Use of Multivariate Optimization to Develop Methods for Direct Copper and Lead Determination in Breast Milk by Graphite Furnace Atomic Absorption Spectrometry Paulo C. P. Lara, Josianne N. Silveira, Waldomiro B. Neto, Mark A. Beinner & José B. B. da Silva Food Analytical Methods ISSN 1936-9751 Food Anal. Methods DOI 10.1007/s12161-013-9682-9
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Author's personal copy Food Anal. Methods DOI 10.1007/s12161-013-9682-9
Use of Multivariate Optimization to Develop Methods for Direct Copper and Lead Determination in Breast Milk by Graphite Furnace Atomic Absorption Spectrometry Paulo C. P. Lara & Josianne N. Silveira & Waldomiro B. Neto & Mark A. Beinner & José B. B. da Silva Received: 2 May 2013 / Accepted: 9 July 2013 # Springer Science+Business Media New York 2013
Abstract A direct method for lead and copper determination in breast milk by graphite furnace atomic absorption spectrometry, using aqueous calibration, was proposed in this study. Samples were diluted with hydroximethylaminomethane 80 %v/v, pH 8. The dilution determination for Pb and Cu was 1:1 and 1:9, respectively. Fractional factorial (24−1) and central composite designs were used to optimize experimental conditions (pyrolysis and atomization temperatures, pyrolysis time, and modifier) using 10 μL samples introduced into a graphite furnace. The methods allowed for copper and lead determination under optimized conditions with an aqueous calibration curve between 0 and 180 μg L−1 for Cu and 0 and 48 μg L−1 for Pb. The detection limits were 0.92 μg L−1 and 6.4 μg L−1 for Pb and Cu, respectively. Intra and interassay studies revealed coefficients of variation of 5.0 and 6.3 %, and 6.4 and 5.5 % for Pb and Cu, respectively. Recovery studies at three concentration levels (three consecutive days, n=7/day) presented results between 107 and 109 % for Pb and 102 and 103 % for Cu. Good accuracy was obtained for both metals through recoveries studies using certified reference P. C. P. Lara : J. B. B. da Silva (*) Department of Chemistry, Federal University of Minas Gerais—UFMG, Av. Antônio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil e-mail:
[email protected] J. N. Silveira Laboratory of Toxicology, Department of Clinical and Toxicological Analysis, UFMG, Av. Antônio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil W. B. Neto Institute of Chemistry, Federal University of Uberlândia—UFU, Av. Engenheiro Diniz, 1178, 38400-902 Uberlândia, MG, Brazil M. A. Beinner School of Nursing and Nutrition, UFMG, Av Prof. Alfredo Balena, 190, Santa Efigenia, 30130-100 Belo Horizonte, MG, Brazil
material (infant formula NIST® 1846). The method determined lead and copper in six samples and the concentrations ranged from 2.90 to 27.9 μg L−1 for Pb and 384 to 1,212 μg L−1 for Cu. While copper is an essential element, lead has no nutritional function and is cumulative at low concentrations. Therefore, safe, efficient, and validated methods should be available to determine its concentration in breast milk. Keywords Breast milk . Copper . Graphite furnace atomic absorption spectrometry . Lead . Multivariate optimization . TRIS
Introduction Lead is a non-essential element. Toxic effects resulting from its extensive use in silver smelting, paint production, jewelry making, ceramic glazes, building construction, and in drinking vessels and water supply systems have been known for many hundreds of years. In the last 150 years, the industrial use of lead has increased enormously, and since the mid1920s, the use of alkyl lead derivatives as anti-knock agents in petrol has resulted in a massive increase in distribution, only recently curbed by the introduction of “unleaded” petrol (Supra-Regional Assay Service: Centres for Analyzes and Clinical Interpretation 2008). Copper is now well established as an essential trace element. The estimated value for safe and adequate adult daily intake ranges from 1.5 to 3 mg/day (Cox 1999). Copper containing metalloenzymes are important for iron and catecholamine metabolism; hemoglobin, elastin and collagen synthesis; and free radical scavenging. About 30 % of the copper ingested by an individual is absorbed in the intestine, bound to albumin and transported to the liver, where it is stored (Cox 1999). The major circulating form of copper is ceruloplasmin, a blue glycoprotein synthesized in the liver containing six to eight
