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Development and feasibility of emulsion breaking method for the extraction of cadmium from omega-3 dietary supplements and determination by flow injection TS-FF-AAS
Development and feasibility of emulsion breaking method for the extraction of cadmium from omega-3 dietary supplements and determination by flow injection TS-FF-AAS Marcela Zanetti Corazza, C´esar Ricardo Teixeira Tarley PII: DOI: Reference:
S0026-265X(16)00054-0 doi: 10.1016/j.microc.2016.02.021 MICROC 2426
To appear in:
Microchemical Journal
Received date: Revised date: Accepted date:
22 January 2016 27 February 2016 27 February 2016
Please cite this article as: Marcela Zanetti Corazza, C´esar Ricardo Teixeira Tarley, Development and feasibility of emulsion breaking method for the extraction of cadmium from omega-3 dietary supplements and determination by flow injection TS-FF-AAS, Microchemical Journal (2016), doi: 10.1016/j.microc.2016.02.021
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ACCEPTED MANUSCRIPT Development and Feasibility of Emulsion Breaking Method for the Extraction of Cadmium from Omega-3 Dietary Supplements and
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Determination by Flow Injection TS-FF-AAS
Universidade Estadual de Londrina (UEL), Departamento de Química, Centro de Ciências Exatas, Rodovia Celso Garcia Cid, PR 445, Km 380, Londrina, PR, 86050-82, Brazil
Instituto Nacional de Ciência e Tecnologia (INCT) de Bioanalítica, Universidade Estadual de Campinas (UNICAMP), Instituto de Química, Departamento de Química Analítica, Cidade Universitária Zeferino Vaz, s/n, Campinas, SP, 13083-970, Brazil.
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Faculdade de Ciências Exatas e Tecnologia, FACET, Universidade Federal da Grande Dourados (UFGD), CEP 79804-970, Dourados-MS, Brazil.
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Marcela Zanetti Corazza1,2, César Ricardo Teixeira Tarley*2,3
* Corresponding author. Tel +55 43 3371 4366; fax +55 43 3371 4286
E-mail address: [email protected] (C. R. T. Tarley) 1
ACCEPTED MANUSCRIPT ABSTRACT In the present study a new method for determination of Cd2+ in omega-3 dietary supplement
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employing extraction induced by emulsion breaking (EIEB) and Thermospray Flame Furnace
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Atomic Absorption Spectrometry (TS-FF-AAS) was described. The method was based on the
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formation of a water-in-oil emulsion by addition of extractor solution constituted by 3.54% (m/v) Triton X-114 and 1.16 mol L-1 HNO3 in the oil sample and further breaking of this emulsion by heating. Two well-defined phases were formed and the acid aqueous one containing the extracted
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cadmium ions was analyzed by TS-FF-AAS using a flow injection system. Different parameters that exert influence on the extraction efficiency of Cd2+ were optimized by means of chemometric tools.
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The limits of detection and quantification were found to be 2.5 and 8.3 ng g-1, respectively using a calibration curve made in aqueous medium. The accuracy of the proposed method was assured by
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good agreement with the results achieved by microwave-assisted acid digestion procedure without statistical differences (confidence interval of 95%) and by spiking the samples with known
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ng g-1.
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concentrations of 50 ng g-1 Cd2+. The content of Cd2+ in the samples was varied from 37.3 up to 54.5
keywords: Fish oil, Cd, factorial design, atomic absorption spectrometry 2
ACCEPTED MANUSCRIPT 1.
Introduction Fatty acids presents in the majority of the oil matrix samples are organic compounds formed
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by a hydrocarbonated chain and by carboxylic groups that are normally bounded with glycerol forming triacylglycerides classified as mono, di or triglycerides [1]. The nature of hydrocarbonated
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chain defines the fatty acids as saturated or unsaturated compounds, which in turn can be monounsaturated or polyunsatured fatty acids (PUFA) [2]. Omega-3 polyunsaturated fatty acids,
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more specifically the EPA (cis-5, 8,11,14,17 – eicosapentaenoic acid) and DHA (cis-4, 7, 10,13,16,19 – docosahexaenoic acid) are essential fatty acids to the human health and known to exert
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beneficial effects to cardiovascular diseases, reducing blood triacylglycerol levels, blood pressure and to inflammatory process, preventing platelet aggregation [3]. However, the amounts of EPA and
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DHA produced by human body through conversion of α-linoleic acid is very low and this deficiency
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can be remedied by the consumption of natural food products, such as fish, sardine, salmon and
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other alternative sources such as marine microalgae or by the ingestion omega-3 gelatin capsules available commercially [4,5].
Nevertheless, the chemical quality of edible oils play important role in the human health and
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commonly are evaluated by means of concentration of peroxidase, fatty acids composition and by presence of trace metals, since it affect the stability of oil matrix, catalyzing oxidative reactions that results in the formation of toxic products [6-8]. The contamination of heavy metals into oils matrix occurs usually from its extraction process or from outer sources to the production process, such as bleaching, hardening, refining and deodorization. In addition, these samples present high levels of carbon and lipids content and can causes serious spectrometric interferences during the measurements for the most analytes, leading to inaccurate determinations [9,10]. Thus, the determination of trace elements in edible oils samples has become a major challenge in the field of analytical chemistry. Some spectroanalytical techniques, such as Flame Atomic Absorption Spectrometry (FAAS), Electrothermal Atomic Absorption Spectrometry (ETAAS), Inductively 3
ACCEPTED MANUSCRIPT Coupled Plasma Mass Spectrometry (ICP-MS) and Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES), have been very popular techniques for the elemental analysis of oil
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samples, due to their high selectively and suitable sensitivity and precision. However, the reliable
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analysis of oil samples by these techniques requires previous sample treatment to avoid matrix effect
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and to minimize possible interferences [6].
In order to overcome these drawbacks, different sample preparation procedures, among them, microwave-assisted digestion and some extraction methods have been proposed in order to eliminate
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the possible interferences. The microwave-assisted digestion is one of the most common method and
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widely used in the preparation of oil samples for inorganic analysis [11-13]. Nevertheless, this procedure requires a relatively long time of analysis besides to need careful temperature control for avoid loss of volatile metals [14]. Alternatively, extraction methods have been employed due to use
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less aggressive reagents, lower time of analyses and the use of aqueous standards for calibration. The procedures that involve the formation of emulsion and microemulsion have received great attention
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in the last years. Microemulsion, composed by a mixture of oil-water stabilized by means surfactant agent and an organic solvent, presents high stability, facility in its preparation, as well as others
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desirable features for direct measurement, such as low viscosity and high sample throughput. Although the use of microemulsion offers some advantages over procedures that involve dilution with organic solvent and some emulsion systems, since they allow the use of aqueous solutions for calibration instead of expensive organomettalics standards, this method can be applied only when atomic spectrometry techniques are used [14-17]. Similarly, detergent less microemulsion has also been proposed to separate and preconcentrate metals from edible oils followed by ICP-MS detection [18], ICP OES [19] and FAAS [20]. In this case, a homogeneous system containing both the aqueous phase and the liquid organic phase resulting in a homogenous and long-term stable three-component solution is formed when a co-solvent is used [16,21]. Furthermore, the determination of metals at trace levels in oil samples with minimal manipulation and high reliability using microextraction as dispersive
liquid-liquid
microextraction
(DLLME)
and
ultrasound-assisted
single-drop 4
ACCEPTED MANUSCRIPT microextraction (UA-SDME) have gained increasing popularity in recent years [22,23]. The main features of DLLME include the low volume of extractor phase, which in turn makes possible to
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achieve high enrichment factor and the high sample throughput [22, 24, 25]. In similar way, the use
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of ultrasonic energy has also been exploited for metal extraction from different oil samples
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[8,22,26]. Trindade and co-workers [27] reported the use of ultrasound assisted liquid-liquid extraction for determination of Cu, Fe, Ni and Zn in edible oil samples using the High-Resolution Continuum Source Flame Atomic Absorption Spectrometry (HR-CS-FAAS). Herein, the amounts of
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studied metals were in agreement with data obtained from a comparative method (wet digestion)
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using ICP OES.
Recently some papers have been reported in the literature regarding the use of extraction induced by emulsion breaking (EIEB) for the extraction of metals from oil samples with posterior
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determination by atomic spectrometric techniques [7,9,10,17]. This procedure is based on the formation and breaking of emulsions prepared by mixture of oil sample and aqueous phase
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containing nitric acid and surfactant. During the emulsion breaking, in which the aqueous phase is separate from oil phase by heating or centrifugation, the analyte is transferred to the aqueous phase.
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This method has shown to be effective, quick, simple, reliable and excellent when compared with other procedures previously reported. Currently, there is no record in the literature regarding the feasibility of using EIEB for extraction of metals in fish oil capsules with further determination through flow injection thermospray flame furnace atomic absorption spectrometry (FI-TS-FF-AAS). According to aforementioned, the present work describes the development of a simple, inexpensive and sensitive method for Cd2+ determination in omega-3 dietary supplement at trace levels using the EIEB associated with TS-FF-AAS, employing external calibration with aqueous standards. The optimization of experimental parameters that exert influences on emulsion breaking procedure was performed by means of factorial design. The accuracy of present method was checked by means of comparison between EIEB and the microwave-assisted acid digestion procedure.
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ACCEPTED MANUSCRIPT 2.
