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Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Improvement of a sample preparation procedure for multi-elemental determination in Brazil nuts by ICPOES a
a
Maja Welna & Anna Szymczycha-Madeja a
Analytical Chemistry Division, Chemistry Department, Wrocław University of Technology, Wrocław, Poland Accepted author version posted online: 03 Jan 2014.Published online: 04 Mar 2014.
Click for updates To cite this article: Maja Welna & Anna Szymczycha-Madeja (2014) Improvement of a sample preparation procedure for multi-elemental determination in Brazil nuts by ICP-OES, Food Additives & Contaminants: Part A, 31:4, 658-665, DOI: 10.1080/19440049.2014.880134 To link to this article: http://dx.doi.org/10.1080/19440049.2014.880134
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Food Additives & Contaminants: Part A, 2014 Vol. 31, No. 4, 658–665, http://dx.doi.org/10.1080/19440049.2014.880134
Improvement of a sample preparation procedure for multi-elemental determination in Brazil nuts by ICP-OES Maja Welna* and Anna Szymczycha-Madeja Analytical Chemistry Division, Chemistry Department, Wrocław University of Technology, Wrocław, Poland
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(Received 1 October 2013; accepted 26 December 2013) Various sample preparation procedures, such as common wet digestions and alternatives based on solubilisation in aqua regia or tetramethyl ammonium hydroxide, were compared for the determination of the total Ba, Ca, Cr, Cd, Cu, Fe, Mg, Mn, Ni, P, Pb, Se, Sr and Zn contents in Brazil nuts using inductively coupled plasma optical emission spectrometry (ICP-OES). For measurement of Se, a hydride generation technique was used. The performance of these procedures was measured in terms of precision, accuracy and limits of detection of the elements. It was found that solubilisation in aqua regia gave the best results, i.e. limits of detection from 0.60 to 41.9 ng ml−1, precision of 1.0–3.9% and accuracy better than 5%. External calibration with simple standard solutions could be applied for the analysis. The proposed procedure is simple, reduces sample handling, and minimises the time and reagent consumption. Thus, this can be a vital alternative to traditional sample treatment approaches based on the total digestion with concentrated reagents. A phenomenon resulting from levels of Ba, Se and Sr in Brazil nuts was also discussed. Keywords: Brazil nuts; sample preparation; minerals; ICP-OES; barium
Introduction Nowadays knowledge about the elemental content of food products is essential in order to define their quality. Nuts are considered one of the most nutritious human foods due to their high amounts of proteins, carbohydrates, unsaturated lipids, vitamins and essential elements. Among a wide variety of nuts, Brazil nuts (Bertholletia excelsa) are worthy of special interest due to their nutritive value as well as some health benefits. The popularity of Brazil nuts is associated with an exceptionally high Se level. A review devoted to the composition and properties of these nuts was presented recently by Yang (2009). On the other hand, surprisingly, Brazil nuts can also accumulate significant amounts of toxic (Ba) and carcinogen (Ra) elements (Parekh et al. 2008; Goncalves et al. 2009). The significance of Se means that all the effort put into the analysis of Brazil nuts is mostly concerned with the determination of Se and Se species (e.g. Vonderheide et al. 2002; Wrobel et al. 2003; Chunhieng et al. 2004; Dumont et al. 2006). Information about their mineral composition, i.e. macro-, micro- and trace elements, can be found only in a few publications (Moodley et al. 2007; Rodushkin et al. 2008; Welna et al. 2008). The assessment of the total concentrations of elements in Brazil nuts is primary performed using spectrochemical methods such as atomic absorption spectrometry (AAS) (Naozuka et al. 2010), inductively coupled plasma optical emission spectrometry (ICP-OES) (Chunhieng et al. 2004; Moodley et al. 2007; Welna et al. 2008), and inductively *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
coupled plasma mass spectrometry (ICP-MS) (Kannamkumarath, Wrobel, et al. 2004; Kannamkumarath, Wuilloud, et al. 2004; Rodushkin et al. 2008). Normally, prior to the atomic spectrometry measurements, samples have to be initially prepared, i.e. brought into the solution by means of the total digestion in oxidative reagents. In this sense, the analysis of Brazil nuts is not an easy task due to complex composition, dominated by high lipid content (> 60%). In addition to lipids, Brazil nuts contain 10–20% proteins and carbohydrates (Yang 2009). Typically, lipids, which are troublesome during sample preparation, are often removed before analysis by solvent extraction. At present, wet digestion in pressurised closed vessel microwave-assisted systems (Kannamkumarath, Wrobel, et al. 2004; Kannamkumarath, Wuilloud, et al. 2004; Moodley et al. 2007; Rodushkin et al. 2008; Goncalves et al. 2009; Naozuka et al. 2010) is preferred. Generally, HNO3 alone (Kannamkumarath, Wrobel, et al. 2004; Kannamkumarath, Wuilloud, et al. 2004; Moodley et al. 2007) as well as its mixtures with H2O2 (Goncalves et al. 2009; Naozuka et al. 2010) is used for sample digestion. The dry ashing at the step of Brazil nuts sample decomposition has also been used (Chunhieng et al. 2004). Unfortunately, although excellent and effective, total wet digestion is time-consuming and requires special tools and equipments, hazardous reagents or laborious procedures. Hence, it is worth examining whether there are simpler alternative approaches of sample preparation that can considerably diminish the time of sample treatment
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Food Additives & Contaminants: Part A and eliminate all inconveniences related to sample digestion. To the best of our knowledge, an alternative to acidbased procedures for Brazil nut sample preparation prior to their multi-element analysis by ICP-OES has not been developed and used. So far, common digestion procedures for different types of nuts have been investigated. Accordingly, Momen et al. (2007) tested on nut samples (peanut, almond, hazelnut, walnut) two wet digestion procedures, i.e. the conventional (hot-plate) heating in HNO3/ H2SO4 and HNO3/H2SO4/H2O2. The proposed procedure, i.e. digestion in the presence of HNO3/H2SO4/H2O2, appeared to be valid for ICP-OES measurements of total concentrations of Al, Ba, Cd, Cr, Cu, Fe, Mg, Mn, Pb and Zn. The optimisation of the sample preparation procedure based on microwave-assisted digestion in HNO3/HF of different nuts (including Brazil nuts) and seeds followed by multi-elemental analysis using ICP-MS was evaluated by Rodushkin et al. (2008). Very recently, Muller et al. (2013) improved the digestion of samples using microwave-induced combustion (MIC) to determine toxic (As, Cd, Hg and Pb) elements in nuts (hazelnuts, almonds, cashew nuts, Brazil nuts and walnuts) by ICP-MS. The aim of this work was to compare various sample preparation procedures and to develop a precise and accurate method of the multi-elemental analysis of Brazil nuts by ICP-OES. According to our knowledge, this work reports for the first time the results of a comparison of several preparation procedures of Brazil nuts, including common and alternative, prior to their analysis by ICP-OES on the content of 14 elements, i.e. Ba, Ca, Cd, Cr, Cu, Fe, Mg, Mn, Ni, P, Pb, Se, Sr and Zn.
Materials and methods Samples Unshelled Brazil nuts were purchased in a local market in Wroclaw, Poland. Before analysis nuts were peeled, grated and mixed. Prior to sampling, lipids were removed from the ground nuts by solvent extraction. Accordingly, a portion of about 25 g of nuts was mixed with 125 ml of petroleum ether and shaken in an ultrasonic bath for 15 min. The extraction procedure was repeated three times. The remaining nut residue was left to dry for 24 h at RT, then its weight was determined (the evaluation of fat content) and finally ground to a fine powder.
