A new method employing extraction induced by emulsion breaking and MIP OES for multi-element determination of inorganic elements in handmade chocolate

A new method employing extraction induced by emulsion breaking and MIP OES for multi-element determination of inorganic elements in handmade chocolate | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A new method employing extraction induced by emulsion breaking and MIP OES for multi-element determination of inorganic elements in handmade chocolate Geovana Brito Guimarães, Leonardo Brito Guimarães, Julia Carneiro Romero, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4365838/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract The objective of this work was the multielement determination of Ca, Zn, Sr, Ba, Cu, Mn, Mg and Cr in chocolate barr samples after extraction induced by emulsion breakage (EIEB) and quantification by optical emission spectrometry whit plasma induced by microwaves (MIP OES). After study of each parameter, the most efficient extraction conditions were obtained using 0.250 g of sample, 5.0 mL of extraction solution consisting of HNO 3 1.5 mol L − 1 and Tween80 1.5% m/v, submitted to an ultrasonic bath for 5 min, followed by breaking the emulsion by heating at 90 ºC in a water bath for 4 min. The detection limits obtained, in mg kg − 1 , were 0,3487 (Cr), 0,0125 (Zn), 0,0635 (Sr), 0,0831 (Ca), 0,4627 (Fe), 0,0101 (Ba), 0,0991, (Cu), 0,0161 (Mg), 0,0356 (Mn). Precision, based on the relative standard deviation (RSD%), was less than 9.8% (N = 7). The accuracy was confirmed by analyzing the SRM Baking Chocolate 2384 and comparing the proposed method with a calcination method. The method was applied to samples of chocolate bars made in the southern region of Bahia and containing cocoa contents at 50, 58, 60, 63, 70, 80 and 85%. The average results obtained were 328 to 1424 mg kg − 1 (Ca), 561 to 2152 mg kg − 1 (Mg), 7.8 to 251 mg kg − 1 (Cu), 8.5 to 304 mg kg − 1 (Mn), 6.22 to 98.32 mg kg − 1 (Fe), 8.38 to 80.2 mg kg − 1 (Zn), 3.4 to 175 mg kg − 1 (Ba), 2.15 to 12.79 mg kg − 1 (Sr). It was observed that as the percentage increases cocoa, there is a tendency to increase the concentration of the studied elements. The developed method has satisfactory precision and accuracy, and is simple, fast and with low consumption of reagents, has good sensitivity, especially when compared to digestion methods. extraction induced by emulsion breaking chocolate MIP OES nutrient elements Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The cocoa bean is an important agricultural input for the food and cosmetics industry, with chocolate being the most nutritionally and economically important by-product (Guriérrez-Macías et al., 2021). Its chemical composition is a rich source of flavonoids, polyphenols and mineral elements, as Ca, Fe, K, Mg, Na, P, Cu, Mn and Zn, essential for the maintenance of the human health. The composition of the natural soil and affected by anthropic actions contributes to the occurrence of inorganic nutrients in plants and consequently in beans and their products, such as: chocolates barr. In addition, other ingredients with milk, sugar and fat can influence the concentrations of these elements (Lewis et al., 2021 ). The determination of inorganic species in chocolate samples is described in the literature using different spectroanalytical techniques, such as the determination of Ca, Cu, Fe, Mn, Ni and Zn by flame atomic absorption spectrometry (F AAS) (Bakircioglu et al., 2022 ), As, Cd, Pb and Sb (Mohamed et al., 2020 ), Al, Ba, Ca, Cu, Fe, K, Mg, Mn, Na, Ni, P, S, Sr and Zn (Karaś et al., 2021 ), Ca, Fe, K, Mg, Na, P, Zn As, Cd and Pb by ICP OES and ICP-MS (Santana et al., 2024 ). Another analytical technique that has been little explored for this type of sample is microwave-induced plasma optical emission spectrometry (MIP OES). MIP OES with N 2 to generate and sustain plasma has gained relevance in recent years as it has superior detection and quantification capabilities compared to F AAS and achieves results comparable to ICP OES at a reduced operating cost. However, N 2 -MIP is more susceptible to interference because it has a lower temperature and less robust plasma than Ar-ICP (Balaram, 2020 ; Fontoura et al., 2022 ). Consequently, the sample treatment step has been used in samples with a high fatty acids/ lipids content (e.g. chocolate bars) to minimize interference in the determination of inorganic species by MIP OES, as observed in the determination of Ba, Ca, Cr, Cu, Fe, Ni, Zn, Mn, Mg, Na and P using microwave-assisted acid digestion to adapt the sample prior to analysis (Oliveira et al., 2021 ). Methods for digesting samples from matrices with a high fat content are complex, labor-intensive, consume excess reagents, are expensive and present high risks (Burguera & Burguera, 2012 ; Garitta et al., 2021 ). The partitioning of analytes from the organic matrix to the aqueous phase, such as emulsion-induced extraction by breaking (EIEB), is an alternative to digestion and its difficulties (Mdluli et al., 2020; Valasques et al, 2018 ). EIEB has been explored for the simplification of complex samples because it is minimalist, has the capacity to reduce interferences and improve the analytical characteristics of the method, and has low operating costs, as described in different analyses of complex samples: Zn determination in diesel oil by FAAS (Cassella et al., 2011 ) and Al, Cu, Mn, Ni, Sn and V by ICP-MS (Cassella et al., 2012 ), Zn edible oils (Bakircioglu et al., 2014 ), Ca and Mg in biodiesel by FAAS (Pereira et al., 2014 ), Hg determination in oil samples by CVAAS (Vicentino, Brum & Cassella, 2015 ), Ba, Ca, Mg and Na in crude oil by ICP OES (Trevelin et al., 2016 ), Ni and V (de Souza, Cassella & Lepri, 2019). Corazza and Tarley studied the use of EIEB for quantification of Cd in omega-3 supplementation by flame-oven and thermal aerosol atomic absorption spectrometry (TS-FF-AAS) (Corazza & Tarley, 2016 ). Vicentino and Cassella proposed a model study for Hg extraction from gasoline (Vicentino & Cassella, 2017 ). More recently, EIEB was applied to complex samples for determination by spectroanalytical techniques: lubricating oil (Al-Dalahmeh et al., 2019 ; Fernandes et al., 2019 ), gasoline mixture with ethanol (Souza et al., 2019 ), palm oil (Valasques et al., 2018 ; Valasques et al., 2020 a; Valasques et al., 2020 b), edible vegetable oils (Robaina et al., 2012 ; He et al., 2014 ; Carneiro, 2020), margarine (Kurtulus et al., 2021 ) and cocoa butter (Guimarães et al., 2022 ). This work proposes the development of an analytical method for the analysis of chocolate bars with different cocoa contents based on emulsion break-induced extraction and microwave-induced plasma optical emission spectrometry (MIP OES) for multielement determination of Ca, Zn, Sr, Ba, Cu, Mn, Mg, and Cr. Experimental Instrumentation The determination of analytes has been performed using a Agilent Technologies 4200 microwave-induced plasma optical emission spectrometer (Agilent, Saint Clara, EUA), equipped with a OneNeb nebulizer, double-pass cyclone spray chamber, a Czerny-Turner monochromator and charge-coupled device (CCD) detector. Nitrogen gas, which feeds the equipment´s plasma, was separated form the ambient air by an nitrogen generator. The operating conditions were as follow: wavelength 285.213 nm (Mg), 393.366 nm (Ca), 371.993 nm (Fe), 455.403 nm (Ba), 407.771 nm (Sr), 403.307 nm (Mn), 213.857 nm (Zn), 425.433 nm (Cr) and 324.754 nm (Cu). Peristaltic pump speed at 15 rpm, stabilization time 10s, absorption time 10s, nebulization flow in L min -1 0.9 (Mg), 0.6 (Ca), 0.65 (Fe), 0.65 (Ba), 0.55 (Sr), 0.9 (Mn), 0.45 (Zn), 0.3 (Cr) and 0.7 (Cu), plasma viewing position, respectively, 10, -20, -10, 0, 0, -20, -10, -10 and − 10. For validation purposes of the proposed method was used the flame atomic absorption spectrometer, model SpectrAA 240FS (Mulgrave, Victoria, Australia) .The instrument was equipped with multi-element hollow cathode lamps (10 mA) and line in 285.2 nm (Mg), 422.7 nm (Ca), 248.3 nm (Fe), 553.6 nm (Ba), 460.7 nm (Sr), 213.9 nm (Mn), 285.2 nm (Zn), 357.9 nm (Cr) and 249.7 nm (Cu), and background correction deuterium lamp, operating under the following conditions: 13.5 L min -1 air flow rate, 2.00 L min -1 acetylene flow, sample aspiration at 6 mL min -1 , and burner height 13.5 mm. Others equipment, such as analytical balance model AUW220D (Shimadzu, Kyoto, Japan) with 10 − 5 g accuracy, thermostatic bath model SL 91 (Solab, Sao Paulo, Brazil), for the digestions blocks models TE-040/25 − 1 and 015 − 1 (Tecnal, Sao Paulo, Brazil) and microwave-assisted digestion using a microwave oven model Mars Xpress (CEM, Carolina North, USA), vortex type shaker model AP59 (Phoenix Luferco, Sao Paulo, Brazil), muffle furnace model SSFM67L (Solidsteel, Sao Paulo, Brazil) were also used. Reagents and samples All laboratory glassware and glass tubes were previously washed with soap and tape water, kept overnight in 10% (v/v) nitric acid solution, rinsed with deionized water supplied by a reverse osmose water purification system (OS10 LX, Gehaka, São Paulo, Brazil), and dried in stove at 65°C. The solutions were prepared with ultrapure water. The reagents used were of analytical grade. Nitric acid 65% (w/w) sulfuric acid 98% (w/w), hydrogen peroxide 30% (Merck, Darmstadt, Germany) were used. Triton X-100 (t-Octylphenoxypolyethoxyethanol), Triton X-114 ((1,1,3,3 tetramethylbutyl)phenyl-polyethylene glycol) surfactants (Sigma-Aldrich, Milwaukee, USA) and Tween 80 (Polysorbate 80) (Labsynth) were used for forming the emulsions. The antifoam used was Lufbem (Natulab, Santo Antonio de Jesus, Brazil) to remove the bubbles from the solution. Analytical curves were prepared from the dilution of multi-element standard (Specsol and QuimLab Química e Metrologia, São José dos Campos, Brazil). Chocolate samples were obtained in stores from Ilhéus, Itacaré and Itabuna cities, Bahia state, Brazil. Extraction induced by emulsion breaking (EIEB) EIEB procedure The emulsions were directly formed in a 10 mL glass tube by mixture of 0.25 g of chocolate sample, 5.0 mL of 1.5% (w v − 1 ) Tween 80 surfactant in 1.5 mol L − 1 nitric acid aqueous solution, and sonication by 5 minutes until the oil/water (o/w) emulsion formation. Then the breakdown of the emulsion has been induced by heating to 80–90°C in a water bath by 4 minutes until the total phase separation and deposition of the enriched aqueous phase at the bottom of the tube. The organic phase was removed and 1mL of the enriched aqueous phase was collected and diluted for determination of each analyte. The procedures have been performed in triplicate, and analytical blanks were performed in the same conditions. Strategy for optimization of method The optimization of the variables that affect the method is necessary to obtain better responses and, consequently, better analytical characteristics. So, the EIEB optimization for Mg, Ca, Ba, Sr, Cr, Mn, Cu, Zn and Fe has been realized with a chocolate sample, using information presented in other works already published in the literature, and previous experiments. The Nitric acid and surfactant Tween 80 solution as extraction phase, temperature of emulsion break, and sonication time were the variables studied by univariate methodology. Tween 80, Triton X-100 and Triton X-114 were studied as surfactants for emulsion formation by univariate methodology, using as analytical response the values of the signals for Mg, Ca, Fe, Sr, Cr, Mn, Cu, Zn and Fe. Univariate optimization of the variables is applied in the development of new analytical methods (Cassella et al., 2010). In this work has been employed to optimize three variables: nitric acid concentration (ac) in 0.25, 0.5, 1.0, 1.5, 2.0 and 2.5 mol L − 1 ; surfactant concentration (sc) in 1.0, 1.5, 2.0, 2.5 and 3.0% w v − 1 ), for the extractor composition; and extraction time (et) in 2, 5, 10, 15, 20 and 25 min. For this work, the influence of the type of surfactant was evaluated where the response obtained by MIP OES (c/s) for each element was converted into values as a function of the time required for the emulsion to break down, followed by normalization (Eq. 1). The data analysis has been performed using the Microssoft Excel® 2013. \(R=\frac{signal}{time}\times s\) −1 On what: \(R=Individual normalized response for each element\) S = \(\left(signal with greater intensity\right)\) Time = \(break time\) (Eq. 1) The influence of acid concentration was studied ranging from 0.25 to 2.5 mol L − 1 , surfactant concentration was studied ranging from 1.0 to 3.0% and extraction time was studied ranging from 2 to 25 minutes. And for the evaluation of the matrix effect calibration strategies were tested as the aqueous curve with external standard (conventional), aqueous curve with external standard plus internal standard (Sc and In), standard addition in the sample extract with dilution 1:5 and 1:10 dilution. Samples calcination The method was adapted from the American Association for Testing Materials (ASTM) and used as reference to verify the accuracy of the proposed method. A mass of 0.500 g of samples has been weighed directly into porcelain crucible and calcinated in oven muffle to 100°C by 1 h, followed of heating 300°C by 2 h. Subsequently, the samples were cooled to room temperature, added about 200 µL of sulfuric acid concentrated and heated to 400°C by 2 h up to a complete ash dry. Finally, ash has been dissolved with 1.0 mol L − 1 HNO 3 solution, quantitatively transferred to a volumetric flask, and completed the final volume of 10.0 mL with some solution. The procedures have been performed in triplicate, and analytical blanks were performed in the same. Determinations were carried by FAAS. Results and Discussion Study of the emulsion formation Comparing the signals of the surfactants, Triton X-100, Triton X-114 and Tween 80, visually the signals were relatively close (Fig. 1 a), however the application of the paired t test proves that there is no significant difference between the values. However, when the signal is divided by the break time, followed by normalization, Eq. 1, it is observed that the tween 80 presents greater responses (Fig. 1 b). This surfactant has a higher molecular