Simultaneous determination of Fe and Ni in guarana (Paullinia cupana Kunth) by HR-CS GF AAS: Comparison of direct solid analysis and wet acid digestion procedures

Journal Pre-proof Simultaneous determination of Fe and Ni in guarana (Paullinia cupana Kunth) by HR-CS GF AAS: comparison of direct solid analysis and wet acid digestion procedures Franciele Rovasi Adolfo, Paulo C´ıcero do Nascimento, Gabriela Camera Leal, Denise Bohrer, Carine Viana, Leandro Machado de Carvalho

PII:

S0889-1575(19)31753-3

DOI:

https://doi.org/10.1016/j.jfca.2020.103459

Reference:

YJFCA 103459

To appear in:

Journal of Food Composition and Analysis

Received Date:

25 November 2019

Revised Date:

13 January 2020

Accepted Date:

18 February 2020

Please cite this article as: Adolfo FR, do Nascimento PC, Leal GC, Bohrer D, Viana C, de Carvalho LM, Simultaneous determination of Fe and Ni in guarana (Paullinia cupana Kunth) by HR-CS GF AAS: comparison of direct solid analysis and wet acid digestion procedures, Journal of Food Composition and Analysis (2020), doi: https://doi.org/10.1016/j.jfca.2020.103459

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Simultaneous determination of Fe and Ni in guarana (Paullinia cupana Kunth) by HRCS GF AAS: comparison of direct solid analysis and wet acid digestion procedures

Franciele Rovasi Adolfo*, Paulo Cícero do Nascimento, Gabriela Camera Leal, Denise Bohrer, Carine Viana, Leandro Machado de Carvalho

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Departamento de Química, Universidade Federal de Santa Maria, 97111-900 Santa Maria, RS, Brasil

E-mail address: [email protected]

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[email protected]

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[email protected] [email protected]

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[email protected]

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* Corresponding author: Franciele Rovasi Adolfo

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E-mail address: [email protected]; Tel..: +55 55 32208870

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Graphical abstract

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Highlights

Simultaneous determination of Fe and Ni in guarana by HR-CS GF AAS;

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Direct solid sample analysis with calibration against aqueous standard solutions;

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Comparison with four acid digestion procedures

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Method simpler and faster than conventional methods of wet digestion of samples;

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Better recovery and reproducibility and more precise and accurate results.

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

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ABSTRACT

Direct solid sampling method was compared with the conventional wet acid digestion

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method to simultaneously assay iron and nickel in guarana by high-resolution continuum source graphite furnace atomic absorption spectrometry. Measurements were done with the

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secondary lines of Fe and Ni. Four digestion procedures using mixtures of HNO3 and H2O2 (procedure A), HNO3 and HCl (procedure B), HNO3, HCl and H2O2 (procedure C) and HNO3, H2SO4 and H2O2 (procedure D) were evaluated. All tested procedures provided quantitative recoveries for Ni, whereas a good recovery for Fe was obtained only with procedure D. Procedure D was chosen for the digestion of all guarana samples. The limit of detection for the direct solid sampling method was 1.004 µg g-1 for Fe and 0.022 µg g-1 for Ni, and the

precision ranged from 3.5 to 20.0% and 2.8 to 8.0% for Fe and Ni, respectively. Method accuracy was evaluated by statistical comparison between analyte concentrations, obtained by measurements in the solid samples by the proposed method and after the digestion of the samples by procedure D. The validation of the analytical results obtained for the solid and the digested sample was performed by Energy Dispersive X-Ray Fluorescence.

Keywords: Food composition; Nutritional supplement; Dietary supplement; Fe and Ni

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contamination

1. Introduction

Guarana (Paullinia cupana Kunth) is a plant of the Sapindaceae family, native to the

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Brazilian Amazon region (Da Silva et al., 2015). Guarana powder is sold as a nutritional

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supplement with the following attributes: stimulant, analgesic, antipyretic, diuretic, antioxidant and vascular tonic (Dalonso & Petkowicz, 2012; Da Silva et al., 2015; Schimpl

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et al., 2013). The versatility of guarana is mainly due to its high content of methylxanthines and polyphenols, as already described in the literature (Dalonso & Petkowicz, 2012; Schimpl

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et al., 2013).