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copper atoms per molecule. The functions of this protein are still unclear; however, it is important for iron metabolism as a ferroxidase and it may also play a role in copper transport regulation. It is an acute phase reactant and its level can increase greatly as a response to infection, injury, chronic inflammatory conditions, or steroid hormones (pregnancy, certain contraceptive pills, and estrogen therapy). Excess copper is excreted in the bile with small amounts found in urine, unless renal damage is present or if substances which bind to copper are excreted. Copper deficiency leads to microcytic hypochromic anemia with marked neutropenia, which is resistant to iron therapy. Children and neonates whose diets are deficient in copper have ineffective collagen synthesis and may develop bone disease (Cox 1999). Milk and dairy products are important components of the human diet from childhood to adulthood. Breast milk is the main source of nutrition in an individual’s first years of life and it can be a source of contamination by lead and other metals if the mother is exposed to them (Mead 2008). One of the limitations of trace analysis in complex matrices is that sample digestion requires several steps, long preparation times, and may result in losses or contamination. Graphite furnace atomic absorption spectrometry (GF AAS) has been employed to analyze complex samples with little or no sample preparation (Amaro and Ferreira 2004; Silveira et al. 2007; Pinto et al. 2006; Bianchin et al. 2006) with the advantage of reducing analysis time, reagent costs, and avoiding contamination. De la Flor et al. (2004), demonstrated the potential of inductively coupled plasma mass spectrometry, using an octopole reaction cell to reduce polyatomic interferences while determining Cr, Mn, Fe, Co, Cu, Zn, Se, I, Al, Cd, and Pb in breast milk and infant formulas after digestion in a microwave oven. In another work, Leotsinidis et al. (2005) determined Cd, Pb, Cu, Zn, Mn, and Fe in colostrum and transitory breast milk from Greek women by flame atomic absorption spectrometry after acid autoclave digestion. Gill et al. (2003) developed a method to determine 18 elements (essential, toxic, and nonessential) in breast milk by radiochemical neutron activation analysis. In another study, Hallén et al. (1995) determined the lead and cadmium levels in blood and breast milk samples by GF AAS. All of the above-mentioned studies included a laborious sample preparation process, the most common of which being digestion in a microwave oven. Folomir et al. (1999) used univariate optimization and evaluated the precision exclusively by intra-assay studies for determination of lead in human milk samples. Furthermore, no tests with permanent modifiers were performed, which generally increases the lifetime of the graphite tube. Krusheva et al. (1996) introduced a new sample preparation procedure incorporating addition of tertiary amines after microwave digestion to dissolve fluorides and neutralize free fluorides; they verified the CFAC improved the analysis.
Quináia and Nóbrega (2000) determined chromium concentration in cow milk using CFAC (a mixture of tertiary amines) as a diluent. CFAC can also be applied to analyze metals in breast milk. However, due to the high costs associated with this reagent, analysis using it is less viable. An alternative is to use others amine reagents for replacing CFAC. In the present work, fractional factorial design followed by central composite design (CCD) was used to develop methods for lead and copper determination in breast milk by GF AAS. Milk samples were diluted 1:1 and 1:9 for lead and copper, respectively, using an 80 %v/v hydroximethylaminomethane (TRIS) solution adjusted for pH=8 with HNO3 (p.a.). Similarly to CFAC, TRIS is also an amine reagent.