Reagents All chemicals were of analytical grade. Concentrated HNO3 (Merck, Darmstadt, Germany), HCl (POCh, Gliwice, Poland) and H2O2 (POCh) solutions, and tetramethyl ammonium hydroxide (TMAH) (Sigma-Aldrich, St Louis, MO, USA) were used for the sample preparation. Aqua regia was prepared by mixing 3:1 (v/v) concentrated
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HCl and HNO3 solutions. Petroleum ether (POCh) was used to remove fat from nuts. Working standard solutions were prepared by dilutions of a multi-element (1000 µg ml−1) ICP standard (Merck). The 1000 μg ml−1 solutions of Se(IV) and Se(VI) were prepared from their salts (POCh). The reductant solutions were made daily by dissolving NaBH4 (Sigma-Aldrich) powder in 0.1 mol l−1 NaOH (POCh) and filtering before being used. A 30% aqueous emulsion of Anti foam A (Sigma-Aldrich) was used as the anti-foaming agent during hydride generation. Deionised water (18.3 MΩ cm) was obtained from an EASYpure™ water purification system (Barnstead, Thermolyne Corporation, Dubuque, IA, USA). Glassware and plastic bottles were cleaned with a 10% (m/v) HNO3 solution and rinsed several times with deionised water. Sample preparation procedures Defatted nuts were brought into solution as follows: ● Microwave(MW)-assisted digestion (P1): about 0.5 g of nut samples were subjected to microwave heating with a maximum power of 600 W for 45 min using concentrated HNO3 + H2O2 (6 + 1 ml). After cooling, residual solutions were quantitatively transferred into 25-ml volumetric flasks and made up to the volume with deionised water. The above procedure was also applied for the decomposition of the certified reference material (TORT-2). ● Hot-plate heating digestion (P2): about 0.5 g of nut samples were placed into 150-ml Pyrex beakers and 20 ml of a concentrated HNO3 solution were added. The beakers were covered with watch glasses and left for pre-digestion overnight. After this time the sample solutions were heated on hot plate at 85°C and then portions of acid were added until all fumes of nitrogen oxides were ceased. They were then cooled and 5 ml of a 30% (v/v) H2O2 solution were added. The heating was continued until clear solutions were obtained. The resulting sample solutions were reduced to about 2 ml, quantitatively transferred into 25-ml volumetric flasks and made up to the volume with deionised water. ● Solubilisation in aqua regia (P3): about 0.25 g of nut samples were weighed into 30-ml polypropylene (PP) centrifuge tubes, then 2 ml of aqua regia were added and left for pre-digestion. Next, the resulting mixtures were sonicated in an ultrasonic bath for 15 min, then made up to 25 ml with deionised water and finally centrifuged for 10 min at 12 000 rpm. ● Solubilisation in TMAH (P4): about 0.25 g of nut samples were weighed into 30-ml PP centrifuge tubes, then 1.0 ml of a 25% (m/v) TMAH aqueous
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M. Welna and A. Szymczycha-Madeja solution was added and pre-digested. After this time the tubes were shaken at 80°C for 30 min, cooled, made up to 25 ml with deionised water and then centrifuged for 10 min at 12 000 rpm.
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Until analysis, sample solutions were kept at 4°C. Three parallel samples were prepared and analysed (n = 3). With each set of sample solutions, procedural blank solutions were prepared and subjected to all sample treatments to correct the final results.
Pretreatment for Se determination Se was determined in a form of hydride based on the reaction between Se(IV) and NaBH4 in acidic medium. To ensure quantification of Se, any Se(VI) was prereduced to Se(IV) with 6 mol l−1 HCl by heating in a water bath at 90°C for 30 min.
Apparatus An inductively coupled plasma sequential spectrometer JY 38S (Jobin Yvon, Longjumeau, France) was used to measure total concentrations of Ba, Ca, Cd, Cr, Cu, Fe, Mg, Mn, Ni, P, Pb, Sr and Zn. The operating parameters were as follows: 1.0 kW of a RF power, 13 l min−1 of a plasma flow rate, 0.2 l min−1 of a sheath-gas flow rate and 0.25 l min−1 of a carrier-gas flow rate. Sample solutions were introduced into the plasma using a parallel nebuliser (Burgener) and a cyclonic spray chamber at a flow rate of 0.75 ml min−1. Analytical lines of Ba 233.5 nm, Ca 317.9 nm, Cd 228.8 nm, Cr 267.7 nm, Cu 324.7 nm, Fe 259.9 nm, Mg 285.2 nm, Mn 259.4 nm, Ni 221.6 nm, P 214.6 nm, Pb 220.3 nm, Se 196.1 nm, Sr 407.8 nm and Zn 213.9 nm were measured. Se measurements were performed by means of a hydride generation ICP-OES hyphenated system with a gas–liquid phase separation (Welna et al. 2011). The same ICP operating condition was used. In the manifold, an acidified sample and a reductant solutions were introduced at a 1.0 ml min−1 flow rate. ICP-OES measurements of samples were interpolated in the external calibration obtained from hydrochloric solutions of Se(IV). A Milestone (Sorisole, BG, Italy) high-pressure microwave digestion system (MLS-1200 MEGA), equipped with a rotor MDR 300/10, was used for the microwaveassisted sample decomposition. An Elpin (Katowice, Poland) thermostatic water bath shaker (type 357) and a JP Selecta (Barcelona, Spain) ultrasonic bath (UltrasonsH, Barcelona, Spain) were used for experiments with aqua regia and TMAH, fat separation and pretreatment for Se determination. A MPW-350 centrifuge (MPW Med. Instruments, Warsaw, Poland) was used to accelerate the separation of liquid phases from solid particles.
Results and discussion Optimisation of the selenium hydride generation procedure The concentration of Se in the nut samples was too low to be measured by pneumatic nebulisation ICP-OES. Therefore, hydride generation was applied for this purpose. Since Se(VI) cannot be reduced easily by NaBH4 into H2Se, firstly the condition for the quantitative prereduction of Se(VI) to Se(IV) with HCl was established. A 1% (m/v) NaBH4 was used. The effects of temperature, time and concentration of HCl were examined. The effect of NaBH4 concentration was then also studied. Both Se (IV) and Se(VI) standards were simultaneously prepared and measured. Three emission lines of Se (196.1, 203.9, 206.3 nm) were tested. The 196.1 nm was chosen because it gave the best results (it was the least noisy and had the best intensity). Concentrations of HCl varied between 1 and 6 mol l−1, pre-reduction time and temperature were from 15–60 min and 30–90°C, respectively. Se emission from Se(IV) increased with acidity to reach a steady signal above 3 mol l−1 HCl, hence it was taken as a reference. Quantitative reduction of Se(VI) was achieved by heating with 6 mol l−1 HCl at 90°C for 30 min. Solutions of NaBH4 in the range of 0.25–1.5% (m/v) (in 0.1 mol l−1 NaOH) applying 6 mol l−1 HCl were considered. To hinder intensive foam formation, anti-foaming agent was added to the reducing agent solution. The signal of Se increased continuously up to 1.5% (m/v) NaBH4, however higher NaBH4 concentrations than 1.0% (m/v) make the plasma unstable. Consequently, 1.0% (m/v) NaBH4 was used in further studies.
Comparison of sample preparation procedures for defatted Brazil nut samples Among the digestion procedures reported for plant matrices, including nuts, the MW-assisted digestion in HNO3/H2O2 is usually suggested. To facilitate decomposition of defatted Brazil nut samples, an alternative (P3, P4) to acid-based methods (P1, P2) was also examined and its analytical characteristic was compared. RSD was used to express the precision. Accuracy was evaluated by comparing total concentrations of elements obtained using different sample preparation procedures (P2, P3, P4) for defatted nut samples with those obtained using the microwave-assisted wet digestion procedure (P1) (reference method) and by analysis of the certified reference material (TORT-2). Additionally, the recovery study of real sample analysis (using a standard addition method) was carried out. LODs were determined using 3σ criterion (3SD of the blank, n = 10).
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Precision The results (average concentrations of elements along with RSDs) are presented in Table 1. The precision for wet digestion procedures (P1, P2) was close and ranged between 0.9% and 6.6% (P1) and between 1.6% and 7.6% (P2). Only for Cr was a higher RSD obtained (8.9%) for P1, probably due to the very low concentration of this element. Notably, Cr was not determined after the hot-plate heating procedure (P2). Meaningful differences in the precision of Ba and Sr were obtained if the hot-plate digestion (P2) was used. Accordingly, much higher RSDs for these elements were obtained. It indicates that digestion of Brazil nut samples in open vessels in an HNO3/ H2O2 mixture may be insufficient for a complete release of Ba and Sr into solution. Considering alternative methods of sample preparation, it can be seen that the solubilisation in aqua regia (P3) provides the best precision ranging between 1.0% and 3.9% for practically all elements. It was nearly twice as good as the results obtained with both wet digestions; the only exception was Cr (6.5% as RSD) for the same reason as for procedure P1. Unfortunately, the precision obtained for the solubilisation in TMAH (P4) was from two to seven times lower than that achieved with the aqua regia procedure (P3). Accordingly, RSDs up to 11% were obtained.
