Atomic spectrometry update: review of advances in elemental speciation

Contents

• 1. Books and topical reviews

• 2. CRMs and metrology

• 3. Elemental speciation analysis
  • 3.1. Antimony
  • 3.2. Arsenic
  • 3.3. Chromium
  • 3.4. Copper
  • 3.5. Gadolinium
  • 3.6. Germanium
  • 3.7. Halogens
  • 3.8. Iron
  • 3.9. Lead
  • 3.10. Manganese
  • 3.11. Mercury
  • 3.12. Phosphorus
  • 3.13. Platinum
  • 3.14. Rare earth elements
  • 3.15. Selenium
  • 3.16. Silver
  • 3.17. Sulfur
  • 3.18. Technetium
  • 3.19. Thallium
  • 3.20. Tin
  • 3.21. Uranium
  • 3.22. Vanadium
  • 3.23. Zinc

• 4. Biomolecular speciation analysis

• 5. Abbreviations

• Conflicts of interest

• References

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Abstract

This is the 16th Atomic Spectrometry Update (ASU) to focus on advances in elemental speciation and covers a period of approximately 12 months from January 2023. This ASU review deals with all aspects of the analytical atomic spectrometry speciation methods developed for: the determination of oxidation states; organometallic compounds; coordination compounds; metal and heteroatom-containing biomolecules, including metalloproteins, proteins, peptides and amino acids; and the use of metal-tagging to facilitate detection via atomic spectrometry. As with all ASU reviews the focus of the research reviewed includes those methods that incorporate atomic spectrometry as the measurement technique. However, because speciation analysis is inherently focused on the relationship between the metal(loid) atom and the organic moiety it is bound to, or incorporated within, atomic spectrometry alone cannot be the sole analytical approach of interest. For this reason, molecular detection techniques are also included where they have provided a complementary approach to speciation analysis. The number of publications covered this year has increased since last year but remains relatively low compared to many of the years that this ASU has been published for. However, there is a good breadth of elements covered, over 20 this year, with the most popular elements still being As, Hg and Se whilst Ge and Tc both make their first appearances this year. Another item to note is the decreasing quality of the abstract for many of the papers, with detail on the methodology, key results with data included, conclusions and implications thereof missing. This is likely to lead to fewer researchers reading the article.

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1. Books and topical reviews

This latest update adds to that from last year¹ and complements the five other annual Atomic Spectrometry Updates, advances in environmental analysis,² advances in the analysis of clinical and biological materials, foods and beverages,³ advances in atomic spectrometry and related techniques,⁴ advances in X-ray fluorescence spectrometry and its special applications⁵ and advances in the analysis of metals, chemicals and materials.⁶

No textbook devoted entirely to elemental speciation has appeared in the current review period; however, several book chapters, or sections in book chapters deal with the topic. A revised version of Thomas's Practical Guide to ICP-MS and Other Atomic Spectroscopy Techniques: A Tutorial for Beginners⁷ contains a chapter (number 19 out of 28 in total) entitled “Coupling ICP-MS with Chromatographic Separation Techniques for Speciation Studies”. An updated version of a text dealing primarily with LC contains a chapter entitled “Speciation and Element-Specific Detection”,⁸ which occupies some 12 pages out of more than 1000 for the entire book. The Royal Society of Chemistry's Evolution of Solid Phase Microextraction Technology contains a chapter entitled Solid Phase Microextraction for Trace Element Determination,⁹ about half of which is devoted to elemental speciation analysis for As, Hg, Pb, Se and Sn as well as the determination of volatile organometallic compounds, transition metals and inorganic anions. The fourth edition of “Chemical and Functional Properties of Food Components”,¹⁰ a text devoted to the roles of chemical compounds in the structure of raw materials, and the various different attributes of food quality, including nutritional value, safety, and sensory properties, contains four new chapters. One of these is entitled Analytical Methods Used for Assessing the Quality of Food Products with elemental speciation also being discussed in an earlier chapter, entitled the Role of Mineral Components. A text devoted to the environmental impacts of Inorganic Contaminants and Radionuclides¹¹ is divided into 15 chapters, each devoted to one analyte, including Ag, As, Cd, Cr, Cu, Fe, Hg, Mn, P, Pb, Se and Zn. The book concluded with a single chapter devoted to radionuclides. “Biofilms,” (volume 53 in the ongoing Methods in Microbiology series from Elsevier),¹² includes a chapter entitled Micro- and Nanoscale Techniques for Studying Biofilm–Mineral Interactions, in which elemental speciation makes a cameo appearance. Another text¹³ from the Royal Society of Chemistry, “Chalcogen Chemistry: Fundamentals and Applications” contains a chapter entitled, “Speciation of Organic and Inorganic Selenium in Food and the Environment”, that features chemical analysis quite extensively. There are no corresponding chapters for the other chalcogens (S and Te). In celebration of the 75th Anniversary of the Vernadsky Institute of the Russian Academy of Sciences, a text entitled, “Advances in Geochemistry, Analytical Chemistry, and Planetary Sciences” has been published¹⁴ containing 41 selected articles written by leading researchers grouped into the following topics: (1) geochemistry, (2) meteoritics, cosmochemistry, lunar and planetary sciences, (3) biogeochemistry and ecology, and (4) analytical chemistry, radiochemistry, and radioecology. The articles cover recent experimental data, theoretical investigations, critical reviews, and the results of computer modelling. The analytical chemistry section features a chapter on the Speciation of Actinides in the Environment.

