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Determination of Co and Cr in wet animal feeds using direct solid sample analysis by HR-CS GF AAS
Microchemical Journal 133 (2017) 524–529
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Determination of Co and Cr in wet animal feeds using direct solid sample analysis by HR-CS GF AAS Dayara Virgínia Lino Ávila a,b, Aline Rocha Borges c, Maria Goreti Rodrigues Vale d,e, Rennan Geovanny Oliveira Araujo a,b,e,⁎, Elisangela Andrade Passos a a
Departamento de Química, Laboratório de Química Analítica Ambiental (LQA), Centro de Ciências Exatas e Tecnologia da Universidade Federal de Sergipe (UFS), 49.100-000 São Cristóvão, SE, Brazil Departamento de Química Analítica, Instituto de Química, Universidade Federal da Bahia (UFBA), 40.170-115 Salvador, BA, Brazil c Instituto Federal do Paraná, Campus Palmas, 85.555-000 Palmas, PR, Brazil d Instituto de Química, Universidade Federal do Rio Grande do Sul (UFGRS), Av. Bento Gonçalves 9500, 91.501-790 Porto Alegre, RS, Brazil e Instituto Nacional de Ciência e Tecnologia do CNPq-INCT de Energia e Ambiente, Universidade Federal da Bahia, Salvador, BA, Brazil b
a r t i c l e
i n f o
Article history: Received 14 November 2016 Received in revised form 5 March 2017 Accepted 18 April 2017 Available online 20 April 2017 Keywords: Wet animal feed Chromium Cobalt Direct solid sample analysis HR-CS GF AAS
a b s t r a c t The present study proposes the determination of Cr and Co in wet animal feeds for employing direct solid sample analysis (SS) and high resolution continuum source graphite furnace atomic absorption spectrometry (HR-CS GF AAS). For determination of Cr and Co, the analytical lines 357.8687 nm and 240.7254 nm were used, respectively. The pyrolysis temperatures were 1500 and 1400 °C and atomization temperatures were 2500 and 2400 °C for Cr and Co, respectively. The limits of quantification (LoQ) obtained for Cr was 0.03 μg g−1 and 0.17 μg g−1 for Co. The characteristic masses (m0) also obtained were 2.2 pg for Cr and 2.9 pg for Co. The accuracy of the proposed methods was performed by analysis of standard reference materials (SRM) of tomato leaves (NIST 1573), bovine muscle powder (NIST 8414) and apple leaves (NIST 1515) for Cr and bovine liver (NIST 1577b) for Co. The trueness obtained were between 102 ± 9 and 116 ± 7% for Cr and 94 ± 8% for Co for analyzed of the SRM. Precision, expressed as relative standard deviation (RSD), was better than 8.7% for Cr and 8.3% for Co (n = 5). Thirteen wet animal feed samples for dogs and cats were analyzed. The concentrations values found for Cr in the samples ranged from 0.17 ± 0.01 to 0.59 ± 0.06 μg g−1 with an average of 0.36 ± 0.13 μg g−1 (n = 13). On the other hand, the obtained concentrations for Co ranged between b 0.18 and 12.57 ± 1.11 μg g−1, with an average of 5.92 ± 3.19 μg. The optimized analytical methods were simple, fast, efficient, precise and accurate for determination of Cr and Co in wet animal feeds for cats and dogs by HR-CS SS-GF AAS. Additional advantage of the method was no dissolution of the samples, no use of harmful reagents and consequently no generation of residues, besides the use of an effective analytical technique that allows optimization of fast and reliable screening procedures. © 2017 Elsevier B.V. All rights reserved.
1. Introduction As result of economic development the population of pets has grown all around the world resulting in an increasing in the demand and in the production of feed [1,2]. Products for dogs and cats are available in a variety of shapes and flavors, including dry and wet feed, sachets, canned, biscuits and sweets, offering the owners a wide range of options regarding what is the best for the health of their pets [2,3]. The Association of American Feed Control Officials (AAFCO), a private organization, comprised by state, federal and foreign representatives, classifies these ⁎ Corresponding author at: Grupo de Pesquisa para Estudos em Química Analítica e Ambiental (GPEQA2), Departamento de Química Analítica, Instituto de Química, Universidade Federal da Bahia (UFBA), 40.170-115 Salvador, BA, Brazil. E-mail addresses: [email protected], [email protected] (R.G.O. Araujo).
http://dx.doi.org/10.1016/j.microc.2017.04.028 0026-265X/© 2017 Elsevier B.V. All rights reserved.
products in three categories according to the moisture content: b 20% of humidity, between 20 and 65% and N65%. Otherwise, the National Research Council (NRC) classifies them in only two categories, dry and wet feed [4,5]. In Brazil, the Ministry of Agriculture, Livestock and Supply (Ministério da Agricultura, Pecuária e Abastecimento - MAPA) is responsible for controlling and establishing criteria and procedures for production of pet food [6]. Increasing interest in the analysis of the chemical composition and nutritional value of feed for animals has been noticeable since 2007, as consequence of the mass death of pets in the USA [7]. Quality control of foodstuff for dogs and cats is important to assure the use of ingredients that are really acknowledge as food and to restrict the use of additives that could be unsafety for them [5,6,8–10]. This work focuses on the determination of the total concentration of Co and Cr in feed for animals, employing direct analysis of solid samples.
D.V.L. Ávila et al. / Microchemical Journal 133 (2017) 524–529
These elements are considered essential when ingested in the proper amounts, but toxic in high concentrations. Cobalt is a vital component of vitamin B12, which has an important role in the synthesis of hemoglobin and in preventing anemia. Only Cr3+ specie is classified as essential, playing an important role in the body weight control, acting as regulator of the blood sugar levels and protein metabolism [2,11]. The determination of different species of an element by spectroanalytical techniques depends on a prior separation of their different oxidation state ions. But since the biological activity of these elements requires trace concentration in the body, information regarding the total amount is highly relevant. Elemental determination in any kind of sample requires sample preparation procedures and technique method development and a lot of options are available. Usually sample treatment includes tedious and laborious steps. Direct solid sampling analysis (SS) has proven to be a feasible alternative and more efficient compared with conventional sample preparation procedures. The use of corrosive substances is completely eliminated and dilution of the sample is not employed, which is especially interesting for the determination of elements in low concentration [12,13]. Besides, since there is no use of a large volume of reagents and consequently no generation of residues, it results in economic and environmental benefits, cooperating with the principles of green chemistry [14–16]. Different analysis techniques have been employed in the determination of chemical elements in general food, such as inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectrometry (ICP OES) have been employed in the analyzes due to their multi-element determination [2,7,10,11]. However, these techniques usually require extensive sample preparation and/or extraction procedures prior the analysis that include the disadvantages described previously [13]. On the other hand, high resolution continuum source graphite furnace atomic absorption spectrometry (HR-CS GF AAS) stands out for trace analysis of several elements in different matrices. It is an extremely valuable tool for the analysis of complex samples, particularly for direct solid sample analysis and highly viscous liquids. Because, the system is equipped with a linear charge-coupled device (CCD) array detector; 200 pixels of this array are used analytically, which means that the equipment works with 200 independent detectors that are illuminated and read out simultaneously. The visibility of the spectral environment at both sides of the analytical line, which is another result of this feature, greatly facilitates method development and optimization. The instrument also shows spectral interferences, making it much easier to avoid this kind of interference or possibility to correct for spectral interference using a least-squares algorithm, if it cannot be avoided [15, 16]. The direct introduction of samples minimizing contamination, it is highly sensitivity, good selectivity and limits of detection, besides promoting faster analysis, are some of advantages of HR-CS SS-GF AAS. Since the use acids or solvents is not required the generation of wastes is almost completely eliminated, making it this technique safer for the analyst, friendly to environment and consequently to humans [13,15– 17]. Thus, the present work aims to optimize analytical methods for the determination of Cr and Co in wet animal feeds using direct solid sample analysis and high-resolution continuum source graphite furnace atomic absorption spectrometry (HR-CS SS-GF AAS). 2. Experimental
