Food Anal. Methods DOI 10.1007/s12161-016-0625-0
Optimization of a Digestion Method Using Diluted Acid in Bee Pollen Samples for Determination of Fe, Mn, and Zn by Flame Atomic Absorption Spectrometry Jean S. Siqueira 1 & João B. Pereira Jr 1 & Michelle S. Lemos 1 & Heronides A. Dantas Filho 1 & Kelly G. Fernandes Dantas 1
Received: 3 May 2016 / Accepted: 19 July 2016 # Springer Science+Business Media New York 2016
Abstract In this study, the determination of Fe, Mn, and Zn by flame atomic absorption spectrometry (FAAS) was performed in nine bee pollen samples from different cities. The efficiency of acid digestion in bee pollen samples using nitric acid at different concentrations (3.5, 7.0, and 14.0 mol L−1) and hydrogen peroxide in a microwave oven was evaluated. The proposed procedure using nitric acid diluted to 3.5 mol L−1 was effective for the digestion of all the pollen samples. The samples showed low levels of Fe. However, Mn and Zn were the elements which presented the highest concentrations in the samples. The bee pollen samples studied showed a great Mn and Zn contribution to the recommended daily intake (RDI) of the human diet, contributing with 71.7 and 15.1 %, respectively. The accuracy of the measurements by FAAS was evaluated by adding aliquots of the elements in the digestates. Recoveries obtained ranged from 89 to 106 % for Fe, 94 to 100 % for Mn, and 88 to 108 % for Zn. Keywords Bee pollen . Inorganic elements . FAAS
Introduction The natural diet of the honeybee is through pollen and nectar. Bee pollen is more important than nectar for the production of young because it provides the necessary nutrients for the development of bees. Bee pollen is the main source of protein in
* Kelly G. Fernandes Dantas
[email protected] 1
Group of Analytical Spectrometry Applied, Faculty of Chemistry, Federal University of Pará, Belém, Pará 66075-110, Brazil
bee larvae and young workers (Crailsheim et al. 1992; SerraBonvehí and Escolà-Jordà 1997; Taha 2015). Bee pollen is a very complex natural material, whose composition may vary significantly depending on environmental conditions such as the region in which it is produced, the preference of bees, the predominant flora, soil characteristics, and between seasons and years (Alcoforado Filho and Gonçalves 2000; O’Rourke and Buchmann 1991; Silva et al. 2012). It is frequently exposed to different elements which can be absorbed by plants from the soil through the root system or water intake, or anthropogenic sources that are directly deposited in pollen (Roman 2007). Bee pollen is highly valued among apiculture products because it has high concentrations of nutrients and other healthpromoting components. According to the report of Morgano et al. (2012), bee pollen has 13–55 % carbohydrates, 10–40 % protein, 1–20 % lipid, and 3–8 % water, and also contains minerals, resins, vitamins, and other compounds such as flavonoids and some antibiotic substances. Because of its nutritional and therapeutic properties, bee pollen is involved in the formulation of many additional products for food. It is also widely used in pollen supplements, for which it is very important to select bee pollen that has a high nutritional value (Iannuzzi 1993; Taha 2015). Mineral nutrients are inorganic elements found in food which the body cannot synthesize. They are essential and vital components of all living cells and are involved in human metabolism. Elements such as Mn and Zn are the active sites of some enzymes, hormones, vitamins, and nucleic acids, which play an important role in maintenance of life metabolism (Dokkum et al. 1989; Jacobson and Wester 1977; Tu et al. 2013). In various countries, many inorganic elements including Ca, Cd, Cu, Fe, K, Mg, Mn, Na, Ni, P, Pb, Se, and Zn have been quantified in bee pollen samples using various analytical
Food Anal. Methods
techniques such as atomic absorption spectrometry (AAS) (Carpes et al. 2007; Conti and Botrè 2001; Formicki et al. 2013; Silva et al. 2012; Taha 2015), electrothermal atomization atomic absorption spectrometry (ET AAS) (Saavedra et al. 2007), inductively coupled plasma optical emission spectrometry (ICP OES) (Morgano et al. 2012; SerraBonvehí and Escolà-Jordà 1997; Somerville and Nicol 2002; Yang et al. 2013) and X-ray fluorescence spectrometry (XRF) (Kump et al. 1996). In this paper, flame atomic absorption spectrometry (FAAS) was used to determine Fe, Mn, and Zn in bee pollen from different cities to evaluate the best procedure after digestion in a microwave oven using diluted nitric acid. The objective of this paper will clarify the distribution of Fe, Mn, and Zn in bee pollen that can provide basic data to evaluate nutritional value and safety, contributing to bee pollen quality control from some species of bees.