A number of review articles featured aspects of elemental speciation analysis, several of which are focused on the instrumental techniques involved. Mass spectrometry for the characterising metal–biomolecule interactions has been reviewed (70 references with titles), and although elemental speciation analysis was not a major component of the review,¹⁵ examples of the information obtained from SEC-ICP-MS were provided, together with the comment that when used in conjunction with proteomics-based MS techniques SEC-ICP-MS will provide a deeper picture of the complexities of metal dynamics and speciation. The reviewers highlighted several outstanding questions, including to what degree does metal speciation allow differentiation of diseases and disease progression? A review (143 references with titles) of the determination of environmental pollutants by IC with MS detection¹⁶ covers several speciation topics, not all of which, such as the determination of inorganic and organic P compounds, are based on elemental analysis. A section on the determination of metalloids and PTEs is mainly concerned with As speciation by IC-ICP-MS. The limitations of ICP-MS for the determination of (halogenated) disinfection by-products are highlighted including (a) the inability to detect chloroacetic acid due to its low sensitivity to chloride ions (b) polyatomic interferences that lead to high background signals and (c) the gradual build-up of salt deposits at the MS interface, which increase the space charge effect, thereby defocusing the ion beam resulting in a continuous decrease in ion signal intensity. The reviewers considered IC-ES-MS methods to be more promising. Pang et al. reviewed (121 references with titles) the applications of IC in urine analysis¹⁷ based on articles published in the last 30 years. They identified several categories of analyte: PTEs (mainly As and radionuclides), halogen ions (of which perchlorate was assigned to a separate sub-section), pesticides and drugs, and others. Elemental speciation analyses with atomic spectrometry detection were featured mostly for the first of these categories, with the most common technique used being IC-ICP-MS. For many of the articles covered, details of the sample pretreatment were given. The researchers do not discuss the relative merits of IC vs. ion pair RP chromatography. The reviewers finished with a special mention of As, concluding that continued research and development of novel columns are expected (to address limitations of low separation efficiency or poor resolution) thereby enabling the separation of other organic As compounds (and other analytes of interest). For readers with an interest in the possible benefits of TLC separation with MS detection, a review (155 references in English without titles) of this combination is available in Chinese.¹⁸ Judicious application of Google Translate enables the sections on TLC with plasma MS to be deciphered. There were no applications concerning elemental speciation in the section on low temperature plasma ion sources, but in the section devoted to other ionisation sources, several examples of elemental analyses were presented in which the coupling is by LA. These examples included the determination of metals in crude oil and distillates, and the determination of As^V in algae, yeast and water. A tutorial review (called a “primer” by the publisher) of ICP-MS¹⁹ covers the contents of 231 sources (with titles) and featured the major analytical applications of ICP-MS: multi-element, sNP, single-cell, LA, speciation, and isotopic analyses; outlined measurement strategies and challenges; and provided examples of applications. The section on speciation analysis under the broad heading of Instrumentation, contained only five references; however, in the Applications section, 11 speciation papers were cited. In the concluding (Outlook) section, the reviewers discussed the possible application of ICP-MS to the detection of microplastics as well as pointing out that LA-sNP-ICP-MS is the only technique capable of simultaneously mapping particulate and dissolved metals, including counting and sizing, directly in soft biological tissues.

The application of techniques based on measurements with synchrotron radiation for the study of the movement of PTEs in the environment has been featured in two publications. Smieska et al. reviewed (122 references with titles) the state of fundamental scientific questions in the cycling of metals, from rocks and soils to plants and animals.²⁰ They included As and Hg in their list of metals and covered several relevant studies for each element. They concluded that there is a demand for a versatile micro-focusing synchrotron beam-line providing 2D and 3D μXRF as well as time- and spatially resolved μXAS and μXRD. Ideally, such a facility would also offer complementary imaging via IR, Raman, or reflectance spectroscopies, as well as access to MS. They also identified that there is a demand for dynamic studies, on a range of length- and time-scales, and a need for training and workshops which would enable more researchers to not only make use of synchrotron-based measurements but also to communicate across traditional disciplinary boundaries. The speciation of PTEs in Pb/Zn smelter slags by XAS has been reported in which results were interpreted in the context of a comprehensive review (87 references with titles) of the literature.²¹ The elements of interest were As, Cu, Fe, P, Pb, S and Zn. The specific slag examined was derived from the Imperial Smelting Process at an anonymous UK source that smelted sulfide ores, containing sphalerite–galena–pyrite-rich bands, interlayered with quartz-carbonate turbidites and mudstones, from the McArthur River zinc-lead mine, Australia. The granulated material containing glassy black particles, which had remained shiny after two to four decades of weathering at their disposal site. Results were discussed in terms of the potential for environmental contamination as well as of the potential for recovery of some of the elements. The researchers concluded that the solubility of the majority of the As, Pb and Zn, could be controlled by a change in pH, while the solubility of some As, most of the Cu and some Zn could be increased by oxidation. They also point out that the element specificity and spatial resolution of XAS provides (a) quantitative information about the distribution of element species in the whole matrix, (b) solves the overlapping problem of quantifying mineral compositions despite the heterogeneity of the slag and (c) significantly decreases the intensive labour that is required for analysis by EPMA or SEM-ED-XRF.

2. CRMs and metrology

The long-term stability of AB, As^Vand DMA, in three CRMs, NMIJ 7901-a, 7912-a, and 7913-a, respectively, has been assessed over the 13 years since their production.²² Quantification of each As species was by HPLC-ICP-MS using a C₁₈ column and a mobile phase of 10 mmol per L sodium butanesulfonate, 4 mmol per L TMAH, 4 mmol per L malonic acid, and 0.05% (v/v) MeOH with a pH of 3. For each CRM the measured and certified mass fractions were in statistical agreement over this time period and no breakdown products or species conversions were detected in the HPCL-ICP-MS chromatograms.