Raw vegetable materials and their derivatives may contain essential and non-

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essential elements from their presence in soil, water or air (Zeiner & Cindrić, 2017). This way, monitoring the contents of the elements present in dietary supplements and medicinal

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plants is important, taking into account the nutritional requirement of minerals and/or the adverse health effects of contaminants. Several spectroscopic methods have been used in the determination of essential and toxic elements in medicinal plants and dietary supplements containing plant materials, such as F- and GF-AAS (Anal & Chase, 2016; Bello et al., 2004), ICP-OES (Marrero et al., 2013; Santos et al., 2019) and ICP-MS (Avula et al., 2010; Filipiak-Szok et al., 2015; Muller et al., 2015; Raman et al., 2004). In general, these

techniques require samples in the form of aqueous solutions, requiring the decomposition of the samples prior to analysis (Santos et al.,2019; Welna et al., 2011). Sample preparation is considered to be the largest source of errors and one of the most critical steps of the analysis (Nemati et al., 2010; Novozamsky et al., 1993; Welna et al., 2011). Ideally, the digestion procedure should ensure that the analytes are completely solubilized and released in a form compatible with the analytical technique used to obtain accurate analytical results (Zeiner & Cindrić, 2017). Several methods of digestion for the analysis of contaminants in dietary supplements

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and medicinal plants have been previously described. Typically, the digestion is carried out by a conventional hot-plate or microwave-assisted heating of samples with oxidizing reagents, including HNO3 (Avula et al., 2010; Filipiak-Szok et al., 2015), HNO3+ H2O2

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(Marrero et al., 2013), HNO3+HCl (Avula et al., 2010), HNO3+HF (Lavilla et al., 1999).

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Unfortunately, due to the large number of analytes and the variability in the matrix composition of different sample types, there is no universal sample preparation technique

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(Welna et al., 2011). Each case requires a different analytical approach that must be selected according to different factors, like the composition of the matrix, concentration

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range of the analytes, level of contamination, detection technique, digestion efficiency and its reproducibility, as well as economic aspects like time and labor investment, reagent

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consumption, cost and availability of equipment (Momen et al., 2006; Welna et al., 2013). The main disadvantages of digestion procedures are the incomplete digestion of

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organic material, precipitation of the insoluble analyte, contamination and/or loss of some elements during heating (Momen et al., 2006; Muller et al., 2015). Additionally, it is well known that plant material also presents problems in the choice of reagents for digestion, because different plant species contain varying amounts of silicate (Nemati et al., 2010; Oliva et al., 2003). Very often, poor analyte recovery (especially Al, Fe, Cu, and Mn) has been associated with their binding with silicates (Novozamsky et al., 1993).

As regards guarana, its chemical composition is complex. Apart from starch, theophylline, theobromine, proteins, polysaccharides, sugars, fibers, fatty acids and minerals, the seeds also contain up to 7.0% caffeine (about 2 to 5 times higher than that found in coffee seeds) and up to 16% of total tannins, formed by the association of monomeric units of catechins and epicatechins (Dalonso & Petkowicz, 2012; Pagliarussi et al., 2002; Schimpl et al., 2013). Compounds such as caffeine and polyphenols have chelating activity and can form stable complexes with certain metal ions (Bittencourt et al., 2016). Thus, considering the relatively complex composition of the matrix and the low

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concentration of the analytes, the preparation of the sample for the determination of trace elements in powdered guarana can still be considered an analytical challenge.

This way, the direct analysis of solid samples (SS) using high-resolution continuum

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source graphite furnace atomic absorption spectrometry (HR-CS GF AAS) can be an

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efficient alternative to the conventional methods of sample preparation (Adolfo et al., 2019). The SS analysis eliminates the sample preparation step, allows the use of small sample

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amounts, reduces the risk of analyte loss and contamination, and offers greater sensitivity, as the sample is not diluted (Gómez-Nieto et al., 2013; Vale et al., 2006). Besides these

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advantages, the efficient background correction of the HR-CS SS-GF AAS also greatly facilitates the direct analysis of a complex solid sample (de Gois et al., 2016).

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It has been demonstrated that several trace elements in a wide variety of complex sample matrices can be determined by HR-CS SS-GF AAS using aqueous standards for

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calibration, after careful optimization of the temperature program (Adolfo et al., 2019; Gómez-Nieto et al., 2013; Resano et al., 2013). The atomization in graphite furnace allows the thermal pretreatment of the sample and facilitates the separation of the analyte from the matrix (de Andrade et al., 2017; Resano et al., 2004). Thus, the present study focuses on the possibility of performing the calibration against aqueous standards for the determination of Fe and Ni in a complex matrix (guarana) with direct analysis of the solid sample.

Additionally, the present work shows the advantages of the direct solid sampling method over the common acid digestion procedures.

2. Materials and Methods 2.1. Instrumentation The absorbance measurements for the solid sampling analysis were carried out using a ContrAA 700 high-resolution continuum source atomic absorption spectrometer (Analytik Jena AG, Jena, Germany) with a graphite furnace atomizer. The ContrAA 700 is equipped

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with a high-intensity xenon short-arc lamp operating in “hot-spot” mode, a high-resolution double monochromator (double-echelle monochromator), and a linear charge-coupled device (CCD) array detector with 200 analytically accessible pixels.

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The samples were weighed onto the graphite platform for solid sampling (SS)

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previously tared using a microbalance integrated in the instrument and automatically inserted into the graphite tube for SS, using the SSA 600 automatic solid sampling

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accessory (Analytik Jena AG). The deposition of the sample on the platform was performed manually using a micro spatula, while the other operations were controlled by the computer.