Materials and Methods Samples Nine bee pollen samples were collected from apiculture producers in different cities of the state of Pará and provided by Embrapa Amazônia Oriental (Table 1). After collection, the samples were packed and transported to the laboratory in polyethylene bags. Instrumentation A Liotop model L101 lyophilizer (São Carlos, SP, Brazil) was used to dry the samples. A SPEX SamplePrep model 6770 cryogenic mill (Metuchen, NJ, USA) was used for grinding the samples. The acid digestion of samples was performed in a Milestone Start E model microwave oven (Sorisole, Italy). An iCE 3300 model AAS flame atomic absorption spectrometer (Thermo Scientific, Cambridge, England) equipped
Table 1 Samples P1 P2 P3 P4 P5 P6 P7 P8 P9
Species, location and color of bee pollen samples Species
Location
Color
Melipona flavolineata Friseomelitta varia Scaptotrigona sp. Tetragonisca angustula Melipona flavolineata Apis mellifera Melipona fasciculate Apis mellifera Melipona fasciculate
Tracuateua, PA Belterra, PA Belterra, PA Belterra, PA Castanhal, PA Castanhal, PA Castanhal, PA Igarapé-Açu, PA Igarapé-Açu, PA
Orange Brown Brown Brown Yellow Brown Brown Brown Yellow
with a deuterium lamp background correction system was employed for Fe, Mn, and Zn determination. Hollow cathode lamps (Photron, Australia) were used as the radiation source for Fe, Mn, and Zn operating at 6.0, 5.0, and 5.0 mA with wavelengths at 248.3, 279.5, and 213.9 nm and spectral bandwidth of 0.2, 0.2, and 1.0 nm, respectively. In all experiments, an oxidizing air-acetylene flame was used. Reagents and Standard Solutions All reagents used were analytical grade. All dilutions were made using ultrapure water (resistivity 18.2 MΩ cm−1) obtained from a Synergy-UV water purification system (Millipore, Bedford, USA). All glassware and polypropylene flasks were soaked in a 10 % (v/v) HNO3 solution for 24 h and rinsed with deionized water prior to use. All solutions and samples were stored in decontaminated polyethylene vials. Nitric acid (Sigma-Aldrich, Germany) previously purified using a sub-boiling distillation system (Berghof, model BSP 929-IR, Germany), and 30 % w/w H2O2 (Impex, Brazil) was used to digest the samples. Calibration standard solutions were prepared by suitable dilution of the stock solutions containing 1000 mg L−1 of each element: 0.25–1.00 mg L−1 for Fe, 1.0–4.0 mg L−1 for Mn, and 0.5–2.0 mg L−1 for Zn in 5.0 % (v/v) nitric acid (Sigma, USA). Sample Preparation and Analysis Procedures The samples were packaged in plastic bags and stored in a freezer at −16 °C until analyses were performed. Prior to analysis, the samples were removed using stainless steel, lyophilized for 72 h, and ground in a cryogenic mill. A two-step program was applied: step I (prefreezing): 10 min, step II (milling): 2 min intercalated by cycles of freezing of 2 min. After lyophilization and grinding, the samples were stored in labeled, tightly sealed polyethylene containers in a desiccator. A mass of 0.25 g was weighed for each sample in re pli cat e (n = 3) a nd d ige ste d with 6. 0 m L of 3.5 mol L−1 HNO3 and 3.0 mL of 30 % (w/w) H2O2 in a microwave oven. The efficiency of the digestion procedure using 3.5 mL of nitric acid was evaluated at the following concentrations: 3.5, 7.0, and 14.0 mol L −1 based on the procedure described by Souza et al. (2013). For all three procedures, HNO3 was added first and left in contact with the sample and then hydrogen peroxide was added. The microwave heating program consisted of three steps: 800 W, 180 °C for 10 min; 800 W, 180 °C for 20 min; and ventilation for 50 min. The digests were transferred to volumetric flasks and diluted to 14 mL with ultrapure water. Blank experiments were carried out in the same way.
