Green and low-cost method for selenium analysis in beans by Photochemical Vapor Generation coupled to Atomic Absorption Spectrometry (PVG-AAS)

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Green and low-cost method for selenium analysis in beans by Photochemical Vapor Generation coupled to Atomic Absorption Spectrometry (PVG-AAS)

https://doi.org/10.21203/rs.3.rs-4829859/v1

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Selenium is an essential element and the main source of it is food. The ingestion of food rich in this element as nuts (Brazil nuts) is important to keep the body healthy. In this work, the development of a green and low-cost methodology for selenium analysis in beans was carried out using the photochemical vapor generation method coupled to atomic absorption spectrometry. The efficiency of the photochemical vapor generation (PVG) was optimized by evaluating the experimental conditions, such as: type and concentration of the organic acid, flow rate of the carrier gas and sample UV irradiation time. The formation of volatile species was more efficient using 6% v v^− 1 acetic acid, carrier gas flow rate of 10 mL min^− 1 and 20 s UV irradiation time. The method of standard addition calibration curves was performed to analyze total Se in green bean samples acquired in the market of different cities of northeast and north regions of Brazil. The accuracy of the proposed method was tested comparing results for Se content with hydride generation (HG) in a sample of Ceara state. The analyzed samples showed total Se content varying from 0.9 to 2.2 mg kg^− 1 in dry matter of green beans. The amount of Se found in green beans is within the expected range when compared to other types of beans reported in the literature.

low-cost method

Selenium

green beans

PVG-AAS

Selenium (Se) is an essential micronutrient for humans due to its antioxidant and anti-inflammatory properties (BRUSA et al., 2023). The amount of ingestion and the chemical form of this element may favor its beneficial characteristics or it can become toxic to the body (BARTOSIAK; GIERSZ; JANKOWSKI, 2019). In addition, selenium deficiency in the body also leads to various diseases (SIGRIST et al., 2012). According to the FAO (Food and Agriculture Organization), the recommended daily intake of selenium should be 55 µg/day for an adult, with a tolerable limit of 300 µg/day.

The main source of selenium for living beings is food. Therefore, due to the importance of selenium for the body, it is interesting to know the nutritional composition of foods in order to have an adequate intake of this element (FERREIRA et al., 2002). Moreover, it is important to know that the selenium content in food will depend on the amount found in the soil where it is present as its inorganic form (iSe) and also that it is converted by plants into selenoaminoacids, such as selenocysteine and selenomethionine (ALCâNTARA et al., 2022; RYBÍNOVÁ et al., 2016).

Beans are considered one of the most consumed foods by the Brazilian population, particularly in the north and northeast regions, as they are a good nutritional source and present high protein content. The so-called "green" bean corresponds to the pod in the pre-ripening phase, a stage in which the production of photosynthates is interrupted and the natural dehydration process begins. In this condition, the pods become swollen and undergo a slight change in tone, turning green or purple, hence the name “green bean”. This type of bean offers the possibility of up to three annual harvests due to the short time in which they can be harvested. The northeast region has a large consume of green beans (Vigna unguiculata L Walp) that is minimally processed, being taken directly from the pod for cooking in Ceara State of Brazil (BORGES DE OLIVEIRA SILVA et al., 2018; MARSARO JÚNIOR; ALCANTARA VILARINHO, 2017). Green beans present a moisture content of 60 to 70% and can be sold in the form of pods or grains (ANDRADE et al., 2010).

As demand grows for the development of new element detection techniques with high sensitivity and precision, there is also a search for efficient sample introduction methods, which will influence the efficiency of the analysis (D’ULIVO, A., STURGEON, R., 2022). Chemical vapor generation (CVG) is one of the most widely used methods for introducing samples, through the generation of volatile species by chemical reactions, such as hydride generation (HG). This method has the following advantages: high sample introduction efficiency, separation of the analyte from the sample matrix, thus reducing the matrix effect (ZOU et al., 2020). However, the use of expensive and unstable reagents and the high generation of waste are the disadvantages of this method. As a result, other sample introduction methods have been developed to overcome these limitations (LIU et al., 2020).