An IUPAC Technical Report on the analytical chemistry of engineered nanomaterials (ENMs) has recently been published. Part 1 (ref. 23) covers the scope, regulation, legislation, and metrology of ENMs whilst Part 2 (ref. 24) covers analysis for ENMs in complex samples. Each paper gives the current situation for the areas discussed and concludes that current spectroscopic methods are capable of analysing the bulk chemical composition and microscopy methods to measure size and shape are available and reasonably straightforward to apply but the selection of analytical techniques required for a specific nanomaterial depends on the composition of the material and the environment in which it is found, as well as the area and purpose of application. The careful selection of appropriate sample preparation methods, which do not modify the ENMs present in the original sample, is required prior to characterisation of ENMs in commercial products and complex biological or environmental matrices is required. The report also states that validated and standardised methods and reference materials are required to support risk assessment for regulation, enable quality control of existing products and to allow inter-laboratory comparability of the data produced. For biological and environmental samples, the development of techniques that can distinguish between naturally occurring and engineered nanomaterials is also required. The authors also point out that the metrological aspect of analysis for ENMs has lagged behind the analytical process. The report contains a table of all of the currently available ENM CRMs and from this it is evident that these are only ‘pure’ standards and that there is an urgent need for matrix matched ENM CRMs to be produced for validation of methods involving real world samples. The report is a must read for workers in this field.

3. Elemental speciation analysis

3.1. Antimony

Interest in Sb speciation methods continues at the modest level seen in the recent past, and as was the case for last year, most of the methods published in the current review period were concerned with non-chromatographic methods for inorganic Sb species.

Two research groups have devised DSPME procedures based on the selective retention of Sb^III on magnetic graphene oxide (GO) nanocomposite materials. Oviedo et al. extracted²⁵ the Sb^III complex with DDTC from 8 mL of sample solution with 3 mg of GO NPs by vortexing for 10 min. Then they added 40 μL of a magnetic ionic liquid (MIL) trihexyl(tetradecyl)phosphonium hexachlorodysprosiate(III) and after vortexing for 1 min, removed the GO NPs by a magnetic rod, and determined the Sb^V remaining in solution by ICP-MS. Total Sb was also determined by ICP-MS and hence the Sb^III concentration was calculated by difference. The LOD values were 5 and 3 ng L⁻¹ for Sb^III and Sb^V, respectively. The total Sb method was validated by the analysis of a CRM, NIST SRM 1643e (trace elements in water) and the speciation method was validated by spike recoveries, at concentrations ranging from 1 to 10 μg L⁻¹, from one tap water, 3 tea extracts and two honeys in which both analytes were found in all samples. In the method devised by Wang et al.,²⁶ the Sb^III was redissolved after separation and determined by ICP-MS. As total Sb was also determined, the Sb^V concentration was determined by difference. To 50 mL of sample solution, adjusted to pH 9, 10 mg of reduced GO/Fe₃O₄ (prepared in house) was added and the mixture shaken at room temperature for 30 min. After magnetic separation and multiple washings, the Sb^III was dissolved in 0.5 mL of concentrated HCl and UAE for 15 min (no information about the UAE power was given). After dilution to 10 mL with water and filtering (0.45 μm), the solution was introduced to the ICP-MS instrument. The LOD for Sb^III was 6 ng L⁻¹, and the method was applied to the analysis of several water samples (tap, river, lake, ground and mine drainage), in all of which it may be deduced that both species were found (apart from the tap water in which no Sb was detected) as the total Sb concentrations given were significantly higher than those of Sb^III. The method was validated by spike recoveries (at 1 μg L⁻¹) of both species to all samples. The total Sb method was validated by the analysis of a CRM GBW(E)080545, which is a standard Sb solution.

A two-step selective HG procedure was devised by Fujihara and Nishimoto²⁷ in which total Sb was determined (by MP-OES) under one set of HG conditions and then Sb^III was determined under a second set of conditions. The Sb^V concentration was obtained by difference. The procedure was applied to the characterisation of Sb species leached from a semiconductor material (undoped and Bi-doped InSb thin films) by 0.1 mol per L sodium acetate solution. At appropriate time intervals, 500 μL of leachate was diluted to 4.5 mL with 1 mol per L HNO₃, and then presumably divided into two portions, though this is not specifically described. To determine total Sb, unspecified volumes of 20% KI and 1 mol per L HCl were added and the solution introduced through the bottom inlet into a multi-mode sample introduction system (MSIS). Stibine was generated by reaction with 3% NaBH₄ solution in 0.2% NaOH introduced via the top inlet. Neither flow rate was given. To determine Sb^III, a 2.4 mL subsample was acidified with 0.6 mL of 1 N HCl and reacted with 0.4% NaBH₄ in 0.2% NaOH. Again, flow rates were not specified. The LOD was 0.02 μg L⁻¹ and the method was validated by spike recoveries (20 μg L⁻¹) from the sodium acetate solution. The researchers found that although Bi doping decreased the amount leached, a large fraction of the Sb leached over 28 days was Sb^III.

A multi-step SPE method has been devised for the pretreatment of iSb species in tea infusions prior to determination by HPLC-ICP-MS.²⁸ In preliminary experiments, the researchers showed that neither Sb^III or Sb^V could be determined directly in tea infusions because (a) the concentrations were too low and (b) the inorganic species were bound to an unknown substance producing compounds that chromatographed poorly. These problems were overcome by the judicious use of a SPE column that would selectively retain the Sb^III species. The researchers investigated a number of Oasis columns and found that the HLB (hydrophilic–lipophilic balance) material (a water-wettable copolymer) was suitable. In the first step, the Sb^III species was retained from 10 mL of tea infusion on the column leaving the Sb^V species in the filtrate. The column was rinsed with 2 mL of water and then the retained Sb^III species eluted by oxidation with 1 mL of 2% (v/v) H₂O₂ + 0.1% (w/v) NaOH and then determined by HPLC-ICP-MS, when a single peak for Sb^V was observed. Meanwhile, the Sb^V in the original filtrate was reduced to Sb^III by the addition of 0.1 mL HCl + 0.05 g L-cysteine (and waiting for 5 min). Then, after adjustment to pH 5.5–7.0 (the pH of the original tea infusion), the solution was again loaded onto the column when the Sb^III was retained. Finally, as before, the column was washed with water, the species eluted with 1.0 mL of the H₂O₂ + NaOH solution and measured by HPLC-ICP-MS. For spike recovery experiments 10 μL of a 500 μg L⁻¹ solution of Sb^III or Sb^V was added to 10 mL of the tea infusion. The LOD was 0.03 μg L⁻¹ and both species were found in 6 different teas at each of four infusion times from 5–20 min. The researchers found that between 50 and 83% of the total Sb in the tea infusion was iSb of which Sb^III was the predominant species.