Food Anal. Methods Table 2 FAAS
Figures of merit in the determination of Fe, Mn and Zn by
Element LOD (mg kg−1) LOQ (mg kg−1) Slope (m) C0
R2
Fe
0.07
0.23
1.9228
0.002 0.997
Mn Zn
0.01 0.09
0.04 0.30
6.2667 2.5928
0.001 0.998 0.001 0.997
LOD limit of detection, LOQ limit of quantification, C0 characteristic concentration, R2 correlation coefficient
Figures of Merit The limit of detection (LOD) and limit of quantification (LOQ) values (Table 2) were calculated using 10 measures of the analytical blank as suggested by Harris (2007); LOD = 3 × SD/m and LOQ = 10 × SD/m, where SD is the standard deviation of 10 measurements of the analytical blank and m is the slope of the analytical calibration curve. Sensitivity by FAAS is given by the characteristic concentration (C0) which is defined as the concentration of analyte that produces an absorbance signal of 0.0044 (Bermejo et al. 2002) (Table 2). The sensitivity obtained was adequate.
Results and Discussion Efficiency of Digestion Procedure At first, to evaluate the best digestion procedure, a bee pollen sample (P1) was selected as it presented a different color. After that, the validated methodology was extended to other samples. The sample was subjected to the microwave heating program using different concentrations of nitric acid (3.5, 7.0, 14.0 mol L−1) (Souza et al. 2013). Hydrogen peroxide was used as an auxiliary oxidizing agent in all digestions. Table 3 shows the concentrations of Fe, Mn, and Zn found in P1 using FAAS. All the results obtained were similar independent of acid concentration. Student’s t test was applied to the values found for the analytes in 3.5 and 14.0 mol L−1. The calculated t values were compared with critical t value for n = 3; t0.05; n−1, crit = 4.303. All calculated t values were lower than critical t value. Therefore, there was no difference at a
Table 3 The levels of Fe, Mn, and Zn digested bee pollen samples using different digestion procedures Procedure
Fe (mg kg−1)
Mn (mg kg−1)
Zn (mg kg−1)
3.5 mol L−1 HNO3 7.0 mol L−1 HNO3 14.0 mol L−1 HNO3
66.4 ± 1.3 62.3 ± 1.9 68.3 ± 4.6
117.6 ± 2.2 119.1 ± 4.2 118.1 ± 4.1
47.5 ± 0.8 44.7 ± 1.9 46.2 ± 1.3
95 % confidence level for concentration values of Fe, Mn, and Zn. Obtaining solutions digested without the presence of residues in the evaluated procedures confirmed the efficiency of diluted nitric acid in the digestion of different samples (Araújo et al. 2002; Gonzalez et al. 2009; Souza et al. 2013). In this study, success achieved in the sample preparation using diluted nitric acid could be achieved only by combining this reaction with hydrogen peroxide, which supplies the oxygen needed for continuation of oxidation-reduction reactions. This in turn ensured the total decomposition of organic matter from the regeneration of nitric acid and consequent increase of pressure and temperature inside in the reaction vessel. Given the success achieved in all the methodologies presented in Table 3, the digestion procedure using 3.5 mol L−1 HNO3 was adopted for other pollen samples due analytical white best analyzes obtained in ensuring excellent limits of detection and quantification. In addition, it decreases the use of concentrated reagents, generates less waste to the environment, has a lower analysis cost, and ensures greater safety for the analyst. Accuracy The accuracy of the sample preparation procedure was evaluated by adding aliquots of 0.3–0.9 mg L−1 for Fe, 0.5– 3.0 mg L−1 for Mn, and 0.2–1.7 mg L−1 for Zn to the samples prior to digestion using diluted nitric acid (3.5 mol L−1). Thereafter, the