volume and more ramifications, consequently forming fewer stable emulsions, influencing the break time (shorter break time). Therefore, Tween 80 was selected for the development of the analytical method proposed for analysis in chocolate by MIP OES. In the evaluation of the choice of the type of agitation, the ultrasound bath and vortex were studied where it was observed that the agitation method with the best response was the extraction assisted by the ultrasound bath, since it is possible that the collapse and implosion of the acoustic cavitation cause an increase in the local temperature and voltage causing damage to substances, that is, acoustic waves increase the interaction of aqueous phases with solid surfaces releasing intracellular substances to the liquid. The effect of ultrasonic cavitation disintegrates the chocolate structure, increasing the contact surface area and consequently improving the extraction of analytes irradiated with ultrasonic waves (Deng, Feng, Qiu, 2009; Júnior et al., 2006). Also depending on the choice, the use of the ultrasonic bath is more practical as it allows us to work with more than one tube at a time, unlike the vortex mixer which only allows us to work with one tube at a time. Univariate optimization of the EIEB The methodology used was univariate, comparing variations due to changes in the levels of variables. The first variable studied was the concentration of nitric acid (Fig. 2 ), which is responsible for displacing metals from organic structures to the aqueous phase as free ions (Robaina, Brum, Cassella, 2012 ). As the concentration of nitric acid increases the extraction efficiency, however, in the experimental region studied, a reduction in the signal is observed from the concentration of 1.5 mol L − 1 . This effect can be explained by the reduction in the stability of the emulsion caused by the increase in the ionic charge, which in turn reduces the contact between the phases and influences the signal of the analytes. The second variable studied was the concentration of the surfactant that acts as an emulsion stabilizer so that there is a greater contact surface between the organic and aqueous phases. Increasing the surfactant concentration improves the extraction efficiency with the contact time between the phases, as there is greater acid-metal interaction. Furthermore, analytes that are strongly bound to the matrix can also be carried by physicochemical interaction in the emulsion breaking process. However, there is a reduction in the experimental region studied from the concentration of 1.5% (Fig. 3 ), which is justified by the change in the physical characteristic of the extract, since the surfactant is also transferred to the aqueous phase due to its high solubility in water (Robaina, Brum, Cassella, 2012 ). The analyzed aliquot becomes more viscous, making it difficult to nebulize the sample and transport it to the plasma. The optimal value selected for the multielement determination was 1.5% Tween 80. Another optimized parameter was the extraction time (Fig. 4 ), that is, the time in which the sample was subjected to the ultrasound bath. The extraction time increases the contact time between the aqueous and organic phases, so the longer the time, the better the stability and efficiency of the extraction. However, this effect is not observed for Zn, Fe and Cu. Chocolate sample ins complex and contains organic compounds that are soluble in water. The extension of contact between the phases extracts these compounds that interfere with the analytical signal and can complex the elements, thus making their determination difficult. Some species are strongly linked to the organic phase, making their extraction difficult. The extraction time selected for the multielement analysis was 5 minutes in an ultrasound bath. Validation and application The proposed method has been validated considering the performance of the analytical feature: detection limit, quantification limit, precision, accuracy, and calibration strategy. The calibration strategy (Table 1 ) was studied considering that the extractor solution has different properties from standard aqueous solutions. Thus, the matrix effect was evaluated by observing the angular coefficients of the analytical curves made so: i ) aqueous curve with external standard; ii ) aqueous curve with external standard plus internal standard (In and/or Sc); iii ) standard addition in the sample extract for dilution 1:5; and iv ) standard addition to the sample extract at 1:10 dilution. Table 1 Calibration strategies for EIEB in the determination of Ca, Zn, Fe, Sr, Ba, Cu, Mn, Mg, Cr in the chocolate sample (Cho A-70) by MIP OES. Values expressed as mean ± standard deviation, n = 3. Calibration strategy Analytical curve Pattern addition curve (1:5) Pattern addition curve (1:10) Internal standard (Sc) Internal standard (In) Ca Coefficients a 482231 ± 16238 472185 ± 24437 475559 ± 37852 692532 ± 50879 684013 ± 39632 b 26911 ± 2164 1131827 ± 37414 714219 ± 37852 54585 ± 7039 -204748 ± 11806 R² 0,993 ± 0,001 0,9945 ± 0,0024 0,9980 ± 0,0016 0,9926 ± 0,0004 0,9943 ± 0,0009 Zn Coefficients a 6760 ± 102,2 5442 ± 125 5568 ± 125 8817 ± 662 5837 ± 365 b -106,8 ± 6,70 975 ± 117 681 ± 77,52 501 ± 37,63 392,01 ± 24,55 R² 0,9990 ± 0,0002 0,9969 ± 0,0034 0,9967 ± 0,0034 0,9994 ± 0,0005 0,9945 ± 0,0003 Fe Coefficients a 15776 ± 1074 17416 ± 2200 16503 ± 2200 2614 ± 261,4 18224 ± 1285 b 34,7 ± 5,09 134,10 ± 22,77 101,4 ± 7,91 140,9 ± 14,10 26,46 ± 1,87 R² 0,9994 ± 0,0020 0,9952 ± 0,0024 0,9973 ± 0,0024 0,9981 ± 0,0007 0,9994 ± 0,0011 Sr Coefficients a 395391 ± 58271 413725 ± 14737 451903 ± 14737 444293 ± 52428 503824 ± 48681 b 2141 ± 198,71 7001 ± 671 8688 ± 459 1708 ± 775 2053 ± 198,4 R² 0,9966 ± 0,0038 0,9987 ± 0,0005 0,9991 ± 0,0005 0,9993 ± 0,0002 0,9998 ± 0,0006 Ba Coefficients a 262618 ± 39063 285375 ± 12043 290545 ± 12043 266360 ± 10208 329945 ± 31688 b 2095 ± 108,58 6386 ± 603 8226 ± 542 1413 ± 362 951,98 ± 817,79 R² 0,9978 ± 0,0015 0,9986 ± 0,0002 0,9974 ± 0,0003 0,9993 ± 0,0002 0,9992 ± 0,0006 Cu Coefficients a 89733 ± 689 97835 ± 9549 82733 ± 433 73187 ± 6245 96750 ± 6292 b 317 ± 24,41 1435 ± 56,50 3142 ± 433 375 ± 159,7 -79,32 ± 1,75 R² 0,9976 ± 0,0003 0,9973 ± 0,0001 0,9948 ± 0,0001 0,9992 ± 0,0006 0,9987 ± 0,0006 Mn Coefficients a 20813 ± 2279 25213 ± 1818 24278 ± 1818 37300 ± 3767 28771 ± 1588 b 116 ± 10,81 455 ± 27,08 769 ± 140,04 351 ± 49,93 -44,80 ± 5,76 R² 0,9992 ± 0,0004 0,9974 ± 0,0002 0,9971 ± 0,0002 0,9973 ± 0,0012 0,9983 ± 0,0012 Mg Coefficients a 120991 ± 20713 111894 ± 27361 107790 ± 27361 212921 ± 16832 19928 ± 1275 b 11727 ± 4159 682487 ± 106392 478835 ± 67365 21365 ± 1689 -83,63 ± 5,56 R² 0,9982 ± 0,0012 0,9976 ± 0,0018 0,9954 ± 0,0018 0,9991 ± 0,0007 0,9992 ± 0,0002 Cr Coefficients a 23047 ± 1573 27393 ± 401 29943 ± 401 30780 ± 2375 31093 ± 2117 b 109 ± 17,51 -329,13 ± 26,65 455 ± 68,25 110,12 ± 6,32 86,36 ± 11,61 R² 0,9996 ± 0,0001 0,9986 ± 0,0005 0,9974 ± 0,0005 0,9991 ± 0,0006 0,9995 ± 0,0001 External calibration, with inorganic standards diluted in water with nitric acid and surfactant, showed a significant difference (t calculatedo > t critical , n 1 = n 2 = 3, p = 0.05) in comparison with sample combustion, calcination, followed by FAAS analysis, reference method. Consequently, the matrix of the extract analyzed by MIP OES influences the analytical signal causing the matrix effect. In the calibration with the addition of a standard in the extracts in the extracts at 1:5 and 1:10 dilutions (extract: water), the solution with the lowest dilution factor (1:5) had a higher concentration when compared to the second dilution (1:10), indicating that the solution with the highest dilution factor presents greater loss of information. In addition, both dilutions when compared to the alternative method show significant differences (t calculated > t critical , n 1 = n 2 = 3, p = 0.05), indicating that despite using the matrix to prepare the curve, there is still an influence of the matrix components and instrumental fluctuations. The two calibration curves with internal standard (Sc and In) showed no significant difference in the concentrations of Ca, Zn, Fe, Cu, Mn and Mg with those found with the reference method, t calculated < t critical , n 1 = n 2 = 3, p = 0.05, t calculated are, respectively, -1.29, -1.63, 0.68, 2.02, 0.63, -1.52 and − 2.00. -0.78, -1.63, -2.17, 1.72, 0.78, except for Sr, Ba and Cr which had a concentration lower than LQ when the combustion is analyzed by FAAS. Both curves were accurate and showed no significant difference between the standard deviations, F calculeted < F critical , n 1 = n 2 = 3, p = 0.05, F calculated for Ca, Zn, fe, Cu, Mn, Mg, Sr Ba and Cr are 11.35, 2.66, 5.49, 1.79, 5.26, 2.62, 1.15, 8.33 and 1.32, respectively. In view of this, we chose to work with the internal standard Sc. The limit of detection (LOD) and limit of quantification (LOQ) were calculated as the concentration of the analyte corresponding to 3 and 10 times, respectively, oh the relative standard deviation of ten independent measurements of the analytical blank, multiplied by the concentration equivalent to the radiation of background (BEC) divided by 100. Then, for the proposed method, the LOD were 0.0831, 0.0125, 0.4627, 0.0635, 0.0101, 0.0991, 0.0356, 0.0161 and 0.03487 mg Kg − 1 for Ca, Zn, Fe, Sr, Ba, Cu, Mn, Mg and Cr, respectively, and the LOQ were 0.2769, 0.0192, 2.103, 0.0800, 0.0696, 0.5011, 0.1356, 0.0347 and 0.5369 mg Kg − 1 for Ca, Zn, Fe, Sr, Ba, cu, Mn, Mg and Cr, respectively, taking into account 0.25 g of the solid sample and 5.0 mL of extractor. Precision was described by the relative standard deviation (RSD %) of seven consecutive measurements from different experiments. For the Cho A-70 sample, the values found for the analytes, in %, were 5.3 (Ca), 9.7 (Zn), 6.4 (Fe), 8.4 (Sr), 6.8 (Ba), 9.7 (Cu), 9.8 (Mn), 8.8 (Mg) and 9.7 (Cr). The Accuracy was evaluated by experiments with SRM Banking Chocolate 2384 (NIST). For the elements present in the certified material, no significant difference was observed, obtained at a confidence level of 95%, therefore, the method is accurate and can be applied in the determination of Ca, Mg, Mn, Cu, Zn and Fe in chocolate samples by MIP OES (Table 2 ). Furthermore, the accuracy was also evaluated by comparing the EIEB with a reference method, in which three samples were subjected to total decomposition to ashy (calcination), followed by dissolution in nitric acid and determination of analytes by FAAS. Comparing the results using the pared t test, t calculated for Ca, Mg, Mn, Cu, Zn and Fe are, respectively, -1.79, 1.01, 1.47, 1.66, 1.64 and 1.58, being obtained t calculated < t critical (4.30), n 1 = n 2 = 3, p = 0.05, demonstrating that there is no significant difference between the methods. Due to FAAS sensitivity, it was not possible to accurately determine for the elements Sr, Cr and Ba by calcination. Table 2 Result of the proposed method applied to SRM Baking Chocolate NIST 2384 (mean ± deviation, n = 3, for 95% confidence). Elements Found by EIEB Reference value Agreement (%) t calculated Ca 847 ± 39 840 ± 74 101 0,33 Mg 2606 ± 226 2610 ± 120 100 -0,030 Mn 21,3 ± 1,3 20,8 ± 1,3 102 0,64 Cu 21,2 ± 0,75 23,9 ± 1,0 89 -3,69 Zn 40,0 ± 1,2 37,6 ± 1,9 106 3,50 Fe 127 ± 9,4 132 ± 11 96 -0,93 t critic = 4.30 (95% confidence) Table 3 Comparison between different strategies for preparing samples of chocolate (Cho A-70) analyzed by FAAS e MIP OES, with analyte concentration expressed with confidence interval (mean ± standard deviation), (n = 3). Elements Reference method Proposed method % Agreement Ca 901 ± 31 820 ± 40 91 Mg 1712 ± 107 1789 ± 77 104 Mn 32,7 ± 1,7 35,0 ± 1,5 107 Cu 21,5 ± 0,82 19,3 ± 1,5 90 Zn 42,1 ± 4,1 36,9 ± 0,98 88 Fe 69,4 ± 4,4 77,0 ± 4,1 111 Ba N.D.* 13,4 ± 0,47 -- Sr N.D. 9,85 ± 0,92 -- Cr N.D. 5,08 ± 0,12 -- * Not determined The extraction induced by emulsion breakage was applied to 10 samples with different cocoa contents, as seen in Fig. 5 . Where the concentrations ranged from 328 to 1424 mg Kg − 1 (Ca), 561 to 2152 mg Kg − 1 (Mg), 7.8 to 251 mg Kg − 1 (Cu), 8.5 to 304 mg Kg − 1 (Mn), 6.22 to 98.32 mg Kg − 1 (Fe), 8.38 to 80.2 mg Kg − 1 (Zn), 3.4 to 175 mg Kg − 1 (Ba) and 2.15 to 12.79 mg Kg − 1 (Sr). It is observed that what determines the concentrations of these elements in chocolate is the soil in which it is grown, the processing of cocoa almonds and the added ingredients, such as milk (Cinquanta el al., 2016; Barraza et al., 2021; Assa et al., 2018). Cocoa beans are important sources of naturally occurring magnesium, which explains the high level in chocolate (Ellam, Williamson, 2013 ). According to the values allowed by the TACO table, 2011, for milk chocolate 1910 mg Kg − 1 (Ca), 570 mg kg − 1 (Mg), 3.0 mg kg − 1 (Mn), 16 mg Kg − 1 (Fe), 3.1 mg kg − 1 (Cu), 11 mg kg − 1 (Zn) and for semisweet chocolate 450 mg kg − 1 (Ca), 1070 mg kg − 1 (Mg), 8.3 mg kg − 1 (Mn), 36 mg kg − 1 (Fe), 7.7 mg kg − 1 (Cu) and 15 mg kg − 1 (Zn) it was observed that for some samples of the present study, the concentrations are within the allowed value and some samples presented values above the allowed limit. Conclusions The application of extraction induced by emulsion breakage in samples with high lipid content allows a simpler, more economical, and faster sample preparation method for application in routine analysis and multielement determination of species such as Ca, Mg, Mn, Fe, Zn, Sr, Ba, Cu and Cr considering economic aspects, health, and well-being of consumers. The proposed method has the advantage of shorter process time, flexibility as a function of mass, less residue formation and non-use of concentrated acids and organic solvents, managing to determine a greater number of elements. In addition, the present method revealed satisfactory detection limits due to the technique employed. Declarations Acknowledgments Authors acknowledge the financial support of the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil) and Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB, Brazil), Financiadora de Estudos e Projetos (FINEP), and Universidade Estadual de Santa Cruz (UESC). Funding information Geovana B. Guimarães, Leonardo B. Guimarães, Julia C. Romero and Sheylla M. S. Queiroz are students of the Programa de Pós-Graduação em Química