Limits of detection LODs of elements achieved are detailed in Table 2. As it can be seen the detectability of elements using both wet acid digestion procedures (P1, P2) and the solubilisation in aqua regia (P3) was better than that obtained using the solubilisation in TMAH (P4). Acidic digestions (P1, P2)
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show comparable LODs, with the exception of Ca, Cd, Cr, P and Se for which LODs are higher for the hot-plate heating (P2). Satisfactorily, the best LODs were obtained using the solubilisation in aqua regia (P3). In contrast, LODs for Ba, Ca, Mn, Ni, Se and Zn resulted from the use of TMAH (P4) were twice as high.
Accuracy Comparing concentrations of elements determined in the Brazil nuts samples (Table 1), it can be seen that the results achieved after the microwave-assisted digestion (P1) and the solubilisation in aqua regia (P3) are Table 2. LODs (ng ml−1) of elements for ICP-OES measurements with different sample preparation procedures: microwaveassisted digestion in HNO3 with H2O2 (P1), hot-plate digestion in HNO3 with H2O2 (P2), solubilisation in aqua regia (P3) and TMAH (P4). Element Ba Ca Cd Cr Cu Fe Mg Mn Ni P Pb Se Sr Zn
P1
P2
P3
P4
0.99 6.88 1.47 1.04 1.29 1.86 3.72 0.46 2.85 45.9 31.6 2.6 0.16 1.07
1.22 12.6 2.45 1.41 1.45 1.89 3.84 0.53 3.25 96.4 35.6 3.50 0.17 1.39
0.94 7.82 1.44 0.95 0.62 1.70 3.61 0.35 2.17 41.9 31.2 0.60 0.14 0.94
1.90 14.0 1.72 0.73 1.12 1.83 3.88 0.58 3.54 54.4 37.5 3.30 0.18 1.45
Table 1. Concentrations (μg g−1) of elements in samples of defatted Brazil nuts prepared using different sample preparation procedures: microwave-assisted digestion in HNO3 with H2O2 (P1), hot-plate digestion in HNO3 with H2O2 (P2), solubilisation in aqua regia (P3) and TMAH (P4). Concentration (μg g−1)a Element Ba Ca Cd Cr Cu Fe Mg Mn Ni P Pb Se Sr Zn
P1 2209 (6.6) 1534 (4.1) < LODb 0.195 (8.9) 17.0 (5.1) 21.9 (4.1) 2748 (0.90) 8.93 (3.8) 4.75 (1.5) 8333 (3.0) < LODb 1.61 (2.3) 188 (2.0) 39.5 (3.0)
Notes: aAverages (n = 3) and (RSDs). b Below the LOD (see Table 2).
P2 115 (36) 1519 (2.1) < LODb < LODb 18.9 (3.5) 18.6 (1.9) 2731 (1.6) 9.29 (6.7) 3.99 (7.6) 7721 (1.9) < LODb 1.62 (4.1) 80.6 (32) 39.7 (3.1)
P3 2228 (2.5) 1492 (1.6) < LODb 0.190 (6.5) 17.8 (1.5) 21.2 (3.9) 2764 (1.0) 8.91 (1.6) 4.76 (1.3) 8346 (1.7) < LODb 1.67 (3.1) 184 (2.0) 38.9 (2.3)
P4 741 (10.9) 396 (9.2) < LODb < LODb 20.8 (6.8) 12.4 (7.5) 950 (7.2) 4.47 (8.6) 4.62 (2.3) 3420 (5.1) < LODb < LODb 72.5 (6.5) 28.4 (4.3)
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practically the same. According to the t-test (p < 0.05), the differences were statistically insignificant, i.e. calculated values of the t-test (tcalculated) are lower than the critical value (tcritical) equal to 4.303. For two remaining procedures (P2, P4) a lowering in elements contents can be observed. Indeed, the use of solubilisation in TMAH (P4) was found ineffective for Se determination and a possible reason is that it could be retained in the solid particles when recommended pre-reduction step with HCl prior to the Se hydride generation was applied. Such effect was not observed for other procedures (P1–P3). Application of the hot-plate heating digestion (P2) seems to be justified but only in the case of selected elements, i.e. Ca, Cu, Mg, Mn, Ni, P, Se and Zn. For these elements differences between their concentrations with the results obtained using the reference procedure (P1) were statistically insignificant. For elements, i.e. Ba, Cr, Fe and Sr, discrepancies were too high and they were significant for Ba and Sr. Concentrations of Ba and Sr were nearly 20and 2.5-times lower, respectively, than obtained using the MW-assisted digestion (P1). For the solubilisation in TMAH (P4) variances vary from 40% (Zn) to 400% (Ca). Therefore, it can lead to analytical errors during determination of 10 out of 12 elements investigated here. Only for Cu and Ni differences were statistically insignificant. In this work the microwave-assisted acidic digestion (P1) was taken as the reference sample preparation procedure. Its accuracy was confirmed by analysis of a certified reference material (TORT-2). Simultaneously, the CRM analysis was also carried out in order to check the accuracy of the alternative procedures (P3, P4) investigated here. The calculated concentrations of all elements in the reference material (as means ± SDs) obtained using different sample processing are given in Table 3. The t-test was used to verify whether any statistically significant differences occurred between the experimental and certified values, assuming a 95% confidence level. Importantly, before the application of the t-test, the F-test was used to compare the SDs, because two means can be compared using the t-test only if irrelevant differences in variance for comparing sets of results (F-test) are present. As can be seen, for the MW digestion procedure (P1) a good agreement between the measured and certified values for all elements was obtained, in practice, i.e. calculated values the t-test (tcalculated) for experimental data are lower than 4.303 (tcritical). The only exception was Pb, where the concentration in the resulting sample solution was too low to be measured. Similarly, using the solubilisation in aqua regia (P3), differences between the results for all elements were statistically insignificant (tcalculated < tcritical). Moreover, the solubilisation in aqua regia (P3) was the only procedure that was adequate to determine Pb in the CRM. Unfortunately, application of the solubilisation in TMAH (P4) seems to justified, however only in case of Cu Ni and Zn (tcalculated < tcritical). For elements, i.e.
Cd, Ni and Zn discrepancies were statistically significant (tcalculated > tcritical) and for Cu, Fe, Mn and Sr the t-test was even not allowed to be used (F > Fcritical). Contents of remaining elements (Cr, Pb and Se) were below their respective LODs. The reason for difficulties during Se measurement may be the same as discussed previously when TMAH was used (formation of solid residue after pre-reduction step with HCl). Additionally, the accuracy of both alternative procedures (P3, P4) was also estimated by the recovery test. For this purpose, Brazil nut samples were spiked with a multielement standard (0.5 and 1.0 μg ml−1) and subjected to the sample preparation procedures under comparison with assess recoveries of elements added. The additions were 0.25 and 0.5 μg ml−1 for the Se determination. It was established (Table 4) that independently of the spike level, the solubilisation in aqua regia (P3) provides practically quantitatively recoveries for all elements studied (96.6–103%). Also, for this procedure, slopes of curves from standard additions and external calibrations were comparable, hence it can be concluded that matrix effects are not observed, in practice. Recoveries obtained for the solubilisation in TMAH (P4) (83.7–148%), similarly to the previous results obtained for the CRM and real samples, indicate that it is not suitable procedure for direct ICP-OES measurements of element concentrations in resulting sample solutions. Moreover, the results for Se (0% as recovery) show that it is lost in its entirety. On considering the analytical characteristic of the sample preparation procedures investigated here (in term of precision, accuracy and detection limit), the solubilisation in aqua regia (P3) is the most suitable procedure for the preparation of defatted Brazil nuts prior to their multielement analysis by ICP-OES. Case of Ba, Se and Sr It is well known that Brazil nuts are rich in Se. The surprising results for Ba and Sr obtained (Table 1) lead us to analyse various seed plants, i.e. walnuts, peanuts, cashew nuts and sunflower seeds for the total concentrations of the aforementioned elements. Prior ICP-OES measurement, raw (fat) samples were prepared by means of wet digestion (hot-plate heating) and dry ashing. The latter method was used to ensure complete destruction of the sample matrix. The results (means of three replicates with RSDs in brackets) are detailed in Table 5. Unlike Brazil nuts, the concentrations of Ba and Sr in examined seeds plants were independent of the digestion method used, i.e. application of conventional hot-plate heating leads to the results comparable with those obtained after dry ashing. However, the precision of the determination of elements using dry ashing was poorer by a factor of 2 than that achieved when wet digestion (3.6– 10%) was used, which could be accounted for mainly by
24.9 ± 1.2 0.64 ± 0.08 101 ± 7 109 ± 11 12.1 ± 0.9 2.26 ± 0.36 < LODc 45.0 ± 2.6 192 ± 11 5.28 ± 0.27
5.63 ± 0.67
± ± ± ± ± ± ± ± ±
P1
0.6 0.15 10 13 1.2 0.19 0.13 1.9 6
26.7 0.77 106 105 13.6 2.50 0.35 45.2 180
Notes: aCritical value of the t-test (tcritical): 4.303 (p = 0.05, n = 3). b Not calculated (F > Fcritical). c Below the LOD.