Experimental
2.1
Apparatus
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The determination of Cd2+ in the solutions (extracts and standards solutions) was carried out
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with a Shimadzu AA 7000 flame atomic absorption spectrometer (Kyoto, Japan) equipped with a
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hollow cathode lamp for cadmium, operated at 8.0 mA and wavelength set at 228 nm. Background correction was performed with a deuterium lamp. For this analysis, the flame was composed of a
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mixture of acetylene and air at flow rate of 1.8 L min-1 and 15.0 L min-1, respectively. The thermospray apparatus was composed of a 99% Ni tube (Camacam, Brazil) of length 10 cm long and
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2.5 cm i.d., containing 6 holes of 2.5 cm i.d. and a 0.5 mm i.d. ceramic capillary tube (99.7% of Al2O3) (Friatec, Mannhein, German). The sample introduction and further transports towards the
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nickel tube was done with the aid of a home-made injector commutator made of Teflon® (PTFE,
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polytetrafluoroethylene) and a peristaltic pump (ICP-08 model, Ismatec, Switzerland). The emulsion
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breaking was induced by heating using a thermostatic bath (Marconi, Piracicaba, Brazil) operating at 88 ºC. The total digestion of the fish oil (omega-3 dietary supplement) samples was performed with a microwave oven Milestone Ethos One (São Paulo, Brazil) equipped with 50.0 mL Teflon®
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vessels.
Reagents and solutions All solutions used in this work were prepared in purified water using a Milli-Q purification
system (Millipore, Bedford, MA, USA) (resistivity higher than 18.2 MΩ cm). Before use, all laboratory glassware was kept overnight in a 10% (v/v) HNO3 solution, in order to avoid any metal contamination. After that, it was rinsed thoroughly with ultra-pure water and dried. A standard Cd2+solution of 1.0 mg L−1 was prepared from a stock standard Cd2+ solution of 1000.0 mg L−1 (Merck, Darmstadt, Germany) using appropriate dilutions. Hydrogen peroxide (35%, v/v) and nitric acid (65%, v/v), used in the digestion procedure, were supplied from Merck (Darmstadt, Germany) 6
ACCEPTED MANUSCRIPT and Sigma Aldrich (St Louis, MO, USA), respectively. Triton X-114 and Triton X-100 solutions (Acros Organics, St. Louis, USA) were prepared by dissolving suitable masses of surfactant
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(according to the experiment) in 25.0 mL of HNO3 solution with desired concentration established
General procedure of extraction induced by emulsion breaking (EIEB)
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2.3
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according to the multivariate experiment.
The extraction of Cd2+ from capsule of omega-3 dietary supplements (fish oil) was performed
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through EIEB procedure using optimized conditions from the multivariate analysis. The procedure is based on formation of stable water-in-oil emulsions, which is obtained by vigorous mixing of 1.0
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mL of fish oil (approximately 0.94 – 0.96 g) with 500.0 µL of a solution containing Triton X-114 (10.6%, m/v) and 3.5 mol L-1 HNO3 in a capped polyethylene flask of 15.0 mL.
The final
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concentration of Triton X-114 and HNO3 was found to be 3.54% (m/v) and 1.16 mol L-1, respectively. Afterwards, the flasks containing the emulsions were transferred to the thermostatic
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water bath at 88 ± 1 ºC and kept until breaking of the emulsion, for approximately 10 min. As result of emulsion breaking, two well-separated phases were formed: (i) the upper organic phase
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containing the omega-3 and (ii) the aqueous lower phase rich in acid-surfactant and ions Cd2+. Finally, the lower aqueous phase was collected with aid of a micropipette and 200 L were inserted in a loop through a flow injection system. The sample was then transported towards TS-FF-AAS system using 1.0 mol L-1HCl/Ethanol (1:1, v/v) as carrier and peak height was taken as analytical response. In order to examine the influence of factors that affect the extraction procedure of Cd2+ from omega-3 dietary supplements (brand 3), a 24 full factorial design was employed. The factors, type and concentration of surfactant, HNO3 concentration and temperature of the thermostatic bath were evaluated and the maximum (+) and minimum (-) levels studied are depicted in Table 1. All experiments were randomly chosen and performed in duplicate. The significance of each factor was evaluated from the analysis of variance (ANOVA) at 95% confidence interval and graphically represented by a Pareto chart. For final optimization of Triton X-114 concentration and temperature 7
ACCEPTED MANUSCRIPT of the thermostatic bath a Doehlert matrix for two variables and response surface methodology were used. All results obtained from multivariate analysis were processed using the STATISTICAL
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package program (Version 7.0).
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2.4 Microwave-assisted acid digestion procedure for total cadmium determination by TS-FFAAS
The total digestion of the omega-3 dietary supplements was carried out in a closed-vessel
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microwave oven. For this task, approximately 1.0 g of each sample was decomposed with 6.0 mL of
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concentrated HNO3 and 4.0 mL of H2O2 (35%, v/v) into the Teflon flasks. The mixture was kept at overnight and subsequently digested using microwave in six steps under following conditions: step 1 – ramp of 80 °C for 5 min, step 2 – 80 °C for 5 min, step 3 – ramp of 120 °C for 7 min, step 4 – 120
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°C for 5 min, step 5 – ramp of 200 °C for 11 min and finally step 6 – 200 °C for 13 min. During overall sample decomposition the magnetron of 2450 MHz was operated with a nominal maximum
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power of 700 W. Afterwards, digested samples were cooled to ambient temperature and transferred to 10.0 mL volumetric flasks, whose volume was made up with ultra-pure water. Two hundred
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milliliters were transported towards TS-FF-AAS detector through a flow injection system and the cadmium determination was carried out using external calibration approach. In order to check possible contamination source, analytical blanks were prepared. All the samples analyzed by the developed method were purchased in drugstore of the Londrina city, Paraná-Brazil and stored at ambient temperature until use.
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Results and Discussion The influence of the most important factors for EIEB procedure including type and
concentration of Triton, acid concentration and temperature of water bath was studied by means of a 24 full factorial design. Table 1 shows the analytical responses for each assay and the results were 8
ACCEPTED MANUSCRIPT assessed through Pareto chart (Figure 1), where the HNO3 concentration and temperature of water bath, as well as the most interactions between the factors were statistically significant at 95%
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confidence interval. Among the studied factors, HNO3 concentration proved to be most significant
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positive effect (12.895), which demonstrates clearly that an increase on the acid concentration
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provides improvements on the extraction efficiency of Cd2+ from the lipidic sample. This finding can be rationalized on the basis of competition of H+ ions with Cd2+ ions by the basic sites present in the organic structure as well as breaking of covalent bonds between metals and organic molecules, thus
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favoring the transference of the metal toward the aqueous medium. According to this result, the
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HNO3 concentration of 1.16 mol L-1 was chosen for the further experiments. Regarding the temperature of water bath, this factor analyzed alone had a slight influence on the extraction efficiency with negative effect (-3.554). On the other hand, its interaction with type of surfactant was
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strongly noticed being the second most important effect (12.488), indicating that both factors cannot be optimized independently of each other. It seems that under lower temperatures in the
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experimental domain (80-95 oC), the emulsion breaking formed from emulsifier agent Triton X-114 is easily obtained, since this emulsion is less stable when compared with emulsion formed from
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Triton X-100 [28]. It is also worth mentioning that although the time required for emulsion breaking was not evaluated in the experimental design, it was observed that 10 min were enough to separate the lipidic and aqueous phases for both surfactants Triton X-110 as Triton X-114, suggesting a quick extraction of Cd2+ ions bound on the organic phase to the aqueous phase. The concentration of the emulsifier agent plays an important role on the extraction induced by emulsion breaking. In general, higher concentration of surfactant forms emulsion highly stable, due to the formation of smaller water droplets, which in turn increase the contact between the two phases (extractor) and organic phases (sample). Nevertheless, as observed in Figure 1, this factor proved not to be a significant factor when analyzed alone, but most of their interactions with other factors, such as temperature of water bath, HNO3 concentration and type of emulsifier agent, are statistically significant at 95% confidence interval. In spite of significant interactions, the concentration and type of surfactant was 9
ACCEPTED MANUSCRIPT the second most significant one with positive effect (9.036), which indicates that the use of Triton X114 with increasing concentrations in the studied range (2.33-5.0% w/v) favors the Cd2+ extraction.
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So, according to achieved results, Triton X-114 and HNO3 concentration of 1.16 mol L-1 were chosen
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for further experiments, while the final optimization of temperature of water bath and Triton X-114
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concentration was carried out by using a Doehlert matrix with a triplicate at central point [29]. The levels of temperature of bath (75, 85 and 95 °C) and Triton X-114 concentration [1.66, 2.5, 3.33, 4.16 and 5.0 % (w/v)] were used for building Doehlert matrix. The results of experimental design
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were evaluated by analysis of variance (ANOVA). The following quadratic model (equation 1) was
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obtained for the analytical response relating temperature of bath and Triton X-114 concentration: Abs = -1.041 + 0.0327.TC – 0.00460.TC2+ 0.02313.TB – 0.00013.TB2 (equation 1) (0.008)
(0.0005)
(0.0018)
(0.00001)
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(0.08)
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According to ANOVA (data not shown), the obtained Fcalculated (3.175) was lower than Ftabulated
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(18.51)] at 95 % confidence, which confirms absence of lack of fit and demonstrates that regression model is considered to be an accurate representation of the experimental data. In addition, the high determination coefficient (R2 =0.9907) also point out that quadratic model fits very well to
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experimental results. Using the quadratic model, the response surface was built (Figure 2), which was able to facilitate a straightforward examination of maximum condition generated by varying the temperature of bath and Triton X-114 concentration. As observed, the maximum analytical response can be achieved when Triton X-114 concentration at 3.54 % (w/v) and temperature of bath of 88.0±0.5 ºC, are employed. These values were further used as the best condition for Cd2+ ions extraction from omega-3 dietary supplement samples. In this work 1.0 mL of omega-3 dietary supplement sample has been subjected to EIEB procedure and under optimized condition 500.0 L of acidified surfactant solution was used as extractor solution. Thus, it was necessary to investigate the effect of extractor on the extraction efficiency in order to obtain improvements on the detectability of method through Cd2+ ions 10
ACCEPTED MANUSCRIPT preconcentration. The achieved results are depicted in Figure 3, where at lowest extractor volume (250.0 L), decreases on the analytical response was observed, which implies the inefficient on the
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extraction of Cd2+ ions. For larger volumes ranging from 750.0 up to 1250.0 L the extraction
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efficiency increases, but due to dilution of rich phase containing Cd2+ ions the analytical signal was
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decreased. Hence, 500 L of extractor volume was selected as optimum.
