Abstract
This study investigated three sample preparation methods for digestion of dietary supplements prior to selenium (Se) determination by graphite furnace atomic absorption spectrometry (GF AAS), such as conductive heating digestion, microwave-assisted wet digestion (MWAD), and microwave-induced combustion (MIC). In wet digestion methods, variable nitric acid and hydrogen peroxide concentrations were evaluated. For MIC, a diluted nitric acid concentration was also investigated as absorbing solution, while microcrystalline cellulose was employed as a combustion aid. The optimized conditions were: 250 mg of the sample with 5 mol L–1 nitric acid and 30% w/w hydrogen peroxide (160 °C, 5 h) for conductive heating; 350 mg with 2.5 mol L–1 nitric acid and 30% w/w hydrogen peroxide (180 °C, 40 min) for MWAD; and 200 mg of sample, 300 mg of microcrystalline cellulose, and 0.25 mol L–1 nitric acid as the absorbing solution (29.2 bar, > 1000 °C during the combustion reaction and 180 °C, 20 min in the reflux step) for MIC. Digestion efficiency was evaluated by residual carbon content (RCC) and residual acidity (RA) in final solutions. The greenness of each method was evaluated by using the AGREEprep software. Selenium was quantified by GF AAS using Pd as the chemical modifier using pyrolysis and atomization temperatures of 1200 and 2000 °C, respectively. The limits of detection (LOD) obtained for Se were 1.71, 0.84, and 0.08 μg g–1 for conductive heating, MWAD, and MIC, respectively, with agreement of 96.7–99.5% for the selenium-enriched yeast certified reference material. Although all methods showed good agreement (ANOVA, 95% confidence level), MIC was the most efficient, providing the lowest RCC (0.1%) and RA (0.5%) values, better LOD, and a greenness score of 0.49.
Introduction
Dietary supplements are over-the-counter products designed to complement the regular diet by providing elevated concentrations of nutrients and bioactive compounds. Common pharmaceutical forms include tablets, capsules, powders, liquids, and chewable gummies. (1) In dietary supplements containing selenium, the main sources of this micronutrient include inorganic salts (selenite and selenate), used in multivitamin and multimineral formulations, and organic sources such as selenomethionine, probiotic strains such as Lactobacillus acidophilus, and selenium-enriched yeast, obtained from the cultivation of Saccharomyces cerevisiae in a medium containing sodium selenite. (2−4)
The recommended daily intake (RDI) of selenium varies according to age, sex, and life stage, and also reflects regional differences in the composition of the food chain. (5) In the United States, the Food and Drug Administration (FDA) (5−7) recommends an RDI of 55 μg for adults, a tolerable upper intake level (UL) of 400 μg, in Europe, the European Food Safety Authority (EFSA) (8,9) establishes an RDI of 70 μg for adults and a UL of 255 μg. In Brazil, the Brazilian Health Regulatory Agency (ANVISA) (10) recommends 34 μg for adults per day, with a maximum limit of 319 μg.
Due to the narrow safety margin for selenium supplementation, even small increases above the recommended concentrations may result in adverse effects. (11) Furthermore, the lack of strict regulatory requirements for dietary supplements, when compared to pharmaceutical products, (12,13) raises concerns regarding the quality, storage conditions, stability, and actual selenium content in these products.