ICP-OES Cd Cr Cu Fe Mn Ni Pb Sr Zn HG-ICP-OES Se
Certified value (μg g−1) ± ± ± ± ± ± ± ± ±
0.3 0.05 4 6 0.2 0.05 0.06 1.0 2
5.12 ± 0.23
26.4 0.83 108 102 13.5 2.47 0.33 45.0 183
P3
Experimental value (μg g−1)
2.598 2.815 1.237 0.630 2.887 1.155 – 0.133 1.890 2.245
< LODc
P1
14.7 ± 0.6 < LODc 46.6 ± 1.9 8.86 ± 0.61 2.01 ± 0.10 2.25 ± 0.11 < LODc 1.23 ± 0.05 173 ± 5
P4
3.841
1.732 2.078 0.866 0.866 0.866 1.039 0.577 0.346 2.598
P3
tcalculateda
–
34.64 – n.c.b n.c.b n.c.b 3.936 – n.c.b 2.425
P4
Table 3. Evaluation of the accuracy of three different sample preparation procedures: microwave-assisted digestion in HNO3 with H2O2 (P1), solubilisation in aqua regia (P3) and TMAH (P4) using the certified reference material (TORT-2).
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the low content of both elements in the analysed samples. Accordingly, RSDs up to 16% were found, respectively. Due to volatility of Se under conditions of dry ashing and in term of the precision of the determination of Ba and Sr, the results from wet digestion were considered.
It was found that the concentration of Se in all analysed nut seeds was below the LOD. The concentrations of Ba of Sr were low and ranged between 0.50 and 5.0 μg g−1 (Ba) and between 1.1 and 3.1 μg g−1 (Sr), respectively. In comparison with the results obtained for Brazil nuts, the content of Ba and Sr in walnuts, peanuts, cashew nuts and sunflower seeds were significantly reduced (two to three orders of magnitude for Ba and one to two orders of magnitude for Sr), while the Se content was too low to be measured (< LOD). This shows that Brazil nuts have much higher total Ba, Sr and Se concentrations among the nuts seeds varieties.
Table 4. Recoveries of the analysed elements in defatted Brazil nuts sample solutions prepared using the solubilisation in aqua regia (P3) and TMAH (P4); n = 3. Recovery (%)
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Element Ba Ca Cd Cr Cu Fe Mg Mn Ni P Pb Sr Zn Se
Added (μg ml−1)
P3
P4
0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.25 0.5
101 99.9 102 99.4 102 99.5 97.2 101 103 99.2 96.6 101 100 99.9 98.9 100 99.2 100 97.3 101 99.7 100 101 99.7 99.3 100 103 99.3
123 94.3 92.6 102 116 96.0 120 94.9 116 96.1 134 91.4 128 92.8 83.7 104 101 97.4 121 87.3 95.6 110 148 87.9 105 98.8 0 0
Effect of lipid removal on total concentrations of elements in Brazil nuts The lipid content of the analysed Brazil nuts was 66.4% ± 2.6%. Our previous work (Welna et al. 2008) found that some elements can be lost during lipid removal from Brazil nut samples. Therefore, in this study the possible loss of analytes during the extraction process was checked. The sample analysis was conducted in the same way as described previously. It was found that about 70% of Cr, 30% of Fe and 20% of Ni were associated with the lipid fraction. All remaining elements were associated with defatted nut residue. Indeed, these results are similar to those reported previously (Welna et al. 2008). Conclusion Various sample preparation procedures (conventional and alternative) of Brazil nuts and their applicability for the multi-elemental analysis by ICP-OES were examined and compared. The digestion of Brazil nuts is not trivial due to complex matrix, mainly its high lipid content. For defatted Brazil nuts, the microwave-assisted digestion in concentrated HNO3/H2O2 (P1) was found to be a reliable procedure based on the total sample decomposition, while the hot-plate heating in a HNO3/H2O2 mixture (P2) is
Table 5. Concentrations (μg g−1) of Ba, Sr and Se in various nuts seeds. Concentration (μg g−1)a Element Ba Sr Se
Cashew nuts
Peanuts
Walnuts
Sunflower seeds
1.08b (5.4) 1.11c (9.2) 1.09b (7.3) 1.23c (5.5) n.d.d < LODe
1.15b (10) 1.17c (16) 2.20b (5.1) 2.32c (12) n.d.d < LODe
5.15b (3.3) 5.06c (11) 3.12b (3.5) 3.14c (8.6) n.d.d < LODe
0.500b (7.7) 0.427c (4.0) 1.37b (6.7) 1.35c (8.0) n.d.d < LODe
Notes: aAverages (n = 3) with (RSDs). b Using hot-plate heating wet digestion in HNO3 with H2O2. c Using dry ashing. d Not detected. Used dry ashing. e Below the LOD. Using hot-plate heating wet digestion in HNO3 with H2O2.
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Food Additives & Contaminants: Part A time- and reagent-consuming and may be insufficient in the case of the determination of such elements as Ba and Sr. Among strategies without the need of previous sample total digestion the method developed here using the solubilisation in aqua regia (P3) can be used prior to the multi-elemental analysis of samples of defatted Brazil nuts. It was found to be reproducible, accurate and matrix-free. Smaller sample and lower reagents amounts can also be used. A much simpler sample handling makes it a viable alternative to wet digestion procedures. Moreover, this procedure is less prone to contamination or to analyte losses, mainly of volatile Se. Unfortunately, the solubilisation of defatted Brazil nuts in TMAH is the least efficient and not applicable in practice. The defatting procedure by solvent extraction before analysis of the Brazil nuts is useful and justified, but cannot be used prior to the determination of Cr, Fe and Ni, which may be partially bonded to lipids. Although the proposed sample treatment (P3) is also effective for these elements, measurements of concentrations of Cr, Fe and Ni in case of Brazil nuts should proceed directly from raw (fat) nut samples. In comparison with other nuts seeds, Brazil nuts contain high contents of Ba, Sr and Se, as demonstrated here. Funding The work was financed by a statutory activity subsidy from the Polish Ministry of Science and Higher Education for the Faculty of Chemistry of Wrocław University of Technology [grant number S30028/Z0302].
References Chunhieng T, Petritis K, Elfakir C, Brochier J, Goli T, Montet D. 2004. Study of selenium distribution in the protein fractions of the Brazil nut, Bertholletia excelsa. J Agric Food Chem. 52:4318–4322. Dumont E, De Pauw L, Vanhaecke F, Cornelis R. 2006. Speciation of Se in bertholletia excelsa (Brazil nut): a hard nut to crack?. Food Chem. 95:684–692. Goncalves AM, Fernandes KG, Ramos LA, Cavalheiro ETG, Nobrega JA. 2009. Determination and fractionation of barium in Brazil nuts. J Braz Chem Soc. 20:760–769.