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3.1 Analytical features of method and application in different kind of omega-3 dietary supplement samples The external calibration approach using standard solutions prepared in water is the most
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common calibration strategy for quantification of metals in extracts obtained from EIEB procedure with further determination by spectrometric techniques. However, in most cases, the use of this
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calibration approach could lead to the systematic errors due to the presence of surfactant in the
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aqueous phase during the emulsion breaking, affecting, for instance, the nebulization process in the
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FAAS technique. In TS-FF-AAS, the high viscosity of solution may severally decrease the thermospray formation. Therefore, in order to evaluate the effect of extractor medium (surfactant and HNO3) in the thermospray formation, external calibration in aqueous medium was compared to
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a calibration curve built in 3.54 % (m/v) Triton X-114 and 1.16 mol L-1 HNO3. The linear equations were found to be Abs = 7.6 x 10-4 + 0.0057[Cd2+, µg-1] for aqueous medium and Abs = 3.6 x 10-3 + 0.0055[Cd2+, µg-1] and for extractor medium. According to obtained results, there was no statistical difference on the sensitivity between curves when evaluated by Student-t test (95 % confidence interval), where tcalculated = 7.00 < ttabulated = 12.706 (n=3). This result proves that the proposed method is free of non-specific interference promoted by surfactant and HNO3, which indicates that external calibration curves prepared with aqueous standards solutions can be successfully used to quantify Cd2+ extracted from lipidic sample. Due to absence of matrix interference, the limits of detection (LD = 2.5 ng g-1) and quantification (LQ = 8.3 ng g-1) of method taking into account the use 1.0 mL of sample and 500.0 L of extractor solution were determined using external calibration 11
ACCEPTED MANUSCRIPT curves prepared with aqueous standards solutions, being calculated as three and ten times the standard deviation of ten measurements of the blank solution, according to IUPAC recommendation,
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respectively [30].
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The accuracy and validity of method was checked by measuring Cd2+ in three omega-3 dietary
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supplement samples, whose results were compared with microwave-assisted acid digestion procedure using paired t-test at 95 % confidence level (Table 2). The tcalculated (0.5786) was lower than critical value ttabulated (4.303), thus indicating the good agreement of results achieved for Cd2+ by
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the method and the reference one. The results also indicate that extraction method is not affected by
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potential interferences and can be successfully applied to the determination of Cd2+ without matrix effect. In addition, the accuracy of microwave-assisted acid digestion procedure was assessed through recovery tests with known amount of Cd2+ added in the omega-3 dietary supplement
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samples, with satisfactory recoveries, as demonstrated in the Table 3. The Cd2+content in the samples was higher than that achieved in vegetable oils, mostly likely due to presence of heavy
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metals in the fish as contamination source [11,31-33]. In the last years, the concentration of heavy metals in food has been identified as the major pathway of human exposure to toxic metals,
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compared with other ways such as inhalation and dermal contact. Although there is not a permissible level of cadmium established specifically for the studied sample (omega-3 dietary supplement samples), agencies such as US Environmental Protection Agency (USEPA) [34], Joint FAO/WHO Expert Committee on Food Additives (JECFA) [35] and Institute of Medicine of the National Academies (IOM) [36] have provided guidelines on the intake of trace elements by humans. According to FAO/WHO Expert Committee on Food Additives, the maximum tolerable weekly intake of cadmium is 7.0 µg Kg-1, corresponding to a dietary intake an average of 65 µg day-1 in a person of 65 Kg. Therefore, determination on the heavy metals levels in edibles oils, from different sources, is extremely important to estimate the potential human health risks. The intra-day and inter-day precision of the developed procedure and total digestion applied to the determination of Cd2+ in omega-3 dietary supplement samples was performed according to ICH 12
ACCEPTED MANUSCRIPT guidelines [37]. Intra-day precision was achieved by three-fold analysis (n=3) of the samples, while the inter-day precision was conducted over a period of two consecutive working days. As can be
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seen from Table 4, the standard relative deviations (RSD) of developed method and the total
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digestion are reduced and similar each other for both intra-day and inter-day analysis, which attest
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the satisfactory precision of method.
A comparison of the proposed extraction method with other procedures based on different approaches for Cd2+ extraction from different kind of samples is given in Table 5. As expected, the
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limit of detection achieved in the proposed method is higher when compared to more sensitive
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spectroanalytical techniques, such as ICP-MS and ETAAS. However, these techniques present high acquisition and operating costs compared to TS-FF-AAS. On the other hand, when compared to direct analysis of Cd2+ by TS-FF-AAS and EIEB procedure coupled to FI-FAAS [40], the proposed
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method presents lower limit of detection. It worth emphasizing that still regarding the figures of merit, the limit of detection of method is only slightly lower than those methods based on solid phase
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extraction procedure on-line coupled to TS-FF-AAS [44,45], but with substantial advantages regarding the microwave-assisted acid digestion required by these methods. In summary, the
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combination of EIBE with TS-FF-AAS result, in general, in a method with over advantages than those ones for Cd2+ determination including, low-cost, absence of organic solvent in the procedure, satisfactory sensitivity and quick analysis.
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Conclusion In this work, we have demonstrated for the first time the association of EIEB with FI-TS-FF-
AAS for determination of Cd2+ in omega-3 dietary supplement samples. The EIEB procedure showed to be a good alternative as previous sample treatment in detriment of microwave-assisted acid digestion procedure. The transference of Cd2+ to aqueous phase allowed the use of standards solutions prepared with ultra-pure water for external calibration, which simplified the procedure and 13
ACCEPTED MANUSCRIPT avoided interferences due to the high content of carbon in the samples. The extraction method is very simple, quick, precise, and economical and when associated to TS-FF-AAS provides
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satisfactory sensitivity to quantify trace levels of Cd2+ in omega-3 dietary supplement matrices,
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being of paramount importance for quality control once the presence of toxic metal ions in these
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samples is not regulated.
Acknowledgments
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The authors are grateful to CNPq (Conselho Nacional de Desenvolvimento Científico Tecnológico), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and
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INCTBio (Instituto Nacional de Ciência e Tecnologia de Bioanalítica), PRÓ-FORENSES (CAPES),
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FUNDAÇÃO ARAUCÁRIA DO PARANÁ and SANEPAR for providing grants and fellowship and
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capillary tube.
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for financial support. We are also grateful to Clésia Cristina Nascentes by supplying the ceramic
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[12] D. Mendil, O.D. Uluozlu, M. Tuzen, M. Soylak, Investigation of the levels of some element in edible oil samples produced in Turkey by atomic absorption spectrometry, J. Hazard Mater. 165 (2009) 724 – 728. [13] M.S. Rahman, A.H. Molla, N. Saha, A. Rahman, Study on heavy metals levels and its risk assessment in some edible fishes from Bangshi River, Savar, Dhaka, Bangladesh, Food Chem. 134 (2012) 1847 – 1854. [14] R.S. Amais, E.E. Garcia, M.R. Monteiro, J.A. Nóbrega, Determination of Ca, Mg, and Zn in biodiesel microemulsions by FAAS using discrete nebulization, Fuel 93 (2012) 167 – 171. [15] M.N.M. Reyes, R.C. Campos, Graphite furnace atomic absorption spectrometric determination of Ni and Pb in diesel and gasoline samples stabilized as microemulsion using conventional and permanent modifiers, Spectrochim. Acta Part B 60 (2005) 615 – 624. [16] R.Q Aucélio, A. Doyle, B.S. Pizzorno, M.L.B. Tristão, R.C Campos, Electrothermal atomic absorption spectrometric method for the determination of vanadium in diesel and asphaltene prepared as detergentless microemulsions, Microchem. J. 78 (2004) 21 – 26.
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ACCEPTED MANUSCRIPT [17] L.F.S. Caldas, D.M. Brum, C.E.R. de Paula, R.J. Casella, Application of the extraction induced by emulsion breaking for the determination of Cu, Fe and Mn in used lubricating oils by flame atomic absorption spectrometry, Talanta 110 (2013) 21 – 27.
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[18] D. Kara, A. Fisher, S. Hill, Detergentless ultrasound-assisted extraction of trace elements from edible oils using lipase as an extractant, Talanta 144 (2015) 219 – 225.