hot-spot mode, emitting a continuous spectrum from 190 nm up to 900 nm. A high-resolution double monochromator and a charge coupled detector (CCD) are used for radiation separation and signal intensity detection, respectively [17]. Determination of Cr and Co was carried out at the main analytical lines, at wavelengths 357.8687 nm and 240.7254 nm, respectively. The integrated absorbance of three pixels, the center pixel (CP) and the two adjacent pixels, i.e. CP ± 1, were summed and used for signal evaluation for both analytes. Absorbance measurements for the analyses were carried out using graphite tubes transversely heated and pyrolytically coated graphite platforms for solid samples (Analytik Jena, Part no. 407-A 81.025). Sample masses of wet feed were measured (0.03 mg to Co and 0.25 mg to Cr) direct into the SS platforms using a M2P microbalance (Sartorius, Göttingen, Germany) with a precision of 0.001 mg. To introduce the SS platform into the graphite tube a pre-adjusted pair of tweezers, which is part of the SSA 5 manual solid sampling accessory (Analytik Jena AG, Jena, Germany), was used. Argon with a purity of 99.996% (White Martins, São Paulo, Brazil) and a flow rate of 2.0 L min−1, was used as the purge and protective gas throughout all steps of the graphite furnace temperature program, except during atomization step, in which the gas flow was interrupted. The optimized temperature programs of the graphite furnace used for all determinations of Cr and Co are shown in Table 1. 2.2. Reagents All reagents were of analytical grade. Nitric acid 65% (w w− 1) suprapure quality (Merck, Germany) was used to prepare standard solutions. Nitric acid went through a bi-distillation process for purification. Deionized water used in the preparation of all solutions was obtained in a purification system Milli-Q (Millipore, Bedford, MA, USA) to a resistivity of 18.2 MΩ cm−1. The intermediate standard solutions of 1000 μg L−1, used for the reference standard solutions of Cr and Co, were prepared from stock solutions of 1000 mg L−1 (Titrisol, Merck). The solutions were used in the calibration curve preparation by a series of dilutions of the intermediate solutions in an acid nitric 0.014 mol L−1. 2.3. Samples and sample preparation of wet feeds Thirteen samples of wet feeds for dogs and cats were analyzed. In their composition, these samples are described as containing: vitamin A (680 UI), vitamin E (15 UI), vitamin D3 (105 UI), vitamin B1 (11 mg), vitamin B2 (1.4 mg), vitamin B6 (1.1 mg), vitamin B12 (5.5 μg), choline (160 mg), niacin (13 mg), biotin (0.02 mg), folic acid (0.2 mg), pantothenic acid (3.5 mg), zinc (14 mg), iron (7 mg), copper (0.6 mg), manganese (1.5 mg), and iodine (0.15 mg), according the packaging labels. Five of the samples were purchased in a supermarket in the city of Aracaju, Sergipe, Brazil, and the other eight samples were acquired in city of Lisbon, Portugal. Previously the analysis, the samples were transferred from the package to polytetrafluoroethylene (PTFE) tubes and submitted to a pretreatment consisting of homogenization and lyophilization drying (Lyophilizer, L 101 LIOTOP model) during a 120 h period. Table 1 Graphite furnace temperature program for Cr and Co determination in wet animal feed samples by HR-CS SS-GF AAS. Step
2.1. Instrumentation All measurements were carried out using a high-resolution continuum source atomic absorption spectrometer (contrAA 700, Analytik Jena, Jena, Germany). The instrument is equipped with a high-intensity xenon short-arc lamp, with a nominal power of 300 W, operating in a
525
Drying 1 Drying 2 Pyrolysis Atomization Cleaning
Temperature/°C a,b
90 130ª/110b 1500ª/1400b 2500ª/2400b 2550ª,b
Pameters for aCr and bCo determinations.
Ramp/°C s−1 b
10ª/3 10ª/5b 300ª,b 3000ª/1500b 300ª/500b
Hold/s 20ª,b 30ª/10b 20ª/45b 10ª/9b 4ª,b
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After lyophilizing, the samples were manually ground in porcelain mortar and pestle, to increase the contact surfaces. After that, to obtain smaller particles and a better homogenization, the samples were ground in ball mill (Model PM 100, Retsch, Düsseldorf, Germany) for 15 min each cycle sample. The particle size was controlled, passing the samples through a 150 μm diameter sieve. Samples were stored in 50 mL falcon tubes and kept in a desiccator until the time of analysis. The measured masses of direct solid sample analysis ranged from 0.040 to 0.045 mg for determination of Cr, and 0.017 to 0.090 mg for determination of Co. 2.4. Analysis of CRMs Standard reference materials (SRM), bovine muscle (NIST 8414), tomato leaves (NIST 1573), apple leaves (NIST 1515) and bovine liver (NIST 1577b), were used to evaluate the accuracy and precision of the proposed analytical methods. Fig. 2. Sample mass study for Co. Pyrolysis temperature: 1400 °C and atomization temperature: 2400 °C.
3. Results and discussion 3.1. Mass study An evaluation of the mass of sample to be introduced into the furnace for the determination of Cr and Co was performed in order to achieve optimal analytical results. A sample of feed for pets (Brand 3cat Chicken for puppies/sachet) was used and the analytical signal was evaluated regarding the symmetrical peak profiles and the absence of spectral interferences. The mass interval for Cr optimization ranged from 0.040 to 0.450 mg and the results of this study is shown in Fig. 1. The optimized mass, whose signal profiles presented symmetrical peaks, no spectral interference and better absorbance, was 0.250 mg of sample. For Co the mass interval ranged from 0.017 to 0.090 mg, Fig. 2, and an optimized mass of 0.030 mg was adopted for the subsequent studies, since this was the condition that better met the evaluation criteria, symmetrical peak profiles and absence of spectral interference. It is noteworthy that the sample used for the mass evaluation was in a particle size smaller than 150 μm in diameter, which increases the contact surface between the samples and the furnace platform, promoting more uniform heating and vaporization, consequently improving the precision of the analysis. The mass of the sample for determination of the analytes was carried out and 0.250 mg for Cr and 0.030 mg for Co were adopted as optimized conditions for the subsequent studies. In the present work chemical modifier was not used for analysis in the determination of the elements
Fig. 1. Sample mass study for Cr. Pyrolysis temperature: 1500 °C and atomization temperature: 2500 °C.
Cr and Co, since in this case these elements were stable at high temperatures [18].
3.2. Pyrolysis temperature optimization Chemical modifier is usually used for the stabilization of the analytes during the temperature program of the graphite furnace. However Cr and Co are stable elements even at high temperatures and the use of modifier was not required for their determination in wet pet food samples [18]. Pyrolysis curves were constructed in order to evaluate the thermal behavior of Cr and Co for removal of the solvent and matrix. The pyrolysis curves were similar for both elements, in which the temperature of pyrolysis ranged from 1100 to 2000 °C for Cr, and between 1000 and 1800 °C for the Co. The pyrolysis curve for the Cr, presented in Fig. 3, shows a significant increase in the signal intensities in temperature between 1300 and 1500 °C. For Co, the increase in absorbance was observed between 1200 and 1400 °C, as shown in Fig. 4. At temperatures above these values the integrated absorbance decreased for both analytes. Also for both elements the signal intensity remained constant throughout the ranges 1300 and 1500 °C, for Cr, and between 1200 and 1400 °C, for Co, due to the thermal stability of these elements [18].
Fig. 3. Pyrolysis curve for (-•-) 60 pg of Cr and (-□-) 0.250 mg of wet feed sample for cat. Atomization temperature of 2500 °C.
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optimized conditions, temperatures of 1500 °C and 2500 °C, for pyrolysis and atomization, respectively. The similar behavior of Cr in aqueous solution and in the sample can be seen by the symmetrical absorption integrated signals, completely free of spectral interferences, assuring that at the optimized conditions excellent results can be achieved for Cr determination in samples and reference materials.
Fig. 4. Pyrolysis curve for (-•-) 80 pg of Co and (-□-) 0.030 mg of wet feed sample for cat. Atomization temperature of 2400 °C.
Pyrolysis temperatures were established for both elements, taking into account the effective removal of most of sample matrix and the increase of the usage life of graphite platforms and furnaces. It was also considered the best atomic absorption peak profiles and absence of spectral interferences, an indication of the elimination of all concomitant of the matrix [19–21]. Pyrolysis temperature adopted for the determination of Cr was 1500 °C and for Co 1400 °C, which presented the best signal intensities, the most symmetrical peaks and a complete absence of interferences.