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Aqueous standards solutions (20 µL) were automatically injected onto the SS platform. Pyrolytically coated graphite tubes with integrated PIN platform and an MPE 60 liquid

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sampler were used for the comparative measurements with digested samples, using a sample volume of 40 μL. The optimized graphite furnace temperature program used for the

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simultaneous determination of Fe and Ni in the solid and digested samples is shown in Table

For validation purposes, the representative sample was also evaluated by Energy

Dispersive X-Ray Fluorescence (ED XRF), using a S2 Ranger ED XFR spectrometer (Bruker, USA), according to the recommendation of the fabricant for solid samples. Insert Table 1

2.2. Reagents, standards and samples All reagents used were at least of analytical grade. Concentrated HNO3 (Vetec, Rio de Janeiro, Brazil), HCl (Neon, São Paulo, Brazil) and H2SO4 (Alphatec, Rio de Janeiro, Brazil) solutions, in addition to a 30% (m/v) H2O2 solution (Synth, São Paulo, Brazil) were used for the sample preparation. A working solution (10 mL) that contained 100 mgL-1 of Fe and 1 mgL-1 of Ni was prepared from a SpecSol (Quimlab, São Paulo, Brazil) standard solution of Fe (1000 mgL-1) and Ni (1000 mgL-1) and acidified with 50 µL of double distillated

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nitric acid 65% (v/v). The calibration solutions were prepared daily through serial dilutions of the working solution with purified water (Milli-Q system – Millipore, Bedford, MA, USA), with a resistivity of 18.2 M Ω cm.

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In the study, five samples of guarana (GUA-01 to GUA-05) obtained from local stores

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were analyzed. GUA-01 was used as a representative sample for method development and

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optimization.

2.3. Procedures

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2.3.1. Determination of Fe and Ni using HR-CS SS-GF AAS Analyses of guarana samples using HR-CS SS-GF AAS were carried out at

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secondary lines of Ni (352.454 nm) and Fe (352.604 nm). These lines of Ni and Fe are, respectively, about 5.3 and 870 times less sensitive than their primary lines, which is

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particularly advantageous in the direct analysis of guarana solid samples, due to the relatively high concentration of the analytes. The measurements were made using the center pixel (CP) and four side pixels (CP±2) for both Fe and Ni, corresponding to a spectral interval of 10 pm; however, the entire spectral range ±0.2 nm around the analytical line of Ni was detected by the 200 pixels used for analytical purposes. Peak volume selected absorbance

(PVSA), which is the integrated absorbance (Aint) summed over five pixels, was used for signal evaluation and quantification for both analytes. Pyrolysis (from 600 to 1400 °C) and atomization (from 2000 to 2700 °C) curves were established using the solid sample GUA-01 and an aqueous standard solution containing 40 ng of Fe and 400 pg of Ni, in order to achieve a compromise condition of the graphite furnace temperature program. The signal profiles were also evaluated. Calibration curves for both analytes were obtained simultaneously using aqueous standard solutions and generalized standard additions method (GSAM) for the comparison

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of both the calibration modes. The GSAM results were obtained through a three-dimensional approach, which considers the added analyte mass, the sample mass and the analytical response (Vale et al., 2006).

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To ensure homogeneity, the solid samples were ground and mixed thoroughly with a

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mortar and pestle, whereas the samples in capsules were opened and the content analyzed directly without pre-treatment. The influence of the sample mass introduced into the graphite

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furnace on the integrated Fe and Ni absorbance was investigated by evaluating the linearity of integrated absorbance versus weighted sample mass. Micro-homogeneity studies were

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performed by analyzing the sample within several mass ranges, and the homogeneity factor (He) was calculated according to Kurfürst (1998). The determination of Fe and Ni in the

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guarana samples was carried out employing a sample mass ranging from 0.25 to 1.433 mg. To compare the measurements performed with different sample masses, the PVSA value

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was normalized for a sample mass of 1 mg. For experiments with comparative purposes, the samples were digested and the

resulting solutions subsequently analyzed using the same conditions as for the simultaneous determination of Fe and Ni. All measurements were performed at least in triplicate.

2.3.2. Digestion procedures For the determination of Fe and Ni in guarana samples, the conventional wet acid digestion method was applied and compared with the direct solid sampling analysis. For this purpose, four sample preparation procedures were tested, all using a digester block. In each test procedure, three portions (approximately 0.2 g) of the guarana sample were accurately weighed in glass tubes (previously decontaminated) and mineralized as follow: A) 2 mL of a concentrated HNO3 solution was added to the sample, and the tube was heated in a digester block at approximately 100 °C. After 24 hours, the flask was removed from

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the heating and 1 mL of H2O2 30% (v/v) was added in to sample. The resulting solution was filtered, quantitatively transferred into a 15 mL volumetric flask and the volume made up with purified water.

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B) The sample was treated with a mixture of 3 mL HNO3 65% (v/v) and 2 mL HCl 37% (v/v)

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and heated in a digester block at approximately 100 °C, until the production of NO2 fumes had ceased. The resulting solution was filtered and diluted to 15 mL with purified water.