A form of SPE, frontal chromatography, was applied for the separation of Sb^IIIand Sb^V in natural waters prior to determination by ICP-MS.²⁹ The sample (adjusted to pH 1) was continuously loaded into a home-made column (60 mm × 2.5 mm id) packed with a strong cation-exchange resin of relatively large particle size (55 ± 20 μm) by a peristaltic pump at 1.7 mL min⁻¹. At pH 1, Sb^III is cationic, whereas Sb^V is neutral and is not retained, and so the Sb signal rises to a plateau corresponding to the Sb^V concentration. As the column becomes saturated with Sb^III, the signal rises to a plateau that corresponds to both species. The analysis time was about 6 min and the LOD values were <1 ng L⁻¹. The method was validated by spike additions to four mineral water samples in each of which both species were found: Sb^III at single-digit ng L⁻¹ concentrations and Sb^V at triple-digit ng L⁻¹ concentrations. No information was given about the regeneration of the column, but it is deduced from other information in the text that washing with 0.5 mol per L HNO₃ for 3 minutes was needed. The researchers pointed out that the analytical Eco-Scale score for the method was 84, higher than the 75 needed for designation as “excellent.”

3.2. Arsenic

There have been few major advances reported for As speciation this year, although a good number of application studies utilising existing techniques with minor modification continue to be recorded.

At least eight reviews on the speciation of As have been published this year, each with a different focus. One of the more comprehensive, with 248 references, is a review by Patel et al.³⁰ which reports on sources and mobility of As in air, water, soil and sediment around the World, together with the associated health hazards. The review provides a good overview of As levels in a wide range of environmental matrices although the focus is on contamination, exposure sources and mobility of As rather than As speciation. Most of the As species specific information is confined to one table showing examples of As speciation in a range of different matrices. The review does however provide a useful insight into the ubiquitous nature of As in the environment. The identification of As containing lipids found in marine organisms such as fish, algae, shellfish, marine oils, and microorganisms has received a lot of attention in recent years, and so a review of strategies for the analysis of arsenolipids in marine foods is timely. The review published by Coniglio et al.³¹ has 107 references and covers occurrence, structure, toxicity, biological role and detection. The use of both ICP-MS and ES-MS coupled with RP-LC has brought significant advancements in the field. In the case of ES-MS, the employment of negative polarity and tandem MS analyses has further enhanced these advancements. The authors highlight that although 4 decades have passed since the earliest structural elucidation of arsenolipids, the lack of accessibility to standard compounds, along with the lack of CRMs, still holds back research. Sadee et al.³² have reported on the use of hyphenated techniques for As speciation in vegetables. The use of different solvents and extraction techniques to extract the As species from the matrix are discussed together with chromatographic and non-chromatographic methods of separation. A table of appropriate CRMs for use with vegetable matrices is also included. The review, with 128 references, includes some useful tables with details of the matrix, extraction and analytical technique for separation and detection. A review summarising current knowledge of As speciation in freshwater fish has been presented by Hoy et al.³³ Fish samples are typically homogenised, and the As species extracted using water/MeOH with the assistance of sonication and enzyme treatment. The As species in the extracts are then commonly determined using HPLC-ICP-MS. The use of ES-MS-MS, in combination with HPLC and ICP-MS, often provides complementary information for the identification and characterisation of As species. However, despite progress made in As speciation analysis, a large fraction of the total As in freshwater fish remains unidentified due to challenges in identifying and quantifying the As species present in complex sample matrices at very low concentrations. These issues are discussed in the review.

Several of the published reviews have focused on specific methodology for As speciation. A critical review of on-site iAs screening methods has been presented by He et al.³⁴ The review has 139 references, although many of the papers described are for techniques to measure total iAs. That said, the review provides a useful insight into the principles, advantages and limitations of commonly used colorimetry, electrochemistry, and biosensing methods for on-site screening and monitoring of As in drinking water. The use of micro-extraction techniques for As speciation have been reviewed.³⁵ The use of single-drop microextraction, DLLME, HFLLME and SPME were covered for a range of matrices. The review focuses on preparing samples and techniques for reducing solvent use and is supported by 121 references. Recent advances in analytical techniques and methods of As-binding proteomes in living systems have been reviewed by Liu et al.³⁶ The review, with 98 references, discussed chromatographic separation and purification, biotin-streptavidin pull-down probes, in situ imaging using novel fluorescent probes, and protein identification. New perspectives on the analysis of As-binding proteomes such as isolation and identification of minor proteins, in vivo targeted protein degradation (TPD) technologies, and spatial As-binding proteomics, are also included.