3.3.2. Cobalt Evaluation of the temperature program for Co was carried out for 80 pg of Co in aqueous solution and for a feed sample throughout a range from 2000 up to 2500 °C. Fig. 6 presents the results of this optimization and similar behavior is observed for the two curves. The atomization temperature considered optimum and adopted for the analysis was 2400 °C, which was the highest analytical signal intensity. Fig. S2-A and -B in Appendix A shows the 3D profile of atomic absorption spectra obtained for a mass of 80 pg of Co and 0.030 mg of cat feed sample at the optimized pyrolysis and atomization temperatures, respectively. The analytical symmetric signals and the complete absence of interferences indicate that the established temperature conditions were suitable for determination of Co. 3.4. Method validation
3.3.1. Chromium Atomization temperature for Cr was evaluated between 2100 and 2500 °C, as shown in Fig. 5, in which it is possible to observe that for both, aqueous standard solution and the cat feed sample, the behavior of the analyte is very similar. The highest analytical signal was achieved at atomization temperature of 2500 °C. This temperature was considered optimal and adopted for the analysis. Time resolved atomic absorption spectra for 60 pg of Cr in aqueous solution and in 0.25 mg of wet cat food sample are presented in Fig. S1, in Appendix A Supplementary data. Fig. S1-A shows the 3D peak profile for aqueous solution and Fig. S1-B the profile for the sample at the
3.4.1. Calibration curves and figures of merit Calibration curve for Cr was prepared as a blank solution and eight standard solutions at concentrations of 3, 6, 12, 15, 25, 30, 40 and 45 μg L−1 (or mass 30–450 pg Cr). For Co a blank solution and six standard solutions at concentrations of 4, 8, 16, 30, 40 and 50 μg L−1 (or mass 40– 500 pg Co), were adopted, and volumes of volume of 10 μL were introduced. The sensitivity of the methods, expressed as the slopes of the linear equations obtained for calibration curves, were 1.46 × 10−3 s pg−1 for Co and 1.76 × 10−3 s pg−1 for Cr. In both cases the values show a similarity between the determination of Co and Cr, in order of magnitude, besides the high sensitivity [18,21,22]. The pyrolytic platform empty, concept of “zero mass” [23] was used as a blank to determine the limits of detection (LoD) and quantification (LoQ) for Cr and Co in wet feed samples, as shown in Table 2. The LoD and LoQ were calculated following the recommendation of the International Union of Pure and Applied Chemistry (IUPAC) [22], in which LoD is calculated as three times the standard deviation (s) of 10 measurements of a blank solution divided by the slope (S) of the calibration curve (LoD = 3 s/S), and LoQ is based on the same calculation, but using 10 times the standard deviation of the blank solution measurements (LoQ = 10 s/S).
Fig. 5. Atomization curve for mass of (-•-) 60 pg of Cr and (-□-) 0.250 mg wet feed sample for cat. Pyrolysis temperature of 1500 °C.
Fig. 6. Atomization curve for mass of (-•-) 80 pg of C0 and (-□-) 0.030 mg wet feed sample for cat. Pyrolysis temperature of 1400 °C.
3.3. Atomization temperature optimization
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Table 2 Figure of merits for Cr and Co determination in wet animal feeds by HR-CS SS-GF AAS. Parameters
Chromium
Cobalt
Linear regression
Aint = 0.00176.mCr (s pg−1) + 0.0048 0.9993 0.01 0.03 2.2
Aint = 0.00146.mCo (s pg−1) + 0.0008 0.9986 0.05 0.17 2.9
Correlation coeficiente (r) LoD (μg g−1) LoQ (μg g−1) m0 (pg)
LoD and LoQ were calculated using the maximum mass of wet animal feed sample measure of 0.45 mg for Cr and 0.09 mg for Co.
The characteristic mass (m0) defined as the mass of analyte equivalent to an integrated absorbance of 0.0044 s [17,20,22] for Cr was 2.2 pg and for Co was 2.9 pg. In our work, the characteristic masses were considered excellent when compared with the values reported in the literature [25], whose found vales were 4.0 pg for Cr (at 357.8687 nm) and 7.0 pg for Co (at 240.7254 nm), asserting that the proposed methods are quite sensitive [24,25]. The LoD values obtained for Cr (0.01 μg g−1) and Co (0.05 μg g−1) in the present work are better than other values found in studies reported in the literature by other spectroanalytical techniques, possibly because no dilution of the sample was performed for determinations by HR-CS SS-GF AAS. The work accomplished by Costa et al. [2], in which a LoD of 0.03 μg g−1 for Cr was obtained, using ICP OES technique, digestion of the samples, using HNO3 and H2O2 in the closed microwave system, was required. Duran et al. [3] obtained a LoD of 0.058 μg g−1 for Cr using FAAS and sample treatment in open system, a hot plate at 150 °C, HNO3 and H2O2 medium. Medeiros et al. [11] obtained a LoD of 0.05 ng g−1 for Co employing ICP-MS technique, in which HNO3 and Mg(NO3)2 were used in a hot plate until drying and then submitted to the muffle furnace. Onsanit et al. [24] obtained LoD of 0.1 μg g−1 for Co by ICP OES, for the samples digested in a digester block with HNO3 and H2O2. Therefore, it is evident that the analytical methods proposed are suitable for analysis of feed for animals. Works describing the determination of Co in animal feed were not found in the literature; however, a comparison of LoD was made with Co determination in several species of fish, ingredient present in the composition of animal feed used in our work. 3.4.2. Precision and accuracy In order to measure the precision and accuracy of the proposed methods, standard reference materials (SRM) of tomato leaves (NIST 1573), apple leaves (SRM NIST 1515) and bovine muscle powder (SRM NIST 8414) were used for Cr and bovine liver (SRM NIST 1577b) was used for Co. The obtained values for all SRM presented values consistent with the certified or informed values. The precision was expressed as the relative standard deviation (RSD) of five measurements of SRMs and the obtained values were better than 8.7% for Cr and 8.3% for Co, as shown in Table 3. These values are considered acceptable for direct solid sample analysis (b 20%) [23–25]. The concentration values obtained for Cr and Co determination in SRM samples by the optimized methods, were compared to the
predicted values through the application of a comparison test (Student's); tcalculated values for the SRM NIST 1573 (tomato leaves), SRM NIST 8414 (bovine muscle powder), SRM NIST 1577b (bovine liver) were 0.60, 2.66 and 1.77, respectively, lower than the ttabulated (2.776; n = 5) for a 95% confidence level, confirming that there is no significant difference between the experimental and the predicted SRM concentration values. For SRM NIST 1515 (apple leaves), used in the evaluation of the accuracy of method proposed for Cr determination, tcalculated value was 5.60, a significant difference when compared to the value ttabulated (2.776; n = 5) for a 95% confidence level. However, this significant difference can be attributed to the homogeneity of sample composition, as stated in the certified document provided by NIST, whose Cr concentration is a reported value and not a certified value. The trueness values for the certified reference materials analyzed in the determination of Cr and Co are enclosed in the acceptable range for quantitative analysis range, 80 to 120% [2].