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C) 1.5 mL of a concentrated HNO3 solution was added to the sample. The solution was heated in a digester block at 100 ºC for about 2 hours. Then 1 mL of HCl 37% (v/v) and

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1 mL of H2O2 30% (v/v) were added. Heating was continued for 12 hours. After cooling, the resulting solution was filtered and diluted to 15 mL with purified water.

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D) The sample was treated with a mixture of 2 mL HNO3 65% (v/v) and 1 mL H2SO4 98.08% (v/v) and heated in a digester block at a temperature of approximately 100 °C. After 24

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hours, the flask was removed from the heat and, when still hot, 1 mL of H2O2 30% (v/v) was added to the sample. The resulting solution was filtered and diluted to 15 mL with purified water.

Procedure A was adapted from Adolfo et al. (2019), while procedures B and C were based on those of Memic et al. (2014), and procedure D on that of Momen et al. (2006). The efficiency of the procedures was evaluated by recovery tests for Fe and Ni, which consisted

of the addition of 2 μg of Fe and 25 ng of Ni in solution in the sample before the acid digestion procedures. The fortified sample and analytical blanks were subjected to the same experimental conditions as the sample, described in procedures A to D.

2.4. Statistical analysis and method validation The proposed method was validated according to the guidelines of the Association of Official Analytical Chemists (AOAC) for dietary supplements and botanicals and in accordance with the guidelines of the National Association of Testing Authorities (NATA) for

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the validation and verification of quantitative and qualitative test methods (AOAC, 2013; NATA, 2012). The analytical parameters evaluated were linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy and matrix effects.

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The linearity of the curves for Fe and Ni was assessed by linear regression analysis.

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Analysis of variance (ANOVA) was used to verify the significance of the regression through the F-Test (95% confidence level). LOD and LOQ were calculated as three and ten times,

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respectively, of the standard deviation of ten measurements of the empty SS platform divided by the slope of the calibration curves (NATA, 2012). Both limits were calculated for

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the maximum sample mass used in this work (1.433 mg). The precision of the proposed method was based on the mean values and standard

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deviation of three replicate measurements (n = 3) of the aqueous standard solutions and the solid sample and was expressed as the relative standard deviation (RSD) (AOAC, 2013).

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Method accuracy was assessed by Fe and Ni recovery experiments in the solid sample, using three fortification levels (5, 7.5 and 10 ng of Fe and 50, 75 and 100 pg of Ni). The tests were performed in triplicate and the recoveries expressed as a percentage. Recovery intervals between 75 to 120% and 85 to 110% are considered satisfactory for the validation of the method when the analyte concentration in the sample is approximately 1 μg g-1 and 100 μg g-1, respectively, according to the guidelines of the AOAC (2013). Method accuracy

was evaluated also by statistical comparison (t-test) between analyte concentrations, obtained by measurements in solid samples by the proposed method and after the digestion of the samples. The representative sample was also evaluated by ED XRF in order to validate the analytical results obtained for the solid and digested samples. For the evaluation of matrix effects, a statistical comparison was made between the slopes of the external calibration curves for Fe and Ni and those obtained by GSAM. According to the NATA (2012), when the slopes are not significantly different (<10%), there is no need to compensate for matrix effects.

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Statistical analysis of data was performed with the OriginPro 8 software (OriginLab Corporation, USA). Mean metal concentrations from different sample preparation procedures were tested for significant difference by t-test and analysis of variance (ANOVA)

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with the multiple comparison Tukey’s test. All statistical evaluations were carried out at 95%

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confidence level.

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3. Results and discussion

3.1. Optimization of HR-CS SS-GF AAS parameters

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3.1.1. Pyrolysis and atomization curves

The pyrolysis and atomization curves (Fig. 1) for Fe and Ni were established using

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the solid sample GUA-01 and an aqueous standard solution containing 40 ng of Fe and 400 pg of Ni, without using a chemical modifier.

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Insert Figure 1

In the aqueous medium, no significant differences (p>0.05) were observed between

the integrated absorbance values of Fe and Ni within the temperature ranges of 700 to 1000 °C and 600 to 1000 °C, respectively. For temperature values above 1000 °C, the integrated absorbance diminished for both analytes. A similar behavior for Ni was observed in the GUA01 sample, for which the highest signal was obtained at the pyrolysis temperature of 1000

°C. For Fe in the solid sample, the behavior was different. Indeed, the pyrolysis curve showed that the sample matrix actually stabilized this analyte at the pyrolysis temperature that was ca. 200 °C higher than the value of the aqueous standard solution since no significant differences (p> 0.05) were observed between the integrated absorbance values of Fe up to 1200 °C. These results suggest a higher sample interaction of Fe in comparison to Ni. A pyrolysis temperature of 1000 °C was adopted for all future determinations, and the addition of a chemical modifier was unnecessary due to the inherent thermal stability of both analytes.

