A group of Chinese researchers has presented a method for the selenium speciation analysis in fortified vegetables providing high extraction yields and good detection power.
Selenium (Se) is an essential element supporting human nutrition and health. Various Se compounds are involved in important metabolic processes playing a vital role in protecting against oxidative stress, improving immune system function, and preventing cancer. Se species being present in plants exist in both inorganic and organic forms, while only the organic Se compounds are responsible for most of the beneficial effects of dietary Se. On the other hand, Se can also produce toxic effects, with organic Se compounds having a higher safety threshold than inorganic species. A sufficient nutritional supply of Se is therefore essential for human health making the use of Se-enriched plant foods an option for populations that do not reach recommended supply. Since the beneficial effects of such Se supplements are highly dependent on the Se speciation, their characterization calls for Se speciation analysis.
The new study:
The most often used analytical technique for Se speciation is the coupling of high-performance liquid chromatography (HPLC) with inductively coupled plasma-mass spectrometry (ICP-MS). Since the HPLC-ICP-MS technique calls for samples in the liquid phase, the sample preparation necessitates an extraction step of the different Se species from the plant matrix. This step is critical with respect to efficiency of extraction as well as degradation of species.
A group of Chinese researchers have now developed a method by investigating especially this extraction step with different enzymes using to liberate selenium species from the plant matrix. Organic Se species exist in plants as seleno-amino acids, selenoproteins, and selenopeptides. Enzymatic hydrolysis, especially proteolysis, is a mild and effective method of Se extraction that ensures the liberation of different Se compounds from complex plant matrices. However, the efficiency of Se extraction by such enzymatic hydrolysis is significantly influenced by the type and dosage of the enzyme, extraction time, and plant matrices calling for optimization.
Photo: The hyperaccumulator Cardamine violifolia.
Se-enriched plant foods investigated included broccoli, cabbage, soybean and bamboo shoots as well as the hyperaccumulator Cardamine violifolia. Total Se was determined after microwave digestion of the plant material using HNO3. Determination was performed by ICP-MS/MS using the oxygen shift mode. The total Se extracted by enzymatic hydrolysis was also determined using the same setup.
For speciation analysis, 0.1-g aliquots of the plant sample materials were added to 15 ml of Tris-HCL buffer (pH=8.5). Simultaneously, 5% alcalase, trypsin, and protease K were added to the sample. The mixtures were stirred for 4 h at 45°C before being centrifuged at 4000 rpm for 30 min. This method is the result of an optimization with respect to enzyme composition and concentration, extraction time and reaction volume. The extracted supernatants were filtered through a 0.22-µm nylon filter for further analysis. Extracted Se species were separated by a reversed-phase ion-pair chromatography technique and detected by ICP-MS/MS. Species were identified by matching their retention times with those of Se standard solutions. Chromatographic conditions were optimized with respect to the composition of the mobile phase, pH, column temperature and flow rate. All five Se species (Se (IV), Se (VI), methylselenocysteine, selenomethionine, and selenocystine) were separated in 8.5 min. The method was validated with respect to working range, detection/quantification limit, species stability, and recovery. Detection limits were in the range of, 0.08-0.15 µg/L and recoveries of the five species ranged from 83.0 to 106%, with RSDs < 6.5 %.
The Se extraction rates of the different plant materials ranged from 69.7 to 97.9%. The composition of Se species was quite different in the different plant materials. While selenocystine is the primary species in C. violifolia and broccoli, selenomethionine is dominant in soybeans and bamboo shoots. Inorganic Se(VI) is the major species in cabbage.
The authors concluded, that the presence of unidentified species necessiates their identification by other MS techniques.
The data on speciation and extraction yield always call for critical evaluation. The hyperaccumulator Cardamine violifolia has the highest Se content (2854 µg/g) of the plant materials investigated here. On the other hand, the extraction rate is the lowest (69.7 %) of all plant materials investigated. While selenocystine showed the highest concentration (2154 µg/g) of all species investigated, other researchers (Both et al. 2018) found that selenolanthioine is the major water-soluble selenium compound in that plant. However this Se species was not part of this analysis.
The original publication
Mei Ye, Jie Li, Ruipeng Yu, Xin Cong, Dejian Huang, Yue Li, Shangwei Chen, Song Zhu, Selenium Speciation in Selenium‑Enriched Plant Foods, Food Anal. Methods, (2022). DOI: 10.1007/s12161-021-02208-9
K. Pyrzynska, A. Sentkowska, Selenium in plant foods: speciation analysis, bioavailability, and factors affecting composition. Crit. Rev. Food Sci. Nutr., 61 (2021) 1340–1352. DOI: 10.1080/10408398.2020.1758027
K. Pyrzynska, A. Sentkowska, Liquid chromatographic analysis of selenium species in plant materials, Trends Anal. Chem., 111 (2019) 128–138. DOI: 10.1016/j.trac.2018.12.011
E.B. Both, S.X. Shao, J.Q. Xiang, Z. Jokai, H.Q. Yin, Y.F. Liu, A. Magyar, M. Dernovics, Selenolanthionine is the major water-soluble selenium compound in the selenium tolerant plant Cardamine violifolia, Biochim Biophys Acta Gen Subj, 11 (2018) 2354–2362. DOI: 10.1016/j.bbagen.2018.01.006
S.X. Shao, X.B. Mi, L. Ouerdane, R. Lobinski, J.F. García-Reyes, A. Molina-Díaz, A. Vass, M. Dernovics, Quantification of Se-methylselenocysteine and its γ-glutamyl derivative from naturally Se-enriched green bean (Phaseolus vulgaris vulgaris) after HPLC-ESI-TOF-MS and orbitrap MSn -based identification, Food Anal. Methods, 7 (2013) 1147–1157. DOI: 10.1007/s12161-013-9728-z