Microwave-assisted wet digestion (MWAD) is a widely utilized method for sample preparation in the determination of selenium concentrations, particularly in atomic spectrometry. It offers high efficiency, minimal analyte loss, and a reduced risk of contamination when utilizing closed vessels. Microwave radiation promotes rapid and homogeneous decomposition of complex matrices, enabling digestion pressures up to 100 bar and temperatures ranging from 200 to 300 °C. (14,15) Nitric acid is one of the most used oxidants in sample digestion due to its strong oxidative capacity, efficiency in organic matrix decomposition, lack of interference with most determinations, and its commercial availability in sufficient purity. (16,17)
In sample digestion, concentrated HNO3 is typically employed, resulting in high residual acid concentrations, which can adversely affect the performance of nebulization systems in inductively coupled plasma-based techniques (ICP) (18,19) or accelerate the degradation of graphite tubes in atomic absorption spectrometry. (20,21) Despite these advantages, concentrated HNO3 is still frequently used, either alone or in combination with auxiliary reagents such as other acids or hydrogen peroxide, for the decomposition of dietary supplement samples. (22−24) For instance, Hirtz and Günther (22) proposed Se determination by inductively coupled plasma mass spectrometry (ICP-MS) in 28 dietary supplements after digestion of 500 mg of sample with 6 mL of concentrated HNO3 and 2 mL of 30% H2O2 at 200 °C for 30 min. Similarly, Krawczyk (23) evaluated MWAD under elevated pressure and temperature conditions for three multivitamin supplements with 4 mL of concentrated HNO3, 1.5 mL of concentrated HF, and 1 mL of 30% H2O2 at 300 W for 20 min before analysis by high-resolution graphite furnace atomic absorption spectrometry (HR-CS GF AAS).
Besides MWAD, other digestion methods can also be employed, ranging from simpler approaches, such as conductive heating digestion, to more advanced systems like microwave-induced combustion (MIC). Augustsson et al. (25) analyzed Se and other elements in 138 dietary supplements by inductively coupled plasma sector field mass spectrometry (ICP-SFMS). The digestion protocol included overnight predigestion with 10 mL of HNO3 and 0.05 mL of HF, both concentrated acids, followed by heating at 110 °C for 120 min. Although effective, the use of HF in wet digestion of dietary supplement samples (23,25) presents several drawbacks, including incompatibility with a wide range of analytical equipment and formation of insoluble or stable fluoride complexes.
An alternative to using HF for the decomposition of samples containing inorganic matter is MIC. This method is characterized by low reagent consumption, minimal contamination, and enables rapid and complete decomposition of high-mass organic samples. (16,26) In the MIC process, the pelletized sample is placed on a quartz support along with filter paper and ammonium nitrate, then pressurized with O2 and ignited by microwave radiation. (26−28) For samples with low organic matter content, microcrystalline cellulose can be employed as a combustion aid to promote analyte volatilization. (26,28) Pereira et al. (29) employed MIC for the decomposition of multivitamin and mineral supplements using 1.0 mol L–1 HNO3 and 1.0 mol L–1 HCl as absorbing solution for the determination of metals by ion chromatography (IC) while Bitencourt et al. (30) used 7.0 mol L–1 HNO3 as the absorbing solution for As determination in dietary supplements samples by ICP-MS. Combustion was performed for 1 min at 900 W. In both works, an additional microwave extraction step was performed to ensure quantitative recovery of the analytes, although the use of cellulose could avoid this additional extraction step.
Previous research has predominantly utilized concentrated nitric acid in MWAD and conductive heating to achieve complete sample decomposition, often followed by dilution before analysis. In this study, diluted HNO3 solutions were investigated to reduce the reagent consumption while maintaining the digestion efficiency. Additionally, MIC was introduced as a novel sample preparation approach for the determination of selenium in dietary supplements, which has not yet been explored for this analyte. The three methods: conductive heating, MWAD, and MIC were systematically evaluated in terms of digestion time, efficiency, reagent consumption, and environmental performance using the AGREEprep metric, as well as their applicability for the quantification of selenium by graphite furnace atomic absorption spectrometry (GF AAS). This technique is particularly advantageous for monoelemental analyses due to its high sensitivity and low detection limits, employing pyrolysis conditions between 1000–1200 °C and atomization conditions between 2000–2300 °C, typically with the use of chemical modifiers such as Pd(NO3)2 or Pd–Mg(NO3)2 in the analysis of dietary supplement samples. (23,24,31−33)
Download the full article as PDF here Investigation of Sample Preparation Methods of Dietary Supplements for Total Selenium Determination
or continue reading here
Investigation of Sample Preparation Methods of Dietary Supplements for Total Selenium Determination, Larissa M. A. Oliveira, Diogo P. Moraes, and Juliana S. F. Pereira, ACS Omega Article ASAP, DOI: 10.1021/acsomega.5c08938
Read more on Weight Management & Metabolism Support here:
