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 (tcalculatedo> tcritical, n1 = n2 = 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 (tcalculated> tcritical, n1 = n2 = 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, tcalculated < tcritical, n1 = n2 = 3, p = 0.05, tcalculated 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, Fcalculeted < Fcritical, n1 = n2 = 3, p = 0.05, Fcalculated 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, tcalculated 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 tcalculated < tcritical (4.30), n1 = n2 = 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.