da UESC and received research grants from Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB). Erik G. P. da Silva, Raildo M. de Jesus and Fábio de S. Dias received research grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). This study is also financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES). Conflict of Interest: Geovana B. Guimarães declares that she has no conflict of interest. Leonardo B. Guimarães declares that he has no conflict of interest. Julia C. Romero declares that he has no conflict of interest. Sheylla M. S. Queiroz declares that she has no conflict of interest. Erik G. P. da Silva declares that he has no conflict of interest. Daniel de C. Lima declares that he has no conflict of interest. Luana N. Santos declares that she has no conflict of interest. Fábio G. Lepri declares that he has no conflict of interest. Fábio S. Dias declares that he has no conflict of interest. Allison G. Santos declares that he has no conflict of interest. Ethical Approval: This article does not contain any studies with human participants or animals performed by any of the authors. Informed Consent: Informed consent was obtained from all individual participants included in the study. Credit author statement Geovana B. Guimarães was responsible for formal analysis, conceptualization; visualization; roles/writing original draft; writing-review and editing; Leonardo B. Guimarães, Julia C. Romero and Sheylla M. S. Queiroz for formal analysis, conceptualization, writing original draft; Erik G. P. da Silva, Daniel C. Lima, Luana N. Santo, Fábio S. Dias, Fábio G. Lepri and Allison G. Sandos were responsible for formal analysis, visualization; writing original draft; writing-review and editing; Fábio A. C. Amorim and Raildo M. de Jesus for formal analysis, funding acquisition, project administration, resources, supervision, roles/writing original draft; visualization, writing-review and editing. References Al-Dalahmeh Y, Al-Swaidan HM, Al-Ghamdi A (2019) Combination of Ultrasonication and Induced Emulsion Breaking for Efficient Extraction of Wear Metals from Lubricating Oils with Inductively Coupled Plasma–Mass Spectrometry Determination. J Anal Chem 74(1):71–80. https://doi.org/10.1134/S1061934819010039 American Section of the International Association for Testing Materials (2015) D7455 – 14 Standard Practice for Sample Preparation of Petroleum and Lubricant Products for Elemental Analysis. B Stand Vol 0504:1–9. https://doi.org/10.1520/D7455-08.1 Bakircioglu D, Bakircioglu Kurtulus Y, Topraksever N (2022) Extraction Induced by Emulsion Breaking for Ca, Cu, Fe, Mn, Ni, and Zn Determination in Chocolate by Flame Atomic Absorption Spectrometry. J AOAC Int 105(5):1299–1308. https://doi.org/10.1093/jaoacint/qsac048 Bakircioglu D, Topraksever N, Kurtulus Y (2014) Determination of zinc in edible oils by flow injection FAAS after extraction induced by emulsion breaking procedure. Food Chem 151:219–224. https://doi.org/10.1016/J.FOODCHEM.2013.11.069 Balaram V (2020) Microwave plasma atomic emission spectrometry (MP-AES) and its applications – A critical review. Microchem J 159(September):105483. https://doi.org/10.1016/j.microc.2020.105483 Burguera JL, Burguera M (2012) Analytical applications of emulsions and microemulsions. Talanta 96:11–20. https://doi.org/10.1016/j.talanta.2012.01.030 Carneiro AF, Carneiro CN, Pires LN, Teixeira LSG, Azcarate SM, Dias FS (2020) D-optimal mixture design for the optimization of extraction induced by emulsion breaking for multielemental determination in edible vegetable oils by microwave-induced plasma optical emission spectrometry. Talanta 219:121218. https://doi.org/10.1016/j.talanta.2020.121218 Cassella R, Brum D, Lima C, Caldas L, De Paula C (2011) Multivariate optimization of the determination of zinc in diesel oil employing a novel extraction strategy based on emulsion breaking. Anal Chim Acta 690(1):79–85. https://doi.org/10.1016/j.aca.2011.01.059 Cassella R, Brum D, Robaina N, Rocha A, Lima C (2012) Extraction induced by emulsion breaking for metals determination in diesel oil by ICP-MS. J Anal Atom Spectrom 27(2):364–370. https://doi.org/10.1039/C2JA10279J Corazza M, Tarley C (2016) 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. Microchem J 127:145–151. https://doi.org/10.1016/J.MICROC.2016.02.021 Costa VC, Babos DV, Aquino FWB, Virgílio A, Amorim FAC, Pereira-Filho ER (2018) Direct Determination of Ca, K and Mg in Cassava Flour Samples by Laser-Induced Breakdown Spectroscopy (LIBS). Food Anal Method 11:1886–1896. https://doi.org/10.1007/s12161-017-1086-9 De Sousa JM, Cassella RJ, Lepri FG (2019) Evaluation of Extraction Induced by Emulsion Breaking for Ni and V Extraction from Off-Shore Brazilian Crude Oils. Energy Fuels 33(11):10435–10441. https://doi.org/10.1021/acs.energyfuels.9b01068 Ellam S, Williamson G (2013) Cocoa and Human Health. Annu Ver Nutr 33:105–128. 10.1146/annurev-nutr-071811-150642 Fernandes A, Vinhal JO, Dutra AJB, Cassella RJ (2019) Study of the extraction of Ca, Mg and Zn from different types of lubricating oils (mineral, semi-synthetic and synthetic) employing the emulsion breaking strategy. Microchem J 145:1112–1118. https://doi.org/10.1016/j.microc.2018.12.043 Fontoura BM, Jofré FC, Williams T, Savio M, Donati GL, Nóbrega JA (2022) Is MIP-OES a suitable alternative to ICP-OES for trace element analysis? J Anal At Spectrom 37(5):966–984. https://doi.org/10.1039/D1JA00375E Garitta JA, Fialho LL, de Oliveira GS, Maria RM, Pirola C, Ferreira AG, Nóbrega JA (2021) Microwave-Assisted Acid Digestion: Evaluation of Reaction Vessel Design and Performance. J Braz Chem Soc 32(4):702–711. https://doi.org/10.21577/0103-5053.20200222 Guimarães LB, Romero JC, Queiroz SMS, Da silva EGP, Lima DC, Coutinho JP, Dias FS, Bezerra MA, Amorim FAC (2022) A new method for determination of Mg, Ca, Zn and Na in cocoa butter by FAAS employing extraction induced by emulsion breaking and multivariate products. Talanta 15:458–467. https://doi.org/10.1007/s12161-021-02123-z Gutiérrez-Macías P, Mirón-Mérida VA, Rodríguez-Nava O, Barragán-Huerta BE (2021) Cocoa: Beyond chocolate, a promising mateiral for potential value-added products. https://doi.org/10.1016/B978-0-12-824044-1.00038-6 . Valorization of Agri-Food Wastes and By-Products.267–268 He Y, Chen J, Zhou Y, Wang X, Liu X (2014) Extraction induced by emulsion breaking for trace multi-element determination in edible vegetable oils by ICP-MS. Anal Methods 6(14):5105–5111. https://doi.org10.1039/C4AY00218K Karaś K, Zioła-Frankowska A, Bartoszewicz M, Krzyśko G, Frankowski M (2021) Investigation of chocolate types on the content of selected metals and non-metals determined by ICP-OES analytical technique. Food Addit Contaminants: Part A 38(2):293–303. https://doi.org/10.1080/19440049.2020.1853821 Kurtulus YB, Bakircioglu D, Babac AC, Yurtsever S, Topraksever N (2021) Extraction induced by emulsion breaking procedure prior to grafite furnace atomic absorption spectrometry and comparison of results to provisional tolerable daily intake values. Food Chem Contaminants 103:1256–1263. 10.1093/jaoacint/qsaa028 Lewis C, Lennon AM, Eudóxie C, Sivapatham P, Umaharan P (2021) Plant metal concentrations in Theobroma cacao as affected by soil metal availability in different soil types. Chemosphere 262. https://doi.org/10.1016/j.chemosphere.2020.127749 Mohamed R, Zainudin BH, Yaakob AS (2020) Method validation and determination of heavy metals in cocoa beans and cocoa products by microwave assisted digestion technique with inductively coupled plasma mass spectrometry. Food Chemistry, 303(August 2019), 125392. https://doi.org/10.1016/j.foodchem.2019.125392 Oliveira LB, de Melo JC, da Boa Morte ES, de Jesus RM, Teixeira LSG, Korn MGA (2021) Multi-element determination in chocolate bars by microwave-induced plasma optical emission spectrometry. Food Chem 351. https://doi.org/10.1016/j.foodchem.2021.129285 Oliveira LB, De Melo jC, Da Boa Morte ES, De Jesus RM, Teixeira LSG, Korn MG (2021) Multi-element determination in chocolate bars by microwave-induced plasma optical emission spectrometry. Food Chem 351. https://doi.org/10.1016/j.foodchem.2021.129285 Pereira F, Brum D, Lepri F, Cassella R (2014) Extraction induced by emulsion breaking as a tool for Ca and Mg determination in biodiesel by fast sequential flame atomic absorption spectrometry (FS-FAAS) using Co as internal standard. Microchem J 117:172–177. https://doi.org/10.1016/j.microc.2014.06.026 Robaina N, Brum D, Cassella R (2012) Application of the extraction induced by emulsion breaking for the determination of chromium and manganese in edible oils by electrothermal atomic absorption spectrometry. Talanta 99:104–112. https://doi.org/155.10.1016/j.talanta.2012.05.025 Santana GFR, Rodrigues RS, Brandão GP, Costa VC, Carneiro MTWD (2024) Multi-element Determination in Craft Chocolate with Different Cocoa Contents Using Ultrasound-Assisted Extraction and Dilute Acid as Sample Preparation. Food Anal Methods 17(3):475–485. https://doi.org/10.1007/s12161-024-02586-w Souza V, Teixeira L, Korn M, Cerqueira U, Bezerra M (2019) Determination of total contents and volatile and non-volatile fractions of nickel and vanadium in gasohol by graphite furnace atomic absorption spectrometry after extraction induced by emulsion-breaking. Fuel 242:479–486. https://doi.org/10.1016/j.fuel.2018.12.129 Tabela Brasileira de Composição de Alimentos (TACO) 4a ed. Campinas: NEPA- UNICAMP (2011) 161 p Trevelin A, Marotto R, De Castro E, Brandão G, Cassella R, Carneiro M (2016) Extraction induced by emulsion breaking for determination of Ba, Ca, Mg and Na in crude oil by inductively coupled plasma optical emission spectrometry. Microchem J 124:338–343. https://doi.org/10.1016/j.microc.2015.09.014 Valasques GS, dos Santos AMP, de Souza VS, Teixeira LSG, Alves JPS, Santos MD, dos Santos WPC, Bezerra MA (2020) Multivariate optimization for the determination of cadmium and lead in crude palm oil by graphite furnace atomic absorption spectrometry after extraction induced by emulsion breaking. Microchem J 153:104401. https://doi.org/10.1016/j.microc.2019.104401 Valasques GS, Dos Santos AM, Silva D, Alves DG, J. P., Bezerra MA (2018) Use of constrained mixture design in the optimization of a method based on extraction induced by emulsion breaking for the determination of Ca, Mg, Mn, Fe and Zn from palm oil by flame atomic absorption spectrometry. J Braz Chem Soc 29(10):2189–2196. https://doi.org/10.21577/0103-5053.20180095 Valasques GS, dos Santos AMP, da Silva DLF, Cerqueira UMFD, Ferreira SLC, dos Santos WNL, Bezerra MA (2020) Extraction induced by emulsion breaking for As, Se and Hg determination in crude palm oil by vapor generation-AFS. Food Chem 318:126473. https://doi.org/10.1016/j.foodchem.2020.126473 Vicentino P, Cassella R (2017) Novel extraction induced by microemulsion breaking: A model study for Hg extraction from Brazilian gasoline. Talanta 162:249–255. https://doi.org/10.1016/j.talanta.2016.10.032 Vicentino P, Brum D, Cassella R (2015) Development of a method for total Hg determination in oil samples by cold vapor atomic absorption spectrometry after its extraction induced by emulsion breaking. Talanta 132:733–738. https://doi.org/10.1016/j.talanta.2014.10.019 Villa JEL, Pereira CD, Cadore S (2015) A novel rapid and simple acid extraction for multielemental determination in chocolate bars. Microchem J 121:199–204 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 04 Jun, 2024 Reviews received at journal 03 Jun, 2024 Reviews received at journal 21 May, 2024 Reviewers agreed at journal 08 May, 2024 Reviewers agreed at journal 07 May, 2024 Reviewers invited by journal 06 May, 2024 Submission checks completed at journal 05 May, 2024 Editor assigned by journal 05 May, 2024 First submitted to journal 03 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4365838","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":300382264,"identity":"0fe1456d-5dc3-492c-9a45-290b8cd8b39e","order_by":0,"name":"Geovana Brito Guimarães","email":"","orcid":"","institution":"Universidade Estadual de Santa Cruz","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Geovana","middleName":"Brito","lastName":"Guimarães","suffix":""},{"id":300382268,"identity":"f769e04b-849c-4a36-ade5-b2d7445b3cf6","order_by":1,"name":"Leonardo Brito 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Carqueija","lastName":"Amorim","suffix":""}],"badges":[],"createdAt":"2024-05-03 20:17:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4365838/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4365838/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56222129,"identity":"252d8203-bb7c-43b3-b9ca-3d3f819ff38d","added_by":"auto","created_at":"2024-05-10 04:51:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":32370,"visible":true,"origin":"","legend":"\u003cp\u003eAnalytical responses (mg kg\u003csup\u003e-1\u003c/sup\u003e and standard deviation) obtained by MIP OES of surfactants and agitation. Extraction induced by emulsion breakage performed with (a) vortex mixer and (b) ultrasonic bath.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4365838/v1/bf20d8e682d59540c121e17e.png"},{"id":56222485,"identity":"4dc8e117-9c0e-4143-8709-e6930491aa64","added_by":"auto","created_at":"2024-05-10 04:59:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":63485,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of the change in the concentration of nitric acid in the EIEB for the determination of Zn, Ca, Fe, Sr, Ba, Cu, Mn, Mg and Cr in chocolate by MIP OES.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4365838/v1/da6b08e75766b24ddb9b35e1.png"},{"id":56222131,"identity":"c4082378-149a-4e59-9bc9-3dc5525820ec","added_by":"auto","created_at":"2024-05-10 04:51:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":63855,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of surfactant concentration variation in EIEB for determination of Zn, Ca, Fe, Sr, Ba, Cu, Mn, Mg and Cr in chocolate by MIP OES.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4365838/v1/2507f0710946fd3efedc41d9.png"},{"id":56222132,"identity":"c034ddb9-c2b3-4ee8-9c19-8c60b08f4d63","added_by":"auto","created_at":"2024-05-10 04:51:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":70018,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluayion of extraction time variation in EIEB for determination of Zn, Ca, Fe, Sr, Ba, Cu, Mn, Mg and Cr in chocolate by MIP OES.