Material and Experiment Instrumentation The integrated absorbances were obtained using an atomic absorption spectrometer SpectrAA Zeeman-220 from Varian (Victoria, Australia) equipped with a graphite furnace, an autosampler (PSD—31–972) and a polarized Zeeman background correction, all from Varian. Hollow cathode lamps (HCLs), also from Varian (Part number: 5610124800 for Pb), were used as a light source. Lead was determined at 10.0 mA with a spectral band-pass of 0.5 nm at 283.3 nm, while copper (HCL—PN-N305-0121) was determined at 15.0 mA with a spectral band-pass of 0.5 at 327.4 nm. Argon, 99.999 % from Air Products® (Contagem, MG, Brazil), was used as a purge gas. Pyrolytic graphite-coated tubes with L’Vov platform and without platform (Varian, part no. 63100023-00, 63-100024-00, and 63-100012-00) were used. Reagents and Solutions All solutions were prepared using deionized water in a Milli-Q system from Millipore (Bedford, MA, USA). Nitric acid 65 % Suprapur®, from Merck (Darmstadt, Germany), was used. The 80 %v/v TRIS (hydroximethylaminomethane) solution was prepared by appropriate dilution of the pure reagent from Vetec (Duque de Caxias, RJ, Brazil) adjusted at pH 8 with HNO3 (p.a.) and buffered with citric acid from Cinética Reagentes e Soluções (São Paulo, SP, Brazil). Calibration solutions were prepared from a stock lead and copper solution at a concentration of 1,000±0.002 mg L−1 (Titrisol—Merck) in 5 %v/v HNO3. One thousand milligrams per liter of a standard solution of tungsten, rhodium, ruthenium, iridium (Titrisol—Merck), titanium, niobium, and tantalum were used as modifiers. Plastic bottles, autosampler cups, and glassware materials were cleaned by immersion into 20 % (v/v) HNO3 for at least 1 day, rinsed several times with Milli-Q water, and dried. An autosampler washing solution containing 0.1 %
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(v/v) Triton X-100 (Merck) plus 0.2 % (v/v) nitric acid was used to avoid clogging the autosampler sampling capillary tip due to analyte adsorption, and also to improve sample solution dispersion onto the platform. Sampling and Sample Treatment Breast milk samples were obtained through a partnership with the Maternidade Odete Valadares Hospital (Belo Horizonte, MG, Brazil). Approximately 100 mL per sample were collected from 15 mothers, using a breast pump. The samples were collected and stored in hermetically sealed glass flasks and stored in a freezer at −10 °C. The samples were removed from the freezer to attain room temperature before being used. The only sample preparation was a 1:1 and 1:9 dilution with a TRIS solution for Pb and Cu, respectively. The resulting solution was an emulsion, stable for at least 20 min. One sample was randomly selected for all subsequent studies (reference sample). Certified reference material (infant formula NIST® 1846) was use to check the accuracy for both metals. Graphite Tube Treatment The L’Vov platform was treated with tungsten, rhodium, ruthenium, iridium, niobium, titanium, or tantalum as permanent modifiers. The treatment with 520 μg of each modifier was made by applying 40 μL of metal solution (1.0 g L−1) onto the tube platform and subjecting it 13 times to a furnace temperature program followed by thirteen 40 μL injections of MilliQ water for tube cleaning (Silva et al. 1999).