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Kannamkumarath SS, Wrobel K, Wrobel K, Caruso JA. 2004. Speciation of arsenic in different types of nuts by ion chromatography-inductively coupled plasma mass spectrometry. J Agric Food Chem. 52:1458–1463. Kannamkumarath SS, Wuilloud RG, Caruso JA. 2004. Studies of various elements of nutritional and toxicological interest associated with different molecular weight fractions in Brazil nuts. J Agric Food Chem. 52:5773–5780. Momen AA, Zachariadis GA, Anthemidis AN, Stratis JA. 2007. Use of fractional factorial design for optimization of digestion procedures followed by multi-element determination of essential and non-essential elements in nuts using ICP-OES technique. Talanta. 71:443–451. Moodley R, Kindness A, Jonnalagadda SB. 2007. Elemental composition and chemical characteristics of five edible nuts (almond, Brazil, pecan, macadamia and walnut) consumed in Southern Africa. J Env Sci Health Part B. 42:585–591. Muller AL, Muller CC, Lyra F, Mello PA, Mesko MF, Muller EI, Flores EMM. 2013. Determination of toxic elements in nuts by inductively coupled plasma mass spectrometry after microwave-induced combustion. Food Anal Meth. 6:258–264. Naozuka J, Marana SR, Oliveira PV. 2010. Water-soluble Cu, Fe, Mn and Zn species in nuts and seeds. J Food Comp Anal. 23:78–85. Parekh PP, Khan AR, Torres MA, Kitto ME. 2008. Concentrations of selenium, barium, and radium in Brazil nuts. J Food Comp Anal. 21:332–335. Rodushkin I, Engstrom E, Sorlin D, Baxter D. 2008. Levels of inorganic constituents in raw nuts and seeds on the Swedish market. Sci. Total Environ. 392:290–304. Vonderheide AP, Wrobel K, Kannamkumarath SS, B’Hymer C, Montes-Bayon M, Ponce De Leon C, Caruso JA. 2002. Characterization of selenium species in Brazil nuts by HPLCICP-MS and ES-MS. J Agric Food Chem. 50:5722–5728. Welna M, Klimpel M, Zyrnicki W. 2008. Investigation of major and trace elements and their distributions between lipid and non-lipid fractions in Brazil nuts by inductively coupled plasma atomic optical spectrometry. Food Chem. 111:1012–1015. Welna M, Lasowska J, Zyrnicki W. 2011. Determination of some inorganic species of Fe, Mn and Cr by chemical vapor generation hyphenated with inductively coupled plasma atomic emission spectrometry. J Braz Chem Soc. 22:1164–1169. Wrobel K, Kannamkumarath SS, Wrobel K, Caruso JA. 2003. Hydrolysis of proteins with methanesulfonic acid for improved HPLC-ICP-MS determination of seleno-methionine in yeast and nuts. Anal Bioanal Chem. 375:133–138. Yang J. 2009. Brazil nuts and associated health benefits: a review. LWT-Food Sci Technol. 42:1573–1580.
Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Improvement of a sample preparation procedure for multi-elemental determination in Brazil nuts by ICPOES a
a
Maja Welna & Anna Szymczycha-Madeja a
Analytical Chemistry Division, Chemistry Department, Wrocław University of Technology, Wrocław, Poland Accepted author version posted online: 03 Jan 2014.Published online: 04 Mar 2014.
Click for updates To cite this article: Maja Welna & Anna Szymczycha-Madeja (2014) Improvement of a sample preparation procedure for multi-elemental determination in Brazil nuts by ICP-OES, Food Additives & Contaminants: Part A, 31:4, 658-665, DOI: 10.1080/19440049.2014.880134 To link to this article: http://dx.doi.org/10.1080/19440049.2014.880134
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Food Additives & Contaminants: Part A, 2014 Vol. 31, No. 4, 658–665, http://dx.doi.org/10.1080/19440049.2014.880134
Improvement of a sample preparation procedure for multi-elemental determination in Brazil nuts by ICP-OES Maja Welna* and Anna Szymczycha-Madeja Analytical Chemistry Division, Chemistry Department, Wrocław University of Technology, Wrocław, Poland
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(Received 1 October 2013; accepted 26 December 2013) Various sample preparation procedures, such as common wet digestions and alternatives based on solubilisation in aqua regia or tetramethyl ammonium hydroxide, were compared for the determination of the total Ba, Ca, Cr, Cd, Cu, Fe, Mg, Mn, Ni, P, Pb, Se, Sr and Zn contents in Brazil nuts using inductively coupled plasma optical emission spectrometry (ICP-OES). For measurement of Se, a hydride generation technique was used. The performance of these procedures was measured in terms of precision, accuracy and limits of detection of the elements. It was found that solubilisation in aqua regia gave the best results, i.e. limits of detection from 0.60 to 41.9 ng ml−1, precision of 1.0–3.9% and accuracy better than 5%. External calibration with simple standard solutions could be applied for the analysis. The proposed procedure is simple, reduces sample handling, and minimises the time and reagent consumption. Thus, this can be a vital alternative to traditional sample treatment approaches based on the total digestion with concentrated reagents. A phenomenon resulting from levels of Ba, Se and Sr in Brazil nuts was also discussed. Keywords: Brazil nuts; sample preparation; minerals; ICP-OES; barium
Introduction Nowadays knowledge about the elemental content of food products is essential in order to define their quality. Nuts are considered one of the most nutritious human foods due to their high amounts of proteins, carbohydrates, unsaturated lipids, vitamins and essential elements. Among a wide variety of nuts, Brazil nuts (Bertholletia excelsa) are worthy of special interest due to their nutritive value as well as some health benefits. The popularity of Brazil nuts is associated with an exceptionally high Se level. A review devoted to the composition and properties of these nuts was presented recently by Yang (2009). On the other hand, surprisingly, Brazil nuts can also accumulate significant amounts of toxic (Ba) and carcinogen (Ra) elements (Parekh et al. 2008; Goncalves et al. 2009). The significance of Se means that all the effort put into the analysis of Brazil nuts is mostly concerned with the determination of Se and Se species (e.g. Vonderheide et al. 2002; Wrobel et al. 2003; Chunhieng et al. 2004; Dumont et al. 2006). Information about their mineral composition, i.e. macro-, micro- and trace elements, can be found only in a few publications (Moodley et al. 2007; Rodushkin et al. 2008; Welna et al. 2008). The assessment of the total concentrations of elements in Brazil nuts is primary performed using spectrochemical methods such as atomic absorption spectrometry (AAS) (Naozuka et al. 2010), inductively coupled plasma optical emission spectrometry (ICP-OES) (Chunhieng et al. 2004; Moodley et al. 2007; Welna et al. 2008), and inductively *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
coupled plasma mass spectrometry (ICP-MS) (Kannamkumarath, Wrobel, et al. 2004; Kannamkumarath, Wuilloud, et al. 2004; Rodushkin et al. 2008). Normally, prior to the atomic spectrometry measurements, samples have to be initially prepared, i.e. brought into the solution by means of the total digestion in oxidative reagents. In this sense, the analysis of Brazil nuts is not an easy task due to complex composition, dominated by high lipid content (> 60%). In addition to lipids, Brazil nuts contain 10–20% proteins and carbohydrates (Yang 2009). Typically, lipids, which are troublesome during sample preparation, are often removed before analysis by solvent extraction. At present, wet digestion in pressurised closed vessel microwave-assisted systems (Kannamkumarath, Wrobel, et al. 2004; Kannamkumarath, Wuilloud, et al. 2004; Moodley et al. 2007; Rodushkin et al. 2008; Goncalves et al. 2009; Naozuka et al. 2010) is preferred. Generally, HNO3 alone (Kannamkumarath, Wrobel, et al. 2004; Kannamkumarath, Wuilloud, et al. 2004; Moodley et al. 2007) as well as its mixtures with H2O2 (Goncalves et al. 2009; Naozuka et al. 2010) is used for sample digestion. The dry ashing at the step of Brazil nuts sample decomposition has also been used (Chunhieng et al. 2004). Unfortunately, although excellent and effective, total wet digestion is time-consuming and requires special tools and equipments, hazardous reagents or laborious procedures. Hence, it is worth examining whether there are simpler alternative approaches of sample preparation that can considerably diminish the time of sample treatment
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Food Additives & Contaminants: Part A and eliminate all inconveniences related to sample digestion. To the best of our knowledge, an alternative to acidbased procedures for Brazil nut sample preparation prior to their multi-element analysis by ICP-OES has not been developed and used. So far, common digestion procedures for different types of nuts have been investigated. Accordingly, Momen et al. (2007) tested on nut samples (peanut, almond, hazelnut, walnut) two wet digestion procedures, i.e. the conventional (hot-plate) heating in HNO3/ H2SO4 and HNO3/H2SO4/H2O2. The proposed procedure, i.e. digestion in the presence of HNO3/H2SO4/H2O2, appeared to be valid for ICP-OES measurements of total concentrations of Al, Ba, Cd, Cr, Cu, Fe, Mg, Mn, Pb and Zn. The optimisation of the sample preparation procedure based on microwave-assisted digestion in HNO3/HF of different nuts (including Brazil nuts) and seeds followed by multi-elemental analysis using ICP-MS was evaluated by Rodushkin et al. (2008). Very recently, Muller et al. (2013) improved the digestion of samples using microwave-induced combustion (MIC) to determine toxic (As, Cd, Hg and Pb) elements in nuts (hazelnuts, almonds, cashew nuts, Brazil nuts and walnuts) by ICP-MS. The aim of this work was to compare various sample preparation procedures and to develop a precise and accurate method of the multi-elemental analysis of Brazil nuts by ICP-OES. According to our knowledge, this work reports for the first time the results of a comparison of several preparation procedures of Brazil nuts, including common and alternative, prior to their analysis by ICP-OES on the content of 14 elements, i.e. Ba, Ca, Cd, Cr, Cu, Fe, Mg, Mn, Ni, P, Pb, Se, Sr and Zn.