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[19] R.M. Souza, B.M. Mathias, C.L.P. da Silveira, R.Q. Aucélio, Inductively coupled plasma optical emission spectrometry for trace multi-element determination in vegetable oils, margarine and butter after stabilization with propan-1-ol and water, Spectrochim. Acta Part B 60 (2005) 711 – 715. [20] A. Jesus, M.M. Silva, M.G.R. Vale, The use of microemulsion for determination of sodium and potassium in biodiesel by flame atomic absorption spectrometry, Talanta 74 (2008) 1378 – 1384.
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[21] L.S. Nunes, J.T.P. Barbosa, A.P. Fernandes, V.A. Lemos, W.N.L. dos Santos, M.G.A. Korn, L.S.G. Teixeira, Multi-element determination of Cu, Fe, Ni and Zn content in vegetable oils samples by high-resolution continuum source atomic absorption spectrometry and microemulsion sample preparation, Food Chem. 127 (2011) 780 – 783.
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[22] I. López-Garcia, Y. Vicente-Martínez, M. Hernández-Córdoba, Determination of cadmium and lead in edible oils by electrothermal atomic absorption spectrometry after reverse dispersive liquidliquid microextraction, Talanta 124 (2014) 106 – 110.
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[23] J.S. Almeida, T.A. Anunciação, G.C. Brandão, A.F. Dantas, V.A. Lemos, L.S.G. Teixeira, Ultrasound-assisted single drop microextraction for the determination of cadmium in vegetable oils using high-resolution continuum source electrothermal atomic absorption spectrometry, Spectrochim. Acta Part B 107 (2015) 159 – 163.
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[24] P. Hashemi, F. Raeisi, A.R. Ghiasvand, A. Rahimi, Reversed-phase dispersive liquid–liquid microextraction with central composite design optimization for preconcentration and HPLC determination of oleuropein, Talanta 80 (2010) 1926 – 1931. [25] J.M. Kokosa, Advances in solvent microextraction techniques, Trends Anal. Chem. 43 (2013) 2 – 12. [26] H. Méndez, F. Alava, I. Lavilla, C. Bendicho, Ultrasonic extraction combined with fast furnace analysis as an improved methodology for total selenium determination in seafood by electrothermalatomic absorption spectrometry, Anal. Chim. Acta 452 (2002) 217 – 222. [27] A.S.N. Trindade, A.F. Dantas, D.C. Lima, S.L.C. Ferreira, L.S.G. Teixeira, Multivariate optimization of ultrasound-assisted extraction for determination of Cu, Fe, Ni and Zn in vegetable oils by high-resolution continuum source atomic absorption spectrometry, Food Chem. 185 (2015) 145 – 150. [28] R.J. Casella, D.M. Brum, C.F. Lima, L.F.S. Caldas, C.E.R de Paula, Multivariate optimization of the determination of zinc in diesel oil employing a novel extraction strategy based on emulsion breaking, Anal. Chim. Acta 690 (2011) 79 – 85. [29] S.L. Ferreira, W.N.L. dos Santos, B.B. Neto, J.M. Bosque-Sendra, Doehlert matrix: a chemometric tool for analytical chemistry-review, Talanta 63 (2004) 1061 –1067. 16
ACCEPTED MANUSCRIPT [30] G.L. Long, J.D. Winefordner, Limit of detection a closer look at the IUPAC definition, Anal. Chem. 55 (1983) 712 – 724.
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[31] M. Martin-Polvillo, T. Albi, A. Guinda, Determination of trace elements in edible vegetables oils by atomic absorption spectrophotometry, J. Am. Oil Chem. Soc. 71 (1994) 347 – 351.
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[32] A.M. Nash, T.L. Mounts, W.F. Kwolek, Determination of ultratrace metals in hydrogenated vegetable oils and fats, J. Am. Oil Chem. Soc. 60 (1983) 811 – 814.
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[33] E. Pehlivan, G. Arslan, F. God, T. Altun, M.M. Ozcan, Determination of some inorganic metals in edible vegetable oils by inductively coupled plasma atomic emission spectroscopy (ICP-AES), Grasas Aceites 59 (2008) 239 – 244. table.
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[34] US EPA Environmental Protection Agency region 3 RBC, 2007 (accessed 21.02.16).
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[35] JECFA Summary and conclusions of the 61st meeting of the Joint FAO/WHO Expert Committee on Food Additives. JECFA/61SC, Rome, Italy, 2003.
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[36] IOM Institute of Medicine. Dietary reference intakes applications in dietary planning subcommittee on interpretation and uses of dietary reference intakes and the standing committee on the scientific evaluation of dietary reference intakes. Washington, DC: Institute of Medicine of the National Academies, The National Academies Press. p. 248, 2003.
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[37] ICH Validation of Analytical Procedures: Methodology, Technical Requirements for the Registration of Pharmaceuticals for Human Use, International Conference on Harmonization, Geneva, Switzerland, 1995. [38] D. Kara, A. Fisher, S. Hill, Extraction of trace elements by ultrasound-assisted emulsification from edible oils producing detergentless microemulsions, Food Chem. 188 (2015) 143 – 148.
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[39] A. Zhuravlev, A. Zacharia, S. Gucer, A. Chebotarev, M. Arabadji, A. Dobrynin, Direct atomic adsorption spectrometry determination of arsenic, cadmium, copper, manganese, lead and zinc in vegetable oil and fat samples with graphite filter furnace atomizer, J. Food Comp. Anal. 38 (2015) 62 – 68. [40] D. Bakircioglu, N. Topraksever, Y.B. Kurtulus, Separation/preconcentration system based on emulsion-induced breaking procedure for determination of cadmium in edible oil samples by flow injection-flame atomic absorption spectrometry, Food Anal. Methods 8 (2015) 2178-2184. [41] K. Miranda, A.G.G. Dionísio, O.D.P. Neto, M.S. Gomes, E.R. Pereira-Filho, Determination of Cd levels in smoke condensate of Brazilian and Paraguayan cigarettes by Thermospray Flame Furnace Atomic Absorption Spectrometry (TS-FF-AAS), Microchem. J. 100 (2012) 27-30 [42] E.R. Pereira-Filho, H. Berndt, M.A.Z. Arruda, Simultaneous sample digestion and determination of Cd, Cu and Pb in biological samples using thermospray flame furnace atomic absorption spectrometry (TS-FF-AAS) with slurry sample introduction, J. Anal. At. Spectrom. 17 (2002) 1308-1315.
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ACCEPTED MANUSCRIPT [43] E. González, R. Ahumada, V. Medina, J. Neira, U. González, Espectrofotometria de Absorción Atómica com tubo em la llama: Aplicación en la determinación total de Cadmio, Plomo y Zinc em aguas frescas, agua de mar y sedimentos marinos, Quim. Nova 27 (2004) 873-877.
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[44] C.R.T. Tarley, A.F. Barbosa, M.G.Segatelli, E.C.Figueiredo, P.O. Luccas, Highly improved sensitivity of TS-FF-AAS for Cd(II) determination at ng L-1 levels using a simple flow injection minicolumn preconcentration system with multiwall carbon nanotubes, J. Anal. At. Spectrom. 21 (2006) 1305–1313.
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[45] C.R.T. Tarley, M.A.Z. Arruda, A sensitive method for cadmium determination using a on-line polyurethane foam preconcentration system and thermospray flame furnace atomic absorption spectrometry, Anal. Sci. 20 (2004) 962-966.
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ACCEPTED MANUSCRIPT Figure Captions Fig. 1. Pareto chart obtained from 24 full factorial design.
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Fig. 2. Surface response for concentration of Triton X-114 (%, w/v) and temperature of water bath (°C).
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Fig. 3. Influence of volume of extractor solution on the extraction of Cd2+ ions by the proposed method. Error bars represent the standard deviation of three independent measurements.
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12.8949
3by4
12.48876
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(2)[HNO3]
9.036581 -4.16292
1by3
4.162919
(3)Temperature of bath
-3.55371 2.944504
2by4
2.944504
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2by3
2.741435
1*2*4
-2.74143
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2*3*4
1*2*3
2.132227
(4)Triton type
-1.11688 .7107423
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1*3*4
.3046039
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(1)[Triton]
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1by2
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1by4
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p=,05 Standardized Effect Estimate (Absolute Value)
Figure 1. Corazza& Tarley
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Figure 2. Corazza& Tarley
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0.040
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0.035
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0.025 0.020 0.015
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Absorbance
0.030
0.010
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0.005 0.000
500
250
750
1000
1250
Figure 3.Corazza& Tarley
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Volume of the extraction solution (L)
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ACCEPTED MANUSCRIPT Table Captions
Table 1. Factors studied in the 24 full factorial design, their levels and results
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TB + + + + + + + +
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[HNO3] + + + + + + + +
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
TC + + + + + + + +
TT + + + + + + + +
Maximum 5.0 1.16 95 X-114 Absorbance (peak height) 0.028/0.029 0.022/0.022 0.037/0.034 0.025/0.029 0.015/0.018 0.013/0.015 0.026/0.021 0.021/0.018 0.013/0.011 0.021/0.022 0.025/0.025 0.021/0.021 0.012/0.015 0.024/0.024 0.027/0.030 0.035/0.036
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Runs
Minimum 2.33 0.7 80 X-100
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Levels Factors Triton concentration (TC) (%) HNO3 concentration (mol L-1) [HNO3] Temperature of water bath (TB) (°C) Triton type (TT)
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ACCEPTED MANUSCRIPT
Brand 2
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Table 2. Concentration of Cd2+ ions in three different omega-3 dietary supplements by TS-FF-AAS after extraction induced by emulsion breaking or microwave-assisted acid digestion procedure Cd2+ concentration (ng g-1) Samples Microwave-assisted acid digestion EIEB* procedure Brand 1 38.6±1.1 39.1±1.2
Brand 3
56.4±2.2
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37.5±1.5
37.3±1.3 54.5±2.3
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EIEB: extraction induced by emulsion breaking. *Results are expressed as mean value ± standard deviation (n=3).