Methods Optimization Strategy of the Furnace Temperature Program The reference sample spiked with 27 μg L−1 of Pb was employed to optimize furnace conditions such as pyrolysis and atomization temperatures, pyrolysis time, and modifier for this Table 1 Fractional factorial design matrix (24−1) for Pb
The signals (+) and (−) indicate levels used in the factorial design
Fig. 1 Pareto graph and the estimated contrast of the optimized variables by fractional factorial design for lead. The horizontal line refers to a 95 % confidence level cutoff. All variables and/or interactions that were beyond the estimated effect’s horizontal line showed some significant effect during optimization
metal in breast milk. According to the manufacturer of the equipment used, 27 μg L−1 of Pb resulted in a 0.2 absorbance unit signal, which was a reasonable signal to make optimizations for lead in this study. There was no need to spike the copper sample because the content was sufficient to obtain a good integrated absorbance in the graphite tube. The total volume introduced into the graphite tube was 10 μL, together with 5 μL of chemical modifier in solution when necessary. For both metals, first, temperature and drying time were optimized, starting from the conditions recommended by the manufacturer and proceeding until no bubbling was observed in the sample inside the furnace. Afterwards, tungsten, rhodium, ruthenium, iridium, niobium, titanium, and tantalum were tested as permanent modifiers, as well as all of the modifiers with a co-injection of 5 μL of the rhodium solution (1 g L−1). This was performed using the temperature program recommended by the manufacturer in order to choose the two modifiers presenting the best performance. Next, a fractional factorial design (24−1)
Experiment
Pyrolysis temperature (°C)
Pyrolysis time (s)
Atomization temperature (°C)
Modifier (permanent+coinjection)
Integrated absorbance (s−1)
1 2 3 4
500 (−) 900 (+) 500 (−) 900 (+)
3 (−) 3 (−) 12 (+) 12 (+)
2,000 (−) 2,000 (−) 2,000 (−) 2,000 (−)
W+co-Rh (−) Ru+co-Rh (+) Ru+co-Rh (+) W+co-Rh (−)
0.0406 0.1120 0.0929 0.1056
5 6 7 8
500 (−) 900 (+) 500 (−) 900 (+)
3 (−) 3 (−) 12 (+) 12 (+)
2,500 (+) 2,500 (+) 2,500 (+) 2,500 (+)
Ru+co-Rh (+) W+co-Rh (−) W+co-Rh (−) Ru+co-Rh (+)
0.1811 0.1090 0.1624 0.1762
Author's personal copy Food Anal. Methods Table 2 Central composite design matrix (with pyrolysis time at 13 s and Ru+co-Rh as modifier) for Pb
Only the central point (CP) was made in five independent analysis for estimate of the model error CP central point (n=5)
Experiment
Pyrolysis temperature (°C)
Pyrolysis time (s)
Atomization temperature (°C)
Integrated absorbance (s−1)
1 2 3 4 5 6 7 8 9 10
700 700 700 700 1,100 1,100 1,100 1,100 560 1,240
8 8 16 16 8 8 16 16 12 12
2,300 2,700 2,300 2,700 2,300 2,700 2,300 2,700 2,500 2,500
0.1479 0.1421 0.1505 0.1413 0.1534 0.1435 0.1514 0.1546 0.1215 0.1395
11 12 13 14 15 (CP)
900 900 900 900 900
5 19 12 12 12
2,500 2,500 2,164 2,800 2,500
0.1525 0.1615 0.0824 0.1294 0.1713
was used to preliminarily evaluate variable pyrolysis and atomization temperatures, pyrolysis time, and modifier. Finally, a CCD was performed to determine the optimal conditions for pyrolysis and atomization temperatures. For all of the experiments, the cleaning temperature was maintained at 2,700 °C for 2 s. The experimental data was processed using STATISTICA 6.0 for Windows (2001).
Results and Discussion Optimization of the Furnace Temperature Program Tungsten and ruthenium modifiers, both with co-injection of rhodium, presented the highest absorbance, smallest relative
standard deviation, and corrected background for Pb in breast milk. Next, a fractional factorial planning (Table 1) was used to preliminarily evaluate the effect of the studied variables. The design revealed that all of the variables examined, as well as the interaction 1:2 and 1:3, produced some significant effects on the absorbance value at a 95 % confidence level, according to the results in the Pareto graph (Fig. 1). The Pareto graph indicated that the atomization and pyrolysis temperatures, time, and modifier variables resulted in a positive effect, with better results at a higher level, namely, 2,500 °C, 900 °C, 12 s, and permanent ruthenium plus coinjection of rhodium as a modifier. Based on the results of the factorial design, a CCD was designed to determine the optimal conditions for the variables pyrolysis and atomization temperatures and time for Pb, maintaining ruthenium plus
