Application of a
MultiNeb® Nebulizer for
the Quantification of
Selenium Species in Se-
enriched Spinach by
HPLC-ID-ICP-MS
1. Introduction
Inductively coupled plasma mass spectrometry (ICP-MS) is the
analytical method of choice for trace element determination due to its high sensitivity and isotopic capabilities1. However, total
metal/metalloid quantification alone is not enough to properly
establish its beneficial or toxic effects. So trace element speciation is required by using HPLC-ICPMS. Isotope dilution mass spectrometry (IDMS), particularly in its non-species-specific form and when coupled to HPLC-ID-ICPMS, enables accurate quantification without the need for individual calibration standards for each individual species, thereby overcoming limitations related to their limited availability .
Traditional IDMS configurations employing auxiliary pumps (Figure
1a) often suffer from incomplete mixing and signal instability2,3.
These limitations are effectively mitigated by the Ingeniatrics
MultiNeb® nebulizer, which features a dual-inlet design that enables direct mixing of the sample and isotopic tracer within the nebulizer itself (Figure 1b), resulting in improved signal stability and analytical precision.
Selenium is not considered essential for plants4,5 but its
supplementation has been shown to enhance plant growth, improve stress tolerance, and increase resistance to pathogens6.
Selenium can be applied as fertilizers in various chemical forms, including inorganic species such as selenate (Se(VI)) and selenite (Se(IV)), organic forms like selenomethionine (SeMet), and selenium nanoparticles (SeNPs)—each exhibiting distinct
uptake, translocation, and assimilation mechanisms that ultimately influence their distribution and speciation within plant tissues. This study focuses on simultaneously investigating the metabolism of
different selenium chemical forms in hydroponically grown Spinacia oleracea (spinach) fertilized with isotopically enriched 76Se-selenomethionine (76SeMet), 77Se-selenite (77Se(IV)), and chitosan-stabilized selenium nanoparticles (Ch-SeNPs) by
using post-column isotope dilution HPLC-ICP-MS, employing the MultiNeb® nebulizer to enhance precision and signal stability.
Figure 1. Workflow used for sample preparation Schematic representation of the two sample introduction systems. (A) MultiNeb-based configuration. (B) Micromist-based configuration
used for speciation analysis by HPLC-ID-ICP-MS
1. Experimental
Reagents and solutions
All reagents used were of the highest available purity. Hydrogen used as reaction gas in an ICP-MS system were of high-purity grade (˃99.999%). Ultra-pure water (resistivity ˃ 18.2 MΩ cm) was
obtained from a Milli-Q water purification system (Millipore, Spain).
Enriched 78Se(IV) was obtained from LGC Standards (LGC Group, UK) as a solution of 10 µg mL-1 in 2% HNO3 and used as tracer to quantify the total selenium and selenium species content. Selenocystine (SeCys2), selenomethylseleno-L-cysteine (SeMeSeCys), SeMet, Se(IV) and Se(VI) were purchased from Sigma-Aldrich (Steinheim, Germany). Protease type XIV, from
Steptomyces griseus and Base Trizma were supplied by Sigma-Aldrich (Steinheim, Germany). Tris-HCl solution was prepared with Base Trizma dissolved in water, and the pH adjusted to 7.5 with HCl
Instrumentation
Selenium species were separated using a Hamilton PRP-X100 anion-exchange column (250 × 4.1 mm, 10 µm particle size). Chromatographic separations coupled to ICP-MS were performed using a JASCO PU-2089 HPLC pump (Tokyo, Japan), with sample
injections introduced via a Rheodyne model 7725i injection valve (Rheodyne, CA, USA) equipped with a 100 µL sample loop. Separation was carried out under isocratic conditions using a mobile phase consisting of 10 mM citric acid (Sigma-Aldrich, Steinheim, Germany) in 2% methanol (Scharlab, Barcelona, Spain). Selenium speciation was monitored using an Agilent 7700x ICP-
MS instrument (Agilent Technologies, Tokyo, Japan), operated as an element-specific detector. The detailed operating conditions for the ICP-MS are summarized in Table 1.
Plant growth
Seeds were germinated in the dark for 20–25 days at 22 °C on moistened filter paper. After germination, 48 seedlings were transferred to perlite-filled trays for hydroponic growth and divided into control and treated groups. At 30 days of growth, treated plants were foliar-sprayed weekly for 45 days with Hoagland’s
solution enriched with selenium (total concentration 4.5 mg Se L-1: 1.5 mg Se L-1 each of 76SeMet, 77Se(IV), and Ch-SeNPs), while controls received only the Hoagland’s solution. Environmental conditions (temperature, light intensity, solar radiation) were
monitored and maintained under consistent conditions throughout the experiment. kept consistent. After the 45 day- treatment, plants were harvested, washed, lyophilized, and ground for analysis, with roots and leaves processed separately.
Table 1. Operational conditions for ID-ICP-MS analysis
Samples preparation
0.0250 g of protease XIV and 2.500 g of 30 mM Tris-HCl solution (pH 7.5) were added to 0.1250 g of root tissue and the mixture was incubated for 48 h at 37ºC. The hydrolyzed samples were then centrifuged (11000 rpm for 5 min) and filtered7. For aerial part, an identical procedure was carried out, but both sample and enzyme weights were double relative to those used for the root
Samples preparation
To determine the total selenium content extracted enzymatically, 1.0000 g of aerial part extract and 0.5000 g of root extract were spiked with an appropriate amount of the 78Se(IV) isotopic standard solution prior to undergoing the enzymatic
extraction procedure described above. Finally, the extracts were diluted 1:200 with ultra-pure water, and selenium content was quantified using ID-ICP-MS, following the instrumental setting detailed in Table 1.