3.5. Analytical application The proposed analytical methods were applied to the determination of Cr and Co in 13 samples of wet feeds for dogs and cats; eight of these samples were obtained in supermarkets in Aracaju city, Sergipe, Brazil, and the other five were acquired in Lisbon city, Portugal, as shown in Table 4, in sachets or canned. The analytical application was performed in the samples after lyophilization. Concentration values obtained for Cr in wet animal feed samples (in dry mass after lyophilization) by the application of the developed method ranged from 0.17 ± 0.01 to 0.59 ± 0.06 μg g−1, as shown in Table 4. The average concentration was of 0.36 ± 0.13 μg g−1 (n = 13). Duran et al. found concentrations ranging from 1.58 ± 0.18 to 2.43 ± 0.26 μg g−1 using the FAAS technique [3]. Determination of Co in the 13 wet animal feed samples presented concentrations values in a range between b0.18 and 12.57 ± 1.11 μg g− 1, as also shown in Table 4. The average concentration of Co in the samples was 5.92 ± 3.19 μg g−1 (n = 12). Medeiros et al. [11] evaluated the concentration of Co in fish samples, ingredients present in feed animals used in this work, and found values between 0.003 and 0.09 μg g−1 by ICP-MS. Neither the American agency (AAFCO) nor the Brazilian Ministry (MAPA) establish concentration limits for Cr and Co in wet animal feed samples. However, Cr3+ is crucial in human and animal diet because of its important role in weight control and in regulating the blood sugar balance, related to the metabolism of glucose, but in excessive concentrations can be toxic [2,7,11]. Likewise, Co is an essential element for both animals and humans, since it is the main component of vitamin B12 complex, and also in high concentration can be a toxic metal for pets, humans and even plants [7]. Considering this, the information about Cr and Co concentrations in pet feed samples obtained in this work is of great relevance. Comparing the concentrations obtained for the Cr and Co in wet feed obtained in this work, we conclude that the values for Co are higher than for Cr, except for beef mousse feed sample, which is an important information considering that the of cobalt is required in as a component of vitamin B12 complex [7].
Table 3 Results obtained for Cr and Co determination in standard reference materials by HR- CS SS-GF AAS and t-test values applied. Elements
Cr Co
SRM
Certified value (μg g−1)
Found value (μg g−1)
Recovery (%)
tcalculated⁎⁎
RSD (%)
Tomato leaves (NIST 1573) Apple leaves (NIST 1515) Bovine muscle powder (NIST 8414) Bovine liver (NIST 1577b)
4.5 ± 0.5 (0.3)* 0.071 ± 0.0038 (0.25)*
4.6 ± 0.4 0.35 ± 0.02 0.079 ± 0.006 0.24 ± 0.02
102 ± 9 116 ± 7 111 ± 8 94 ± 8
0.60 5.60 2.66 1.17
8.7 5.7 7.6 8.3
Results expressed as average ± standard deviation (n = 5)//*Informed value//**ttabelated (2.776) for a 95% confidence level and n = 5.
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Table 4 Concentration values obtained for Cr and Co in wet animal feed samples using HR-CS SS-GF AASa. Manufacturer
Origin
Flavor/packing
Co (μg g−1)
Cr (μg g−1)
Brand 1cat Brand 2cat Brand 1cat Brand 2cat Brand 2cat Brand 1cat Brand 2cat Brand 2cat Brand 3cat Brand 3cat Brand 3cat Brand 4dog Brand 3cat
Brazil Portugal Brazil Portugal Portugal Brazil Portugal Portugal Brazil Brazil Brazil Brazil Brazil
Trout and spinach to the sauce/canned Salmon mousse/canned Salmon to the sauce/canned Beef mousse/canned Veal mousse/canned Chicken and carrots to the sauce/canned Seafood (selection)/canned Chicken mousse/canned Chicken for puppies/sachet Meat/sachet Lamb/sachet Meat/sachet Fish/sachet
5.91 ± 1.20 6.30 ± 0.55 4.15 ± 0.69 b0.18 12.57 ± 1.11 1.83 ± 0.20 1.09 ± 0.23 8.97 ± 0.98 5.98 ± 0.43 6.59 ± 0.99 8.51 ± 1.98 6.00 ± 0.91 3.10 ± 0.45
0.40 ± 0.04 0.30 ± 0.02 0.17 ± 0.01 0.40 ± 0.03 0.23 ± 0.02 0.18 ± 0.01 0.35 ± 0.03 0.45 ± 0.03 0.36 ± 0.02 0.35 ± 0.03 0.59 ± 0.06 0.41 ± 0.03 0.54 ± 0.05
a
Results expressed as average concentration ± standard deviation (n = 5) in dry mass.
4. Conclusion High resolution continuum source graphite furnace atomic absorption spectrometry employing direct solid sample analysis (HR-CS SSGF AAS) proved to be an effective analytical tool in optimizing a fast and reliable screening procedure for the determination of Cr and Co in wet animal feed samples. Two analytical methods, simple, fast, efficient and precise, were optimized for Cr and Co concentration determination in wet animal feed samples. The optimal temperature conditions of graphite furnace promoted the thermal stability of both elements without the use of chemical modifier. It is worth to mention that good accuracy and precision were achieved for the methods by HR-CS SS-GF AAS, besides to be free of spectral interferences, resulting in better specificity and selectivity for determination of Co and Cr in feed animals. Since direct solid sample analysis were carried out, the method presented some advantages, such as, no use of concentrated acids and, consequently, no generation of toxic wastes, when compared to other methods using different techniques. Acknowledgments Financial support and scholarship were provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq: R.G.O.A - process no 482416/2013-0 and 308917/2015-4; and M.G.R.V process no 305679/2015-5), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo a Pesquisa do Estado da Bahia (FAPESB: PRONEX and process no APP0065/2016/Universal). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.microc.2017.04.028. References [1] A. Thompson, Ingredients: where pet food starts, Top. Companion Anim. Med. 23 (2008) 127–132. [2] S.S.L. Costa, A.C.L. Pereira, E.A. Passos, J.P.H. Alves, C.A.B. Garcia, R.G.O. Araujo, Multivariate optimization of an analytical method for the analysis of dog and cat foods by ICP OES, Talanta 108 (2013) 157–164. [3] A. Duran, M. Tuzen, M. Soylak, Trace element concentrations of some pet foods commercially available in Turkey, Food Chem. Toxicol. 48 (2010) 2833–2837. [4] National Research Council, Nutrient Requirements of Dogs and Cats, National Academies, Washington, 2006 398. [5] AAFCO, Inspectors Manual 6th Edition, Association of American Feed Control Officials, Oxford, 2017. [6] MAPA – Ministério da Agricultura, Pecuária e AbastecimentoPortaria N° 3, de 22 de Janeiro de 2009.