Fig. 2 Response surfaces generated by CCD for lead. The left surface represents the combination of pyrolysis temperature and pyrolysis time, while the right surface represents the combination between the pyrolysis and atomization temperatures
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rhodium as permanent modifier. Table 2 presents the matrix for experiments associated with this design. The surface response generated for Pb (Fig. 2) revealed that the optimum values for pyrolysis time and temperature, and atomization temperature were 17 s, 950 °C, and 2,520 °C, respectively. For copper, permanent Rh, and the condition without modifier, presented the same advantages as previously commented for Pb. A fractional factorial design (Table 3) was constructed to preliminary evaluate the effect of the studied variables. Evaluation of this planning indicated that all the studied variables, as well as all possible interactions between them, had a significant effect on the integrated absorbance values at a 95 % confidence level when TRIS was used as milk diluent. This is shown in the Pareto graphs in Fig. 3. The positive effect values indicate that increasing the variable values increases the integrated absorbance. The Pareto graphs also illustrate permanent Rh as the best modifier. Considering the results of the factorial design, a CCD was done to refine the optimized conditions for the variables pyrolysis and atomization temperatures, and pyrolysis time, using the same modifiers determined by the factorial planning. The matrices for this design are shown in Table 4. Analysis of the CCD indicated that, at the studied levels, only atomization temperature was significantly positive. This suggests that higher values for this variable provide better results. The optimized values for each of the variables were chosen according to the Pareto graph for this design (Fig. 3). The lowest values were selected for the significantly negative variables: 800 °C and 6.0 seconds for pyrolysis temperature and pyrolysis time, respectively. As far as the atomization temperature was concerned, the tendency, indicated by the surface (Fig. 4—high values), was followed, and a temperature of 2,700 °C was used in the proposed method. Analytical Parameters of Merit Three curves, using matrix matching calibration (in breast milk) and three aqueous curves, were obtained within a range Table 3 Matrix for factorial fractional planning (24−1) for preliminary evaluation of the optimized variables for Cu
(+) and (−) represent the high and low level evaluated for each variable
Fig. 3 Pareto graph and the estimated contrast of variables optimized by the planning for copper using TRIS as diluents. The horizontal line refers to a 95 % confidence level cutoff. All variables and/or interactions that were beyond the estimated effect’s horizontal line showed some significant effect during optimization
of 0 to 48 μg L−1 for Pb using their optimized temperatures and times. For Cu, the same curves were made in the range of 0 to 180 μg L−1. The range of the calibration curves were thus chosen for covering the entire linear range by the equipment, according to the manufacturer. The aqueous calibration curves were made by appropriate dilution of the lead and copper standard stock solution (1,000 μg L−1) in deionized water, keeping the final solution at 3 %v/v HNO3. The matrix effect was evaluated by comparing the averages of the angular and linear coefficients of these curves using statistical tools (F and Student’s t tests). These tests revealed that the curves did not present statistically significant differences at a 95 % confidence level, which indicates the absence of matrix effect for Pb. Therefore, all of the subsequent studies were conducted using aqueous calibration curves for Pb. For copper, the tests revealed that the curves present statistically significant differences at a 95 % confidence level, which indicates the presence of matrix effect. Therefore, all of the
Experiment
Pyrolysis temperature (°C)
Pyrolysis time (s)
Atomization temperature (°C)