Photochemical vapor generation has emerged as an alternative to solve the limitations of CVG, because in addition to having the same advantages as CVG, it uses low molecular weight organic acids (LMWOA) which are degraded by UV radiation, thus generating less waste. This method is based on the photolysis reaction of the ions of the analyte of interest with the radicals produced by the organic acids in the presence of UV radiation (FROIS et al., 2020).

Both methods can be coupled with a low-cost technique as atomic absorption spectrometry (AAS) to determine selenium presenting good sensitivity. The aim of this study was to develop a green and low-cost method for selenium analysis in beans using photochemical vapor generation coupled with flame atomic absorption spectrometry (PVG-AAS).

2.1. Instrumentation

The measurements were performed using an atomic absorption spectrometer (Varian AS 240FS, Mulgrave, Australia) equipped with a deuterium lamp for background correction and a selenium hollow cathode lamp (Agilent Technologies) as radiation source. A quartz T cell (166 mm long and 7 mm in internal diameter) was used and positioned over the burner aligned with the optical path of the spectrometer. The volatile selenium species were transported from the GLS to the cell with the aid of N₂ gas (99.996%, White Martins, São Paulo, Brazil) in a continuous flow controlled by a flowmeter (VFA-22, HygroTherm).

A low-pressure Hg lamp emitting UV-C flow-through radiation of 19 W (Beijing Titan Instrument Co., Beijing, China) was used for the photochemical reactor, containing an internal synthetic quartz analytical path located in the center of the lamp. The radiation emitted is at wavelengths (λ) 185 and 254 nm and radiates at 360°. The reactor was placed in an aluminum-covered box to protect the operator from ultraviolet radiation.

A microwave oven (Master-100, Milestone) and an ultrasonic bath (7lab, Brazil) were used for the sample preparation procedures. A freeze dryer (Liotop L101, Liobrás®, Brazil) was used to dry samples of beans.

2.2. Sample and solutions

All reagents used in this study were of analytical grade. Ultra-pure water (18 MΩ cm) obtained in the Milli-Q water purification system (Millipore, Bedford, USA) was used to prepare standards, solutions and samples. All vials were washed and immersed for 24 hours in 10% nitric acid for decontamination, and then rinsed with ultra-pure water.

Samples of fresh green beans (Vigna unguiculata L Walp) were obtained from local markets in different cities of Ceara state (Caucaia and Fortaleza) and Para state (Belém), and were stored in a freezer (-18 ^oC). Samples were freeze dried and macerated before solubilization and/or digested in the microwave oven.

Standard solution of 1000 mg L^− 1 of selenium (IV) was prepared by dissolving sodium selenite salt (Sigma-Aldrich, USA) in ultrapure water and then stored at 4°C for later use. The working solutions were prepared daily from the selenium stock solution previously prepared with the corresponding dilution.

Formic acid 98% (Sigma-Aldrich, USA) was used as low molecular weight organic acid (LMWC). Hydrochloric acid 37% (w/w) (Dinâmica, Brazil) was used in the hydride generation system and for the sample preparation procedures. Nitric acid 65% (v/v) (Sigma-Aldrich, USA) and hydrogen peroxide 30% (v/v) (Sigma-Aldrich, USA) were used for the digestion of the samples in the microwave oven. Sodium borohydride (Vetec, Brazil) was used as a reducing agent in the hydride generation system and sodium hydroxide (Vetec, Brazil) was used to stabilize it.

2.3. Procedures

2.3.1. Sample Preparation

250 mg of sub-samples were microwave-assisted digested using 3 mL of nitric acid (65% w w^− 1) and 2 mL of hydrochloric acid (37% w w^− 1). Sample solutions were diluted to 50 mL after digestion before the Hydride Generation (HG-AAS) procedure.