Materials and methods Samples Unshelled Brazil nuts were purchased in a local market in Wroclaw, Poland. Before analysis nuts were peeled, grated and mixed. Prior to sampling, lipids were removed from the ground nuts by solvent extraction. Accordingly, a portion of about 25 g of nuts was mixed with 125 ml of petroleum ether and shaken in an ultrasonic bath for 15 min. The extraction procedure was repeated three times. The remaining nut residue was left to dry for 24 h at RT, then its weight was determined (the evaluation of fat content) and finally ground to a fine powder.
Reagents All chemicals were of analytical grade. Concentrated HNO3 (Merck, Darmstadt, Germany), HCl (POCh, Gliwice, Poland) and H2O2 (POCh) solutions, and tetramethyl ammonium hydroxide (TMAH) (Sigma-Aldrich, St Louis, MO, USA) were used for the sample preparation. Aqua regia was prepared by mixing 3:1 (v/v) concentrated
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HCl and HNO3 solutions. Petroleum ether (POCh) was used to remove fat from nuts. Working standard solutions were prepared by dilutions of a multi-element (1000 µg ml−1) ICP standard (Merck). The 1000 μg ml−1 solutions of Se(IV) and Se(VI) were prepared from their salts (POCh). The reductant solutions were made daily by dissolving NaBH4 (Sigma-Aldrich) powder in 0.1 mol l−1 NaOH (POCh) and filtering before being used. A 30% aqueous emulsion of Anti foam A (Sigma-Aldrich) was used as the anti-foaming agent during hydride generation. Deionised water (18.3 MΩ cm) was obtained from an EASYpure™ water purification system (Barnstead, Thermolyne Corporation, Dubuque, IA, USA). Glassware and plastic bottles were cleaned with a 10% (m/v) HNO3 solution and rinsed several times with deionised water. Sample preparation procedures Defatted nuts were brought into solution as follows: ● Microwave(MW)-assisted digestion (P1): about 0.5 g of nut samples were subjected to microwave heating with a maximum power of 600 W for 45 min using concentrated HNO3 + H2O2 (6 + 1 ml). After cooling, residual solutions were quantitatively transferred into 25-ml volumetric flasks and made up to the volume with deionised water. The above procedure was also applied for the decomposition of the certified reference material (TORT-2). ● Hot-plate heating digestion (P2): about 0.5 g of nut samples were placed into 150-ml Pyrex beakers and 20 ml of a concentrated HNO3 solution were added. The beakers were covered with watch glasses and left for pre-digestion overnight. After this time the sample solutions were heated on hot plate at 85°C and then portions of acid were added until all fumes of nitrogen oxides were ceased. They were then cooled and 5 ml of a 30% (v/v) H2O2 solution were added. The heating was continued until clear solutions were obtained. The resulting sample solutions were reduced to about 2 ml, quantitatively transferred into 25-ml volumetric flasks and made up to the volume with deionised water. ● Solubilisation in aqua regia (P3): about 0.25 g of nut samples were weighed into 30-ml polypropylene (PP) centrifuge tubes, then 2 ml of aqua regia were added and left for pre-digestion. Next, the resulting mixtures were sonicated in an ultrasonic bath for 15 min, then made up to 25 ml with deionised water and finally centrifuged for 10 min at 12 000 rpm. ● Solubilisation in TMAH (P4): about 0.25 g of nut samples were weighed into 30-ml PP centrifuge tubes, then 1.0 ml of a 25% (m/v) TMAH aqueous
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M. Welna and A. Szymczycha-Madeja solution was added and pre-digested. After this time the tubes were shaken at 80°C for 30 min, cooled, made up to 25 ml with deionised water and then centrifuged for 10 min at 12 000 rpm.
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Until analysis, sample solutions were kept at 4°C. Three parallel samples were prepared and analysed (n = 3). With each set of sample solutions, procedural blank solutions were prepared and subjected to all sample treatments to correct the final results.
Pretreatment for Se determination Se was determined in a form of hydride based on the reaction between Se(IV) and NaBH4 in acidic medium. To ensure quantification of Se, any Se(VI) was prereduced to Se(IV) with 6 mol l−1 HCl by heating in a water bath at 90°C for 30 min.
Apparatus An inductively coupled plasma sequential spectrometer JY 38S (Jobin Yvon, Longjumeau, France) was used to measure total concentrations of Ba, Ca, Cd, Cr, Cu, Fe, Mg, Mn, Ni, P, Pb, Sr and Zn. The operating parameters were as follows: 1.0 kW of a RF power, 13 l min−1 of a plasma flow rate, 0.2 l min−1 of a sheath-gas flow rate and 0.25 l min−1 of a carrier-gas flow rate. Sample solutions were introduced into the plasma using a parallel nebuliser (Burgener) and a cyclonic spray chamber at a flow rate of 0.75 ml min−1. Analytical lines of Ba 233.5 nm, Ca 317.9 nm, Cd 228.8 nm, Cr 267.7 nm, Cu 324.7 nm, Fe 259.9 nm, Mg 285.2 nm, Mn 259.4 nm, Ni 221.6 nm, P 214.6 nm, Pb 220.3 nm, Se 196.1 nm, Sr 407.8 nm and Zn 213.9 nm were measured. Se measurements were performed by means of a hydride generation ICP-OES hyphenated system with a gas–liquid phase separation (Welna et al. 2011). The same ICP operating condition was used. In the manifold, an acidified sample and a reductant solutions were introduced at a 1.0 ml min−1 flow rate. ICP-OES measurements of samples were interpolated in the external calibration obtained from hydrochloric solutions of Se(IV). A Milestone (Sorisole, BG, Italy) high-pressure microwave digestion system (MLS-1200 MEGA), equipped with a rotor MDR 300/10, was used for the microwaveassisted sample decomposition. An Elpin (Katowice, Poland) thermostatic water bath shaker (type 357) and a JP Selecta (Barcelona, Spain) ultrasonic bath (UltrasonsH, Barcelona, Spain) were used for experiments with aqua regia and TMAH, fat separation and pretreatment for Se determination. A MPW-350 centrifuge (MPW Med. Instruments, Warsaw, Poland) was used to accelerate the separation of liquid phases from solid particles.
Results and discussion Optimisation of the selenium hydride generation procedure The concentration of Se in the nut samples was too low to be measured by pneumatic nebulisation ICP-OES. Therefore, hydride generation was applied for this purpose. Since Se(VI) cannot be reduced easily by NaBH4 into H2Se, firstly the condition for the quantitative prereduction of Se(VI) to Se(IV) with HCl was established. A 1% (m/v) NaBH4 was used. The effects of temperature, time and concentration of HCl were examined. The effect of NaBH4 concentration was then also studied. Both Se (IV) and Se(VI) standards were simultaneously prepared and measured. Three emission lines of Se (196.1, 203.9, 206.3 nm) were tested. The 196.1 nm was chosen because it gave the best results (it was the least noisy and had the best intensity). Concentrations of HCl varied between 1 and 6 mol l−1, pre-reduction time and temperature were from 15–60 min and 30–90°C, respectively. Se emission from Se(IV) increased with acidity to reach a steady signal above 3 mol l−1 HCl, hence it was taken as a reference. Quantitative reduction of Se(VI) was achieved by heating with 6 mol l−1 HCl at 90°C for 30 min. Solutions of NaBH4 in the range of 0.25–1.5% (m/v) (in 0.1 mol l−1 NaOH) applying 6 mol l−1 HCl were considered. To hinder intensive foam formation, anti-foaming agent was added to the reducing agent solution. The signal of Se increased continuously up to 1.5% (m/v) NaBH4, however higher NaBH4 concentrations than 1.0% (m/v) make the plasma unstable. Consequently, 1.0% (m/v) NaBH4 was used in further studies.