A tutorial review with 169 references has discussed existing methods for the analysis of As^III and As^V in water samples.³⁷ Chromatographic, spectroscopic, colorimetric, biological (whole cell biosensors and aptasensors), electroanalytical and coupled techniques were included. The discussion also covered some current challenges and potential avenues for future research (the need for enhanced analytical performance, rapid analysis and on-site technologies for remote water analysis and environmental applications). In the final review covered here, the applications of PVG coupled to analytical atomic spectrometry combined with various chromatographic separation techniques and non-chromatographic analysis methods for As, Hg and Se have been summarised by Li et al.³⁸ The review provides a comprehensive overview of recent developments and is supported with 95 references. However, it concludes that although PVG-analytical atomic spectrometry remains a powerful analytical technique for As speciation, even with the help of sensitisers (e.g. Fe^III, Cd^II and nanomaterials) to significantly improve the PVG efficiency for As, reported applications of PVG-atomic spectrometry for As speciation are relative rare at present.

There have been several publications looking at analytical developments for As speciation, although most papers in this review period have focused on a specific matrix. The development of a new analytical method for the quantitative determination of As-containing hydrocarbons in environmental samples by GC-MS, as an alternative approach to the use of HPLC-ICP-MS, has been reported by Raber et al.³⁹ The work was prompted by the lack of commercially available standards and complexity of some ICP-MS methods. The As-containing hydrocarbons were extracted and purified from fish oil and seaweed samples. Due to the thermal conversion of oxo-As-containing hydrocarbons to their trivalent forms in the injection port of the GC, the conversion was incomplete resulting in low signal intensities and large carry overs. The authors therefore chemically reduced the As-containing hydrocarbons to their trivalent forms and analysed them with GC-MS, GC-MS-MS and HPLC-ICP-MS. The possibility of compound independent quantification with commercially available standards was also investigated. After optimisation of the conditions for reduction of the As-containing hydrocarbons, the compounds could be determined in marine samples using triphenylarsine as a commercially available standard. The results showed good agreement with results from HPLC-ICP-MS. The method was relatively fast, with run times of <10 min, which compares well with HPLC-ICP-MS methods which can take 30–40 min using gradient elution techniques. Cotton fibres and thiol-ligands, including APDC, L-cysteine, and DDTC, have been used for the determination of As using SPE with ICP-OES for detection.⁴⁰ The separation parameters, including pH, thiol-ligand concentration, mass of cotton fibre, type of eluents, and flow rate were all optimised. It was found that As^III could be efficiently separated from As^V using a column containing 0.5 g cotton fibres and 0.1% APDC at pH 2. The As^III present was then eluted using 5 mL of mixed solvents of 4 mol per L HNO₃, 4 mol per L HCl, and 0.5 mol per L H₂O₂ (1 : 1 : 1 v/v). Using the optimal conditions, the method was then used to speciate As^III and As^V in model solutions with concentrations of both species ranging from 10 to 1000 μg L⁻¹. The results showed good recoveries of both As species at 83–112% with RSDs up to 7.8% (As^V generally better than As^III). The LOD and LOQ were 1.5 and 4.5 μg L⁻¹ (n = 9) respectively. The method was subsequently applied to As speciation analysis in natural and wastewater samples. An on-line internal standard correction technique for HPLC-ICP-MS has designed and reported.⁴¹ The system used an autosampler system equipped with HPLC to improve the analytical precision. The autosampler was programmed to operate in the following sequence: first it took up a portion of sample solution, rinsed the nozzle, sucked up air as a spacer, took up the internal standard solution and finally injected all of them into a sampling loop through an injection valve. The repeatability of the sampling (20 μL) was improved from 2.5 to 1.2% using the on-line internal standard technique. Rhenium was used as the internal standard element due to its retention time, peak shape and water solubility. The technique was applied to As speciation in food safety monitoring since this requires high precision and high sample throughput.

The use of imprinted polymers and 3D printing techniques for As speciation is becoming more common. A method for the speciation of iAs (As^III and As^V) by μTLC-LA-ICP-MS based on an ion imprinted polymer has been developed by Shushtarian et al.⁴² To characterise the fabricated ion imprinted polymer, FTIR and field emission SEM were used. The TLC plate was prepared by incorporating CaSO₄ (as a binder) with the polymer. The surface of the TLC plate was then scanned using LA, which volatilised the As species from the TLC plate for introduction into the ICP-MS. Various parameters effecting the isolation efficiency, such as the ion imprinted polymer/CaSO₄ mass ratio, mobile phase composition, and pH, were examined. Under optimal conditions, the developed method gave a LOD of 0.3 μg L⁻¹ with a wide LDR of 0.2–100 μg L⁻¹, and a RSD of 3.8%. The performance of the developed method was utilised for the isolation of As^III and As^V in mine wastewater and human blood plasma. To extract As^III and As^V ions from water, a polymer inclusion membrane has been fabricated by studying the effect of varying the compositions of the base polymer, carrier and plasticiser.⁴³ The highest transportation efficiency of ions was found for a polymer inclusion membrane containing 50% cellulose triacetate and 50% Aliquat 336. Whilst the plasticiser is an important component of the membrane, helping in the transportation of ions, in this case the polymer inclusion membrane without plasticiser exhibited the highest efficiency. The transportation efficiency obtained for As^V was 99.7 ± 0.2% and the As^V was separated from As^III in 5 hours. The membrane was found to be resistant to biofouling for a period of 10 days and both FAAS and ICP-MS were used for As detection. Applications included its use to differentiate between oxidation states of As species and identify the redox potential of water in geochemical explorations. Post-printing functionalisation can enhance the functionality and applicability of analytical devices manufactured using three-dimensional printing. In a study by Chen et al.⁴⁴ 3D-printed SPE columns incorporating TiO₂ NP coated porous polyamide monoliths were used to develop a highly sensitive SPE-based method for the speciation of iAs (and Cr and Se) in high salt content samples such as natural water and human urine samples. Treatments with a formic acid (30%, v/v) solution and a NaHCO₃ (0.5%, w/v) solution TiO₂ NPs (1.0%, w/v) were used for in situ fabrication of the SPE columns. After optimising the experimental conditions, the 3D-printed SPE columns extracted the species of interest with a 5 to 22 fold enhancement, relative to those obtained with the uncoated monolith, and absolute extraction efficiencies ranging from 85 to 98%. The method LOD values ranged from 0.7 to 32 ng L⁻¹ using ICP-MS. The method was validated using four CRMs [CASS-4 (nearshore seawater), SLRS-5 (river water), 1643f (fresh water), and Seronorm Trace Elements Urine L-2 (human urine)]. Spike analyses of seawater, river water, agriculture waste, and human urine samples gave spike recoveries of 96–104% with an RSD of <4.3%.