Solubilization using formic acid for Se in the PVG was performed following the previous procedure reported in the literature (LOPES et al, 2016). 250 mg of sub-samples were solubilized using 8 mL of formic acid (98% w w^− 1) and placed in the ultrasonic bath at 50 ^oC during 3h, then at 60°C during 15h. Solubilized samples were filtered and diluted to 50 mL.

2.3.2. Hydride and photochemical generation of Se

Hydride Generation (HG-AAS) procedure was implemented using a flow system (peristaltic pump) with teflon tubings and T connections for the introduction of the reagent NaBH₄ 0.2% (NaOH 0.05%), HCl 3,0 mol L^− 1 and sample solution, then pump to the gas-liquid separator (GLS). The volatile species (Hydride) was carried to the T-quartz cell (AAS) using N₂ gas at 30 mL min^− 1 flow rate.

The scheme of photochemical vapor generation (PVG-AAS) system is shown in Fig. 1. The system was implemented using a flow system (peristaltic pump) with teflon tubings for the introduction of the sample into the PVG reactor (flow through lamp, 19W), transportation to the gas-liquid separator (GLS) and to the T quartz cell in the atomic absorption spectrometer. The volatile species formed were carried to the T-quartz cell using a mixture in the flow system of N₂ gas at 10 mL min^− 1 flow rate and an auxiliary gas (Ar/H2) at 10 ml min^− 1 flow rate. The conditions of the PVG were optimized using a Box-behnken experimental design.

Figure 1 here

2.4. Figures of merit

The parameters estimated in the figures of merit of the proposed method were: Linearity and Working Range, Limits of Detection and Quantification, Precision (RSD) and Accuracy (using the HG-AAS method). These parameters were determined in accordance with Resolution 899/2003 of ANVISA - National Health Surveillance Agency. The linear working range was estimated by adding Se (IV) in a range of 0 to 175 µg L^− 1 to the bean samples after the sample preparation procedures for each method (PVG-AAS and HG-AAS). The accuracy of the method was determined by comparing the results of analysis of Se in one sample of Ceara state (Caucaia/CE) of both methods HG-AAS (consolidated method in the literature) and the proposed method PVG-AAS. Precision was determined by the repeatability, calculated by the relative standard deviation (%RSD) in which the same operating conditions, the same operator and the same measurement system are used, and measurements can be made in the same solution over a short period of time (INMETRO, 2016). The precision calculation was based on the determination of 8 replicates of 100 µg L^− 1, obtaining 15% RSD.

The limits of detection (LOD) and limits of quantification (LOQ) were calculated as 3 and 10 times, respectively, the standard deviation of 10 measurements of the analytical blank solution of the selenium (IV) method and divided by the slope of the calibration curves.

The first parameter studied in the univariate method was the type of the carboxylic acid (formic acid and acetic acid) in different concentrations evaluating the magnitude of the Se absorbance signal. Figure 2 shows that the better response for Se (IV) was obtained using acetic acid. This result is in agreement with the obtained by NOVÁKOVÁ et al. (2017). In this study, acetic acid showed an increase of almost 50% in the maximum absorbance of Se when compared to formic acid. Therefore, acetic acid was chosen for the measurements. Regarding the concentration of the organic acid, the range between 2.0% and 6.0% of acetic acid was chosen, due to the better absorbance signal for Se observed in Fig. 2.

Figure 2 here

The parameters "irradiation time, acetic acid concentration and flow rate of the carrier gas" were optimized using the multivariate method of surface methodology known as Box Behnken Design.

Table 1 shows the independent variables and levels used in the Box-Behnken experimental design. The response variable used was the absorbance value. The symbol − 1 indicates the minimum level, 0 the central point and + 1 the maximum level.