Comparison of sample preparation procedures for defatted Brazil nut samples Among the digestion procedures reported for plant matrices, including nuts, the MW-assisted digestion in HNO3/H2O2 is usually suggested. To facilitate decomposition of defatted Brazil nut samples, an alternative (P3, P4) to acid-based methods (P1, P2) was also examined and its analytical characteristic was compared. RSD was used to express the precision. Accuracy was evaluated by comparing total concentrations of elements obtained using different sample preparation procedures (P2, P3, P4) for defatted nut samples with those obtained using the microwave-assisted wet digestion procedure (P1) (reference method) and by analysis of the certified reference material (TORT-2). Additionally, the recovery study of real sample analysis (using a standard addition method) was carried out. LODs were determined using 3σ criterion (3SD of the blank, n = 10).
Food Additives & Contaminants: Part A
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Precision The results (average concentrations of elements along with RSDs) are presented in Table 1. The precision for wet digestion procedures (P1, P2) was close and ranged between 0.9% and 6.6% (P1) and between 1.6% and 7.6% (P2). Only for Cr was a higher RSD obtained (8.9%) for P1, probably due to the very low concentration of this element. Notably, Cr was not determined after the hot-plate heating procedure (P2). Meaningful differences in the precision of Ba and Sr were obtained if the hot-plate digestion (P2) was used. Accordingly, much higher RSDs for these elements were obtained. It indicates that digestion of Brazil nut samples in open vessels in an HNO3/ H2O2 mixture may be insufficient for a complete release of Ba and Sr into solution. Considering alternative methods of sample preparation, it can be seen that the solubilisation in aqua regia (P3) provides the best precision ranging between 1.0% and 3.9% for practically all elements. It was nearly twice as good as the results obtained with both wet digestions; the only exception was Cr (6.5% as RSD) for the same reason as for procedure P1. Unfortunately, the precision obtained for the solubilisation in TMAH (P4) was from two to seven times lower than that achieved with the aqua regia procedure (P3). Accordingly, RSDs up to 11% were obtained.
Limits of detection LODs of elements achieved are detailed in Table 2. As it can be seen the detectability of elements using both wet acid digestion procedures (P1, P2) and the solubilisation in aqua regia (P3) was better than that obtained using the solubilisation in TMAH (P4). Acidic digestions (P1, P2)
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show comparable LODs, with the exception of Ca, Cd, Cr, P and Se for which LODs are higher for the hot-plate heating (P2). Satisfactorily, the best LODs were obtained using the solubilisation in aqua regia (P3). In contrast, LODs for Ba, Ca, Mn, Ni, Se and Zn resulted from the use of TMAH (P4) were twice as high.
Accuracy Comparing concentrations of elements determined in the Brazil nuts samples (Table 1), it can be seen that the results achieved after the microwave-assisted digestion (P1) and the solubilisation in aqua regia (P3) are Table 2. LODs (ng ml−1) of elements for ICP-OES measurements with different sample preparation procedures: microwaveassisted digestion in HNO3 with H2O2 (P1), hot-plate digestion in HNO3 with H2O2 (P2), solubilisation in aqua regia (P3) and TMAH (P4). Element Ba Ca Cd Cr Cu Fe Mg Mn Ni P Pb Se Sr Zn
P1
P2
P3
P4
0.99 6.88 1.47 1.04 1.29 1.86 3.72 0.46 2.85 45.9 31.6 2.6 0.16 1.07
1.22 12.6 2.45 1.41 1.45 1.89 3.84 0.53 3.25 96.4 35.6 3.50 0.17 1.39
0.94 7.82 1.44 0.95 0.62 1.70 3.61 0.35 2.17 41.9 31.2 0.60 0.14 0.94
1.90 14.0 1.72 0.73 1.12 1.83 3.88 0.58 3.54 54.4 37.5 3.30 0.18 1.45
Table 1. Concentrations (μg g−1) of elements in samples of defatted Brazil nuts prepared using different sample preparation procedures: microwave-assisted digestion in HNO3 with H2O2 (P1), hot-plate digestion in HNO3 with H2O2 (P2), solubilisation in aqua regia (P3) and TMAH (P4). Concentration (μg g−1)a Element Ba Ca Cd Cr Cu Fe Mg Mn Ni P Pb Se Sr Zn
P1 2209 (6.6) 1534 (4.1) < LODb 0.195 (8.9) 17.0 (5.1) 21.9 (4.1) 2748 (0.90) 8.93 (3.8) 4.75 (1.5) 8333 (3.0) < LODb 1.61 (2.3) 188 (2.0) 39.5 (3.0)
Notes: aAverages (n = 3) and (RSDs). b Below the LOD (see Table 2).
P2 115 (36) 1519 (2.1) < LODb < LODb 18.9 (3.5) 18.6 (1.9) 2731 (1.6) 9.29 (6.7) 3.99 (7.6) 7721 (1.9) < LODb 1.62 (4.1) 80.6 (32) 39.7 (3.1)
P3 2228 (2.5) 1492 (1.6) < LODb 0.190 (6.5) 17.8 (1.5) 21.2 (3.9) 2764 (1.0) 8.91 (1.6) 4.76 (1.3) 8346 (1.7) < LODb 1.67 (3.1) 184 (2.0) 38.9 (2.3)
P4 741 (10.9) 396 (9.2) < LODb < LODb 20.8 (6.8) 12.4 (7.5) 950 (7.2) 4.47 (8.6) 4.62 (2.3) 3420 (5.1) < LODb < LODb 72.5 (6.5) 28.4 (4.3)
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practically the same. According to the t-test (p < 0.05), the differences were statistically insignificant, i.e. calculated values of the t-test (tcalculated) are lower than the critical value (tcritical) equal to 4.303. For two remaining procedures (P2, P4) a lowering in elements contents can be observed. Indeed, the use of solubilisation in TMAH (P4) was found ineffective for Se determination and a possible reason is that it could be retained in the solid particles when recommended pre-reduction step with HCl prior to the Se hydride generation was applied. Such effect was not observed for other procedures (P1–P3). Application of the hot-plate heating digestion (P2) seems to be justified but only in the case of selected elements, i.e. Ca, Cu, Mg, Mn, Ni, P, Se and Zn. For these elements differences between their concentrations with the results obtained using the reference procedure (P1) were statistically insignificant. For elements, i.e. Ba, Cr, Fe and Sr, discrepancies were too high and they were significant for Ba and Sr. Concentrations of Ba and Sr were nearly 20and 2.5-times lower, respectively, than obtained using the MW-assisted digestion (P1). For the solubilisation in TMAH (P4) variances vary from 40% (Zn) to 400% (Ca). Therefore, it can lead to analytical errors during determination of 10 out of 12 elements investigated here. Only for Cu and Ni differences were statistically insignificant. In this work the microwave-assisted acidic digestion (P1) was taken as the reference sample preparation procedure. Its accuracy was confirmed by analysis of a certified reference material (TORT-2). Simultaneously, the CRM analysis was also carried out in order to check the accuracy of the alternative procedures (P3, P4) investigated here. The calculated concentrations of all elements in the reference material (as means ± SDs) obtained using different sample processing are given in Table 3. The t-test was used to verify whether any statistically significant differences occurred between the experimental and certified values, assuming a 95% confidence level. Importantly, before the application of the t-test, the F-test was used to compare the SDs, because two means can be compared using the t-test only if irrelevant differences in variance for comparing sets of results (F-test) are present. As can be seen, for the MW digestion procedure (P1) a good agreement between the measured and certified values for all elements was obtained, in practice, i.e. calculated values the t-test (tcalculated) for experimental data are lower than 4.303 (tcritical). The only exception was Pb, where the concentration in the resulting sample solution was too low to be measured. Similarly, using the solubilisation in aqua regia (P3), differences between the results for all elements were statistically insignificant (tcalculated < tcritical). Moreover, the solubilisation in aqua regia (P3) was the only procedure that was adequate to determine Pb in the CRM. Unfortunately, application of the solubilisation in TMAH (P4) seems to justified, however only in case of Cu Ni and Zn (tcalculated < tcritical). For elements, i.e.
