (1985): Copper deficiency in infants fed cow     Steijns J.M. (2001): Milk ingredients as nutraceuticals.
  milk. J. Pediatr., 106, 786–788.                           Int. J. Dairy Technol., 54, 81.
Moreno-Rojas R., Zurera-Cosano G., Amaro-López M.          Zurera-Cosano G., Moreno-Rojas R., Amaro-López M.
  (1993): Micronutrients in natural cow, ewe and goat        (1994): Effect of processing on contents and relation-
  milk. Int. J. Food Sci. Nutr., 44, 37–46.                  ships of mineral elements of milk. Food Chem., 51,
Narodne Novine (1994): Regulation of allowed pesti-          75–78.
  cides, toxins, mycotoxins, metals and histamine and                                          Received: 03–01–21
  similar substances in food. 46, 1583.                                        Accepted after corrections:03–10–15


ABSTRAKT

Stanovení obsahu kovů v kravském mléce pomocí plamenové atomové absorpční spektrofometrie

Obsahy základních kovů (Ca, Cu, Fe, Mg, Mn, Zn) a toxických kovů (Pb a Cd) v syrovém kravském mléce byly
stanoveny plamenovou atomovou absorpční spektrofotometrií (FA-AAS). Vzorky mléka byly odebrány čtyřikrát
v březnu v roce 2000 na 15 farmách poblíž Križe v záhřebské oblasti ze směsného mléka přímo do plastových lahví
(aby se předešlo kontaminaci). Vzorky syrového kravského mléka byly podrobeny mineralizaci v mikrovlnném
digesčním systému MLS-1200 Mega pomocí technologie MDR; obsahy kovů byly stanoveny přímo pomocí FA-AAS
v roztocích vzorků, které prošly mineralizací. Ke statistickým analýzám byl použit statistický software SAS v. 8.0.
Obsahy základních kovů (Ca, Cu, Fe, Mg, Zn) v mléce z chorvatských farem jsou ve srovnání s údaji z ostatních
zemí na normální hladině. Hladina toxických kovů (Pb a Cd) byla nižší než povolené limity, které definují chor-
vatské předpisy (Pb < 100 µg/l a Cd < 10 µg/l).

Klíčová slova: olovo; kadmium; měď; železo; mangan; hořčík; vápník; syrové kravské mléko; plamenová atomová
absorpční spektrofotometrie




Corresponding Author

Dr. Nataša Brajenović, Ruđer Bošković 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

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