The UV irradiation time of the sample solution was observed by varying the sample flow rate of the peristaltic pump in the photochemical reactor (flow through lamp). The results were obtained with the irradiation time varying between 16.42 and 22.56 seconds, and were estimated by rotation per minute (rpm) of the peristaltic pump, varying between 3 and 4 rpm.

Table 1

Variables and levels used in the box-behnken design.
Variable	Variable code	Level
Variable	Variable code	-1	0	+ 1
UV Irradiation time (s)	X₁	22.56	20.31	16.42
Acetic acid % (v v^− 1)	X₂	2.0	4.0	6.0
Carrier flow rate (mL min^− 1)	X₃	10	20	30

The response surface plots (Fig. 3) were built using the data obtained by the Box Behnken experimental design. Figures 3(I) and 3(II) show the acetic acid concentration versus irradiation time and carrier gas flow rate versus acetic acid concentration. The response (absorbance signal of Se) is increasing as the acetic acid concentration increases and the irradiation time and carrier gas flow decrease. The same occurs in Fig. 3(III), which represents the carrier gas flow rate versus irradiation time, in which the maximum response is obtained with the minimum values obtained for these variables. Therefore, the maximum absorbance response can be obtained for the optimized values of the variables, which are: 6.0% v v^− 1 of acetic acid, 10 mL min^− 1 for the carrier gas flow and 16.42 s for the irradiation time. However, due to the high solution consumption, the irradiation time used was 20.31 s.

Figure 3 here

Table 2 summarizes the estimated values of the analytical figures of merit of the method for Se analysis by PVG-AAS developed in this work. The accuracy was tested using the green beans sample purchased in Ceara state (Caucaia) and analyzed by the proposed PVG-AAS and the HG-AAS methods.

Table 3 shows a comparison between some methods described in the literature and the one presented in this work. It is worth mentioning that even though the estimated values were close (LOD and LOQ) or even slightly worse than those obtained for HG-AAS, the proposed PVG-AAS method confirms itself as a green analytical method when we consider the 12 parameters developed by by PENA-PEREIRA et al. (2020) known as “Analytical GREEnness metric approach”.

Table 2

Analytical figures of merit for Se determination by PVG-AAS and HG-AAS.
Figures of merit	PVG-AAS	HG-AAS
Linear range	50–175	25–125
RSD (%)	(n = 8) 15.1	(n = 9) 7.57
LOD (µg kg^− 1)	1.60	1.07
LOQ (µg kg^− 1)	5.33	3.57
Accuracy (mg kg^− 1)	2.24 ± 0.38 ^a	2.84 ± 0.24^a
^a standard deviation for triplicate analysis

Table 3

Limits of detection for methods of total Se analysis using different techniques.
Method	Reagent	LOD (µg kg^− 1)	Reference
PVG-AAS	Acetic acid	1.60	This work
HG-AAS	NaBH₄	1.07	This work
HG-AAS	NaBH₄	7.0	SIGRIST et al. (2012)
HG-AFS	NaBH₄	0.7	MATOS-REYES et al. (2010)
PVG-GFAAS	Formic acid	0.65	DA LUZ POTES et al. (2019)

The detection and quantification limits obtained in this work (PVG-AAS) are better when compared to methodologies that also use flame atomic absorption (AAS) techniques. When using higher sensitivity techniques, we observed lower LOD, which is expected; however, the costs related to analyses using more sensitive techniques must be considered. Methods using hydride generation also present slightly better sensitivity than that obtained by PVG-AAS; however, it should be noted that these methods use unstable reducing agents and, therefore, must be prepared daily, and thus, present higher reagent costs, as well as generate more waste.

The GREEnness method was proposed in 2020 by PENA-PEREIRA et al. and it aims to evaluate, with 12 questions to be answered, how green the analytical method in question is. We evaluated the proposed PVG-AAS methods and the HG-AAS used to estimate its accuracy. The result is shown in Fig. 4, which has a pictogram with a number that the closer to 1, the greener the method evaluated. An analytical method with a value above 0.59, such as the PVG-AAS (0.69) proposed in this work, can be considered a green analytical method and the pictogram presents a green color. It is worth mentioning that the HG-AAS method presented a value of 0.49 in the pictogram (yellow).