[7] D.G. Kelly, S.D. White, R.D. Weir, Elemental composition of dog foods using nitric acid and simulated gastric digestions, Food Chem. Toxicol. 55 (2013) 568–577. [8] S. Saracoglu, K.O. Saygi, O.D. Uluozlu, M. Tuzen, S. Mustafa, Determination of trace element contents of baby foods from Turkey, Food Chem. 105 (2007) 280–285. [9] J.C.S. Filho, M.J.A. Armelin, A.G. Silva, Determinação da Composição mineral de subprodutos agroindustriais utilizados na alimentação animal, pela técnica de ativação neutrônica, Pesq. Agrop. Brasileira 34 (1999) 235–241. [10] H. Altundag, M. Tuzen, Comparison of dry, wet and microwave digestion methods for the multi element determination in some dried fruit samples by ICP-OES, Food Chem. Toxicol. 49 (2011) 2800–2807. [11] R.J. Medeiros, L.M.G. dos Santos, A.S. Freire, R.E. Santelli, A.M.C.B. Braga, T.M. Krauss, S.C. Jacob, Determination of inorganic trace elements in edible marine fish from Rio de Janeiro State, Brazil, Food Control 23 (2012) 535–541. [12] G.C.L. Araújo, M.H. Gonzalez, A.G. Ferreira, A.R.A. Nogueira, J.A. Nóbrega, Effect of acid concentration on closed-vessel microwave-assisted digestion of plant materials, Spectrochim. Acta B 57 (2002) 2121–2132. [13] A.R. Borges, E.M. Becker, M.B. Dessuy, M.G.R. Vale, B. Welz, Investigation of chemical modifiers for the determination of lead in fertilizers and limestone using graphite furnace atomic absorption spectrometry with Zeeman-effect background correction and slurry sampling, Spectrochim. Acta B 92 (2014) 1–8. [14] J.L. Eder, A.F. Rogério, J.D. Miguel, C.F.B. Antônio, C.S. Claudio, “Green Chemistry” – Os 12 princípios da Química Verde e sua inserção nas atividades de ensino e pesquisa, Quim Nova 26 (2003) 123–129. [15] I.M. Dittert, J.S.A. Silva, R.G.O. Araujo, A.J. Curtius, B. Welz, H.B. Ross, Simultaneous determination of cobalt and vanadium in undiluted crude oil using high-resolution continuum source graphite furnace atomic absorption spectrometry, J. Anal. Atom. Spectrom. 25 (2010) 590–595. [16] A. Virgilio, J.A. Nóbrega, J.F. Rêgo, J.A.G. Neto, Evaluation of solid sampling high-resolution continuum source grafite furnace atomic absorption spectrometry for direct determination of chromium in medicinal plants, Spectrochim. Acta B 78 (2012) 58–61. [17] A.R. Borges, L.L. François, E.M. Becker, M.G.R. Vale, B. Welz, Method development for the determination of chromium and thalium in fertilizer samples using graphite furnace atomic absorption spectrometry and direct solid sample analysis, Microchem. J. 119 (2015) 169–175. [18] B. Welz, M. Sperling, Atomic Absorption Spectrometry, third ed. Wiley-VCH, Weinheim, 1999. [19] B. Gómez-Nieto, M.J. Gismera, M.T. Sevilla, J.R. Procopio, Simultaneous and direct determination of iron and nickel in biological solid samples by high-resolution continuum source grafite furnace atomic absorption spectrometry, Talanta 116 (2013) 860–865. [20] I.U.P.A.C. Analytical Chemistry Division, Spectrochim. Acta B 33 (1978) 247–269. [21] U. Kurfurst, Solid Sample Analysis: Direct and Slurry Sampling Using GF-AAS and ETV-ICP, first ed. Springer, Berlin, 1998. [22] B. Welz, H. Becker-Ross, S. Florek, U. Heitmann, High-resolution Continuum Source AAS, Weinheim, Wiley-VCH, 2005. [23] R.G.O. Araujo, F. Vignola, I.N.B. Castilho, D.L.G. Borges, B. Welz, M.G.R. Vale, P. Smichowski, S.L.C. Ferreira, H. Becker-Ross, Determination of mercury in airborne particulate matter collected on glass fiber filters using high-resolution continuum source graphite furnace atomic absorption spectrometry and direct solid sampling, Spectrochim. Acta B 66 (2011) 378–382. [24] S. Onsanit, C. Ke, X. Wang, K.J. Wang, W.X. Wang, Trace elements in two marine fish cultured in fish cages in Fujian province, China, Environ. Pollut. 158 (2010) 1334–1342. [25] R.G.O. Araujo, F. Vignola, I.N.B. Castilho, B. Welz, M.G.R. Vale, P. Smichowski, S.L.C. Ferreira, H. Becker-Ross, Determination of silver in airborne particulate matter collected on glass fiber filters using high-resolution continuum source graphite furnace atomic absorption spectrometry and direct solid sampling, Microchem. J. 109 (2013) 36–40.
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Determination of Co and Cr in wet animal feeds using direct solid sample analysis by HR-CS GF AAS Dayara Virgínia Lino Ávila a,b, Aline Rocha Borges c, Maria Goreti Rodrigues Vale d,e, Rennan Geovanny Oliveira Araujo a,b,e,⁎, Elisangela Andrade Passos a a
Departamento de Química, Laboratório de Química Analítica Ambiental (LQA), Centro de Ciências Exatas e Tecnologia da Universidade Federal de Sergipe (UFS), 49.100-000 São Cristóvão, SE, Brazil Departamento de Química Analítica, Instituto de Química, Universidade Federal da Bahia (UFBA), 40.170-115 Salvador, BA, Brazil c Instituto Federal do Paraná, Campus Palmas, 85.555-000 Palmas, PR, Brazil d Instituto de Química, Universidade Federal do Rio Grande do Sul (UFGRS), Av. Bento Gonçalves 9500, 91.501-790 Porto Alegre, RS, Brazil e Instituto Nacional de Ciência e Tecnologia do CNPq-INCT de Energia e Ambiente, Universidade Federal da Bahia, Salvador, BA, Brazil b
a r t i c l e
i n f o
Article history: Received 14 November 2016 Received in revised form 5 March 2017 Accepted 18 April 2017 Available online 20 April 2017 Keywords: Wet animal feed Chromium Cobalt Direct solid sample analysis HR-CS GF AAS
a b s t r a c t The present study proposes the determination of Cr and Co in wet animal feeds for employing direct solid sample analysis (SS) and high resolution continuum source graphite furnace atomic absorption spectrometry (HR-CS GF AAS). For determination of Cr and Co, the analytical lines 357.8687 nm and 240.7254 nm were used, respectively. The pyrolysis temperatures were 1500 and 1400 °C and atomization temperatures were 2500 and 2400 °C for Cr and Co, respectively. The limits of quantification (LoQ) obtained for Cr was 0.03 μg g−1 and 0.17 μg g−1 for Co. The characteristic masses (m0) also obtained were 2.2 pg for Cr and 2.9 pg for Co. The accuracy of the proposed methods was performed by analysis of standard reference materials (SRM) of tomato leaves (NIST 1573), bovine muscle powder (NIST 8414) and apple leaves (NIST 1515) for Cr and bovine liver (NIST 1577b) for Co. The trueness obtained were between 102 ± 9 and 116 ± 7% for Cr and 94 ± 8% for Co for analyzed of the SRM. Precision, expressed as relative standard deviation (RSD), was better than 8.7% for Cr and 8.3% for Co (n = 5). Thirteen wet animal feed samples for dogs and cats were analyzed. The concentrations values found for Cr in the samples ranged from 0.17 ± 0.01 to 0.59 ± 0.06 μg g−1 with an average of 0.36 ± 0.13 μg g−1 (n = 13). On the other hand, the obtained concentrations for Co ranged between b 0.18 and 12.57 ± 1.11 μg g−1, with an average of 5.92 ± 3.19 μg. The optimized analytical methods were simple, fast, efficient, precise and accurate for determination of Cr and Co in wet animal feeds for cats and dogs by HR-CS SS-GF AAS. Additional advantage of the method was no dissolution of the samples, no use of harmful reagents and consequently no generation of residues, besides the use of an effective analytical technique that allows optimization of fast and reliable screening procedures. © 2017 Elsevier B.V. All rights reserved.
1. Introduction As result of economic development the population of pets has grown all around the world resulting in an increasing in the demand and in the production of feed [1,2]. Products for dogs and cats are available in a variety of shapes and flavors, including dry and wet feed, sachets, canned, biscuits and sweets, offering the owners a wide range of options regarding what is the best for the health of their pets [2,3]. The Association of American Feed Control Officials (AAFCO), a private organization, comprised by state, federal and foreign representatives, classifies these ⁎ Corresponding author at: Grupo de Pesquisa para Estudos em Química Analítica e Ambiental (GPEQA2), Departamento de Química Analítica, Instituto de Química, Universidade Federal da Bahia (UFBA), 40.170-115 Salvador, BA, Brazil. E-mail addresses: [email protected], [email protected] (R.G.O. Araujo).
http://dx.doi.org/10.1016/j.microc.2017.04.028 0026-265X/© 2017 Elsevier B.V. All rights reserved.
products in three categories according to the moisture content: b 20% of humidity, between 20 and 65% and N65%. Otherwise, the National Research Council (NRC) classifies them in only two categories, dry and wet feed [4,5]. In Brazil, the Ministry of Agriculture, Livestock and Supply (Ministério da Agricultura, Pecuária e Abastecimento - MAPA) is responsible for controlling and establishing criteria and procedures for production of pet food [6]. Increasing interest in the analysis of the chemical composition and nutritional value of feed for animals has been noticeable since 2007, as consequence of the mass death of pets in the USA [7]. Quality control of foodstuff for dogs and cats is important to assure the use of ingredients that are really acknowledge as food and to restrict the use of additives that could be unsafety for them [5,6,8–10]. This work focuses on the determination of the total concentration of Co and Cr in feed for animals, employing direct analysis of solid samples.