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Table 3. Results of the addition and recovery test for omega-3 dietary supplements after microwaveassisted acid digestion procedure Cd2+ concentration Cd2+ concentration Samples Recovery (%) -1 (ng g ) added (ng g-1) found* ---
---
39.1±1.2
50
84.8±0.91
95
---
86.9±4.6
99
54.5±2.3
---
97.0±3.1
93
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50
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50
Brand 3
---
37.3±1.3
Brand 2
---
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Brand 1
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*Results are expressed as mean value ± standard deviation (n=3).
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ACCEPTED MANUSCRIPT Table 4. Intra-day and inter-day precision for the proposed method and microwave-assisted acid digestion procedure Cd2+ concentration RSD (%) (ng g-1) Samples Days
Brand 3
Digestion
1
38.6
39.9
2.9
5.4
2
38.8
38.9
3.4
3.9
1
38.4
37.3
2.8
4.1
2
36.9
37.7
4.5
3.4
1
55.4
56.0
1.8
3.6
2
57.6
53.1
4.5
3.3
Intra-day (n=2)
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EIEB
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Digestion
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Brand 1
EIEB*
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Cd2+ concentration
RSD (%)
(ng g-1)
EIEB
Digestion
EIEB
Digestion
38.7
39.4
2.9
4.4
Brand 2
37.7
37.5
3.9
3.4
56.5
54.6
3.8
4.2
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Brand 1
Brand 3
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RSD: Relative Standard Deviation, EIEB: extraction induced by emulsion breaking. *Results are expressed as mean value ± standard deviation (n=3).
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ACCEPTED MANUSCRIPT Table 5. Comparison of reported method for determination of Cd2+ using different extraction approaches, direct analysis and different spectroanalytical techniques
Ultrasoundassisted extraction
lipase
ICP-MS
Vegetable oil
DLLME
4:1 isopropyl alcohol:3% v/v HNO3 solution
ET AAS
Vegetable oil
UA-SDME
0.1 mol L-1 HNO3
UA-SDME
0.01 mol L-1 EDTA
Direct determination
hexane
Vegetable oil
EIBE
Triton X-114/ HNO3
Smoke condensate
Direct
Direct
Fresh Waters, seawater, marine sediment
Direct
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Water, cigarette, bovine liver and rye grass
0.023
[18]
18
0.006
[22]
HR-CS ET AAS
15
0.007
[23]
ICP-MS
30
0.013
[38]
(FF) ET AAS
3-5
0.3
[39]
FAAS
50-84
5.3
[40]
---
TS-FF-AAS
---
5.8
[41]
TS-FF-AAS
---
500
[42]
---
TS-FF-AAS
---
0.32*
[43]
Carbon nanotubes
TS-FF-AAS
12
0.5
[44]
TS-FF-AAS
13.7
1.2
[45]
TS-FF-AAS
10
2.5
This work
Solid phase extraction
Water samples, pig kidney, rye grass
Solid phase extraction
Omega-3 supplements
EIEB
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0.014 mol L-1 HNO3/0.05% (v/v) Triton X-100
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Biological matrices
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Vegetable oil
Ref.
Polyurethane foam using ammonium O,Odiethyl dithiophosphate (DDTP) as chelating agent Triton X-114/ HNO3
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Vegetable oil
Vegetable oil
LOD (ng g-1)
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Technique
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Extractor
Time required for analysis (min)
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Samples
Extraction approach
LOD – limit of detection; DLLME – dispersive liquid–liquid microextraction; UA-SDME – ultrasound-assisted single-drop microextraction; EIEB extraction induced by emulsion breaking; EDTA – ethylenediaminetetraacetic acid; ET AAS – electrothermal atomic absorption spectroscopy; ICP-MS – Inductively Coupled Plasma-Mass Spectrometry; HR-CS ET AAS – high-resolution continuum source electrothermal atomic absorption spectrometry; (FF) ET AAS – filter furnace-electrothermal atomic absorption spectroscopy; TS-FF-AAS – thermospray flame furnace atomic absorption spectrometry. * LOD in µg L-1
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ACCEPTED MANUSCRIPT Highlights
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EIEB is a very simple method for the extraction of Cd2+ from the fish oil Similar results were achieved by EIEB and microwave-assisted acid digestion procedure The EIEB associated to TS-FF-AAS is rapid and sensitive for Cd2+ determination in fish oil The limit of detection of method was found to be 2.5 ng g-1
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S0026-265X(16)00054-0 doi: 10.1016/j.microc.2016.02.021 MICROC 2426
To appear in:
Microchemical Journal
Received date: Revised date: Accepted date:
22 January 2016 27 February 2016 27 February 2016
Please cite this article as: Marcela Zanetti Corazza, C´esar Ricardo Teixeira Tarley, Development and feasibility of emulsion breaking method for the extraction of cadmium from omega-3 dietary supplements and determination by flow injection TS-FF-AAS, Microchemical Journal (2016), doi: 10.1016/j.microc.2016.02.021
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ACCEPTED MANUSCRIPT Development and Feasibility of Emulsion Breaking Method for the Extraction of Cadmium from Omega-3 Dietary Supplements and
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Determination by Flow Injection TS-FF-AAS
Universidade Estadual de Londrina (UEL), Departamento de Química, Centro de Ciências Exatas, Rodovia Celso Garcia Cid, PR 445, Km 380, Londrina, PR, 86050-82, Brazil
Instituto Nacional de Ciência e Tecnologia (INCT) de Bioanalítica, Universidade Estadual de Campinas (UNICAMP), Instituto de Química, Departamento de Química Analítica, Cidade Universitária Zeferino Vaz, s/n, Campinas, SP, 13083-970, Brazil.
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Faculdade de Ciências Exatas e Tecnologia, FACET, Universidade Federal da Grande Dourados (UFGD), CEP 79804-970, Dourados-MS, Brazil.
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Marcela Zanetti Corazza1,2, César Ricardo Teixeira Tarley*2,3
* Corresponding author. Tel +55 43 3371 4366; fax +55 43 3371 4286
E-mail address: [email protected] (C. R. T. Tarley) 1
ACCEPTED MANUSCRIPT ABSTRACT In the present study a new method for determination of Cd2+ in omega-3 dietary supplement
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employing extraction induced by emulsion breaking (EIEB) and Thermospray Flame Furnace
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Atomic Absorption Spectrometry (TS-FF-AAS) was described. The method was based on the
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formation of a water-in-oil emulsion by addition of extractor solution constituted by 3.54% (m/v) Triton X-114 and 1.16 mol L-1 HNO3 in the oil sample and further breaking of this emulsion by heating. Two well-defined phases were formed and the acid aqueous one containing the extracted
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cadmium ions was analyzed by TS-FF-AAS using a flow injection system. Different parameters that exert influence on the extraction efficiency of Cd2+ were optimized by means of chemometric tools.
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The limits of detection and quantification were found to be 2.5 and 8.3 ng g-1, respectively using a calibration curve made in aqueous medium. The accuracy of the proposed method was assured by
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good agreement with the results achieved by microwave-assisted acid digestion procedure without statistical differences (confidence interval of 95%) and by spiking the samples with known
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ng g-1.
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concentrations of 50 ng g-1 Cd2+. The content of Cd2+ in the samples was varied from 37.3 up to 54.5
keywords: Fish oil, Cd, factorial design, atomic absorption spectrometry 2
ACCEPTED MANUSCRIPT 1.
Introduction Fatty acids presents in the majority of the oil matrix samples are organic compounds formed
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by a hydrocarbonated chain and by carboxylic groups that are normally bounded with glycerol forming triacylglycerides classified as mono, di or triglycerides [1]. The nature of hydrocarbonated
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chain defines the fatty acids as saturated or unsaturated compounds, which in turn can be monounsaturated or polyunsatured fatty acids (PUFA) [2]. Omega-3 polyunsaturated fatty acids,
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more specifically the EPA (cis-5, 8,11,14,17 – eicosapentaenoic acid) and DHA (cis-4, 7, 10,13,16,19 – docosahexaenoic acid) are essential fatty acids to the human health and known to exert
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beneficial effects to cardiovascular diseases, reducing blood triacylglycerol levels, blood pressure and to inflammatory process, preventing platelet aggregation [3]. However, the amounts of EPA and
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DHA produced by human body through conversion of α-linoleic acid is very low and this deficiency
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can be remedied by the consumption of natural food products, such as fish, sardine, salmon and
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other alternative sources such as marine microalgae or by the ingestion omega-3 gelatin capsules available commercially [4,5].
Nevertheless, the chemical quality of edible oils play important role in the human health and
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commonly are evaluated by means of concentration of peroxidase, fatty acids composition and by presence of trace metals, since it affect the stability of oil matrix, catalyzing oxidative reactions that results in the formation of toxic products [6-8]. The contamination of heavy metals into oils matrix occurs usually from its extraction process or from outer sources to the production process, such as bleaching, hardening, refining and deodorization. In addition, these samples present high levels of carbon and lipids content and can causes serious spectrometric interferences during the measurements for the most analytes, leading to inaccurate determinations [9,10]. Thus, the determination of trace elements in edible oils samples has become a major challenge in the field of analytical chemistry. Some spectroanalytical techniques, such as Flame Atomic Absorption Spectrometry (FAAS), Electrothermal Atomic Absorption Spectrometry (ETAAS), Inductively 3
ACCEPTED MANUSCRIPT Coupled Plasma Mass Spectrometry (ICP-MS) and Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES), have been very popular techniques for the elemental analysis of oil
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samples, due to their high selectively and suitable sensitivity and precision. However, the reliable
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analysis of oil samples by these techniques requires previous sample treatment to avoid matrix effect
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and to minimize possible interferences [6].