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The profiles of the atomization curves are very similar in both the aqueous standard solution and the Ni sample. The PVSA increased steeply with the increase of the atomization temperature up to 2700 °C for this analyte. Similarly, for Fe in the solid sample, the maximum

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value of integrated absorbance was observed at 2700 °C. Thus, although the atomization

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curve for Fe in the aqueous medium showed a small decrease in integrated absorbance at 2700 °C, this temperature was the best compromise between the signal intensities for both

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analytes in the solid sample.

The signal profiles for Fe and Ni in aqueous standard solution as well as in solid

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sample at different atomization temperatures were also evaluated, in order to demonstrate that the temperature of 2700 °C was the best choice in this case. For Fe, a unimodal well-

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defined signal profile was obtained above 2400 °C and a stable baseline attained, even for direct sampling of solids (data not shown). As can be seen in Fig. 2, the Ni signal presented

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a tailed profile at 2400 °C, which became less pronounced with increasing temperature. At 2700 °C, more symmetrical peaks were obtained for Ni in both the standard solution (Fig. 2A) and the solid sample (Fig. 2B). Thus, this temperature was considered optimal for Fe and Ni determination in guarana sample. Insert Figure 2

3.1.2. Influence of the sample mass and homogeneity factor Sample homogeneity is of primary importance in direct solid sample analysis, considering that the small sample sizes may not represent the entire sample (Virgilio et al., 2012). However, high masses introduced into the atomizer may also influence the results obtained. The use of masses larger than ideal (critical mass) may cause inefficient matrix removal, increased background values and deviate the integrated absorbance from linearity, which may also decrease the precision (Gómez-Nieto et al., 2013; Zmozinski et al., 2015). Therefore, the influence of the sample mass introduced into the furnace on the integrated

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absorbance of Fe and Ni was evaluated. A good linear correlation for Fe (R = 0.985) and Ni (R = 0.988) was obtained from 0.25 to 1.433 mg. Although for masses above 0.8 mg a residue persisted after the application of the temperature program, no significant influence

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of this residue on the atomization of both elements was observed. Additionally, no signal

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was obtained for the analytes in the residue analysis or memory effect for Fe and Ni. In order to attest the sample’s homogeneity, the PVSA obtained for both analytes in

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the mass range evaluated was normalized to 1 mg and the results were separated into seven groups of five masses each. The RSD values of the normalized signals for each mass

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interval were used to calculate the homogeneity factor (He), which was introduced by Kurfürst (1998). The calculated He values for Fe and Ni varied from 4.07 to 8.90 and 2.74 to

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9.90 respectively, suggesting that the sample was sufficiently homogeneous (He < 10) within

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the evaluated mass range.

3.1.3. Matrix effects, calibration and figures of merit

The evaluation of the matrix effect on the analytical signal is extremely important for

choosing the most appropriate calibration method. Due to the absence of a pre-treatment step, the analytes are chemically bound in the sample, which may differ from the chemical form of the analytes in aqueous standards. As a consequence, the solid sample matrix may

influence the atomization process of the analytes, resulting in a difference in the atomization mechanism compared to the analytes in aqueous medium. This factor results in differences in sensitivity, which may compromise the use of calibration against aqueous standards (de Andrade et al., 2017). In a study for Ni determination in guarana samples, the slopes obtained by calibration against aqueous standards were compared to those obtained by calibration against two different solid certified reference materials. According to the authors, Ni atomization was significantly influenced by the solid matrix, since the slope obtained by the calibration against

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aqueous standards lies between the slopes obtained by calibration against NIST 1515 (apple leaves) and NIST 8433 (corn bran) (de Gois et al., 2016). In practice, in addition to increasing the cost of the analysis, the application of this technique is restricted, since

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adequate CRM is not always available for each type of sample to be analyzed (Nomura et

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al., 2008). Fortunately, it has been demonstrated that several trace elements in a wide variety of complex matrices can be determined by HR-CS SS-GF AAS using aqueous

et al., 2017; Welz et al., 2007).

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standards for calibration, after careful optimization of the temperature program (de Andrade

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Thus, after the optimization of pyrolysis and atomization temperatures, the signals for both analytes in solid sample and reference solution were overlapped. The transient signals

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for Fe (Fig. 3A) and Ni (Fig. 3B) were found to be very similar in shapes and time profiles, when the solid sample and the aqueous standard were compared, indicating that the solid

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matrix did not influence the atomization efficiency. Additionally, no spectral interferences of the sample matrix or interferences of the diatomic molecules with the fine rotational structure within the monitored spectral range were observed. Therefore, the calibration procedure was performed with aqueous standard solutions in the concentration range of 0.50 to 4.25 mg L-1 for Fe (mass range 10 to 85 ng) and 5.0 to 42.5 μg L-1 for Ni (mass range 0.10 to 0.85 ng).