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4365838/v1/a9b99f8ed8da68dce6c90380.png"},{"id":56222133,"identity":"29b5990b-6112-4962-9169-09935049cdac","added_by":"auto","created_at":"2024-05-10 04:51:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":27002,"visible":true,"origin":"","legend":"\u003cp\u003eResults found in real samples using the proposed method (mg kg\u003csup\u003e-1\u003c/sup\u003e).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4365838/v1/141754c5fdc4b8510b3defe2.png"},{"id":56222827,"identity":"768d24b7-a42d-47e2-911b-19578f91d307","added_by":"auto","created_at":"2024-05-10 05:07:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":945889,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4365838/v1/47642224-ed5c-4ffb-88ec-b8b03596a5a3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A new method employing extraction induced by emulsion breaking and MIP OES for multi-element determination of inorganic elements in handmade chocolate","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe cocoa bean is an important agricultural input for the food and cosmetics industry, with chocolate being the most nutritionally and economically important by-product (Guri\u0026eacute;rrez-Mac\u0026iacute;as et al., 2021). Its chemical composition is a rich source of flavonoids, polyphenols and mineral elements, as Ca, Fe, K, Mg, Na, P, Cu, Mn and Zn, essential for the maintenance of the human health. The composition of the natural soil and affected by anthropic actions contributes to the occurrence of inorganic nutrients in plants and consequently in beans and their products, such as: chocolates barr. In addition, other ingredients with milk, sugar and fat can influence the concentrations of these elements (Lewis et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe determination of inorganic species in chocolate samples is described in the literature using different spectroanalytical techniques, such as the determination of Ca, Cu, Fe, Mn, Ni and Zn by flame atomic absorption spectrometry (F AAS) (Bakircioglu et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), As, Cd, Pb and Sb (Mohamed et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), Al, Ba, Ca, Cu, Fe, K, Mg, Mn, Na, Ni, P, S, Sr and Zn (Karaś et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), Ca, Fe, K, Mg, Na, P, Zn As, Cd and Pb by ICP OES and ICP-MS (Santana et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Another analytical technique that has been little explored for this type of sample is microwave-induced plasma optical emission spectrometry (MIP OES). MIP OES with N\u003csub\u003e2\u003c/sub\u003e to generate and sustain plasma has gained relevance in recent years as it has superior detection and quantification capabilities compared to F AAS and achieves results comparable to ICP OES at a reduced operating cost. However, N\u003csub\u003e2\u003c/sub\u003e-MIP is more susceptible to interference because it has a lower temperature and less robust plasma than Ar-ICP (Balaram, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Fontoura et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Consequently, the sample treatment step has been used in samples with a high fatty acids/ lipids content (e.g. chocolate bars) to minimize interference in the determination of inorganic species by MIP OES, as observed in the determination of Ba, Ca, Cr, Cu, Fe, Ni, Zn, Mn, Mg, Na and P using microwave-assisted acid digestion to adapt the sample prior to analysis (Oliveira et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMethods for digesting samples from matrices with a high fat content are complex, labor-intensive, consume excess reagents, are expensive and present high risks (Burguera \u0026amp; Burguera, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Garitta et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The partitioning of analytes from the organic matrix to the aqueous phase, such as emulsion-induced extraction by breaking (EIEB), is an alternative to digestion and its difficulties (Mdluli et al., 2020; Valasques et al, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). EIEB has been explored for the simplification of complex samples because it is minimalist, has the capacity to reduce interferences and improve the analytical characteristics of the method, and has low operating costs, as described in different analyses of complex samples: Zn determination in diesel oil by FAAS (Cassella et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and Al, Cu, Mn, Ni, Sn and V by ICP-MS (Cassella et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), Zn edible oils (Bakircioglu et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), Ca and Mg in biodiesel by FAAS (Pereira et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), Hg determination in oil samples by CVAAS (Vicentino, Brum \u0026amp; Cassella, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), Ba, Ca, Mg and Na in crude oil by ICP OES (Trevelin et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), Ni and V (de Souza, Cassella \u0026amp; Lepri, 2019). Corazza and Tarley studied the use of EIEB for quantification of Cd in omega-3 supplementation by flame-oven and thermal aerosol atomic absorption spectrometry (TS-FF-AAS) (Corazza \u0026amp; Tarley, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Vicentino and Cassella proposed a model study for Hg extraction from gasoline (Vicentino \u0026amp; Cassella, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). More recently, EIEB was applied to complex samples for determination by spectroanalytical techniques: lubricating oil (Al-Dalahmeh et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Fernandes et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), gasoline mixture with ethanol (Souza et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), palm oil (Valasques et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Valasques et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003ea; Valasques et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003eb), edible vegetable oils (Robaina et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; He et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Carneiro, 2020), margarine (Kurtulus et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and cocoa butter (Guimar\u0026atilde;es et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis work proposes the development of an analytical method for the analysis of chocolate bars with different cocoa contents based on emulsion break-induced extraction and microwave-induced plasma optical emission spectrometry (MIP OES) for multielement determination of Ca, Zn, Sr, Ba, Cu, Mn, Mg, and Cr.\u003c/p\u003e"},{"header":"Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eInstrumentation\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eThe determination of analytes has been performed using a Agilent Technologies 4200 microwave-induced plasma optical emission spectrometer (Agilent, Saint Clara, EUA), equipped with a OneNeb nebulizer, double-pass cyclone spray chamber, a Czerny-Turner monochromator and charge-coupled device (CCD) detector. Nitrogen gas, which feeds the equipment\u0026acute;s plasma, was separated form the ambient air by an nitrogen generator. The operating conditions were as follow: wavelength 285.213 nm (Mg), 393.366 nm (Ca), 371.993 nm (Fe), 455.403 nm (Ba), 407.771 nm (Sr), 403.307 nm (Mn), 213.857 nm (Zn), 425.433 nm (Cr) and 324.754 nm (Cu). Peristaltic pump speed at 15 rpm, stabilization time 10s, absorption time 10s, nebulization flow in L min\u003csup\u003e-1\u003c/sup\u003e 0.9 (Mg), 0.6 (Ca), 0.65 (Fe), 0.65 (Ba), 0.55 (Sr), 0.9 (Mn), 0.45 (Zn), 0.3 (Cr) and 0.7 (Cu), plasma viewing position, respectively, 10, -20, -10, 0, 0, -20, -10, -10 and \u0026minus;\u0026thinsp;10.\u003c/p\u003e\n\u003cp\u003eFor validation purposes of the proposed method was used the flame atomic absorption spectrometer, model SpectrAA 240FS (Mulgrave, Victoria, Australia) .The instrument was equipped with multi-element hollow cathode lamps (10 mA) and line in 285.2 nm (Mg), 422.7 nm (Ca), 248.3 nm (Fe), 553.6 nm (Ba), 460.7 nm (Sr), 213.9 nm (Mn), 285.2 nm (Zn), 357.9 nm (Cr) and 249.7 nm (Cu), and background correction deuterium lamp, operating under the following conditions: 13.5 L min\u003csup\u003e-1\u003c/sup\u003e air flow rate, 2.00 L min\u003csup\u003e-1\u003c/sup\u003e acetylene flow, sample aspiration at 6 mL min\u003csup\u003e-1\u003c/sup\u003e, and burner height 13.5 mm.\u003c/p\u003e\n\u003cp\u003eOthers equipment, such as analytical balance model AUW220D (Shimadzu, Kyoto, Japan) with 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e g accuracy, thermostatic bath model SL 91 (Solab, Sao Paulo, Brazil), for the digestions blocks models TE-040/25\u0026thinsp;\u0026minus;\u0026thinsp;1 and 015\u0026thinsp;\u0026minus;\u0026thinsp;1 (Tecnal, Sao Paulo, Brazil) and microwave-assisted digestion using a microwave oven model Mars Xpress (CEM, Carolina North, USA), vortex type shaker model AP59 (Phoenix Luferco, Sao Paulo, Brazil), muffle furnace model SSFM67L (Solidsteel, Sao Paulo, Brazil) were also used.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eReagents and samples\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eAll laboratory glassware and glass tubes were previously washed with soap and tape water, kept overnight in 10% (v/v) nitric acid solution, rinsed with deionized water supplied by a reverse osmose water purification system (OS10 LX, Gehaka, S\u0026atilde;o Paulo, Brazil), and dried in stove at 65\u0026deg;C. The solutions were prepared with ultrapure water. The reagents used were of analytical grade. Nitric acid 65% (w/w) sulfuric acid 98% (w/w), hydrogen peroxide 30% (Merck, Darmstadt, Germany) were used. Triton X-100 (t-Octylphenoxypolyethoxyethanol), Triton X-114 ((1,1,3,3 tetramethylbutyl)phenyl-polyethylene glycol) surfactants (Sigma-Aldrich, Milwaukee, USA) and Tween 80 (Polysorbate 80) (Labsynth) were used for forming the emulsions. The antifoam used was Lufbem (Natulab, Santo Antonio de Jesus, Brazil) to remove the bubbles from the solution. Analytical curves were prepared from the dilution of multi-element standard (Specsol and QuimLab Qu\u0026iacute;mica e Metrologia, S\u0026atilde;o Jos\u0026eacute; dos Campos, Brazil). Chocolate samples were obtained in stores from Ilh\u0026eacute;us, Itacar\u0026eacute; and Itabuna cities, Bahia state, Brazil.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eExtraction induced by emulsion breaking (EIEB)\u003c/h2\u003e\n\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n\u003ch2\u003eEIEB procedure\u003c/h2\u003e\n\u003cp\u003eThe emulsions were directly formed in a 10 mL glass tube by mixture of 0.25 g of chocolate sample, 5.0 mL of 1.5% (w v\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) Tween 80 surfactant in 1.5 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e nitric acid aqueous solution, and sonication by 5 minutes until the oil/water (o/w) emulsion formation. Then the breakdown of the emulsion has been induced by heating to 80\u0026ndash;90\u0026deg;C in a water bath by 4 minutes until the total phase separation and deposition of the enriched aqueous phase at the bottom of the tube. The organic phase was removed and 1mL of the enriched aqueous phase was collected and diluted for determination of each analyte. The procedures have been performed in triplicate, and analytical blanks were performed in the same conditions.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eStrategy for optimization of method\u003c/h2\u003e\n\u003cp\u003eThe optimization of the variables that affect the method is necessary to obtain better responses and, consequently, better analytical characteristics. So, the EIEB optimization for Mg, Ca, Ba, Sr, Cr, Mn, Cu, Zn and Fe has been realized with a chocolate sample, using information presented in other works already published in the literature, and previous experiments. The Nitric acid and surfactant Tween 80 solution as extraction phase, temperature of emulsion break, and sonication time were the variables studied by univariate methodology.\u003c/p\u003e\n\u003cp\u003eTween 80, Triton X-100 and Triton X-114 were studied as surfactants for emulsion formation by univariate methodology, using as analytical response the values of the signals for Mg, Ca, Fe, Sr, Cr, Mn, Cu, Zn and Fe.\u003c/p\u003e\n\u003cp\u003eUnivariate optimization of the variables is applied in the development of new analytical methods (Cassella et al., 2010). In this work has been employed to optimize three variables: nitric acid concentration (ac) in 0.25, 0.5, 1.0, 1.5, 2.0 and 2.5 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; surfactant concentration (sc) in 1.0, 1.5, 2.0, 2.5 and 3.0% w v\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), for the extractor composition; and extraction time (et) in 2, 5, 10, 15, 20 and 25 min. For this work, the influence of the type of surfactant was evaluated where the response obtained by MIP OES (c/s) for each element was converted into values as a function of the time required for the emulsion to break down, followed by normalization (Eq.\u0026nbsp;1). The data analysis has been performed using the Microssoft Excel\u0026reg; 2013.\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(R=\\frac{signal}{time}\\times s\\)\u003c/span\u003e \u003c/span\u003e \u003csup\u003e\u0026minus;1\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eOn what: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(R=Individual normalized response for each element\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n\u003cp\u003eS = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\left(signal with greater intensity\\right)\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eTime = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(break time\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e(Eq.