Selenium species extracted from the samples were separated chromatographically using an anion exchange column connected online to the ICP-MS system. The specific chromatographic conditions and instrumental settings employed for the separation are detailed in Table 1. For quantification,
post-column ID-HPLC-ICP-MS was utilized. A 78Se(IV) standard solution at the appropriate concentration was continuously delivered at a flow rate of 0.85 mL min-1 through the MultiNeb nebulizer, driven by the ICP-MS peristaltic pump. To determine
the selenium concentration of each species resolved
by the column, the raw intensity chromatograms (counts per second) were converted into mass flow chromatograms expressed in nanograms per minute8,9. Noise reduction was achieved by applying a Savitzky-Golay smoothing filter, followed by mathematical corrections to compensate for potential interferences caused by BrH⁺ and SeH⁺ ions10. Subsequently, the isotope ratios 76Se/78Se, 77Se/78Se, and 80Se/78Se were calculated and adjusted for mass bias using an exponential
correction model. The isotope dilution calculation was then applied point-by-point along the chromatogram to generate mass flow chromatograms. Finally, selenium quantities in each
chromatographic fraction were obtained by integrating the peaks using Origin 9.6.0.172 software (Microcal Software Inc.,Northampton, MA, USA).
2. Results and discussion
To investigate the metabolic transformation of 76SeMet, 77Se(IV), and Ch-SeNPs within spinach plants, enzymatic extracts from both roots and aerial parts were analyzed by post column isotope dilution HPLC-ICP-MS following proteolytic
hydrolysis. This approach enabled both the identification and quantification of selenium species, and providing insight into the chemical forms present after uptake and assimilation. The total
selenium content recovered from each tissue, corresponding to the three selenium sources used for fertilization, is summarized in Table 2. In both roots and aerial parts, the highest levels of selenium accumulated were obtained for samples treated with
76SeMet, followed by 77Se(IV) and Ch-SeNPs.
The chromatographic profiles obtained for aerial part extracts (Figure 2a) revealed that both 76SeMet and 77Se(IV) were
predominantly metabolized to SeMet, which was confirmed based on retention time matching with the SeMet standard.
In contrast, Ch-SeNPs were largely transformed into an unidentified compound, hereafter referred to as compound A. An
early-eluting selenium peak, appearing at around 2 minutes, was observed in all treatments and is likely attributable to
either SeCys2 or SeMetO, as these species coelute under the applied chromatographic conditions. However, due to the
presence of SeMet, it is more plausible that this peak corresponds to SeMetO, a known oxidation product formed during
enzymatic digestion. Although 76SeMet mostly remain its original form, a minor fraction (~8%) was converted to
SeMetSeCys in both aerial part and root. Meanwhile, samples treated with 77Se(IV) and Ch-SeNPs also exhibited the
presence of Se(IV), Se(VI), and the unidentified compound A, suggesting suggesting more extensive biotransformation
pathways.
Figure 2. Mass flow chromatograms 76Se/78Se, 77Se/78Se and 80Se/78Se isotope ratios in a) aerial part and b) root. Lowercase letters above the chromatographic peaks identify different selenium compound (a: SeMetO/SeCys; b: SeMetSeCys; c: Se(IV); d: SeMet; e: Se(VI); f: Unknown).
In root extracts (Figure 2b), SeMet again emerged as the dominant species, except in the Ch-SeNPs treated samples,
where Se(VI) was most abundant. This pattern indicates that SeMet largely remains unaltered during translocation to root tissues. No significant transformation of 76SeMet was observed in roots, whereas 77Se(IV) showed partial conversion to SeMetSeCys but not to compound A. Ch-SeNPs-treated roots only contained SeMet and Se(VI) with no detectable levels of compound A in agreement with the pattern observed for 77Se(IV). These observations underscore that the distribution and transformation of selenium species vary depending on the chemical form and plant tissue.
Quantification of individual selenium species in both aerial part and root tissues was performed using post-column IDMS (Table 2). Excellent column recoveries, exceeding 95%, were achieved for most treatments, confirming that neither chromatographic separation nor aerosol generation prior to ICP-MS detection resulted in significant analyte loss. The only exception was the Ch-SeNPs extract from aerial tissues, which showed a slightly lower recovery of 87%. These high recovery rates underscore the robustness and reliability of the analytical method. In particular, the efficient and consistent tracer–sample mixing achieved by the MultiNeb® nebulizer played a critical role in preserving signal stability and ensuring accurate isotope ratio measurements throughout the analysis
Table 2. Total selenium extracted and selenium species content in aerial part and roots of Spinacia oleracea.
3. Conclusions
This study demonstrates the utility of isotopically
enriched selenium tracers for simultaneously investigating the uptake, distribution, and biochemical transformation of multiple selenium species in plants within a single experimental set-up. The combination of chromatographic separation with post-column HPLC-ID-ICP-MS enabled accurate quantification of selenium
species in spinach tissues, offering valuable insights into the metabolic fate of each selenium form.
A key advancement in this analytical approach was the implementation of the MultiNeb® nebulizer. Its built-in dual-inlet mixing system eliminated the need for external mixing components and auxiliary pumps, ensuring efficient and consistent
homogenization of the tracer and sample streams immediately prior to nebulization. This contributed significantly to improved signal stability and precision in isotope ratio measurements. Overall,
this configuration facilitated robust selenium speciation analysis and highlights the MultiNeb®nebulizer as an effective tool for enhancing the reliability and efficiency of isotope dilution-based
speciation studies.
Funding
The authors thank financial support through the PID2023-148425NB-I00 project funded by
MCIU/AEI/10.13039/501100011033 and Feder, UE.
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