Insert Figure 3 The calibration functions were y = 0.00383 + 2.7 × 10−6 x (r=0.999) and y = 0.00260 + 1.65 × 10−4 x (r=0.997) for Fe and Ni, respectively, where y is the analytical signal and x the analyte mass expressed in pg. Both curves showed significant linearity at a 95% confidence level, as Fregression for Fe (3944) and Ni (1199) was greater than the Fcritical (6.608) and the p values were less than 0.05 for both curves. The LOD and LOQ were calculated according to the guidelines of the NATA, where the values obtained were 1.004 µg g-1 and 3.011 µg g-1, for Fe, and 0.022 µg g-1 and 0.066

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µg g-1, for Ni, respectively. The characteristic mass (m0), defined as the mass of the analyte that produces a PVSA of 0.0044 s, was 1597 pg for Fe and 26.64 for Ni.

Recoveries ranging from 98 to 106% for Fe and from 84 to 106% for Ni were obtained

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by the proposed method, by measuring the spiked sample with 5, 7.5, and 10 ng of Fe and

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50, 75, and 100 pg of Ni. These results were suitable, because the recoveries lie within the range proposed by AOAC (2013) (85 – 110% and 75 – 120% over the concentration ranges

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studied for Fe and Ni, respectively).

The feasibility of calibration with aqueous standards for solid sampling was also

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evaluated by comparing the slopes obtained for Fe and Ni in an aqueous medium to those obtained by GSAM for both analytes. For this purpose, the guarana sample was initially

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analyzed without the addition of the analytes. Subsequently, increased amounts of the analytes (5 ng to 10 ng Fe and 50 pg to 100 pg Ni) in standard solution were added to the

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previously weighed sample mass on the SS platform. The measurements were performed in triplicate, adding up the twelve data points distributed in the four concentration levels. As a result, low precisions were obtained for both Fe and Ni. A study found in the literature reported that the results obtained by the addition of aqueous standard to the solid sample differed from those obtained without the presence of the matrix. This error was attributed to the presence at the same time of the analyte in two

different phases (standard and sample). The problem could be corrected by first adding the standard solution to the empty platform and then, after applying a drying step, weighing the sample on the platform and subjecting it to the temperature program (Coşkun & Akman, 2004). This procedure was also performed in the present work, resulting in lower RSD values for Fe and Ni. The relationship of the absorbance with the two independent variables (sample mass and amount of analyte added) was obtained through multiple linear regression analysis, resulting in a plane in three dimensional space. Figure 4 shows the calibration curve

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obtained for Fe by GSAM, where the filled circles represent the experimental data on the adjusted plane surface for Fe (R2 = 0.994). Similar results were obtained for Ni (data not shown).

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Insert Figure 4

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The slopes of the external calibration curves for Fe (2.71 x10-6 s pg-1) and Ni (1.65 x10-4 s pg-1) were in agreement with those obtained by the GSAM (2.96 x10-6 s pg-1 for Fe

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and 1.58 x10-4 s pg-1 for Ni), since the difference of sensitivity was lower than 10% for the two analytes. According to the NATA (2012), this indicates that there is no matrix effect to

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overcome and confirms that aqueous standard solutions can be successfully used for

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calibration.

3.2. Comparison of sample preparation procedures

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The applicability of direct analysis method and HR-CS GF AAS was evaluated by

comparing the results obtained for Fe and Ni determination in GUA-01 sample with the results using sample digestion procedures (Table 2). Insert Table 2 For Ni determination by HR-CS SS-GF AAS, a good agreement with results derived from the analyses of solution digests was obtained, with uncertainty intervals overlapping in

all cases. The data obtained were evaluated by an ANOVA test (Table 3) and no significant differences (Fcal < Ftab) between the Ni concentration determined by solid sampling analysis and after procedures A–D were observed for 95% confidence level. However, the choice of a suitable procedure for the sample digestion for Fe determination was slightly more complex. In Table 2, the mean values from procedures SS and D were found to be higher than those from procedures A, B and C. In fact, the hypothesis of the equality of the results was rejected by ANOVA (Fcal > Ftab) at the 0.05 significance level (Table 3). Tukey’s test confirmed that the Fe concentration from procedures SS and D was statistically equivalent

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(qcal (1.43) < qtab (4.65); p =0.842) but differed significantly from the other digestion procedures as qcal for SS-A (11.5), SS-B (7.79) and SS-C (16.9) were greater than the qtab (4.65) and the p values were less than 0.05.

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Insert Table 3

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The precision of the procedures was calculated from three consecutive measurements (n = 3) of the digested solutions and the solid sample (Fig. 5A). The precision

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values obtained for the SS, expressed as the relative standard deviation (RSD), were 3.53 for Fe and 8.05 for Ni, considering the normalized absorbance. These results were suitable

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because the RSD values approximated those proposed by AOAC (2013). The same was observed for the RSD values for Fe and Ni from the digestion procedures tested, except

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procedure C that presented a high RSD value for Fe according to the level of concentration of this element in the sample.