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These elements are considered essential when ingested in the proper amounts, but toxic in high concentrations. Cobalt is a vital component of vitamin B12, which has an important role in the synthesis of hemoglobin and in preventing anemia. Only Cr3+ specie is classified as essential, playing an important role in the body weight control, acting as regulator of the blood sugar levels and protein metabolism [2,11]. The determination of different species of an element by spectroanalytical techniques depends on a prior separation of their different oxidation state ions. But since the biological activity of these elements requires trace concentration in the body, information regarding the total amount is highly relevant. Elemental determination in any kind of sample requires sample preparation procedures and technique method development and a lot of options are available. Usually sample treatment includes tedious and laborious steps. Direct solid sampling analysis (SS) has proven to be a feasible alternative and more efficient compared with conventional sample preparation procedures. The use of corrosive substances is completely eliminated and dilution of the sample is not employed, which is especially interesting for the determination of elements in low concentration [12,13]. Besides, since there is no use of a large volume of reagents and consequently no generation of residues, it results in economic and environmental benefits, cooperating with the principles of green chemistry [14–16]. Different analysis techniques have been employed in the determination of chemical elements in general food, such as inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectrometry (ICP OES) have been employed in the analyzes due to their multi-element determination [2,7,10,11]. However, these techniques usually require extensive sample preparation and/or extraction procedures prior the analysis that include the disadvantages described previously [13]. On the other hand, high resolution continuum source graphite furnace atomic absorption spectrometry (HR-CS GF AAS) stands out for trace analysis of several elements in different matrices. It is an extremely valuable tool for the analysis of complex samples, particularly for direct solid sample analysis and highly viscous liquids. Because, the system is equipped with a linear charge-coupled device (CCD) array detector; 200 pixels of this array are used analytically, which means that the equipment works with 200 independent detectors that are illuminated and read out simultaneously. The visibility of the spectral environment at both sides of the analytical line, which is another result of this feature, greatly facilitates method development and optimization. The instrument also shows spectral interferences, making it much easier to avoid this kind of interference or possibility to correct for spectral interference using a least-squares algorithm, if it cannot be avoided [15, 16]. The direct introduction of samples minimizing contamination, it is highly sensitivity, good selectivity and limits of detection, besides promoting faster analysis, are some of advantages of HR-CS SS-GF AAS. Since the use acids or solvents is not required the generation of wastes is almost completely eliminated, making it this technique safer for the analyst, friendly to environment and consequently to humans [13,15– 17]. Thus, the present work aims to optimize analytical methods for the determination of Cr and Co in wet animal feeds using direct solid sample analysis and high-resolution continuum source graphite furnace atomic absorption spectrometry (HR-CS SS-GF AAS). 2. Experimental
hot-spot mode, emitting a continuous spectrum from 190 nm up to 900 nm. A high-resolution double monochromator and a charge coupled detector (CCD) are used for radiation separation and signal intensity detection, respectively [17]. Determination of Cr and Co was carried out at the main analytical lines, at wavelengths 357.8687 nm and 240.7254 nm, respectively. The integrated absorbance of three pixels, the center pixel (CP) and the two adjacent pixels, i.e. CP ± 1, were summed and used for signal evaluation for both analytes. Absorbance measurements for the analyses were carried out using graphite tubes transversely heated and pyrolytically coated graphite platforms for solid samples (Analytik Jena, Part no. 407-A 81.025). Sample masses of wet feed were measured (0.03 mg to Co and 0.25 mg to Cr) direct into the SS platforms using a M2P microbalance (Sartorius, Göttingen, Germany) with a precision of 0.001 mg. To introduce the SS platform into the graphite tube a pre-adjusted pair of tweezers, which is part of the SSA 5 manual solid sampling accessory (Analytik Jena AG, Jena, Germany), was used. Argon with a purity of 99.996% (White Martins, São Paulo, Brazil) and a flow rate of 2.0 L min−1, was used as the purge and protective gas throughout all steps of the graphite furnace temperature program, except during atomization step, in which the gas flow was interrupted. The optimized temperature programs of the graphite furnace used for all determinations of Cr and Co are shown in Table 1. 2.2. Reagents All reagents were of analytical grade. Nitric acid 65% (w w− 1) suprapure quality (Merck, Germany) was used to prepare standard solutions. Nitric acid went through a bi-distillation process for purification. Deionized water used in the preparation of all solutions was obtained in a purification system Milli-Q (Millipore, Bedford, MA, USA) to a resistivity of 18.2 MΩ cm−1. The intermediate standard solutions of 1000 μg L−1, used for the reference standard solutions of Cr and Co, were prepared from stock solutions of 1000 mg L−1 (Titrisol, Merck). The solutions were used in the calibration curve preparation by a series of dilutions of the intermediate solutions in an acid nitric 0.014 mol L−1. 2.3. Samples and sample preparation of wet feeds Thirteen samples of wet feeds for dogs and cats were analyzed. In their composition, these samples are described as containing: vitamin A (680 UI), vitamin E (15 UI), vitamin D3 (105 UI), vitamin B1 (11 mg), vitamin B2 (1.4 mg), vitamin B6 (1.1 mg), vitamin B12 (5.5 μg), choline (160 mg), niacin (13 mg), biotin (0.02 mg), folic acid (0.2 mg), pantothenic acid (3.5 mg), zinc (14 mg), iron (7 mg), copper (0.6 mg), manganese (1.5 mg), and iodine (0.15 mg), according the packaging labels. Five of the samples were purchased in a supermarket in the city of Aracaju, Sergipe, Brazil, and the other eight samples were acquired in city of Lisbon, Portugal. Previously the analysis, the samples were transferred from the package to polytetrafluoroethylene (PTFE) tubes and submitted to a pretreatment consisting of homogenization and lyophilization drying (Lyophilizer, L 101 LIOTOP model) during a 120 h period. Table 1 Graphite furnace temperature program for Cr and Co determination in wet animal feed samples by HR-CS SS-GF AAS. Step
2.1. Instrumentation All measurements were carried out using a high-resolution continuum source atomic absorption spectrometer (contrAA 700, Analytik Jena, Jena, Germany). The instrument is equipped with a high-intensity xenon short-arc lamp, with a nominal power of 300 W, operating in a
525
Drying 1 Drying 2 Pyrolysis Atomization Cleaning
Temperature/°C a,b
90 130ª/110b 1500ª/1400b 2500ª/2400b 2550ª,b
Pameters for aCr and bCo determinations.
Ramp/°C s−1 b
10ª/3 10ª/5b 300ª,b 3000ª/1500b 300ª/500b
Hold/s 20ª,b 30ª/10b 20ª/45b 10ª/9b 4ª,b
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After lyophilizing, the samples were manually ground in porcelain mortar and pestle, to increase the contact surfaces. After that, to obtain smaller particles and a better homogenization, the samples were ground in ball mill (Model PM 100, Retsch, Düsseldorf, Germany) for 15 min each cycle sample. The particle size was controlled, passing the samples through a 150 μm diameter sieve. Samples were stored in 50 mL falcon tubes and kept in a desiccator until the time of analysis. The measured masses of direct solid sample analysis ranged from 0.040 to 0.045 mg for determination of Cr, and 0.017 to 0.090 mg for determination of Co. 2.4. Analysis of CRMs Standard reference materials (SRM), bovine muscle (NIST 8414), tomato leaves (NIST 1573), apple leaves (NIST 1515) and bovine liver (NIST 1577b), were used to evaluate the accuracy and precision of the proposed analytical methods. Fig. 2. Sample mass study for Co. Pyrolysis temperature: 1400 °C and atomization temperature: 2400 °C.
3. Results and discussion 3.1. Mass study An evaluation of the mass of sample to be introduced into the furnace for the determination of Cr and Co was performed in order to achieve optimal analytical results. A sample of feed for pets (Brand 3cat Chicken for puppies/sachet) was used and the analytical signal was evaluated regarding the symmetrical peak profiles and the absence of spectral interferences. The mass interval for Cr optimization ranged from 0.040 to 0.450 mg and the results of this study is shown in Fig. 1. The optimized mass, whose signal profiles presented symmetrical peaks, no spectral interference and better absorbance, was 0.250 mg of sample. For Co the mass interval ranged from 0.017 to 0.090 mg, Fig. 2, and an optimized mass of 0.030 mg was adopted for the subsequent studies, since this was the condition that better met the evaluation criteria, symmetrical peak profiles and absence of spectral interference. It is noteworthy that the sample used for the mass evaluation was in a particle size smaller than 150 μm in diameter, which increases the contact surface between the samples and the furnace platform, promoting more uniform heating and vaporization, consequently improving the precision of the analysis. The mass of the sample for determination of the analytes was carried out and 0.250 mg for Cr and 0.030 mg for Co were adopted as optimized conditions for the subsequent studies. In the present work chemical modifier was not used for analysis in the determination of the elements
Fig. 1. Sample mass study for Cr. Pyrolysis temperature: 1500 °C and atomization temperature: 2500 °C.