Modifier
Integrated absorbance (s−1)
1
800 (−)
7 (−)
2,000 (−)
0.0850
2 3 4
1,500 (+) 800 (−) 1,500 (+)
7 (−) 16 (+) 7 (+)
2,000 (−) 2,000 (−) 2,000 (−)
5 6
800 (−) 1,500 (+)
7 (−) 7 (−)
2,500 (+) 2,500 (+)
7
800 (−)
16 (+)
2,500 (+)
8
1,500 (+)
16 (+)
2,500 (+)
Without (−) Rh (+) Rh (+) Without (−) Rh (+) Without (−) Without (−) Rh (+)
0.1008 0.0645 0.0636 0.2149 0.1576 0.1750 0.1910
Author's personal copy Food Anal. Methods Table 4 Central composite design matrix for each of the diluents for Cu
The central point (CP) was made in five independent analyzes to estimate the model error CP central point (n=5)
Experiment
Pyrolysis temperature (°C)
Pyrolysis time (s)
Atomization temperature (°C)
Integrated absorbance (s)
1 2 3 4 5 6 7 8 9 10
600 600 600 600 1,000 1,000 1,000 1,000 460 1,140
4.0 4.0 8.0 8.0 4.0 4.0 8.0 8.0 6.0 6.0
2,300 2,700 2,300 2,700 2,300 2,700 2,300 2,700 2,500 2,500
0.1060 0.2770 0.3774 0.4108 0.3450 0.4024 0.1556 0.4088 0.3954 0.3622
11 12 13 14 15 (CP)
800 800 800 800 800
3.0 9.0 6.0 6.0 6.0
2,500 2,500 2,160 2,800 2,500
0.4594 0.3766 0.0558 0.4228 0.3530
subsequent studies were carried out using matrix matching calibration curves. Table 5 presents the findings for the main parameters of merit for the proposed methods. The limits of detection and quantification of the method were defined by the equations LOD=3S/b and LOQ=10S/b, where S is the standard deviation of ten independent samples of the lowest point on the matrix matching calibration curve and b is the slope of the curve. LOD and LOQ were 0.92 and 3.07 μg L−1 and 6.4 and 21.4 μg L−1 for Pb and Cu, respectively. Turan et al. (2001), determining Pb, Cu, and other metals in human colostrum samples, obtained a LOD of 0.92 μg L−1 for Pb and 0.71 μg L−1 for Cu. Precision was evaluated through intra- and inter-assay
studies. The intra-assay coefficient of variation (CV) was calculated using the standard deviation of the reference sample spiked with 8, 24, and 40 μg L−1 of Pb and with 30, 90, and 150 μg L−1 of Cu divided by the average of the respective concentrations, and then multiplied by 100. Such determinations were performed separately in seven replicates on the same day and read in triplicate. To calculate the inter-assay coefficient of variation, the same procedure was repeated with seven replicates of the sample and readings in triplicates on three different days. The average CV for the intra- and inter-assay precision was 5.0±1.0 % and 6.3±0.3 % and 6.4±0.8 % and 5.5±4.7 % for Pb and Cu, respectively. The characteristic mass (1 %
Fig. 4 Response surfaces generated by CCD for copper. The left surface represents the combination of pyrolysis temperature and pyrolysis time, while the right surface represents the combination between the pyrolysis and atomization temperatures
Author's personal copy Food Anal. Methods Table 5 Parameters of merit for the proposed methods
Regression equation (n=7) R2 (n=3) Linear range (μg L−1) Intra-assay precision (%, n=7) Inter-assay precision (%, n=21) Recovery (%, n=21) Certified sample (μg L−1, n=3)a LOD (μg L−1) LOQ (μg L−1) Characteristic mass (pg)c (n=7)
Pb
Cu
Abs (absorbance)=(0.0061±0.0006)CPb +(0.002±0.001) 0.999±0.005 0–48 4.0–6.0 6.0–6.6 107–109 267±4b (average recovery of 112.4 %) 0.92 3.07 7.1±0.3
Abs=(0.0027±0.0002)CCu +(−0.02±0.01) 0.996±0.007 0–180 5.6–7.2 0.1–9.0 102–103 5.21±0.20b (average recovery of 104.2 %) 6.4 21.4 19±2
a
Certified 202 to 273 μg L−1 for Pb and 4.97 to 5.51 for Cu
b
95 % confidence level
c
Recommended values of 5.5 pg f or Pb and 20.0 pg for Cu
absorption) for Pb was 7.1±0.3 pg and 19±2 pg for Cu, calculated using the data from the calibration curve (recommended mass of 5.5 pg for Pb and 20.0 pg for Cu). Accuracy was evaluated for Pb and Cu using certified reference material (CRM-infant formula NIST® 1846), and by recovery studies with breast milk samples spiked with 8, 24, and 40 μg L−1 of Pb and 30, 90, and 150 μg L−1 of Cu. Recoveries showed results ranging from 107 to 109 % and 102 to 103 % for Pb and Cu, respectively. The results obtained for lead and copper in the CRM are in agreement with the certified values for both metals. Determination of Lead and Copper in Breast Milk Lead and copper concentrations were determined in six breast milk samples using the optimized experimental conditions and aqueous calibration curves or matrix matching calibration, respectively, for metals. An 80 %v/v TRIS solution, diluted at 1:1 with deionized water, was used as blank. The Pb and Cu concentration in the blank solution were less than the Table 6 Lead and copper concentrations in six breast milk samples (value ± standard deviation, n=3) Sample