samples were submitted to the digestion process assisted using microwave radiation (Pereira Junior and Dantas 2016). Then, the elements were determined in the digestates by FAAS. This procedure aimed to evaluate whether there were losses of elements by volatilization during the sample preparation procedure. Good recoveries were obtained for all analytes ranging from 92 to 107 %. The accuracy of the measurements by FAAS was also verified by the recovery test. Firstly, the digest samples were spiked with four different concentration levels: 0.35, 0.65, 0.85, and 0.90 mg L−1 for Fe, 0.5, 1.5, 2.5, and 3.5 mg L−1 for Mn, 0.25, 0.75, 1.25, and 1.75 mg L−1 for Zn and then, the resulting solutions analyzed by FAAS. Recoveries obtained ranged from 89 to 106 % for Fe, 94 to 100 % for Mn, and 88 to 108 % for Zn. Quantification of Bee Pollen Samples Table 4 presents the quantification values of Fe, Mn, and Zn in bee pollen samples. The results revealed good precision under the analytical conditions used, since the relative standard deviations (RSD) were lower than 5 %. Fe is considered an essential micronutrient but presents smaller concentrations in bee pollen samples ranging from 6.6 to 66.4 mg kg−1 in the samples collected in the city of Igarapé-Açu (P8) and Tracuateua (P1), respectively. In this study, Fe levels obtained were
Food Anal. Methods Table 4 Fe, Mn, and Zn levels in nine bee pollen samples determined by FAAS (mean ± SD, n = 3) Samples
Fe (mg kg−1)
Mn (mg kg−1)
Zn (mg kg−1)
Table 5 Contributions of Fe, Mn, and Zn to recommended daily intake (RDI) (for adults consuming 25 mg day−1) compared to average levels found in bee pollen samples Medium levels (mg kg−1)
RDIa (mg dia−1)
Fe
22
14
Mn Zn
66 42
Element P1
66.4 ± 1.3
117.6 ± 2.2
47.5 ± 0.8
P2 P3
34.8 ± 1.2 13.7 ± 0.2
8.1 ± 0.2 22.9 ± 0.6
14.3 ± 0.3 39.9 ± 0.3
P4
15.7 ± 0.8
5.7 ± 0.4
21.2 ± 1.4
P5
12.9 ± 0.3
178.5 ± 2.3
68.1 ± 1.8
P6 P7
28.0 ± 2.1 10.9 ± 0.2
27.9 ± 1.5 14.0 ± 0.2
37.8 ± 1.4 45.9 ± 0.5
P8
6.6 ± 0.2
88.5 ± 0.3
47.8 ± 0.3
P9
14.7 ± 0.6
130.4 ± 1.1
59.2 ± 0.2
lower than the levels found by Morgano et al. (2012) in a study of 154 bee pollen samples from different regions of Brazil. The authors found levels ranging from 11.1 to 551.6 mg kg−1. Except for one sample (P8), the other samples showed Fe concentrations similar to the levels found by Silva et al. (2014). However, Yang et al. (2013) found concentrations higher than the values o b t a i n e d i n t h i s s t u d y, r a n g i n g f r o m 7 5 . 2 t o 207.8 mg kg−1. Mn was the element which presented the highest concentrations. The lowest and highest Mn levels observed were 5.7 mg kg−1 for sample P4 and 178.5 mg kg−1 for sample P5. According to Harmsen and Vlek (1985), Mn is among the most abundant 12 elements of the lithosphere and can reach about 3000 mg kg−1 in the environment. In other bee pollen from Brazil, Mn values ranged from 12 to 211 mg kg −1 (Morgano et al. 2012). Other Mn concentrations reported in the literature varied from 8.7 to 357 mg kg−1 in bee pollen in China (Yang et al. 2013) and from 16.6 to 38.6 mg kg−1 in bee pollen from Saudi Arabia (Taha 2015). The levels of Mn in 27 bee pollen samples from different countries were reported in the range of 13.2 to 429.8 mg kg−1 by Szczêsna (2007). The Zn content in the bee pollen studied varied in the range of 14.3 mg kg−1 (P4) to 68.1 mg kg−1 (P5). The Zn levels in 320 bee pollen samples from Poland (Formicki et al. 2013), 34 bee pollen samples from Australia (Somerville and Nicol 2002) and 37 bee pollen samples from Argentina (BaldiCoronel et al. 2004) were reported in the ranges 19– 81 mg kg−1, 16–340 mg kg−1 and 23–106 mg kg−1, respectively. The Zn content obtained in this study corroborated the levels found by Morgano et al. (2012). According to the report of Martins (2010), the mineral composition of bee pollen varies by floral origin, soil composition, and geographical origin. Thus, the high Mn and Zn levels found in sample P5 can be explained because city soil