\u0026nbsp;1)\u003c/p\u003e\n\u003cp\u003eThe influence of acid concentration was studied ranging from 0.25 to 2.5 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, surfactant concentration was studied ranging from 1.0 to 3.0% and extraction time was studied ranging from 2 to 25 minutes. And for the evaluation of the matrix effect calibration strategies were tested as the aqueous curve with external standard (conventional), aqueous curve with external standard plus internal standard (Sc and In), standard addition in the sample extract with dilution 1:5 and 1:10 dilution.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\n\u003ch2\u003eSamples calcination\u003c/h2\u003e\n\u003cp\u003eThe method was adapted from the American Association for Testing Materials (ASTM) and used as reference to verify the accuracy of the proposed method. A mass of 0.500 g of samples has been weighed directly into porcelain crucible and calcinated in oven muffle to 100\u0026deg;C by 1 h, followed of heating 300\u0026deg;C by 2 h. Subsequently, the samples were cooled to room temperature, added about 200 \u0026micro;L of sulfuric acid concentrated and heated to 400\u0026deg;C by 2 h up to a complete ash dry. Finally, ash has been dissolved with 1.0 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e HNO\u003csub\u003e3\u003c/sub\u003e solution, quantitatively transferred to a volumetric flask, and completed the final volume of 10.0 mL with some solution. The procedures have been performed in triplicate, and analytical blanks were performed in the same. Determinations were carried by FAAS.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStudy of the emulsion formation\u003c/h2\u003e \u003cp\u003eComparing the signals of the surfactants, Triton X-100, Triton X-114 and Tween 80, visually the signals were relatively close (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), however the application of the paired t test proves that there is no significant difference between the values. However, when the signal is divided by the break time, followed by normalization, Eq.\u0026nbsp;1, it is observed that the tween 80 presents greater responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). This surfactant has a higher molecular volume and more ramifications, consequently forming fewer stable emulsions, influencing the break time (shorter break time). Therefore, Tween 80 was selected for the development of the analytical method proposed for analysis in chocolate by MIP OES.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the evaluation of the choice of the type of agitation, the ultrasound bath and vortex were studied where it was observed that the agitation method with the best response was the extraction assisted by the ultrasound bath, since it is possible that the collapse and implosion of the acoustic cavitation cause an increase in the local temperature and voltage causing damage to substances, that is, acoustic waves increase the interaction of aqueous phases with solid surfaces releasing intracellular substances to the liquid. The effect of ultrasonic cavitation disintegrates the chocolate structure, increasing the contact surface area and consequently improving the extraction of analytes irradiated with ultrasonic waves (Deng, Feng, Qiu, 2009; J\u0026uacute;nior et al., 2006). Also depending on the choice, the use of the ultrasonic bath is more practical as it allows us to work with more than one tube at a time, unlike the vortex mixer which only allows us to work with one tube at a time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eUnivariate optimization of the EIEB\u003c/h2\u003e \u003cp\u003eThe methodology used was univariate, comparing variations due to changes in the levels of variables. The first variable studied was the concentration of nitric acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), which is responsible for displacing metals from organic structures to the aqueous phase as free ions (Robaina, Brum, Cassella, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). As the concentration of nitric acid increases the extraction efficiency, however, in the experimental region studied, a reduction in the signal is observed from the concentration of 1.5 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. This effect can be explained by the reduction in the stability of the emulsion caused by the increase in the ionic charge, which in turn reduces the contact between the phases and influences the signal of the analytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe second variable studied was the concentration of the surfactant that acts as an emulsion stabilizer so that there is a greater contact surface between the organic and aqueous phases. Increasing the surfactant concentration improves the extraction efficiency with the contact time between the phases, as there is greater acid-metal interaction. Furthermore, analytes that are strongly bound to the matrix can also be carried by physicochemical interaction in the emulsion breaking process. However, there is a reduction in the experimental region studied from the concentration of 1.5% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), which is justified by the change in the physical characteristic of the extract, since the surfactant is also transferred to the aqueous phase due to its high solubility in water (Robaina, Brum, Cassella, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The analyzed aliquot becomes more viscous, making it difficult to nebulize the sample and transport it to the plasma. The optimal value selected for the multielement determination was 1.5% Tween 80.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnother optimized parameter was the extraction time (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), that is, the time in which the sample was subjected to the ultrasound bath. The extraction time increases the contact time between the aqueous and organic phases, so the longer the time, the better the stability and efficiency of the extraction. However, this effect is not observed for Zn, Fe and Cu. Chocolate sample ins complex and contains organic compounds that are soluble in water. The extension of contact between the phases extracts these compounds that interfere with the analytical signal and can complex the elements, thus making their determination difficult. Some species are strongly linked to the organic phase, making their extraction difficult. The extraction time selected for the multielement analysis was 5 minutes in an ultrasound bath.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eValidation and application\u003c/h2\u003e \u003cp\u003eThe proposed method has been validated considering the performance of the analytical feature: detection limit, quantification limit, precision, accuracy, and calibration strategy.\u003c/p\u003e \u003cp\u003eThe calibration strategy (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) was studied considering that the extractor solution has different properties from standard aqueous solutions. Thus, the matrix effect was evaluated by observing the angular coefficients of the analytical curves made so: \u003cem\u003ei\u003c/em\u003e) aqueous curve with external standard; \u003cem\u003eii\u003c/em\u003e) aqueous curve with external standard plus internal standard (In and/or Sc); \u003cem\u003eiii\u003c/em\u003e) standard addition in the sample extract for dilution 1:5; and \u003cem\u003eiv\u003c/em\u003e) standard addition to the sample extract at 1:10 dilution.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCalibration strategies for EIEB in the determination of Ca, Zn, Fe, Sr, Ba, Cu, Mn, Mg, Cr in the chocolate sample (Cho A-70) by MIP OES. Values expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, n\u0026thinsp;=\u0026thinsp;3.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eCalibration strategy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAnalytical curve\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePattern addition curve (1:5)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePattern addition curve (1:10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eInternal standard (Sc)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eInternal standard (In)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eCa\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCoefficients\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e482231\u0026thinsp;\u0026plusmn;\u0026thinsp;16238\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e472185\u0026thinsp;\u0026plusmn;\u0026thinsp;24437\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e475559\u0026thinsp;\u0026plusmn;\u0026thinsp;37852\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e692532\u0026thinsp;\u0026plusmn;\u0026thinsp;50879\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e684013\u0026thinsp;\u0026plusmn;\u0026thinsp;39632\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e26911\u0026thinsp;\u0026plusmn;\u0026thinsp;2164\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1131827\u0026thinsp;\u0026plusmn;\u0026thinsp;37414\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e714219\u0026thinsp;\u0026plusmn;\u0026thinsp;37852\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e54585\u0026thinsp;\u0026plusmn;\u0026thinsp;7039\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e-204748\u0026thinsp;\u0026plusmn;\u0026thinsp;11806\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u0026sup2;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0,993\u0026thinsp;\u0026plusmn;\u0026thinsp;0,001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0,9945\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0,9980\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0,9926\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0,9943\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0009\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eZn\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCoefficients\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6760\u0026thinsp;\u0026plusmn;\u0026thinsp;102,2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e5442\u0026thinsp;\u0026plusmn;\u0026thinsp;125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e5568\u0026thinsp;\u0026plusmn;\u0026thinsp;125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e8817\u0026thinsp;\u0026plusmn;\u0026thinsp;662\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e5837\u0026thinsp;\u0026plusmn;\u0026thinsp;365\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-106,8\u0026thinsp;\u0026plusmn;\u0026thinsp;6,70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e975\u0026thinsp;\u0026plusmn;\u0026thinsp;117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e681\u0026thinsp;\u0026plusmn;\u0026thinsp;77,52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e501\u0026thinsp;\u0026plusmn;\u0026thinsp;37,63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e392,01\u0026thinsp;\u0026plusmn;\u0026thinsp;24,55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u0026sup2;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0,9990\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0,9969\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0034\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0,9967\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0034\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0,9994\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0,9945\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eFe\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCoefficients\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e15776\u0026thinsp;\u0026plusmn;\u0026thinsp;1074\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e17416\u0026thinsp;\u0026plusmn;\u0026thinsp;2200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e16503\u0026thinsp;\u0026plusmn;\u0026thinsp;2200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e2614\u0026thinsp;\u0026plusmn;\u0026thinsp;261,4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e18224\u0026thinsp;\u0026plusmn;\u0026thinsp;1285\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e34,7\u0026thinsp;\u0026plusmn;\u0026thinsp;5,09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e134,10\u0026thinsp;\u0026plusmn;\u0026thinsp;22,77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e101,4\u0026thinsp;\u0026plusmn;\u0026thinsp;7,91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e140,9\u0026thinsp;\u0026plusmn;\u0026thinsp;14,10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e26,46\u0026thinsp;\u0026plusmn;\u0026thinsp;1,87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u0026sup2;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0,9994\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0,9952\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0,9973\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0,9981\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0,9994\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0011\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eSr\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCoefficients\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e395391\u0026thinsp;\u0026plusmn;\u0026thinsp;58271\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e413725\u0026thinsp;\u0026plusmn;\u0026thinsp;14737\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e451903\u0026thinsp;\u0026plusmn;\u0026thinsp;14737\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e444293\u0026thinsp;\u0026plusmn;\u0026thinsp;52428\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e503824\u0026thinsp;\u0026plusmn;\u0026thinsp;48681\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2141\u0026thinsp;\u0026plusmn;\u0026thinsp;198,71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e7001\u0026thinsp;\u0026plusmn;\u0026thinsp;671\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e8688\u0026thinsp;\u0026plusmn;\u0026thinsp;459\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1708\u0026thinsp;\u0026plusmn;\u0026thinsp;775\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e2053\u0026thinsp;\u0026plusmn;\u0026thinsp;198,4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u0026sup2;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0,9966\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0038\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0,9987\