Figure 4 here

Bean samples from the states of Ceará and Pará were analyzed using the proposed PVG-AAS method, and the results are summarized in Table 4. We compared the total Se levels found in the samples with those obtained in studies presented in the literature for other types of beans. By comparing the data obtained with those from other studies, it is possible to note that the selenium content in the samples is quite varied, due to several factors, such as the region, the climate, and the soil used. According to the FAO (Food and Agriculture Organization) and the US Institute of Medicine, it was established that the maximum content for the acceptable daily intake of selenium for an adult is 55 µg/day, with a tolerable limit of up to 400 µg/day (INSTITUTE OF MEDICINE, 2006). Therefore, the values obtained fit within these limits and are in agreement with the concentrations found by other authors.

Table 4

Results of the analysis of total Se of selenium in different beans from Brazil.
	Se (mg kg^− 1)	Region	Reference
Green beans	2.24 ± 0.38	Ceara state (Caucaia)	This work
Green beans	2.00 ± 0.15	Ceara state (Fortaleza)	This work
Green beans	0.88 ± 0.02	Para state (Belem)	This work
Beans	1.73 ± 0.06	North region	SHALTOUT et al. (2011)
Beans	< 5 µg kg^− 1	Nine states	SHALTOUT et al. (2020)

The difficulty in analyzing low Se concentrations in complex samples (high protein contents) limits the number of articles reporting on such subjects. SHALTOUT et al. (2020) carried out a study using a high-resolution, continuous-source graphite furnace atomic absorption spectrometry (HS-CS-GF-AAS) system to implement a method for routine analysis of beans. The authors used 14 different samples of beans (Faseolus Vulgaris L.) collected in 9 states of Brazil and found concentrations lower than 5 µg kg^− 1. LODs of 30 ng kg^− 1 were estimated and even so, the authors concluded that methods with in situ trapping hydride generation for pre-concentration and subsequent analysis of Se should be used to achieve sufficient sensitivity. It is worth mentioning that this is another type of bean that, unlike green beans, is harvested ripe.

The results obtained allow us to conclude that the green and low-cost PVG-AAS method developed for selenium analysis in bean samples is efficient and accurate.

The experiments performed to estimate each figure of merit parameter in this study showed good selectivity and accuracy for the analysis of Se in beans, indicating proportionality between the concentrations analyzed in the working range used.

The proposed PVG-AAS method is considered “green” according to the GREEnness assessment performed.

Therefore, all the parameters analyzed are in compliance with Brazilian standards and legislation and thus, it can be stated that the method evaluated is valid for the analysis of selenium concentration in bean samples.

Author Contribution

A. C. C. Aragão and F. S. Ferreira: Conceptualization, Data curation,Formal analysis, Investigation, Methodology, Validation, Writing –original draft; J. K. S. de Brito: Conceptualization, Data curation,Formal analysis, Investigation, Methodology and G. S. Lopes: Conceptualization,Funding acquisition, Investigation, Project administration,Resources, Supervision, Validation, Visualization, Writing – review &editing.

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No competing interests reported.

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You are reading this latest preprint version

Green and low-cost method for selenium analysis in beans by Photochemical Vapor Generation coupled to Atomic Absorption Spectrometry (PVG-AAS)

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Version 1

Abstract

Figures

1. Introduction

2. Experimental

2.1. Instrumentation

2.2. Sample and solutions

2.3. Procedures

2.3.1. Sample Preparation

2.3.2. Hydride and photochemical generation of Se

2.4. Figures of merit

3. Results and Discussion

Conclusion

Declarations

Author Contribution

References

Additional Declarations

Status:

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