Cr and Co, since in this case these elements were stable at high temperatures [18].
3.2. Pyrolysis temperature optimization Chemical modifier is usually used for the stabilization of the analytes during the temperature program of the graphite furnace. However Cr and Co are stable elements even at high temperatures and the use of modifier was not required for their determination in wet pet food samples [18]. Pyrolysis curves were constructed in order to evaluate the thermal behavior of Cr and Co for removal of the solvent and matrix. The pyrolysis curves were similar for both elements, in which the temperature of pyrolysis ranged from 1100 to 2000 °C for Cr, and between 1000 and 1800 °C for the Co. The pyrolysis curve for the Cr, presented in Fig. 3, shows a significant increase in the signal intensities in temperature between 1300 and 1500 °C. For Co, the increase in absorbance was observed between 1200 and 1400 °C, as shown in Fig. 4. At temperatures above these values the integrated absorbance decreased for both analytes. Also for both elements the signal intensity remained constant throughout the ranges 1300 and 1500 °C, for Cr, and between 1200 and 1400 °C, for Co, due to the thermal stability of these elements [18].
Fig. 3. Pyrolysis curve for (-•-) 60 pg of Cr and (-□-) 0.250 mg of wet feed sample for cat. Atomization temperature of 2500 °C.
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optimized conditions, temperatures of 1500 °C and 2500 °C, for pyrolysis and atomization, respectively. The similar behavior of Cr in aqueous solution and in the sample can be seen by the symmetrical absorption integrated signals, completely free of spectral interferences, assuring that at the optimized conditions excellent results can be achieved for Cr determination in samples and reference materials.
Fig. 4. Pyrolysis curve for (-•-) 80 pg of Co and (-□-) 0.030 mg of wet feed sample for cat. Atomization temperature of 2400 °C.
Pyrolysis temperatures were established for both elements, taking into account the effective removal of most of sample matrix and the increase of the usage life of graphite platforms and furnaces. It was also considered the best atomic absorption peak profiles and absence of spectral interferences, an indication of the elimination of all concomitant of the matrix [19–21]. Pyrolysis temperature adopted for the determination of Cr was 1500 °C and for Co 1400 °C, which presented the best signal intensities, the most symmetrical peaks and a complete absence of interferences.
3.3.2. Cobalt Evaluation of the temperature program for Co was carried out for 80 pg of Co in aqueous solution and for a feed sample throughout a range from 2000 up to 2500 °C. Fig. 6 presents the results of this optimization and similar behavior is observed for the two curves. The atomization temperature considered optimum and adopted for the analysis was 2400 °C, which was the highest analytical signal intensity. Fig. S2-A and -B in Appendix A shows the 3D profile of atomic absorption spectra obtained for a mass of 80 pg of Co and 0.030 mg of cat feed sample at the optimized pyrolysis and atomization temperatures, respectively. The analytical symmetric signals and the complete absence of interferences indicate that the established temperature conditions were suitable for determination of Co. 3.4. Method validation
3.3.1. Chromium Atomization temperature for Cr was evaluated between 2100 and 2500 °C, as shown in Fig. 5, in which it is possible to observe that for both, aqueous standard solution and the cat feed sample, the behavior of the analyte is very similar. The highest analytical signal was achieved at atomization temperature of 2500 °C. This temperature was considered optimal and adopted for the analysis. Time resolved atomic absorption spectra for 60 pg of Cr in aqueous solution and in 0.25 mg of wet cat food sample are presented in Fig. S1, in Appendix A Supplementary data. Fig. S1-A shows the 3D peak profile for aqueous solution and Fig. S1-B the profile for the sample at the
3.4.1. Calibration curves and figures of merit Calibration curve for Cr was prepared as a blank solution and eight standard solutions at concentrations of 3, 6, 12, 15, 25, 30, 40 and 45 μg L−1 (or mass 30–450 pg Cr). For Co a blank solution and six standard solutions at concentrations of 4, 8, 16, 30, 40 and 50 μg L−1 (or mass 40– 500 pg Co), were adopted, and volumes of volume of 10 μL were introduced. The sensitivity of the methods, expressed as the slopes of the linear equations obtained for calibration curves, were 1.46 × 10−3 s pg−1 for Co and 1.76 × 10−3 s pg−1 for Cr. In both cases the values show a similarity between the determination of Co and Cr, in order of magnitude, besides the high sensitivity [18,21,22]. The pyrolytic platform empty, concept of “zero mass” [23] was used as a blank to determine the limits of detection (LoD) and quantification (LoQ) for Cr and Co in wet feed samples, as shown in Table 2. The LoD and LoQ were calculated following the recommendation of the International Union of Pure and Applied Chemistry (IUPAC) [22], in which LoD is calculated as three times the standard deviation (s) of 10 measurements of a blank solution divided by the slope (S) of the calibration curve (LoD = 3 s/S), and LoQ is based on the same calculation, but using 10 times the standard deviation of the blank solution measurements (LoQ = 10 s/S).
Fig. 5. Atomization curve for mass of (-•-) 60 pg of Cr and (-□-) 0.250 mg wet feed sample for cat. Pyrolysis temperature of 1500 °C.
Fig. 6. Atomization curve for mass of (-•-) 80 pg of C0 and (-□-) 0.030 mg wet feed sample for cat. Pyrolysis temperature of 1400 °C.
3.3. Atomization temperature optimization
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Table 2 Figure of merits for Cr and Co determination in wet animal feeds by HR-CS SS-GF AAS. Parameters
Chromium
Cobalt
Linear regression
Aint = 0.00176.mCr (s pg−1) + 0.0048 0.9993 0.01 0.03 2.2
Aint = 0.00146.mCo (s pg−1) + 0.0008 0.9986 0.05 0.17 2.9
Correlation coeficiente (r) LoD (μg g−1) LoQ (μg g−1) m0 (pg)
LoD and LoQ were calculated using the maximum mass of wet animal feed sample measure of 0.45 mg for Cr and 0.09 mg for Co.
The characteristic mass (m0) defined as the mass of analyte equivalent to an integrated absorbance of 0.0044 s [17,20,22] for Cr was 2.2 pg and for Co was 2.9 pg. In our work, the characteristic masses were considered excellent when compared with the values reported in the literature [25], whose found vales were 4.0 pg for Cr (at 357.8687 nm) and 7.0 pg for Co (at 240.7254 nm), asserting that the proposed methods are quite sensitive [24,25]. The LoD values obtained for Cr (0.01 μg g−1) and Co (0.05 μg g−1) in the present work are better than other values found in studies reported in the literature by other spectroanalytical techniques, possibly because no dilution of the sample was performed for determinations by HR-CS SS-GF AAS. The work accomplished by Costa et al. [2], in which a LoD of 0.03 μg g−1 for Cr was obtained, using ICP OES technique, digestion of the samples, using HNO3 and H2O2 in the closed microwave system, was required. Duran et al. [3] obtained a LoD of 0.058 μg g−1 for Cr using FAAS and sample treatment in open system, a hot plate at 150 °C, HNO3 and H2O2 medium. Medeiros et al. [11] obtained a LoD of 0.05 ng g−1 for Co employing ICP-MS technique, in which HNO3 and Mg(NO3)2 were used in a hot plate until drying and then submitted to the muffle furnace. Onsanit et al. [24] obtained LoD of 0.1 μg g−1 for Co by ICP OES, for the samples digested in a digester block with HNO3 and H2O2. Therefore, it is evident that the analytical methods proposed are suitable for analysis of feed for animals. Works describing the determination of Co in animal feed were not found in the literature; however, a comparison of LoD was made with Co determination in several species of fish, ingredient present in the composition of animal feed used in our work. 3.4.2. Precision and accuracy In order to measure the precision and accuracy of the proposed methods, standard reference materials (SRM) of tomato leaves (NIST 1573), apple leaves (SRM NIST 1515) and bovine muscle powder (SRM NIST 8414) were used for Cr and bovine liver (SRM NIST 1577b) was used for Co. The obtained values for all SRM presented values consistent with the certified or informed values. The precision was expressed as the relative standard deviation (RSD) of five measurements of SRMs and the obtained values were better than 8.7% for Cr and 8.3% for Co, as shown in Table 3. These values are considered acceptable for direct solid sample analysis (b 20%) [23–25]. The concentration values obtained for Cr and Co determination in SRM samples by the optimized methods, were compared to the
predicted values through the application of a comparison test (Student's); tcalculated values for the SRM NIST 1573 (tomato leaves), SRM NIST 8414 (bovine muscle powder), SRM NIST 1577b (bovine liver) were 0.60, 2.66 and 1.77, respectively, lower than the ttabulated (2.776; n = 5) for a 95% confidence level, confirming that there is no significant difference between the experimental and the predicted SRM concentration values. For SRM NIST 1515 (apple leaves), used in the evaluation of the accuracy of method proposed for Cr determination, tcalculated value was 5.60, a significant difference when compared to the value ttabulated (2.776; n = 5) for a 95% confidence level. However, this significant difference can be attributed to the homogeneity of sample composition, as stated in the certified document provided by NIST, whose Cr concentration is a reported value and not a certified value. The trueness values for the certified reference materials analyzed in the determination of Cr and Co are enclosed in the acceptable range for quantitative analysis range, 80 to 120% [2].