1 2 3 4 5 6
Value (μg L−1) Pb
Cu
5 μg L−1) (1995). Turan et al. (2001) obtained lead and copper in human colostrum samples that ranged between 8.8 and 35.4 μg L−1 and 179 and 454 μg L−1, respectively. Goudarzi et al. (2012) reported mercury, cadmium, and lead concentrations in breast milk of healthy lactating women living in Isfahan, Iran. For lead, the obtained values ranged between 3.06 and 19.47 μg L−1. In another study (Isaac et al. 2012), 25 healthy breast-feeding mothers in the Ranipet Industrial area of the Vellore District of Tamil, Nadu, and from 25 lactating mothers in the non-industrial areas of the same district, had their breast milk analyzed. The levels of Pb ranged from 5 to 25 μg L−1 in the non-industrial area, whereas samples from mothers in the industrial area showed higher lead levels ranging between 15 and 44.5 μg L−1. Orun et al. (2011) investigated breast milk lead and cadmium levels from suburban areas of Ankara by inductively coupled plasma mass spectrometry. They obtained a Pb median breast milk concentration of 20.59 μg L−1. Gundacker et al. (2002), analyzing breast milk provided by healthy mothers in Vienna (urban; n=59), an industrial area in Linz (n=47), and a rural area in Tulln (n=59), obtained low concentrations of Hg and Pb (Pb, 1.63±1.66 μg L−1). Ursinyova and Masanova (2005) investigated Cd, Pb, and Hg levels in the breast milk of 158 lactating healthy women who were not occupationally exposed and living in eight environmentally different polluted locations of the Slovak Republic. The average concentration of Pb in the breast milk samples, taken on the fourth day postpartum, was 0.94 μg Kg−1
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Conclusion The chemometric methods employed allowed for the optimization of the analytical conditions for lead and copper determinations by GF AAS using TRIS as diluent and performing only a small number of experiments. The multivariate optimization was a simple and fast method to achieve the best values for the evaluated parameters. For Pb, the values obtained for the optimal conditions were 17 s, 950 °C, and 2520 °C for pyrolysis time and pyrolysis and atomization temperatures, respectively, using ruthenium with co-injection of rhodium as modifier. The aqueous calibration presents sufficient linear correlation among the data. For Cu, the optimized conditions were 800 °C and 6.0 s for temperature and pyrolysis time, and 2,700 °C for the atomization temperature, using rhodium as modifier. The parameters of merit presented in the proposed methods, such as limits of detection and quantification, linearity, intra- and inter-assay precision, recovery, accuracy, and characteristic mass, were appropriate for this purpose. Simple sample preparation, as well as the possibility of using aqueous calibration for Pb and matrix matching calibration for Cu makes these methods fast, simple, and accurate. Acknowledgments The authors wish to thank the Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for their financial support. We are grateful to Maternidade Odete Valadares (Belo Horizonte—MG, Brazil) for providing the maternal milk breast samples. J.B.B. Silva is scholarship recipient of CNPq. Conflict of Interest The authors declare that there were no conflicts of interests before, during, and after the execution of the study. Paulo CP Lara has no conflict of interest. Josianne N Silveira has no conflict of interest. Waldomiro B Neto has no conflict of interest. Mark A Beinner has no conflict of interest. José BB da Silva has no conflict of interest. Finally, the authors also declare that all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (FHEMIG—Fundação Hospitalar do Estado de Minas Gerais, Brazil) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all subjects to use their breast milk samples included in this study.
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