In order to overcome these drawbacks, different sample preparation procedures, among them, microwave-assisted digestion and some extraction methods have been proposed in order to eliminate
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the possible interferences. The microwave-assisted digestion is one of the most common method and
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widely used in the preparation of oil samples for inorganic analysis [11-13]. Nevertheless, this procedure requires a relatively long time of analysis besides to need careful temperature control for avoid loss of volatile metals [14]. Alternatively, extraction methods have been employed due to use
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less aggressive reagents, lower time of analyses and the use of aqueous standards for calibration. The procedures that involve the formation of emulsion and microemulsion have received great attention
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in the last years. Microemulsion, composed by a mixture of oil-water stabilized by means surfactant agent and an organic solvent, presents high stability, facility in its preparation, as well as others
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desirable features for direct measurement, such as low viscosity and high sample throughput. Although the use of microemulsion offers some advantages over procedures that involve dilution with organic solvent and some emulsion systems, since they allow the use of aqueous solutions for calibration instead of expensive organomettalics standards, this method can be applied only when atomic spectrometry techniques are used [14-17]. Similarly, detergent less microemulsion has also been proposed to separate and preconcentrate metals from edible oils followed by ICP-MS detection [18], ICP OES [19] and FAAS [20]. In this case, a homogeneous system containing both the aqueous phase and the liquid organic phase resulting in a homogenous and long-term stable three-component solution is formed when a co-solvent is used [16,21]. Furthermore, the determination of metals at trace levels in oil samples with minimal manipulation and high reliability using microextraction as dispersive
liquid-liquid
microextraction
(DLLME)
and
ultrasound-assisted
single-drop 4
ACCEPTED MANUSCRIPT microextraction (UA-SDME) have gained increasing popularity in recent years [22,23]. The main features of DLLME include the low volume of extractor phase, which in turn makes possible to
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achieve high enrichment factor and the high sample throughput [22, 24, 25]. In similar way, the use
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of ultrasonic energy has also been exploited for metal extraction from different oil samples
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[8,22,26]. Trindade and co-workers [27] reported the use of ultrasound assisted liquid-liquid extraction for determination of Cu, Fe, Ni and Zn in edible oil samples using the High-Resolution Continuum Source Flame Atomic Absorption Spectrometry (HR-CS-FAAS). Herein, the amounts of
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studied metals were in agreement with data obtained from a comparative method (wet digestion)
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using ICP OES.
Recently some papers have been reported in the literature regarding the use of extraction induced by emulsion breaking (EIEB) for the extraction of metals from oil samples with posterior
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determination by atomic spectrometric techniques [7,9,10,17]. This procedure is based on the formation and breaking of emulsions prepared by mixture of oil sample and aqueous phase
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containing nitric acid and surfactant. During the emulsion breaking, in which the aqueous phase is separate from oil phase by heating or centrifugation, the analyte is transferred to the aqueous phase.
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This method has shown to be effective, quick, simple, reliable and excellent when compared with other procedures previously reported. Currently, there is no record in the literature regarding the feasibility of using EIEB for extraction of metals in fish oil capsules with further determination through flow injection thermospray flame furnace atomic absorption spectrometry (FI-TS-FF-AAS). According to aforementioned, the present work describes the development of a simple, inexpensive and sensitive method for Cd2+ determination in omega-3 dietary supplement at trace levels using the EIEB associated with TS-FF-AAS, employing external calibration with aqueous standards. The optimization of experimental parameters that exert influences on emulsion breaking procedure was performed by means of factorial design. The accuracy of present method was checked by means of comparison between EIEB and the microwave-assisted acid digestion procedure.
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Experimental
2.1
Apparatus
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The determination of Cd2+ in the solutions (extracts and standards solutions) was carried out
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with a Shimadzu AA 7000 flame atomic absorption spectrometer (Kyoto, Japan) equipped with a
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hollow cathode lamp for cadmium, operated at 8.0 mA and wavelength set at 228 nm. Background correction was performed with a deuterium lamp. For this analysis, the flame was composed of a
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mixture of acetylene and air at flow rate of 1.8 L min-1 and 15.0 L min-1, respectively. The thermospray apparatus was composed of a 99% Ni tube (Camacam, Brazil) of length 10 cm long and
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2.5 cm i.d., containing 6 holes of 2.5 cm i.d. and a 0.5 mm i.d. ceramic capillary tube (99.7% of Al2O3) (Friatec, Mannhein, German). The sample introduction and further transports towards the
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nickel tube was done with the aid of a home-made injector commutator made of Teflon® (PTFE,
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polytetrafluoroethylene) and a peristaltic pump (ICP-08 model, Ismatec, Switzerland). The emulsion
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breaking was induced by heating using a thermostatic bath (Marconi, Piracicaba, Brazil) operating at 88 ºC. The total digestion of the fish oil (omega-3 dietary supplement) samples was performed with a microwave oven Milestone Ethos One (São Paulo, Brazil) equipped with 50.0 mL Teflon®
2.2
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vessels.
Reagents and solutions All solutions used in this work were prepared in purified water using a Milli-Q purification
system (Millipore, Bedford, MA, USA) (resistivity higher than 18.2 MΩ cm). Before use, all laboratory glassware was kept overnight in a 10% (v/v) HNO3 solution, in order to avoid any metal contamination. After that, it was rinsed thoroughly with ultra-pure water and dried. A standard Cd2+solution of 1.0 mg L−1 was prepared from a stock standard Cd2+ solution of 1000.0 mg L−1 (Merck, Darmstadt, Germany) using appropriate dilutions. Hydrogen peroxide (35%, v/v) and nitric acid (65%, v/v), used in the digestion procedure, were supplied from Merck (Darmstadt, Germany) 6
ACCEPTED MANUSCRIPT and Sigma Aldrich (St Louis, MO, USA), respectively. Triton X-114 and Triton X-100 solutions (Acros Organics, St. Louis, USA) were prepared by dissolving suitable masses of surfactant
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(according to the experiment) in 25.0 mL of HNO3 solution with desired concentration established
General procedure of extraction induced by emulsion breaking (EIEB)
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2.3
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according to the multivariate experiment.
The extraction of Cd2+ from capsule of omega-3 dietary supplements (fish oil) was performed
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through EIEB procedure using optimized conditions from the multivariate analysis. The procedure is based on formation of stable water-in-oil emulsions, which is obtained by vigorous mixing of 1.0
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mL of fish oil (approximately 0.94 – 0.96 g) with 500.0 µL of a solution containing Triton X-114 (10.6%, m/v) and 3.5 mol L-1 HNO3 in a capped polyethylene flask of 15.0 mL.
The final
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concentration of Triton X-114 and HNO3 was found to be 3.54% (m/v) and 1.16 mol L-1, respectively. Afterwards, the flasks containing the emulsions were transferred to the thermostatic
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water bath at 88 ± 1 ºC and kept until breaking of the emulsion, for approximately 10 min. As result of emulsion breaking, two well-separated phases were formed: (i) the upper organic phase
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containing the omega-3 and (ii) the aqueous lower phase rich in acid-surfactant and ions Cd2+. Finally, the lower aqueous phase was collected with aid of a micropipette and 200 L were inserted in a loop through a flow injection system. The sample was then transported towards TS-FF-AAS system using 1.0 mol L-1HCl/Ethanol (1:1, v/v) as carrier and peak height was taken as analytical response. In order to examine the influence of factors that affect the extraction procedure of Cd2+ from omega-3 dietary supplements (brand 3), a 24 full factorial design was employed. The factors, type and concentration of surfactant, HNO3 concentration and temperature of the thermostatic bath were evaluated and the maximum (+) and minimum (-) levels studied are depicted in Table 1. All experiments were randomly chosen and performed in duplicate. The significance of each factor was evaluated from the analysis of variance (ANOVA) at 95% confidence interval and graphically represented by a Pareto chart. For final optimization of Triton X-114 concentration and temperature 7
ACCEPTED MANUSCRIPT of the thermostatic bath a Doehlert matrix for two variables and response surface methodology were used. All results obtained from multivariate analysis were processed using the STATISTICAL
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package program (Version 7.0).
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2.4 Microwave-assisted acid digestion procedure for total cadmium determination by TS-FFAAS
The total digestion of the omega-3 dietary supplements was carried out in a closed-vessel
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microwave oven. For this task, approximately 1.0 g of each sample was decomposed with 6.0 mL of
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concentrated HNO3 and 4.0 mL of H2O2 (35%, v/v) into the Teflon flasks. The mixture was kept at overnight and subsequently digested using microwave in six steps under following conditions: step 1 – ramp of 80 °C for 5 min, step 2 – 80 °C for 5 min, step 3 – ramp of 120 °C for 7 min, step 4 – 120
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°C for 5 min, step 5 – ramp of 200 °C for 11 min and finally step 6 – 200 °C for 13 min. During overall sample decomposition the magnetron of 2450 MHz was operated with a nominal maximum
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power of 700 W. Afterwards, digested samples were cooled to ambient temperature and transferred to 10.0 mL volumetric flasks, whose volume was made up with ultra-pure water. Two hundred
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milliliters were transported towards TS-FF-AAS detector through a flow injection system and the cadmium determination was carried out using external calibration approach. In order to check possible contamination source, analytical blanks were prepared. All the samples analyzed by the developed method were purchased in drugstore of the Londrina city, Paraná-Brazil and stored at ambient temperature until use.