3.5. Analytical application The proposed analytical methods were applied to the determination of Cr and Co in 13 samples of wet feeds for dogs and cats; eight of these samples were obtained in supermarkets in Aracaju city, Sergipe, Brazil, and the other five were acquired in Lisbon city, Portugal, as shown in Table 4, in sachets or canned. The analytical application was performed in the samples after lyophilization. Concentration values obtained for Cr in wet animal feed samples (in dry mass after lyophilization) by the application of the developed method ranged from 0.17 ± 0.01 to 0.59 ± 0.06 μg g−1, as shown in Table 4. The average concentration was of 0.36 ± 0.13 μg g−1 (n = 13). Duran et al. found concentrations ranging from 1.58 ± 0.18 to 2.43 ± 0.26 μg g−1 using the FAAS technique [3]. Determination of Co in the 13 wet animal feed samples presented concentrations values in a range between b0.18 and 12.57 ± 1.11 μg g− 1, as also shown in Table 4. The average concentration of Co in the samples was 5.92 ± 3.19 μg g−1 (n = 12). Medeiros et al. [11] evaluated the concentration of Co in fish samples, ingredients present in feed animals used in this work, and found values between 0.003 and 0.09 μg g−1 by ICP-MS. Neither the American agency (AAFCO) nor the Brazilian Ministry (MAPA) establish concentration limits for Cr and Co in wet animal feed samples. However, Cr3+ is crucial in human and animal diet because of its important role in weight control and in regulating the blood sugar balance, related to the metabolism of glucose, but in excessive concentrations can be toxic [2,7,11]. Likewise, Co is an essential element for both animals and humans, since it is the main component of vitamin B12 complex, and also in high concentration can be a toxic metal for pets, humans and even plants [7]. Considering this, the information about Cr and Co concentrations in pet feed samples obtained in this work is of great relevance. Comparing the concentrations obtained for the Cr and Co in wet feed obtained in this work, we conclude that the values for Co are higher than for Cr, except for beef mousse feed sample, which is an important information considering that the of cobalt is required in as a component of vitamin B12 complex [7].
Table 3 Results obtained for Cr and Co determination in standard reference materials by HR- CS SS-GF AAS and t-test values applied. Elements
Cr Co
SRM
Certified value (μg g−1)
Found value (μg g−1)
Recovery (%)
tcalculated⁎⁎
RSD (%)
Tomato leaves (NIST 1573) Apple leaves (NIST 1515) Bovine muscle powder (NIST 8414) Bovine liver (NIST 1577b)
4.5 ± 0.5 (0.3)* 0.071 ± 0.0038 (0.25)*
4.6 ± 0.4 0.35 ± 0.02 0.079 ± 0.006 0.24 ± 0.02
102 ± 9 116 ± 7 111 ± 8 94 ± 8
0.60 5.60 2.66 1.17
8.7 5.7 7.6 8.3
Results expressed as average ± standard deviation (n = 5)//*Informed value//**ttabelated (2.776) for a 95% confidence level and n = 5.
D.V.L. Ávila et al. / Microchemical Journal 133 (2017) 524–529
529
Table 4 Concentration values obtained for Cr and Co in wet animal feed samples using HR-CS SS-GF AASa. Manufacturer
Origin
Flavor/packing
Co (μg g−1)
Cr (μg g−1)
Brand 1cat Brand 2cat Brand 1cat Brand 2cat Brand 2cat Brand 1cat Brand 2cat Brand 2cat Brand 3cat Brand 3cat Brand 3cat Brand 4dog Brand 3cat
Brazil Portugal Brazil Portugal Portugal Brazil Portugal Portugal Brazil Brazil Brazil Brazil Brazil
Trout and spinach to the sauce/canned Salmon mousse/canned Salmon to the sauce/canned Beef mousse/canned Veal mousse/canned Chicken and carrots to the sauce/canned Seafood (selection)/canned Chicken mousse/canned Chicken for puppies/sachet Meat/sachet Lamb/sachet Meat/sachet Fish/sachet
5.91 ± 1.20 6.30 ± 0.55 4.15 ± 0.69 b0.18 12.57 ± 1.11 1.83 ± 0.20 1.09 ± 0.23 8.97 ± 0.98 5.98 ± 0.43 6.59 ± 0.99 8.51 ± 1.98 6.00 ± 0.91 3.10 ± 0.45
0.40 ± 0.04 0.30 ± 0.02 0.17 ± 0.01 0.40 ± 0.03 0.23 ± 0.02 0.18 ± 0.01 0.35 ± 0.03 0.45 ± 0.03 0.36 ± 0.02 0.35 ± 0.03 0.59 ± 0.06 0.41 ± 0.03 0.54 ± 0.05
a
Results expressed as average concentration ± standard deviation (n = 5) in dry mass.
4. Conclusion High resolution continuum source graphite furnace atomic absorption spectrometry employing direct solid sample analysis (HR-CS SSGF AAS) proved to be an effective analytical tool in optimizing a fast and reliable screening procedure for the determination of Cr and Co in wet animal feed samples. Two analytical methods, simple, fast, efficient and precise, were optimized for Cr and Co concentration determination in wet animal feed samples. The optimal temperature conditions of graphite furnace promoted the thermal stability of both elements without the use of chemical modifier. It is worth to mention that good accuracy and precision were achieved for the methods by HR-CS SS-GF AAS, besides to be free of spectral interferences, resulting in better specificity and selectivity for determination of Co and Cr in feed animals. Since direct solid sample analysis were carried out, the method presented some advantages, such as, no use of concentrated acids and, consequently, no generation of toxic wastes, when compared to other methods using different techniques. Acknowledgments Financial support and scholarship were provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq: R.G.O.A - process no 482416/2013-0 and 308917/2015-4; and M.G.R.V process no 305679/2015-5), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo a Pesquisa do Estado da Bahia (FAPESB: PRONEX and process no APP0065/2016/Universal). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.microc.2017.04.028. References [1] A. Thompson, Ingredients: where pet food starts, Top. Companion Anim. Med. 23 (2008) 127–132. [2] S.S.L. Costa, A.C.L. Pereira, E.A. Passos, J.P.H. Alves, C.A.B. Garcia, R.G.O. Araujo, Multivariate optimization of an analytical method for the analysis of dog and cat foods by ICP OES, Talanta 108 (2013) 157–164. [3] A. Duran, M. Tuzen, M. Soylak, Trace element concentrations of some pet foods commercially available in Turkey, Food Chem. Toxicol. 48 (2010) 2833–2837. [4] National Research Council, Nutrient Requirements of Dogs and Cats, National Academies, Washington, 2006 398. [5] AAFCO, Inspectors Manual 6th Edition, Association of American Feed Control Officials, Oxford, 2017. [6] MAPA – Ministério da Agricultura, Pecuária e AbastecimentoPortaria N° 3, de 22 de Janeiro de 2009.
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