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Insert Figure 5

The efficiency of the digestion procedures and the accuracy of the direct solid

sampling were evaluated by recovery tests (Fig. 5B). Recoveries of around 98 and 99 % for Fe and Ni, respectively, were obtained by the SS procedure by measuring the spiked sample with 5 ng of Fe and 50 pg of Ni. These results were suitable because the recoveries were within the range proposed by AOAC (2013): 85 – 110% and 75 – 120% over the

concentration ranges studied for Fe and Ni, respectively. Similarly, all tested procedures provided quantitative recoveries for Ni, i.e., within the acceptable range for the concentration level of this element. Thus, the procedures A–D were suitable for the solubilization of this element from the guarana sample matrix. However, the low recovery values for Fe through the application of procedures A–C make it evident that the guarana sample requires stronger digestion conditions to guarantee the complete solubilization of this element. In our opinion, guarana seeds contain a high concentration of methylxanthines, mainly caffeine and polyphenols, which have chelating activity and can form complexes with

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transitions metals. The major class of polyphenols found in guarana seeds is the flavan-3ols, such as catechins and epicatechins (Bittencourt et al., 2016; Pagliarussi et al., 2002). Catechol moieties present in polyphenols have a high affinity for metal ions (Fraga et al.,

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2010). When deprotonated, the catecholate binder has electron donors of high electron

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density and, for this reason, behaves as a hard Lewis base. This way, the interaction with cations of high density of charge is favored (hard Lewis acids), such as Fe3+ (Hider et al.,

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2001; Perron & Brumaghim, 2009). On the other hand, Ni2+ is a moderate Lewis acid and does not bind strongly with catechol (Perron & Brumaghim, 2009). Similarly, some studies

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found in the literature indicate that there is a strong interaction between Fe and caffeine, leading to the formation of stable complexes (Jabbar et al., 2012; Pohl et al., 2013). It is

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noteworthy that the caffeine content in guarana seeds is 2 to 5 times higher than that found in coffee beans (Schimpl et al., 2013).

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This explains why low recovery values were obtained for Fe, while quantitative

recoveries for Ni were observed. The total decomposition of the siliceous and organic based matrix samples containing polyphenols and caffeine is difficult, because of their distinctive constitution. Possibly, the strong interaction of Fe with the matrix of the guarana sample has made it difficult to completely release this element to the solution with the application of procedures A–C. On the other hand, the use of procedure D resulted in an improvement in

the efficiency of the decomposition of the sample GUA-01, attributed to the combination of H2SO4 with the other reagents. In this case, recovery was 92.6% for Fe and 102.3% for Ni. Thus, procedure D was chosen for the wet digestion of all the guarana samples in this study. It should be noted that microwave-assisted digestion procedures are more efficient than open systems for the preparation of different samples, but also require optimization of the methodology employed. Mierzwa et al. (1998) evaluated different microwave-assisted digestion procedures for the determination of Fe and other elements in tea leaves. Initially, concentrated nitric acid was tested, but the recoveries were not good, and the method was

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considered ineffective. Thereafter, they investigated three combinations of acids (HNO3/HF, HNO3/HCl and HNO3/HCl/HF), so that better recoveries for Fe were obtained only by using a combination of HNO3, HCl and HF. Additionally, microwave-digestion system has some

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drawbacks that limit its wide adoption for routine digestion of a large number of samples,

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including the high cost of equipment and the relatively low number of vessels processed in each batch.

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For the validation of the analytical results obtained by the proposed method for the solid sample and after the digestion of the sample by procedure D, the GUA-01 sample was

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analyzed by ED XRF. The concentrations of Fe and Ni determined were 38.0 ± 3.5 µg g-1 and 1.66 ± 0.6 µg g-1, respectively, and no significant differences between the data obtained

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for both elements in GUA-01 sample were observed according to ANOVA, since Fcal for Fe (0.685) and Ni (1.429) were smaller than the Fcritical (5.143) and the p value was greater than

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0.05 in both cases.

3.3. Analytical application The proposed method was applied for Fe and Ni determination in five guarana

samples. For validation purposes, the analytes concentrations in these samples were also determined by HR-CS GF AAS after the wet digestion of the samples with HNO3, H2SO4

and H2O2. The results obtained (Table 4) were statistically compared by t-test at the 0.05 significance level, and no significant difference between the data obtained for both Fe and Ni in guarana samples was observed using the proposed solid method and the conventional wet digestion procedure. Insert Table 4 The concentrations of Fe and Ni determined in the guarana samples analyzed in this study ranged from 21.3 to 144 μg g-1 and 0.46 to 2.28 μg g-1, respectively. These values were similar to those found in the literature. Santos et al. (2019) reported that the

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concentration of Fe determined by ICP-OES in seventy-two guarana samples ranged from 25.9 to 462 µg g-1. De Gois et al. (2016) analyzed eight samples of guarana using direct solid analysis. According to the authors, the concentration of Ni in the guarana samples

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ranged from 1.58 to 3.57 μg g-1.