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0,9991\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0,9993\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0,9998\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eBa\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCoefficients\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e262618\u0026thinsp;\u0026plusmn;\u0026thinsp;39063\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e285375\u0026thinsp;\u0026plusmn;\u0026thinsp;12043\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e290545\u0026thinsp;\u0026plusmn;\u0026thinsp;12043\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e266360\u0026thinsp;\u0026plusmn;\u0026thinsp;10208\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e329945\u0026thinsp;\u0026plusmn;\u0026thinsp;31688\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2095\u0026thinsp;\u0026plusmn;\u0026thinsp;108,58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e6386\u0026thinsp;\u0026plusmn;\u0026thinsp;603\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e8226\u0026thinsp;\u0026plusmn;\u0026thinsp;542\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1413\u0026thinsp;\u0026plusmn;\u0026thinsp;362\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e951,98\u0026thinsp;\u0026plusmn;\u0026thinsp;817,79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u0026sup2;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0,9978\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0,9986\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0,9974\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0,9993\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0,9992\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eCu\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCoefficients\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e89733\u0026thinsp;\u0026plusmn;\u0026thinsp;689\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e97835\u0026thinsp;\u0026plusmn;\u0026thinsp;9549\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e82733\u0026thinsp;\u0026plusmn;\u0026thinsp;433\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e73187\u0026thinsp;\u0026plusmn;\u0026thinsp;6245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e96750\u0026thinsp;\u0026plusmn;\u0026thinsp;6292\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e317\u0026thinsp;\u0026plusmn;\u0026thinsp;24,41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1435\u0026thinsp;\u0026plusmn;\u0026thinsp;56,50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e3142\u0026thinsp;\u0026plusmn;\u0026thinsp;433\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e375\u0026thinsp;\u0026plusmn;\u0026thinsp;159,7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e-79,32\u0026thinsp;\u0026plusmn;\u0026thinsp;1,75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u0026sup2;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0,9976\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0,9973\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0,9948\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0,9992\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0,9987\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eMn\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCoefficients\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e20813\u0026thinsp;\u0026plusmn;\u0026thinsp;2279\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e25213\u0026thinsp;\u0026plusmn;\u0026thinsp;1818\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e24278\u0026thinsp;\u0026plusmn;\u0026thinsp;1818\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e37300\u0026thinsp;\u0026plusmn;\u0026thinsp;3767\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e28771\u0026thinsp;\u0026plusmn;\u0026thinsp;1588\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e116\u0026thinsp;\u0026plusmn;\u0026thinsp;10,81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e455\u0026thinsp;\u0026plusmn;\u0026thinsp;27,08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e769\u0026thinsp;\u0026plusmn;\u0026thinsp;140,04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e351\u0026thinsp;\u0026plusmn;\u0026thinsp;49,93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e-44,80\u0026thinsp;\u0026plusmn;\u0026thinsp;5,76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u0026sup2;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0,9992\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0,9974\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0,9971\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0,9973\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0,9983\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eMg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCoefficients\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e120991\u0026thinsp;\u0026plusmn;\u0026thinsp;20713\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e111894\u0026thinsp;\u0026plusmn;\u0026thinsp;27361\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e107790\u0026thinsp;\u0026plusmn;\u0026thinsp;27361\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e212921\u0026thinsp;\u0026plusmn;\u0026thinsp;16832\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e19928\u0026thinsp;\u0026plusmn;\u0026thinsp;1275\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e11727\u0026thinsp;\u0026plusmn;\u0026thinsp;4159\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e682487\u0026thinsp;\u0026plusmn;\u0026thinsp;106392\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e478835\u0026thinsp;\u0026plusmn;\u0026thinsp;67365\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e21365\u0026thinsp;\u0026plusmn;\u0026thinsp;1689\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e-83,63\u0026thinsp;\u0026plusmn;\u0026thinsp;5,56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u0026sup2;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0,9982\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0,9976\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0,9954\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0,9991\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0,9992\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eCr\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCoefficients\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e23047\u0026thinsp;\u0026plusmn;\u0026thinsp;1573\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e27393\u0026thinsp;\u0026plusmn;\u0026thinsp;401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e29943\u0026thinsp;\u0026plusmn;\u0026thinsp;401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e30780\u0026thinsp;\u0026plusmn;\u0026thinsp;2375\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e31093\u0026thinsp;\u0026plusmn;\u0026thinsp;2117\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e109\u0026thinsp;\u0026plusmn;\u0026thinsp;17,51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e-329,13\u0026thinsp;\u0026plusmn;\u0026thinsp;26,65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e455\u0026thinsp;\u0026plusmn;\u0026thinsp;68,25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e110,12\u0026thinsp;\u0026plusmn;\u0026thinsp;6,32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e86,36\u0026thinsp;\u0026plusmn;\u0026thinsp;11,61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u0026sup2;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0,9996\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0,9986\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0,9974\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0,9991\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0,9995\u0026thinsp;\u0026plusmn;\u0026thinsp;0,0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eExternal calibration, with inorganic standards diluted in water with nitric acid and surfactant, showed a significant difference (t\u003csub\u003ecalculatedo\u003c/sub\u003e\u0026gt; t\u003csub\u003ecritical\u003c/sub\u003e, n\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;n\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;=\u0026thinsp;0.05) in comparison with sample combustion, calcination, followed by FAAS analysis, reference method. Consequently, the matrix of the extract analyzed by MIP OES influences the analytical signal causing the matrix effect. In the calibration with the addition of a standard in the extracts in the extracts at 1:5 and 1:10 dilutions (extract: water), the solution with the lowest dilution factor (1:5) had a higher concentration when compared to the second dilution (1:10), indicating that the solution with the highest dilution factor presents greater loss of information. In addition, both dilutions when compared to the alternative method show significant differences (t\u003csub\u003ecalculated\u003c/sub\u003e\u0026gt; t\u003csub\u003ecritical\u003c/sub\u003e, n\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;n\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;=\u0026thinsp;0.05), indicating that despite using the matrix to prepare the curve, there is still an influence of the matrix components and instrumental fluctuations.\u003c/p\u003e \u003cp\u003eThe two calibration curves with internal standard (Sc and In) showed no significant difference in the concentrations of Ca, Zn, Fe, Cu, Mn and Mg with those found with the reference method, t\u003csub\u003ecalculated\u003c/sub\u003e \u0026lt; t\u003csub\u003ecritical\u003c/sub\u003e, n\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;n\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;=\u0026thinsp;0.05, t\u003csub\u003ecalculated\u003c/sub\u003e are, respectively, -1.29, -1.63, 0.68, 2.02, 0.63, -1.52 and \u0026minus;\u0026thinsp;2.00. -0.78, -1.63, -2.17, 1.72, 0.78, except for Sr, Ba and Cr which had a concentration lower than LQ when the combustion is analyzed by FAAS. Both curves were accurate and showed no significant difference between the standard deviations, F\u003csub\u003ecalculeted\u003c/sub\u003e \u0026lt; F\u003csub\u003ecritical\u003c/sub\u003e, n\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;n\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;=\u0026thinsp;0.05, F\u003csub\u003ecalculated\u003c/sub\u003e for Ca, Zn, fe, Cu, Mn, Mg, Sr Ba and Cr are 11.35, 2.66, 5.49, 1.79, 5.26, 2.62, 1.15, 8.33 and 1.32, respectively. In view of this, we chose to work with the internal standard Sc.\u003c/p\u003e \u003cp\u003eThe limit of detection (LOD) and limit of quantification (LOQ) were calculated as the concentration of the analyte corresponding to 3 and 10 times, respectively, oh the relative standard deviation of ten independent measurements of the analytical blank, multiplied by the concentration equivalent to the radiation of background (BEC) divided by 100. Then, for the proposed method, the LOD were 0.0831, 0.0125, 0.4627, 0.0635, 0.0101, 0.0991, 0.0356, 0.0161 and 0.03487 mg Kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for Ca, Zn, Fe, Sr, Ba, Cu, Mn, Mg and Cr, respectively, and the LOQ were 0.2769, 0.0192, 2.103, 0.0800, 0.0696, 0.5011, 0.1356, 0.0347 and 0.5369 mg Kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for Ca, Zn, Fe, Sr, Ba, cu, Mn, Mg and Cr, respectively, taking into account 0.25 g of the solid sample and 5.0 mL of extractor.\u003c/p\u003e \u003cp\u003ePrecision was described by the relative standard deviation (RSD %) of seven consecutive measurements from different experiments. For the Cho A-70 sample, the values found for the analytes, in %, were 5.3 (Ca), 9.7 (Zn), 6.4 (Fe), 8.4 (Sr), 6.8 (Ba), 9.7 (Cu), 9.8 (Mn), 8.8 (Mg) and 9.7 (Cr).\u003c/p\u003e \u003cp\u003eThe Accuracy was evaluated by experiments with SRM Banking Chocolate 2384 (NIST). For the elements present in the certified material, no significant difference was observed, obtained at a confidence level of 95%, therefore, the method is accurate and can be applied in the determination of Ca, Mg, Mn, Cu, Zn and Fe in chocolate samples by MIP OES (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Furthermore, the accuracy was also evaluated by comparing the EIEB with a reference method, in which three samples were subjected to total decomposition to ashy (calcination), followed by dissolution in nitric acid and determination of analytes by FAAS. Comparing the results using the pared \u003cem\u003et\u003c/em\u003e test, t\u003csub\u003ecalculated\u003c/sub\u003e for Ca, Mg, Mn, Cu, Zn and Fe are, respectively, -1.79, 1.01, 1.47, 1.66, 1.64 and 1.58, being obtained t\u003csub\u003ecalculated\u003c/sub\u003e \u0026lt; t\u003csub\u003ecritical\u003c/sub\u003e (4.30), n\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;n\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;=\u0026thinsp;0.05, demonstrating that there is no significant difference between the methods. Due to FAAS sensitivity, it was not possible to accurately determine for the elements Sr, Cr and Ba by calcination.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResult of the proposed method applied to SRM Baking Chocolate NIST 2384 (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;deviation, n\u0026thinsp;=\u0026thinsp;3, for 95% confidence).