There have been several studies reporting on As speciation in biological and environmental samples. The results from an international round-robin study to identify the possible sources of discrepancies in the quantitation of several As species in different matrices of biological reference materials: one plant tissue, three marine and three terrestrial biological tissues have been published [WOS:000981895400001]. Each participating laboratory was provided with a set of identical calibration standard solutions and analysed the samples following a prescribed extraction method as well as using an in-house protocol. The results showed that significant biases could be introduced by insufficient verification of the analyte's mass fractions in the calibration standard solutions. The choice of extraction method seemed to have very little impact on the quantitation of As species in the plant and terrestrial biological tissues studied. However, it was found that following the prescribed extraction method led to significant reduction of uncertainties in more complex samples such as marine animal tissues. Some differences in the mass fractions of As species extracted from marine animal tissues in water (with and without H₂O₂) were observed and this needs to be further investigated. Despite the variety of extraction methods used, very good agreement between reported mass fractions was achieved and the combined consensus values will be published as CRM values of As species in the studied materials. A study using single cell ICP-MS to quantify the cellular uptake and biotransformation of As^V by freshwater phytoplankton under a salinity gradient has been published by Alam et al.⁴⁵ Three freshwater phytoplankton cells, Staurastrum paradoxum (S. paradoxum), Pediastrum duplex (P. duplex) and Scenedesmus acutus (S. acutus), were incubated in 0.1 μmol L⁻¹ As^V solution for 14 days at varying salinity. A single cell device was used to introduce a cell suspension solution into the ICP-MS whilst cold trap HG-AAS was used to investigate the biotransformation of As^V under varying salinity conditions. The results revealed that cellular As levels decreased as salinity increased in P. duplex and S. paradoxum, but increased in S. acutus. The single cell-ICP-MS data showed uptake of As^V by freshwater phytoplankton and was in good agreement with the ICP-MS analysis. Various As management strategies were seen in the phytoplankton species: P. duplex converted it to methylated forms; S. acutus produced organoarsenicals; and S. paradoxum reduced As^V to As^III which was excreted. The study also showed changes in the physiological status of phytoplankton following salt stress and As exposure.

The speciation of As in drinking water remains an active topic of research. The accurate determination of As species in waters can still be an environmental challenge because of the rapid redox transformation of As. Procedures for on-site separation of As^III, As^V, MMA, and DMA in natural waters are therefore attractive. This year another method to achieve this has been reported, in this case passing the water sample through tandem IE resin columns whilst on site.⁴⁶ Selective retention of As species occurred on the resin cartridges which was then followed by a selective elution step. A cation-exchange column (SCX, 500 mg, 55 μm particle size) containing sulfo and hydroxyl groups was used with an anion-exchange column (SAX, 500 mg, 55 μm particle size) containing quaternary amine and hydroxyl groups. The columns were used in tandem and the eluate was collected and analysed by ICP-MS. Since the pH of solution plays an important role in As species distribution, this was also investigated. The results showed that the recoveries of As^III, As^V, MMA, and DMA were in the range of 93–108% between pH 4–10. Thus, the sample pH did not need to be adjusted. The CRM CASS-5 was diluted 10 times and analysed directly to validate accuracy of measurements, and SLRS-2 was used to validate on-site separation procedure, respectively. The results obtained agreed well with the certified values of the two CRMs. The determination and speciation of As in drinking water samples using WD-XRF has been reported.⁴⁷ Valence to core electronic transitions, i.e. AsK beta(2,5) fluorescence lines were used for the As speciation. The study entailed direct measurement of activated alumina pellets containing As^V and As^III species adsorbed from water sample without separation of the trivalent and pentavalent species. The biggest advantage of the method is reported to be the direct analysis of synthesised nanotubes or other solid-phase extraction sorbents which are used to entrap both the As species. For the determination of total As using activated alumina as adsorbent, the most intense AsK alpha(1,2) analytical lines were used and the instrumental LOD and the LOQ were 0.23 μg L⁻¹ and 0.89 μg L⁻¹, respectively. For As speciation, the LOD and the LOQ were calculated to be 50 μg L⁻¹ and 200 μg L⁻¹, respectively.

The As speciation in freshwater lakes has also been reported. Lake Geneva is presently an oligo–mesotrophic lake, and not polluted by As. The water column never becomes anoxic but the oxygen saturation at the bottom of the Grand Lac is now below 30% owing to lack of water column mixing since 2012. Thus, this lake offers excellent conditions to study As behaviour in an unpolluted, oxic freshwater body. Filella et al.⁴⁸ have undertaken long term monitoring and redox and methylation speciation studies to understand the As behaviour in the lake. The ‘dissolved’ As species: As^III, total iAs, MMA^III, MA^(III+V), DMA^(III+V), and TMAO were analysed by HG-CT-ICP-MS-MS. Water column measurements were also complemented with occasional sampling in the main rivers feeding into the lake and in the interstitial waters of a sediment core. Arsenic speciation was followed monthly through the spring productivity period (January–June 2021) in the Petit Lac (76 m deep) and in April and June 2021 in the Grand Lac (309.7 m deep). The presence of MMA^III and TMAO and the predominance of iAs^V in lake and river samples was confirmed as well as the key role of algae in the formation of organic As species. While the total ‘dissolved’ As concentrations showed nearly vertical profiles in the Petit Lac, the As concentrations steadily increased at deeper depths in the Grand Lac due to the lack of mixing and accumulation in bottom waters. Evaluation of 25 years of monthly data of ‘dissolved’ As concentrations showed no significant temporal trends between 1997 and 2021. The observed seasonal character of the ‘dissolved’ As through this period coincided with a lack of seasonality in As mass inventories, pointing to a seasonal internal cycling of As species in the water column with exchanges between the ‘dissolved’ and ‘particulate’ (i.e., algae) fractions.