4. Conclusion

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The comparison of sample preparation procedures demonstrated the importance of using adequate reagents for sample digestion. More relevantly, the results highlight the

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advantages of direct SS with calibration against aqueous standard solutions for Fe and Ni determination in guarana samples by HR-CS AAS. Despite the matrix complexity, the

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adequate optimization of the instrumental parameters for SS analysis allows reliable and accurate results for both elements to be obtained. The simultaneous determination of Fe

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and Ni is carried out in a few minutes by using a minimum sample mass, without the use of additional reagents, which makes the proposed method faster and simpler than the conventional methods based on the acid digestion of the samples.

Conflict of interest The authors declare that no conflict of interest exist.

Acknowledgements

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We are grateful to the Brazilian foundations CAPES and CNPq for their financial support.

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Figure Captions

Figure 1. Pyrolysis (Ta= 2500 °C) (black) and atomization (Tp= 1000 °C) (grey) curves for Fe (circle) and Ni (square) for an aqueous standard solution containing 40 ng of Fe and 400 pg of Ni (opened symbols) and for the GUA-01 solid sample (filled symbols), n = 3. Analytical

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signals were normalized for 1 mg of sample.

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Figure 2. Absorbance signals for 400 pg of Ni (352.454 nm) in an aqueous standard solution

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(A) and for Ni in the GUA-01 solid sample (B), Tp = 1000 °C and Ta = 2400 °C (▬), 2500 °C

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(▬), 2600 °C (▬) and 2700 °C (▬).

Figure 3. Absorbance signals for 0.611 mg of the GUA-01 solid sample (solid line) and for (A) 25 ng of Fe (352.604 nm) and (B) 700 pg of Ni (352.454 nm) in aqueous standard solution (dashed line). Tp = 1000 °C and Ta = 2700 °C.

Figure 4. Calibration curve obtained for Fe in the GUA-01 solid sample by GSAM. The filled

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circles represent the experimental data on the adjusted plane surface.

Figure 5. RSD (A) and recovery (B) values for Fe and Ni in the GUA-01 sample obtained by

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HR-CS GF AAS with direct solid sampling (SS) and after the digestion of the sample with the reagent mixtures HNO3/H2O2 (A), HNO3/HCl (B), HNO3/HCl/H2O2 (C) and

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HNO3/H2SO4/H2O2 (D).

Table 1 Temperature program for simultaneous determination of Fe and Ni in guarana by HR-CS GF AAS. Temperature (°C)

Ramp (°C s-1)

Hold time (s)

Drying

120

5

10

Pyrolysis

1000

150

10

Autozero

1000

0

10

Atomization

2700

1200

10

Cleaning

2720

500

4

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Stage

Table 2 Results obtained for Fe and Ni in guarana sample by HR-CS GF AAS and after sample digestion. Results expressed as mean ± standard deviation (n = 3). Fe (ng mg-1)

Ni (pg mg-1)

SS

38.6 ± 1.4

1230 ± 99

A

25.4 ± 1.5

1290 ± 190

B

29.9 ± 2.3

1260 ± 12

C

19.6 ± 3.3

1400 ± 140

D

40.2 ± 1.7

1300 ± 73

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Procedure

SS = Solid Sampling; A = HNO3/H2O2; B = HNO3/HCl; C = HNO3/HCl/H2O2; D =

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HNO3/H2SO4/H2O2.

Table 3 Analysis of variance for Fe and Ni mean values in guarana sample. Source of

Sum of

Degree of

Mean

variation

squares

freedom

squares

Between groups

912.0

4

228.0

Within groups

37.82

10

3.782

Total

949.8

14

Between groups

49879

4

Within groups

114234

10

Total

164113

14

Fcal

P

Fe 60.28 <0.0001

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Ni 12470

1.092

0.4117

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11423

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Ftab = 3.478.

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Table 4 Results obtained for Fe and Ni in guarana samples by HR-CS SS-GF AAS and after samples digestion. Results expressed as mean ± standard deviation (n = 3).

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HR-CS SS-GF AAS Fe

Ni

Fe

Ni

(µg g-1)

(µg g-1)

(µg g-1)

(µg g-1)

38.6 ± 1.4

1.23 ± 0.09

40.2 ± 0.8

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Sample

t- values Fe

Ni

1.30 ± 0.07

0.99

1.25

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GUA-01

Comparison value

GUA-02

186 ± 15

2.28 ± 0.06

166 ± 3.6

2.22 ± 0.05

2.10

1.35

GUA-03

21.3 ± 4.3

0.46 ± 0.02

22.8 ± 1.8

0.46 ± 0.06

1.01

0.06

GUA-04

144 ± 18

1.94 ± 0.1

122 ± 6.1

1.98 ± 0.1

2.04

0.28

GUA-05

43.3 ± 3.1

1.50 ± 0.1

42.2 ± 2.8

1.51 ± 0.06

0.40

0.14

tcritical (0.05, 2) (4.30).

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