Cd, Ni and Zn discrepancies were statistically significant (tcalculated > tcritical) and for Cu, Fe, Mn and Sr the t-test was even not allowed to be used (F > Fcritical). Contents of remaining elements (Cr, Pb and Se) were below their respective LODs. The reason for difficulties during Se measurement may be the same as discussed previously when TMAH was used (formation of solid residue after pre-reduction step with HCl). Additionally, the accuracy of both alternative procedures (P3, P4) was also estimated by the recovery test. For this purpose, Brazil nut samples were spiked with a multielement standard (0.5 and 1.0 μg ml−1) and subjected to the sample preparation procedures under comparison with assess recoveries of elements added. The additions were 0.25 and 0.5 μg ml−1 for the Se determination. It was established (Table 4) that independently of the spike level, the solubilisation in aqua regia (P3) provides practically quantitatively recoveries for all elements studied (96.6–103%). Also, for this procedure, slopes of curves from standard additions and external calibrations were comparable, hence it can be concluded that matrix effects are not observed, in practice. Recoveries obtained for the solubilisation in TMAH (P4) (83.7–148%), similarly to the previous results obtained for the CRM and real samples, indicate that it is not suitable procedure for direct ICP-OES measurements of element concentrations in resulting sample solutions. Moreover, the results for Se (0% as recovery) show that it is lost in its entirety. On considering the analytical characteristic of the sample preparation procedures investigated here (in term of precision, accuracy and detection limit), the solubilisation in aqua regia (P3) is the most suitable procedure for the preparation of defatted Brazil nuts prior to their multielement analysis by ICP-OES. Case of Ba, Se and Sr It is well known that Brazil nuts are rich in Se. The surprising results for Ba and Sr obtained (Table 1) lead us to analyse various seed plants, i.e. walnuts, peanuts, cashew nuts and sunflower seeds for the total concentrations of the aforementioned elements. Prior ICP-OES measurement, raw (fat) samples were prepared by means of wet digestion (hot-plate heating) and dry ashing. The latter method was used to ensure complete destruction of the sample matrix. The results (means of three replicates with RSDs in brackets) are detailed in Table 5. Unlike Brazil nuts, the concentrations of Ba and Sr in examined seeds plants were independent of the digestion method used, i.e. application of conventional hot-plate heating leads to the results comparable with those obtained after dry ashing. However, the precision of the determination of elements using dry ashing was poorer by a factor of 2 than that achieved when wet digestion (3.6– 10%) was used, which could be accounted for mainly by
24.9 ± 1.2 0.64 ± 0.08 101 ± 7 109 ± 11 12.1 ± 0.9 2.26 ± 0.36 < LODc 45.0 ± 2.6 192 ± 11 5.28 ± 0.27
5.63 ± 0.67
± ± ± ± ± ± ± ± ±
P1
0.6 0.15 10 13 1.2 0.19 0.13 1.9 6
26.7 0.77 106 105 13.6 2.50 0.35 45.2 180
Notes: aCritical value of the t-test (tcritical): 4.303 (p = 0.05, n = 3). b Not calculated (F > Fcritical). c Below the LOD.
ICP-OES Cd Cr Cu Fe Mn Ni Pb Sr Zn HG-ICP-OES Se
Certified value (μg g−1) ± ± ± ± ± ± ± ± ±
0.3 0.05 4 6 0.2 0.05 0.06 1.0 2
5.12 ± 0.23
26.4 0.83 108 102 13.5 2.47 0.33 45.0 183
P3
Experimental value (μg g−1)
2.598 2.815 1.237 0.630 2.887 1.155 – 0.133 1.890 2.245
< LODc
P1
14.7 ± 0.6 < LODc 46.6 ± 1.9 8.86 ± 0.61 2.01 ± 0.10 2.25 ± 0.11 < LODc 1.23 ± 0.05 173 ± 5
P4
3.841
1.732 2.078 0.866 0.866 0.866 1.039 0.577 0.346 2.598
P3
tcalculateda
–
34.64 – n.c.b n.c.b n.c.b 3.936 – n.c.b 2.425
P4
Table 3. Evaluation of the accuracy of three different sample preparation procedures: microwave-assisted digestion in HNO3 with H2O2 (P1), solubilisation in aqua regia (P3) and TMAH (P4) using the certified reference material (TORT-2).
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the low content of both elements in the analysed samples. Accordingly, RSDs up to 16% were found, respectively. Due to volatility of Se under conditions of dry ashing and in term of the precision of the determination of Ba and Sr, the results from wet digestion were considered.
It was found that the concentration of Se in all analysed nut seeds was below the LOD. The concentrations of Ba of Sr were low and ranged between 0.50 and 5.0 μg g−1 (Ba) and between 1.1 and 3.1 μg g−1 (Sr), respectively. In comparison with the results obtained for Brazil nuts, the content of Ba and Sr in walnuts, peanuts, cashew nuts and sunflower seeds were significantly reduced (two to three orders of magnitude for Ba and one to two orders of magnitude for Sr), while the Se content was too low to be measured (< LOD). This shows that Brazil nuts have much higher total Ba, Sr and Se concentrations among the nuts seeds varieties.
Table 4. Recoveries of the analysed elements in defatted Brazil nuts sample solutions prepared using the solubilisation in aqua regia (P3) and TMAH (P4); n = 3. Recovery (%)
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Element Ba Ca Cd Cr Cu Fe Mg Mn Ni P Pb Sr Zn Se
Added (μg ml−1)
P3
P4
0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.25 0.5
101 99.9 102 99.4 102 99.5 97.2 101 103 99.2 96.6 101 100 99.9 98.9 100 99.2 100 97.3 101 99.7 100 101 99.7 99.3 100 103 99.3
123 94.3 92.6 102 116 96.0 120 94.9 116 96.1 134 91.4 128 92.8 83.7 104 101 97.4 121 87.3 95.6 110 148 87.9 105 98.8 0 0
Effect of lipid removal on total concentrations of elements in Brazil nuts The lipid content of the analysed Brazil nuts was 66.4% ± 2.6%. Our previous work (Welna et al. 2008) found that some elements can be lost during lipid removal from Brazil nut samples. Therefore, in this study the possible loss of analytes during the extraction process was checked. The sample analysis was conducted in the same way as described previously. It was found that about 70% of Cr, 30% of Fe and 20% of Ni were associated with the lipid fraction. All remaining elements were associated with defatted nut residue. Indeed, these results are similar to those reported previously (Welna et al. 2008). Conclusion Various sample preparation procedures (conventional and alternative) of Brazil nuts and their applicability for the multi-elemental analysis by ICP-OES were examined and compared. The digestion of Brazil nuts is not trivial due to complex matrix, mainly its high lipid content. For defatted Brazil nuts, the microwave-assisted digestion in concentrated HNO3/H2O2 (P1) was found to be a reliable procedure based on the total sample decomposition, while the hot-plate heating in a HNO3/H2O2 mixture (P2) is
Table 5. Concentrations (μg g−1) of Ba, Sr and Se in various nuts seeds. Concentration (μg g−1)a Element Ba Sr Se
Cashew nuts
Peanuts
Walnuts
Sunflower seeds
1.08b (5.4) 1.11c (9.2) 1.09b (7.3) 1.23c (5.5) n.d.d < LODe
1.15b (10) 1.17c (16) 2.20b (5.1) 2.32c (12) n.d.d < LODe
5.15b (3.3) 5.06c (11) 3.12b (3.5) 3.14c (8.6) n.d.d < LODe
0.500b (7.7) 0.427c (4.0) 1.37b (6.7) 1.35c (8.0) n.d.d < LODe
Notes: aAverages (n = 3) with (RSDs). b Using hot-plate heating wet digestion in HNO3 with H2O2. c Using dry ashing. d Not detected. Used dry ashing. e Below the LOD. Using hot-plate heating wet digestion in HNO3 with H2O2.
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Food Additives & Contaminants: Part A time- and reagent-consuming and may be insufficient in the case of the determination of such elements as Ba and Sr. Among strategies without the need of previous sample total digestion the method developed here using the solubilisation in aqua regia (P3) can be used prior to the multi-elemental analysis of samples of defatted Brazil nuts. It was found to be reproducible, accurate and matrix-free. Smaller sample and lower reagents amounts can also be used. A much simpler sample handling makes it a viable alternative to wet digestion procedures. Moreover, this procedure is less prone to contamination or to analyte losses, mainly of volatile Se. Unfortunately, the solubilisation of defatted Brazil nuts in TMAH is the least efficient and not applicable in practice. The defatting procedure by solvent extraction before analysis of the Brazil nuts is useful and justified, but cannot be used prior to the determination of Cr, Fe and Ni, which may be partially bonded to lipids. Although the proposed sample treatment (P3) is also effective for these elements, measurements of concentrations of Cr, Fe and Ni in case of Brazil nuts should proceed directly from raw (fat) nut samples. In comparison with other nuts seeds, Brazil nuts contain high contents of Ba, Sr and Se, as demonstrated here. Funding The work was financed by a statutory activity subsidy from the Polish Ministry of Science and Higher Education for the Faculty of Chemistry of Wrocław University of Technology [grant number S30028/Z0302].
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