The remediation of As contaminated soils and waters is widely studied. Biochar is widely used for this, in part because of its local availability and low production cost. However, its effectiveness depends on physicochemical properties related to its feedstock and pyrolysis temperature, as well as the environmental conditions at the site where it is used. The effects of pyrolysis temperature and biochar application on the release and transformations of As in contaminated sediments subjected to redox fluctuations have been studied by Soares et al.⁴⁹ The ordered phases in the Biochar was investigated using SR-XRD, XPS was used to characterise the sediments and ICP-MS coupled to customised HG and cryotrapping units for the As speciation. Biochar application and pyrolysis temperature were found to play an important role in As species (As^III, As^V, MMA and DMA) availability, As methylation, and DOC concentration. Furthermore, successive flooding cycles which induced reductive conditions in the sediments increased the As content in the solution by up to seven times. In the solid phase, the application of biochar and the flooding cycle altered the spatial distribution and speciation of As, C, and Fe. In general, the application of Biochar decreased the reduction of Fe^III and As^V after the first cycle of flooding indicating that the flooding cycle play an important role in the reoxidation of biochar enhancing the immobilisation of As. The As hyperaccumulator plants Pityrogramma calomelanos and Pteris vittate have been widely studied, but the chemical transformations of As in the rhizosphere-root interface of these plants is less well known. Remigio et al.⁵⁰ have used a combination of synchrotron μXRF and XANES imaging to reveal chemical transformations of As in the rhizosphere-root interface of these ferns. The dominant form of As in soils was found to be As^V, even in As^III dosed soils. In roots, the major form in P. calomelanos roots was As^III, while it was As^V in P. vittata roots. Arsenic was cycled from roots growing in As-rich soil to roots growing in a control soil. The study may have application in phytotechnologies used to remediated As-polluted soils.

Several studies have reported on the speciation of As in food this year. Zhao et al.⁵¹ have reported the simultaneous determination of 11 As species in vegetables, although the title of the publication is misleading as only six species were actually found in the vegetable samples. The high performance IEC-ICP-MS method reported in the paper was however capable of separating and detecting eleven As species (AB, DMA, As^III, AC, MMA, 4-aminobenzenearsonic acid, As^V, carbarsone, 4-nitrophenylarsonic acid, p-hydroxyphenylarsonic acid and ROX) with LOQ values of between 0.10 and 0.39 μg kg⁻¹. The extraction efficiency and transformation of As species in vegetables was investigated using five extraction methods and eight solvents. The total As content in different vegetables varied greatly. The As in the edible parts of tomatoes (1.24–3.17 μg kg⁻¹) and peppers (2.10–6.74 μg kg⁻¹) was low. In canola (4.72–66.9 μg kg⁻¹), celery (8.25–46.3 μg kg⁻¹), leek (11.6–67.0 μg kg⁻¹), spinach (9.30–117.6 μg kg⁻¹) and total As content was significantly higher than that in tomato and pepper. The authors suggest that his may be due to the different enrichment abilities of different parts of the plants for As. However, the total As content in the same vegetables also varied significantly. The highest As content in canola was 14 times higher than that of the lowest, which may possibly be due to high As content in the growing media, or the use of pesticides containing As during growth. A vortex assisted RP spray-based fine droplet formation LPME has been combined with HPLC-CF-HG-FAAS for iAs speciation in olive oil by Bodur et al.⁵² The LOD and LOQ for As^III were calculated (as As) as 2.86 μg kg⁻¹ and 9.52 μg kg⁻¹, respectively. After the extraction/preconcentration process, a prereduction was carried out in the presence of 0.010 mol per L HCl (containing 0.39% L-cysteine) with heat (90 ± 5 °C) for the determination of total As. Any As^III present was directly determined via the developed system. The recovery for As^III and total iAs was in the range of 92 to 120% and 90 to 104%, respectively. An analytical method was been validated to determine As species in edible insects (Asian forest scorpion, diving beetles, giant water bug, grasshoppers, June beetles, mole crickets, male rhino beetle, female rhino beetle, sago worms, and silkworm pupae).⁵³ The As species were extracted from the insects by heating at 100 °C in 0.3 mol per L nitric acid and then the concentrations of As^III, As^V, MMA, DMA and AB were determined by LC-ICP-MS employing an ODS column with a mobile phase of 1.458 g 25% TMAH, 1.602 g sodium 1-butane sulfonate, 0.416 g malonic acid and 5 mL of MeOH in 1 L of water with the pH adjusted to 3.0 with nitric acid. THE CRMs NRCC DORM-4 (fish protein), NMIJ CRM 7405-a (Hijiki seaweed), and NMIJ CRM 7533-a (brown rice flour) were used for method validation. The proposed method gave a LOD of 0.007–0.012 mg kg⁻¹, LOQ of 0.021–0.038 mg kg⁻¹, repeatability of 1.2–3.2%, intermediate precision of 2.8–4.5%, and trueness of 97–102% (based on spiked samples) for the insects studied. The results indicated that the majority of the As was iAs. Diving beetles and scorpions contained characteristic organic As compounds such as arsenosugars and AB. Based on previous studies it was assumed that most As species in the insects were feed dependent. However, for AB found in scorpions, the authors suggested that it may be convert to AB in vivo because they are arthropods like lobsters. Both the grasshoppers and mole crickets were the same species of edible insects approved as novel foods by the EU, and these had low levels of As (<0.1 mg kg⁻¹).