3.
Results and Discussion The influence of the most important factors for EIEB procedure including type and
concentration of Triton, acid concentration and temperature of water bath was studied by means of a 24 full factorial design. Table 1 shows the analytical responses for each assay and the results were 8
ACCEPTED MANUSCRIPT assessed through Pareto chart (Figure 1), where the HNO3 concentration and temperature of water bath, as well as the most interactions between the factors were statistically significant at 95%
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confidence interval. Among the studied factors, HNO3 concentration proved to be most significant
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positive effect (12.895), which demonstrates clearly that an increase on the acid concentration
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provides improvements on the extraction efficiency of Cd2+ from the lipidic sample. This finding can be rationalized on the basis of competition of H+ ions with Cd2+ ions by the basic sites present in the organic structure as well as breaking of covalent bonds between metals and organic molecules, thus
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favoring the transference of the metal toward the aqueous medium. According to this result, the
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HNO3 concentration of 1.16 mol L-1 was chosen for the further experiments. Regarding the temperature of water bath, this factor analyzed alone had a slight influence on the extraction efficiency with negative effect (-3.554). On the other hand, its interaction with type of surfactant was
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strongly noticed being the second most important effect (12.488), indicating that both factors cannot be optimized independently of each other. It seems that under lower temperatures in the
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experimental domain (80-95 oC), the emulsion breaking formed from emulsifier agent Triton X-114 is easily obtained, since this emulsion is less stable when compared with emulsion formed from
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Triton X-100 [28]. It is also worth mentioning that although the time required for emulsion breaking was not evaluated in the experimental design, it was observed that 10 min were enough to separate the lipidic and aqueous phases for both surfactants Triton X-110 as Triton X-114, suggesting a quick extraction of Cd2+ ions bound on the organic phase to the aqueous phase. The concentration of the emulsifier agent plays an important role on the extraction induced by emulsion breaking. In general, higher concentration of surfactant forms emulsion highly stable, due to the formation of smaller water droplets, which in turn increase the contact between the two phases (extractor) and organic phases (sample). Nevertheless, as observed in Figure 1, this factor proved not to be a significant factor when analyzed alone, but most of their interactions with other factors, such as temperature of water bath, HNO3 concentration and type of emulsifier agent, are statistically significant at 95% confidence interval. In spite of significant interactions, the concentration and type of surfactant was 9
ACCEPTED MANUSCRIPT the second most significant one with positive effect (9.036), which indicates that the use of Triton X114 with increasing concentrations in the studied range (2.33-5.0% w/v) favors the Cd2+ extraction.
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So, according to achieved results, Triton X-114 and HNO3 concentration of 1.16 mol L-1 were chosen
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for further experiments, while the final optimization of temperature of water bath and Triton X-114
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concentration was carried out by using a Doehlert matrix with a triplicate at central point [29]. The levels of temperature of bath (75, 85 and 95 °C) and Triton X-114 concentration [1.66, 2.5, 3.33, 4.16 and 5.0 % (w/v)] were used for building Doehlert matrix. The results of experimental design
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were evaluated by analysis of variance (ANOVA). The following quadratic model (equation 1) was
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obtained for the analytical response relating temperature of bath and Triton X-114 concentration: Abs = -1.041 + 0.0327.TC – 0.00460.TC2+ 0.02313.TB – 0.00013.TB2 (equation 1) (0.008)
(0.0005)
(0.0018)
(0.00001)
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(0.08)
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According to ANOVA (data not shown), the obtained Fcalculated (3.175) was lower than Ftabulated
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(18.51)] at 95 % confidence, which confirms absence of lack of fit and demonstrates that regression model is considered to be an accurate representation of the experimental data. In addition, the high determination coefficient (R2 =0.9907) also point out that quadratic model fits very well to
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experimental results. Using the quadratic model, the response surface was built (Figure 2), which was able to facilitate a straightforward examination of maximum condition generated by varying the temperature of bath and Triton X-114 concentration. As observed, the maximum analytical response can be achieved when Triton X-114 concentration at 3.54 % (w/v) and temperature of bath of 88.0±0.5 ºC, are employed. These values were further used as the best condition for Cd2+ ions extraction from omega-3 dietary supplement samples. In this work 1.0 mL of omega-3 dietary supplement sample has been subjected to EIEB procedure and under optimized condition 500.0 L of acidified surfactant solution was used as extractor solution. Thus, it was necessary to investigate the effect of extractor on the extraction efficiency in order to obtain improvements on the detectability of method through Cd2+ ions 10
ACCEPTED MANUSCRIPT preconcentration. The achieved results are depicted in Figure 3, where at lowest extractor volume (250.0 L), decreases on the analytical response was observed, which implies the inefficient on the
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extraction of Cd2+ ions. For larger volumes ranging from 750.0 up to 1250.0 L the extraction
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efficiency increases, but due to dilution of rich phase containing Cd2+ ions the analytical signal was
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decreased. Hence, 500 L of extractor volume was selected as optimum.
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3.1 Analytical features of method and application in different kind of omega-3 dietary supplement samples The external calibration approach using standard solutions prepared in water is the most
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common calibration strategy for quantification of metals in extracts obtained from EIEB procedure with further determination by spectrometric techniques. However, in most cases, the use of this
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calibration approach could lead to the systematic errors due to the presence of surfactant in the
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aqueous phase during the emulsion breaking, affecting, for instance, the nebulization process in the
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FAAS technique. In TS-FF-AAS, the high viscosity of solution may severally decrease the thermospray formation. Therefore, in order to evaluate the effect of extractor medium (surfactant and HNO3) in the thermospray formation, external calibration in aqueous medium was compared to
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a calibration curve built in 3.54 % (m/v) Triton X-114 and 1.16 mol L-1 HNO3. The linear equations were found to be Abs = 7.6 x 10-4 + 0.0057[Cd2+, µg-1] for aqueous medium and Abs = 3.6 x 10-3 + 0.0055[Cd2+, µg-1] and for extractor medium. According to obtained results, there was no statistical difference on the sensitivity between curves when evaluated by Student-t test (95 % confidence interval), where tcalculated = 7.00 < ttabulated = 12.706 (n=3). This result proves that the proposed method is free of non-specific interference promoted by surfactant and HNO3, which indicates that external calibration curves prepared with aqueous standards solutions can be successfully used to quantify Cd2+ extracted from lipidic sample. Due to absence of matrix interference, the limits of detection (LD = 2.5 ng g-1) and quantification (LQ = 8.3 ng g-1) of method taking into account the use 1.0 mL of sample and 500.0 L of extractor solution were determined using external calibration 11
ACCEPTED MANUSCRIPT curves prepared with aqueous standards solutions, being calculated as three and ten times the standard deviation of ten measurements of the blank solution, according to IUPAC recommendation,
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respectively [30].
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The accuracy and validity of method was checked by measuring Cd2+ in three omega-3 dietary
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supplement samples, whose results were compared with microwave-assisted acid digestion procedure using paired t-test at 95 % confidence level (Table 2). The tcalculated (0.5786) was lower than critical value ttabulated (4.303), thus indicating the good agreement of results achieved for Cd2+ by
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the method and the reference one. The results also indicate that extraction method is not affected by
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potential interferences and can be successfully applied to the determination of Cd2+ without matrix effect. In addition, the accuracy of microwave-assisted acid digestion procedure was assessed through recovery tests with known amount of Cd2+ added in the omega-3 dietary supplement
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samples, with satisfactory recoveries, as demonstrated in the Table 3. The Cd2+content in the samples was higher than that achieved in vegetable oils, mostly likely due to presence of heavy
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metals in the fish as contamination source [11,31-33]. In the last years, the concentration of heavy metals in food has been identified as the major pathway of human exposure to toxic metals,
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compared with other ways such as inhalation and dermal contact. Although there is not a permissible level of cadmium established specifically for the studied sample (omega-3 dietary supplement samples), agencies such as US Environmental Protection Agency (USEPA) [34], Joint FAO/WHO Expert Committee on Food Additives (JECFA) [35] and Institute of Medicine of the National Academies (IOM) [36] have provided guidelines on the intake of trace elements by humans. According to FAO/WHO Expert Committee on Food Additives, the maximum tolerable weekly intake of cadmium is 7.0 µg Kg-1, corresponding to a dietary intake an average of 65 µg day-1 in a person of 65 Kg. Therefore, determination on the heavy metals levels in edibles oils, from different sources, is extremely important to estimate the potential human health risks. The intra-day and inter-day precision of the developed procedure and total digestion applied to the determination of Cd2+ in omega-3 dietary supplement samples was performed according to ICH 12
ACCEPTED MANUSCRIPT guidelines [37]. Intra-day precision was achieved by three-fold analysis (n=3) of the samples, while the inter-day precision was conducted over a period of two consecutive working days. As can be
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seen from Table 4, the standard relative deviations (RSD) of developed method and the total
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digestion are reduced and similar each other for both intra-day and inter-day analysis, which attest
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the satisfactory precision of method.