a
2.3 7
RDI (%) 3.9 71.7 15.1
Anvisa (2005)
presents a high humidity and low pH, favoring the availability of Mn in the soil in soluble form (Mn2+) and being easily absorbed by the production of plant pollen (Valente et al. 2001). On the other hand, Igarapé-Açu is located in the area with the largest agricultural production of the State of Pará, where soil fertilization is an inherent process factor, and this could be related to the significant levels of Mn and Zn obtained in samples collected in this city (Hayden and De França, 2013).
Estimated Intake of Elements in Bee Pollen Compared with the Daily Recommended Intake The contribution of the levels of Fe, Mn, and Zn found in the pollen to the recommended daily intake (RDI) of minerals established by Brazilian legislation (Anvisa 2005) is shown in Table 5. According to Lengler (2002), the recommended dose of pollen consumption is 25 g day−1 for adults and 5– 10 g day−1 for children, depending on age. With respect to mean levels, Mn has a large contribution to daily intake (71.7 % of RDI). A contribution of more than 30 % classifies the product as a food with high content and over 15 % as a source of minerals (Anvisa 2005). Thus, the studied pollens, in addition to having the high doses required for daily intake of Mn, can be classified as a potential source of Zn in the human diet.
Conclusions The procedure of microwave digestion developed using diluted nitric acid (3.5 mol L−1) for determination of Fe, Mn, and Zn in bee pollen samples was shown to be simple, efficient, and low cost. As can be seen from the results, Mn and Zn elements were present at the highest concentrations in the samples. However, Fe showed the lowest levels. Despite the absence of specific legislation, the studied pollen samples revealed that they have a great Mn and Zn contribution to the RDI for the human diet, contributing with 71.7 and 15.1 %, respectively.
Food Anal. Methods Compliance with Ethical Standards This is an original research article that has neither been published previously nor considered presently for publication elsewhere. All authors named in the manuscript are entitled to the authorship and have approved the final version of the submitted manuscript. Funding This study was funded by Fundação Amazônia de Amparo a Estudos e Pesquisas (FAPESPA) (Processo ICAAF N° 012/2012), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Processo CNPq –REPENSA n° 562994/2010–6), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Conflict of Interest Jean S. Siqueira has received research grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). João B. Pereira Junior, Michelle S. Lemos have received research grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Jean S. Siqueira declares that he has no conflict of interest. João B. Pereira Junior declares that he has no conflict of interest. Michelle S. Lemos declares that she has no conflict of interest. Heronides A. Dantas Filho declares that he has no conflict of interest. Kelly G. Fernandes Dantas declares that she 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.
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