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElements\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFound by EIEB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAgreement\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003et \u003csub\u003ecalculated\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e847\u0026thinsp;\u0026plusmn;\u0026thinsp;39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e840\u0026thinsp;\u0026plusmn;\u0026thinsp;74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2606\u0026thinsp;\u0026plusmn;\u0026thinsp;226\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2610\u0026thinsp;\u0026plusmn;\u0026thinsp;120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0,030\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e21,3\u0026thinsp;\u0026plusmn;\u0026thinsp;1,3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e20,8\u0026thinsp;\u0026plusmn;\u0026thinsp;1,3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e102\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e21,2\u0026thinsp;\u0026plusmn;\u0026thinsp;0,75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e23,9\u0026thinsp;\u0026plusmn;\u0026thinsp;1,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3,69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e40,0\u0026thinsp;\u0026plusmn;\u0026thinsp;1,2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e37,6\u0026thinsp;\u0026plusmn;\u0026thinsp;1,9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3,50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e127\u0026thinsp;\u0026plusmn;\u0026thinsp;9,4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e132\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0,93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003et \u003csub\u003ecritic\u003c/sub\u003e = 4.30 (95% confidence)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison between different strategies for preparing samples of chocolate (Cho A-70) analyzed by FAAS e MIP OES, with analyte concentration expressed with confidence interval (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation), (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElements\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReference method\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProposed method\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e% Agreement\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e901\u0026thinsp;\u0026plusmn;\u0026thinsp;31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e820\u0026thinsp;\u0026plusmn;\u0026thinsp;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1712\u0026thinsp;\u0026plusmn;\u0026thinsp;107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1789\u0026thinsp;\u0026plusmn;\u0026thinsp;77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e104\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32,7\u0026thinsp;\u0026plusmn;\u0026thinsp;1,7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e35,0\u0026thinsp;\u0026plusmn;\u0026thinsp;1,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e107\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21,5\u0026thinsp;\u0026plusmn;\u0026thinsp;0,82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e19,3\u0026thinsp;\u0026plusmn;\u0026thinsp;1,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42,1\u0026thinsp;\u0026plusmn;\u0026thinsp;4,1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e36,9\u0026thinsp;\u0026plusmn;\u0026thinsp;0,98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e69,4\u0026thinsp;\u0026plusmn;\u0026thinsp;4,4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e77,0\u0026thinsp;\u0026plusmn;\u0026thinsp;4,1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e111\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN.D.*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e13,4\u0026thinsp;\u0026plusmn;\u0026thinsp;0,47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN.D.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e9,85\u0026thinsp;\u0026plusmn;\u0026thinsp;0,92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN.D.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5,08\u0026thinsp;\u0026plusmn;\u0026thinsp;0,12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e* Not determined\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe extraction induced by emulsion breakage was applied to 10 samples with different cocoa contents, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Where the concentrations ranged from 328 to 1424 mg Kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e(Ca), 561 to 2152 mg Kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Mg), 7.8 to 251 mg Kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Cu), 8.5 to 304 mg Kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Mn), 6.22 to 98.32 mg Kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fe), 8.38 to 80.2 mg Kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Zn), 3.4 to 175 mg Kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Ba) and 2.15 to 12.79 mg Kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Sr). It is observed that what determines the concentrations of these elements in chocolate is the soil in which it is grown, the processing of cocoa almonds and the added ingredients, such as milk (Cinquanta el al., 2016; Barraza et al., 2021; Assa et al., 2018). Cocoa beans are important sources of naturally occurring magnesium, which explains the high level in chocolate (Ellam, Williamson, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAccording to the values allowed by the TACO table, 2011, for milk chocolate 1910 mg Kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Ca), 570 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Mg), 3.0 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Mn), 16 mg Kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fe), 3.1 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Cu), 11 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Zn) and for semisweet chocolate 450 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Ca), 1070 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Mg), 8.3 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Mn), 36 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fe), 7.7 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Cu) and 15 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Zn) it was observed that for some samples of the present study, the concentrations are within the allowed value and some samples presented values above the allowed limit.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe application of extraction induced by emulsion breakage in samples with high lipid content allows a simpler, more economical, and faster sample preparation method for application in routine analysis and multielement determination of species such as Ca, Mg, Mn, Fe, Zn, Sr, Ba, Cu and Cr considering economic aspects, health, and well-being of consumers. The proposed method has the advantage of shorter process time, flexibility as a function of mass, less residue formation and non-use of concentrated acids and organic solvents, managing to determine a greater number of elements. In addition, the present method revealed satisfactory detection limits due to the technique employed.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors acknowledge the financial support of the Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq, Brazil), Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES, Brazil) and Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado da Bahia (FAPESB, Brazil), Financiadora de Estudos e Projetos (FINEP), and Universidade Estadual de Santa Cruz (UESC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGeovana B. Guimar\u0026atilde;es, Leonardo B. Guimar\u0026atilde;es, Julia C. Romero and Sheylla M. S. Queiroz are students of the Programa de P\u0026oacute;s-Gradua\u0026ccedil;\u0026atilde;o em Qu\u0026iacute;mica da UESC and received research grants from Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado da Bahia (FAPESB). Erik G. P. da Silva, Raildo M. de Jesus and F\u0026aacute;bio de S. Dias received research grants from Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq). This study is also financed in part by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior-Brasil (CAPES).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest: \u003c/strong\u003eGeovana B. Guimar\u0026atilde;es declares that she has no conflict of interest. Leonardo B. Guimar\u0026atilde;es declares that he has no conflict of interest. Julia C. Romero declares that he has no conflict of interest. Sheylla M. S. Queiroz declares that she has no conflict of interest.\u003c/p\u003e\n\u003cp\u003eErik G. P. da Silva declares that he has no conflict of interest. Daniel de C. Lima declares that he has no conflict of interest. Luana N. Santos declares that she has no conflict of interest. F\u0026aacute;bio G. Lepri declares that he has no conflict of interest. F\u0026aacute;bio S. Dias declares that he has no conflict of interest. Allison G. Santos declares that he has no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval: \u003c/strong\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent: \u003c/strong\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCredit author statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGeovana B. Guimar\u0026atilde;es was responsible for formal analysis, conceptualization; visualization; roles/writing original draft; writing-review and editing; Leonardo B. Guimar\u0026atilde;es, Julia C. Romero and Sheylla M. S. Queiroz for formal analysis, conceptualization, writing original draft; Erik G. P. da Silva, Daniel C. Lima, Luana N. Santo, F\u0026aacute;bio S. Dias, F\u0026aacute;bio G. Lepri and Allison G. Sandos were responsible for formal analysis, visualization; writing original draft; writing-review and editing; F\u0026aacute;bio A. C. Amorim and Raildo M. de Jesus for formal analysis, funding acquisition, project administration, resources, supervision, roles/writing original draft; visualization, writing-review and editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAl-Dalahmeh Y, Al-Swaidan HM, Al-Ghamdi A (2019) Combination of Ultrasonication and Induced Emulsion Breaking for Efficient Extraction of Wear Metals from Lubricating Oils with Inductively Coupled Plasma\u0026ndash;Mass Spectrometry Determination. J Anal Chem 74(1):71\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1134/S1061934819010039\u003c/span\u003e\u003cspan address=\"10.1134/S1061934819010039\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmerican Section of the International Association for Testing Materials (2015) D7455\u0026thinsp;\u0026ndash;\u0026thinsp;14 Standard Practice for Sample Preparation of Petroleum and Lubricant Products for Elemental Analysis. B Stand Vol 0504:1\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1520/D7455-08.1\u003c/span\u003e\u003cspan address=\"10.1520/D7455-08.1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBakircioglu D, Bakircioglu Kurtulus Y, Topraksever N (2022) Extraction Induced by Emulsion Breaking for Ca, Cu, Fe, Mn, Ni, and Zn Determination in Chocolate by Flame Atomic Absorption Spectrometry. J AOAC Int 105(5):1299\u0026ndash;1308. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jaoacint/qsac048\u003c/span\u003e\u003cspan address=\"10.1093/jaoacint/qsac048\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBakircioglu D, Topraksever N, Kurtulus Y (2014) Determination of zinc in edible oils by flow injection FAAS after extraction induced by emulsion breaking procedure. Food Chem 151:219\u0026ndash;224. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.FOODCHEM.2013.11.069\u003c/span\u003e\u003cspan address=\"10.1016/J.FOODCHEM.2013.11.069\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalaram V (2020) Microwave plasma atomic emission spectrometry (MP-AES) and its applications \u0026ndash; A critical review. Microchem J 159(September):105483. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.microc.2020.105483\u003c/span\u003e\u003cspan address=\"10.1016/j.microc.2020.105483\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurguera JL, Burguera M (2012) Analytical applications of emulsions and microemulsions. Talanta 96:11\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.talanta.2012.01.030\u003c/span\u003e\u003cspan address=\"10.1016/j.talanta.2012.01.030\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarneiro AF, Carneiro CN, Pires LN, Teixeira LSG, Azcarate SM, Dias FS (2020) D-optimal mixture design for the optimization of extraction induced by emulsion breaking for multielemental determination in edible vegetable oils by microwave-induced plasma optical emission spectrometry. Talanta 219:121218. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.talanta.2020.121218\u003c/span\u003e\u003cspan address=\"10.1016/j.talanta.2020.121218\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCassella R, Brum D, Lima C, Caldas L, De Paula C (2011) Multivariate optimization of the determination of zinc in diesel oil employing a novel extraction strategy based on emulsion breaking. Anal Chim Acta 690(1):79\u0026ndash;85. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.aca.2011.01.059\u003c/span\u003e\u003cspan address=\"10.1016/j.aca.2011.01.059\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCassella R, Brum D, Robaina N, Rocha A, Lima C (2012) Extraction induced by emulsion breaking for metals determination in diesel oil by ICP-MS. J Anal Atom Spectrom 27(2):364\u0026ndash;370. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/C2JA10279J\u003c/span\u003e\u003cspan address=\"10.1039/C2JA10279J\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorazza M, Tarley C (2016) 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. Microchem J 127:145\u0026ndash;151. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.MICROC.2016.02.021\u003c/span\u003e\u003cspan address=\"10.1016/J.MICROC.2016.02.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCosta VC, Babos DV, Aquino FWB, Virg\u0026iacute;lio A, Amorim FAC, Pereira-Filho ER (2018) Direct Determination of Ca, K and Mg in Cassava Flour Samples by Laser-Induced Breakdown Spectroscopy (LIBS). Food Anal Method 11:1886\u0026ndash;1896. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12161-017-1086-9\u003c/span\u003e\u003cspan address=\"10.1007/s12161-017-1086-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Sousa JM, Cassella RJ, Lepri FG (2019) Evaluation of Extraction Induced by Emulsion Breaking for Ni and V Extraction from Off-Shore Brazilian Crude Oils. Energy Fuels 33(11):10435\u0026ndash;10441. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.energyfuels.9b01068\u003c/span\u003e\u003cspan address=\"10.1021/acs.energyfuels.9b01068\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEllam S, Williamson G (2013) Cocoa and Human Health. Annu Ver Nutr 33:105\u0026ndash;128. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1146/annurev-nutr-071811-150642\u003c/span\u003e\u003cspan address=\"10.1146/annurev-nutr-071811-150642\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFernandes A, Vinhal JO, Dutra AJB, Cassella RJ (2019) Study of the extraction of Ca, Mg and Zn from different types of lubricating oils (mineral, semi-synthetic and synthetic) employing the emulsion breaking strategy. Microchem J 145:1112\u0026ndash;1118. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.microc.2018.12.043\u003c/span\u003e\u003cspan address=\"10.1016/j.microc.2018.12.043\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFontoura BM, Jofr\u0026eacute; FC, Williams T, Savio M, Donati GL, N\u0026oacute;brega JA (2022) Is MIP-OES a suitable alternative to ICP-OES for trace element analysis? J Anal At Spectrom 37(5):966\u0026ndash;984. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/D1JA00375E\u003c/span\u003e\u003cspan address=\"10.1039/D1JA00375E\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGaritta JA, Fialho LL, de Oliveira GS, Maria RM, Pirola C, Ferreira AG, N\u0026oacute;brega JA (2021) Microwave-Assisted Acid Digestion: Evaluation of Reaction Vessel Design and Performance. J Braz Chem Soc 32(4):702\u0026ndash;711. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21577/0103-5053.20200222\u003c/span\u003e\u003cspan address=\"10.21577/0103-5053.20200222\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuimar\u0026atilde;es LB, Romero JC, Queiroz SMS, Da silva EGP, Lima DC, Coutinho JP, Dias FS, Bezerra MA, Amorim FAC (2022) A new method for determination of Mg, Ca, Zn and Na in cocoa butter by FAAS employing extraction induced by emulsion breaking and multivariate products. Talanta 15:458\u0026ndash;467. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12161-021-02123-z\u003c/span\u003e\u003cspan address=\"10.1007/s12161-021-02123-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuti\u0026eacute;rrez-Mac\u0026iacute;as P, Mir\u0026oacute;n-M\u0026eacute;rida VA, Rodr\u0026iacute;guez-Nava O, Barrag\u0026aacute;n-Huerta BE (2021) Cocoa: Beyond chocolate, a promising mateiral for potential value-added products. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/B978-0-12-824044-1.00038-6\u003c/span\u003e\u003cspan address=\"10.1016/B978-0-12-824044-1.00038-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Valorization of Agri-Food Wastes and By-Products.267\u0026ndash;268\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe Y, Chen J, Zhou Y, Wang X, Liu X (2014) Extraction induced by emulsion breaking for trace multi-element determination in edible vegetable oils by ICP-MS. Anal Methods 6(14):5105\u0026ndash;5111. https://doi.org10.1039/C4AY00218K\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaraś K, Zioła-Frankowska A, Bartoszewicz M, Krzyśko G, Frankowski M (2021) Investigation of chocolate types on the content of selected metals and non-metals determined by ICP-OES analytical technique. Food Addit Contaminants: Part A 38(2):293\u0026ndash;303. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/19440049.2020.1853821\u003c/span\u003e\u003cspan address=\"10.1080/19440049.2020.1853821\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurtulus YB, Bakircioglu D, Babac AC, Yurtsever S, Topraksever N (2021) Extraction induced by emulsion breaking procedure prior to grafite furnace atomic absorption spectrometry and comparison of results to provisional tolerable daily intake values. Food Chem Contaminants 103:1256\u0026ndash;1263. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jaoacint/qsaa028\u003c/span\u003e\u003cspan address=\"10.1093/jaoacint/qsaa028\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLewis C, Lennon AM, Eud\u0026oacute;xie C, Sivapatham P, Umaharan P (2021) Plant metal concentrations in Theobroma cacao as affected by soil metal availability in different soil types. Chemosphere 262. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.chemosphere.2020.127749\u003c/span\u003e\u003cspan address=\"10.1016/j.chemosphere.2020.127749\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohamed R, Zainudin BH, Yaakob AS (2020) Method validation and determination of heavy metals in cocoa beans and cocoa products by microwave assisted digestion technique with inductively coupled plasma mass spectrometry. Food Chemistry, 303(August 2019), 125392. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodchem.2019.125392\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2019.125392\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOliveira LB, de Melo JC, da Boa Morte ES, de Jesus RM, Teixeira LSG, Korn MGA (2021) Multi-element determination in chocolate bars by microwave-induced plasma optical emission spectrometry. Food Chem 351. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodchem.2021.129285\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2021.129285\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOliveira LB, De Melo jC, Da Boa Morte ES, De Jesus RM, Teixeira LSG, Korn MG (2021) Multi-element determination in chocolate bars by microwave-induced plasma optical emission spectrometry. Food Chem 351. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodchem.2021.129285\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2021.129285\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePereira F, Brum D, Lepri F, Cassella R (2014) Extraction induced by emulsion breaking as a tool for Ca and Mg determination in biodiesel by fast sequential flame atomic absorption spectrometry (FS-FAAS) using Co as internal standard. Microchem J 117:172\u0026ndash;177. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.microc.2014.06.026\u003c/span\u003e\u003cspan address=\"10.1016/j.microc.2014.06.026\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobaina N, Brum D, Cassella R (2012) Application of the extraction induced by emulsion breaking for the determination of chromium and manganese in edible oils by electrothermal atomic absorption spectrometry. Talanta 99:104\u0026ndash;112. https://doi.org/155.10.1016/j.talanta.2012.05.025\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantana GFR, Rodrigues RS, Brand\u0026atilde;o GP, Costa VC, Carneiro MTWD (2024) Multi-element Determination in Craft Chocolate with Different Cocoa Contents Using Ultrasound-Assisted Extraction and Dilute Acid as Sample Preparation. Food Anal Methods 17(3):475\u0026ndash;485. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12161-024-02586-w\u003c/span\u003e\u003cspan address=\"10.1007/s12161-024-02586-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSouza V, Teixeira L, Korn M, Cerqueira U, Bezerra M (2019) Determination of total contents and volatile and non-volatile fractions of nickel and vanadium in gasohol by graphite furnace atomic absorption spectrometry after extraction induced by emulsion-breaking. Fuel 242:479\u0026ndash;486. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fuel.2018.12.129\u003c/span\u003e\u003cspan address=\"10.1016/j.fuel.2018.12.129\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTabela Brasileira de Composi\u0026ccedil;\u0026atilde;o de Alimentos (TACO) 4a ed. Campinas: NEPA- UNICAMP (2011) 161 p\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrevelin A, Marotto R, De Castro E, Brand\u0026atilde;o G, Cassella R, Carneiro M (2016) Extraction induced by emulsion breaking for determination of Ba, Ca, Mg and Na in crude oil by inductively coupled plasma optical emission spectrometry. Microchem J 124:338\u0026ndash;343. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.microc.2015.09.014\u003c/span\u003e\u003cspan address=\"10.1016/j.microc.2015.09.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValasques GS, dos Santos AMP, de Souza VS, Teixeira LSG, Alves JPS, Santos MD, dos Santos WPC, Bezerra MA (2020) Multivariate optimization for the determination of cadmium and lead in crude palm oil by graphite furnace atomic absorption spectrometry after extraction induced by emulsion breaking. Microchem J 153:104401. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.microc.2019.104401\u003c/span\u003e\u003cspan address=\"10.1016/j.microc.2019.104401\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValasques GS, Dos Santos AM, Silva D, Alves DG, J. P., Bezerra MA (2018) Use of constrained mixture design in the optimization of a method based on extraction induced by emulsion breaking for the determination of Ca, Mg, Mn, Fe and Zn from palm oil by flame atomic absorption spectrometry. J Braz Chem Soc 29(10):2189\u0026ndash;2196. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21577/0103-5053.20180095\u003c/span\u003e\u003cspan address=\"10.21577/0103-5053.20180095\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValasques GS, dos Santos AMP, da Silva DLF, Cerqueira UMFD, Ferreira SLC, dos Santos WNL, Bezerra MA (2020) Extraction induced by emulsion breaking for As, Se and Hg determination in crude palm oil by vapor generation-AFS. Food Chem 318:126473. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodchem.2020.126473\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2020.126473\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVicentino P, Cassella R (2017) Novel extraction induced by microemulsion breaking: A model study for Hg extraction from Brazilian gasoline. Talanta 162:249\u0026ndash;255. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.talanta.2016.10.032\u003c/span\u003e\u003cspan address=\"10.1016/j.talanta.2016.10.032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVicentino P, Brum D, Cassella R (2015) Development of a method for total Hg determination in oil samples by cold vapor atomic absorption spectrometry after its extraction induced by emulsion breaking. Talanta 132:733\u0026ndash;738. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.talanta.2014.10.019\u003c/span\u003e\u003cspan address=\"10.1016/j.talanta.2014.10.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVilla JEL, Pereira CD, Cadore S (2015) A novel rapid and simple acid extraction for multielemental determination in chocolate bars. Microchem J 121:199\u0026ndash;204\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-analytical-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Analytical Methods](https://www.springer.com/journal/12161)","snPcode":"12161","submissionUrl":"https://submission.nature.com/new-submission/12161/3","title":"Food Analytical Methods","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"extraction induced by emulsion breaking, chocolate, MIP OES, nutrient elements","lastPublishedDoi":"10.21203/rs.3.rs-4365838/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4365838/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe objective of this work was the multielement determination of Ca, Zn, Sr, Ba, Cu, Mn, Mg and Cr in chocolate barr samples after extraction induced by emulsion breakage (EIEB) and quantification by optical emission spectrometry whit plasma induced by microwaves (MIP OES). After study of each parameter, the most efficient extraction conditions were obtained using 0.250 g of sample, 5.0 mL of extraction solution consisting of HNO\u003csub\u003e3\u003c/sub\u003e 1.5 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and Tween80 1.5% m/v, submitted to an ultrasonic bath for 5 min, followed by breaking the emulsion by heating at 90 \u0026ordm;C in a water bath for 4 min. The detection limits obtained, in mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, were 0,3487 (Cr), 0,0125 (Zn), 0,0635 (Sr), 0,0831 (Ca), 0,4627 (Fe), 0,0101 (Ba), 0,0991, (Cu), 0,0161 (Mg), 0,0356 (Mn). Precision, based on the relative standard deviation (RSD%), was less than 9.8% (N\u0026thinsp;=\u0026thinsp;7). The accuracy was confirmed by analyzing the SRM Baking Chocolate 2384 and comparing the proposed method with a calcination method. The method was applied to samples of chocolate bars made in the southern region of Bahia and containing cocoa contents at 50, 58, 60, 63, 70, 80 and 85%. The average results obtained were 328 to 1424 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Ca), 561 to 2152 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Mg), 7.8 to 251 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Cu), 8.5 to 304 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Mn), 6.22 to 98.32 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fe), 8.38 to 80.2 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Zn), 3.4 to 175 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Ba), 2.15 to 12.79 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Sr). It was observed that as the percentage increases cocoa, there is a tendency to increase the concentration of the studied elements. The developed method has satisfactory precision and accuracy, and is simple, fast and with low consumption of reagents, has good sensitivity, especially when compared to digestion methods.\u003c/p\u003e","manuscriptTitle":"A new method employing extraction induced by emulsion breaking and MIP OES for multi-element determination of inorganic elements in handmade chocolate","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-10 04:51:24","doi":"10.21203/rs.3.rs-4365838/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-04T15:22:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-04T00:10:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-21T08:53:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"230952100710211478203329916076992169502","date":"2024-05-09T00:06:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"240012611618139670539736398332507450805","date":"2024-05-07T10:22:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-06T13:40:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-05T22:48:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-05T22:48:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food Analytical Methods","date":"2024-05-03T20:16:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-analytical-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Analytical Methods](https://www.springer.com/journal/12161)","snPcode":"12161","submissionUrl":"https://submission.nature.com/new-submission/12161/3","title":"Food Analytical Methods","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"995f2660-e63a-4d4a-8e8c-670718d378c0","owner":[],"postedDate":"May 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-08-28T09:35:29+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-10 04:51:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4365838","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4365838","identity":"rs-4365838","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}