A comparison of the proposed extraction method with other procedures based on different approaches for Cd2+ extraction from different kind of samples is given in Table 5. As expected, the
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limit of detection achieved in the proposed method is higher when compared to more sensitive
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spectroanalytical techniques, such as ICP-MS and ETAAS. However, these techniques present high acquisition and operating costs compared to TS-FF-AAS. On the other hand, when compared to direct analysis of Cd2+ by TS-FF-AAS and EIEB procedure coupled to FI-FAAS [40], the proposed
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method presents lower limit of detection. It worth emphasizing that still regarding the figures of merit, the limit of detection of method is only slightly lower than those methods based on solid phase
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extraction procedure on-line coupled to TS-FF-AAS [44,45], but with substantial advantages regarding the microwave-assisted acid digestion required by these methods. In summary, the
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combination of EIBE with TS-FF-AAS result, in general, in a method with over advantages than those ones for Cd2+ determination including, low-cost, absence of organic solvent in the procedure, satisfactory sensitivity and quick analysis.
4.
Conclusion In this work, we have demonstrated for the first time the association of EIEB with FI-TS-FF-
AAS for determination of Cd2+ in omega-3 dietary supplement samples. The EIEB procedure showed to be a good alternative as previous sample treatment in detriment of microwave-assisted acid digestion procedure. The transference of Cd2+ to aqueous phase allowed the use of standards solutions prepared with ultra-pure water for external calibration, which simplified the procedure and 13
ACCEPTED MANUSCRIPT avoided interferences due to the high content of carbon in the samples. The extraction method is very simple, quick, precise, and economical and when associated to TS-FF-AAS provides
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satisfactory sensitivity to quantify trace levels of Cd2+ in omega-3 dietary supplement matrices,
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being of paramount importance for quality control once the presence of toxic metal ions in these
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samples is not regulated.
Acknowledgments
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The authors are grateful to CNPq (Conselho Nacional de Desenvolvimento Científico Tecnológico), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and
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INCTBio (Instituto Nacional de Ciência e Tecnologia de Bioanalítica), PRÓ-FORENSES (CAPES),
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FUNDAÇÃO ARAUCÁRIA DO PARANÁ and SANEPAR for providing grants and fellowship and
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capillary tube.
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for financial support. We are also grateful to Clésia Cristina Nascentes by supplying the ceramic
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ACCEPTED MANUSCRIPT Figure Captions Fig. 1. Pareto chart obtained from 24 full factorial design.
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Fig. 2. Surface response for concentration of Triton X-114 (%, w/v) and temperature of water bath (°C).
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Fig. 3. Influence of volume of extractor solution on the extraction of Cd2+ ions by the proposed method. Error bars represent the standard deviation of three independent measurements.
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12.8949
3by4
12.48876
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(2)[HNO3]
9.036581 -4.16292
1by3
4.162919
(3)Temperature of bath
-3.55371 2.944504
2by4
2.944504
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2by3
2.741435
1*2*4
-2.74143
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2*3*4
1*2*3
2.132227
(4)Triton type
-1.11688 .7107423
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1*3*4
.3046039
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(1)[Triton]
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1by2
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1by4
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p=,05 Standardized Effect Estimate (Absolute Value)
Figure 1. Corazza& Tarley
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Figure 2. Corazza& Tarley
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0.040
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0.035
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0.025 0.020 0.015
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Absorbance
0.030
0.010
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0.005 0.000
500
250
750
1000
1250
Figure 3.Corazza& Tarley
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Volume of the extraction solution (L)
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ACCEPTED MANUSCRIPT Table Captions
Table 1. Factors studied in the 24 full factorial design, their levels and results
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TB + + + + + + + +
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[HNO3] + + + + + + + +
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
TC + + + + + + + +
TT + + + + + + + +
Maximum 5.0 1.16 95 X-114 Absorbance (peak height) 0.028/0.029 0.022/0.022 0.037/0.034 0.025/0.029 0.015/0.018 0.013/0.015 0.026/0.021 0.021/0.018 0.013/0.011 0.021/0.022 0.025/0.025 0.021/0.021 0.012/0.015 0.024/0.024 0.027/0.030 0.035/0.036
AC
Runs
Minimum 2.33 0.7 80 X-100
T
Levels Factors Triton concentration (TC) (%) HNO3 concentration (mol L-1) [HNO3] Temperature of water bath (TB) (°C) Triton type (TT)
23
ACCEPTED MANUSCRIPT
Brand 2
IP
T
Table 2. Concentration of Cd2+ ions in three different omega-3 dietary supplements by TS-FF-AAS after extraction induced by emulsion breaking or microwave-assisted acid digestion procedure Cd2+ concentration (ng g-1) Samples Microwave-assisted acid digestion EIEB* procedure Brand 1 38.6±1.1 39.1±1.2
Brand 3
56.4±2.2
SC R
37.5±1.5
37.3±1.3 54.5±2.3
AC
CE P
TE
D
MA
NU
EIEB: extraction induced by emulsion breaking. *Results are expressed as mean value ± standard deviation (n=3).
24
ACCEPTED MANUSCRIPT
T
Table 3. Results of the addition and recovery test for omega-3 dietary supplements after microwaveassisted acid digestion procedure Cd2+ concentration Cd2+ concentration Samples Recovery (%) -1 (ng g ) added (ng g-1) found* ---
---
39.1±1.2
50
84.8±0.91
95
---
86.9±4.6
99
54.5±2.3
---
97.0±3.1
93
NU
D
50
MA
50
Brand 3
---
37.3±1.3
Brand 2
---
SC R
IP
Brand 1
AC
CE P
TE
*Results are expressed as mean value ± standard deviation (n=3).
25
ACCEPTED MANUSCRIPT Table 4. Intra-day and inter-day precision for the proposed method and microwave-assisted acid digestion procedure Cd2+ concentration RSD (%) (ng g-1) Samples Days
Brand 3
Digestion
1
38.6
39.9
2.9
5.4
2
38.8
38.9
3.4
3.9
1
38.4
37.3
2.8
4.1
2
36.9
37.7
4.5
3.4
1
55.4
56.0
1.8
3.6
2
57.6
53.1
4.5
3.3
Intra-day (n=2)
IP
T
EIEB
SC R
Brand 2
Digestion
NU
Brand 1
EIEB*
MA
Cd2+ concentration
RSD (%)
(ng g-1)
EIEB
Digestion
EIEB
Digestion
38.7
39.4
2.9
4.4
Brand 2
37.7
37.5
3.9
3.4
56.5
54.6
3.8
4.2
TE
D
Brand 1
Brand 3
AC
CE P
RSD: Relative Standard Deviation, EIEB: extraction induced by emulsion breaking. *Results are expressed as mean value ± standard deviation (n=3).
26
ACCEPTED MANUSCRIPT Table 5. Comparison of reported method for determination of Cd2+ using different extraction approaches, direct analysis and different spectroanalytical techniques
Ultrasoundassisted extraction
lipase
ICP-MS
Vegetable oil
DLLME
4:1 isopropyl alcohol:3% v/v HNO3 solution
ET AAS
Vegetable oil
UA-SDME
0.1 mol L-1 HNO3
UA-SDME
0.01 mol L-1 EDTA
Direct determination
hexane
Vegetable oil
EIBE
Triton X-114/ HNO3
Smoke condensate
Direct
Direct
Fresh Waters, seawater, marine sediment
Direct
AC
Water, cigarette, bovine liver and rye grass
0.023
[18]
18
0.006
[22]
HR-CS ET AAS
15
0.007
[23]
ICP-MS
30
0.013
[38]
(FF) ET AAS
3-5
0.3
[39]
FAAS
50-84
5.3
[40]
---
TS-FF-AAS
---
5.8
[41]
TS-FF-AAS
---
500
[42]
---
TS-FF-AAS
---
0.32*
[43]
Carbon nanotubes
TS-FF-AAS
12
0.5
[44]
TS-FF-AAS
13.7
1.2
[45]
TS-FF-AAS
10
2.5
This work
Solid phase extraction
Water samples, pig kidney, rye grass
Solid phase extraction
Omega-3 supplements
EIEB
NU
MA
0.014 mol L-1 HNO3/0.05% (v/v) Triton X-100
CE P
Biological matrices
30
D
Vegetable oil
Ref.
Polyurethane foam using ammonium O,Odiethyl dithiophosphate (DDTP) as chelating agent Triton X-114/ HNO3
T
Vegetable oil
Vegetable oil
LOD (ng g-1)
IP
Technique
SC R
Extractor
Time required for analysis (min)
TE
Samples
Extraction approach
LOD – limit of detection; DLLME – dispersive liquid–liquid microextraction; UA-SDME – ultrasound-assisted single-drop microextraction; EIEB extraction induced by emulsion breaking; EDTA – ethylenediaminetetraacetic acid; ET AAS – electrothermal atomic absorption spectroscopy; ICP-MS – Inductively Coupled Plasma-Mass Spectrometry; HR-CS ET AAS – high-resolution continuum source electrothermal atomic absorption spectrometry; (FF) ET AAS – filter furnace-electrothermal atomic absorption spectroscopy; TS-FF-AAS – thermospray flame furnace atomic absorption spectrometry. * LOD in µg L-1
27
ACCEPTED MANUSCRIPT Highlights
T
IP
SC R NU MA D TE
CE P
EIEB is a very simple method for the extraction of Cd2+ from the fish oil Similar results were achieved by EIEB and microwave-assisted acid digestion procedure The EIEB associated to TS-FF-AAS is rapid and sensitive for Cd2+ determination in fish oil The limit of detection of method was found to be 2.5 ng g-1
AC
28