Once again Rice has proved a popular matrix for As speciation studies, reflecting its importance as a staple food crop in many countries. Field-testing kits for detecting As based on the Gutzeit method and previously reported for water testing, have been shown to be able to screen iAs in rice. Such kits are useful when assessing food safety and enable quick decision-making without the need for laboratory analysis. However, the As extraction within the field method utilises nitric acid. To make the field method safer, cost-effective and easier to handle, the method has been further adapted by Wehmeier et al.⁵⁴ using Cola in the extraction process, with the phosphoric acid in the drink presumably being the active extractant. The method was tested by screening a total of 30 rice and rice products (a variety of rice for infants, polished and parboiled rice as well as unpolished rice and rice crackers) obtained from the Austrian market. To verify the results obtained using the Cola extraction method, the iAs concentrations were compared to an HPLC-ICP-MS method employing the CRM NIST 1568b Rice Flour and the found and certified values were in good agreement. The Cola extraction method gave an LOD of 39 μg iAs per kg rice, and an average RSD of 14%. The method did not give false-negative results and a relatively small number (7%) of false-positives compared to the validated reference method. However, a limited number of samples showed a large difference in the comparison with the nitric acid HPLC-ICP-MS method, and a few samples showed poor recoveries. The authors note the semi-quantitative nature of the method, but it is a useful method to screen rice and rice-based commodities in a relative short time in remote field sites without access to concentrated acids and trained personnel. In a second paper by the same group, As Cd and Pb were determined 51 rice containing commercial products in Austrian (25 rice varieties, 8 rice products, and 18 rice containing baby foods).⁵⁵ Some products were found to be higher than the limits for iAs in rice five years after the introduction of regulation (EU) 2015/1006. It was also shown that rice grown under upland (aerobic) agricultural conditions in Austria, had both low iAs (<19 μg kg⁻¹) and low Cd levels, supporting food safety and sourcing for infant foods. The mean concentrations for iAs in rice was 120 μg kg⁻¹, 191 μg kg⁻¹ in rice products, and 77 μg kg⁻¹ in baby foods. The average DMA and MMA concentrations were 56 μg kg⁻¹ and 2 μg kg⁻¹, respectively. The highest iAs concentration was found in rice flakes (237 ± 15 μg kg⁻¹), close to the Maximum Level set by the EU regulation for husked rice (250 μg kg⁻¹). As noted above, an acid extraction is commonly used when determining As species in rice, although it has been observed that slight differences in the heating and extraction conditions may result in MMA being transformed into different As compounds. A similar phenomenon is observed in the As speciation analysis of seafood. To identify these compounds Matsumoto et al.⁵⁶ have analysed a number of extracts using LC-TOF-MS and LC-ICP-MS. The transformed compound was identified as MMMTA, a thioarsenical, which is estimated to be more cytotoxic than MMA. In further experimental work, MMMTA was readily produced by bubbling hydrogen sulfide through MMA, and this suggested that MMA may react with S in rice during the extraction process. The authors suggested that DMA could also be transformed into another unidentified compound, although the generation rate was low. Magnetic SPE has been used as a nonchromatographic method for the quantification of ultratrace iAs in rice by ICP-AES.⁵⁷ Iron nanoparticles were modified with an organophosphorus compound and used as the solid phase for MSPE of iAs from the rice matrix. The MSPE procedure was performed using 4 mL of a buffer solution with pH 4.0, 20 mg of the nanomaterial, and a 15 min extraction time. The total As, determined by ICP-OES, was also quantified using the same MSPE procedure after solubilisation of the samples using a block digester. The accuracy of total As and iAs quantification was verified using CRM NIST 1568b (97% and 101% recovery, respectively) whilst the LOD was 1.08 and the LOQ 3.70 μg kg⁻¹. The rice samples used in the study had total As contents between 0.090 and 0.3 mg kg⁻¹ and iAs mass fractions between 0.055 and 0.11 mg kg⁻¹. A natural bacterial biosensor utilising Escherichia coli AW3110 (pBB-ArarsR-mCherry), in conjunction with amylase hydrolysis for extraction has been used for the quantitative detection of iAs in rice grains.⁵⁸ The biosensor exhibited high specificity for As and it was possible to distinguished between As^III and As^V by modulating the concentration of PO₄³⁻ in the detection system. The iAs concentrations were determined in 19 rice grain samples with varying total As concentrations and compared with a reference technique using enzymatic hydrolysis and microwave digestion coupled with HPLC-ICP-MS. The CRM SRN1568b rice grain was also used for quality assurance. Both methods exhibited comparable results, without significant bias in the concentrations of As^III and As^V. The whole-cell biosensor demonstrated good repeatability and a high signal-to-noise ratio. A LOD of 16 μg kg⁻¹ for As^III and 29 μg kg⁻¹ for As^V were obtained.

Two publications have reported on studies to assess the health risks associated with the consumption of regionally grown rice. The first of these was based in Hunan Province, the major rice-producing region in China.⁵⁹ The total As content in rice, paddy soils, and irrigation water was determined by ICP-MS following microwave digestion of the solid samples. The As speciation was preforming using AEC for separation and HG-AFS for detection. The results showed that the mean values of total As were 132 ± 42, 198 ± 95, 521 ± 390, and 2050 ± 3300 μg kg⁻¹ in milled rice, brown rice, rice bran, and rice husk, respectively. The maximum As concentration in the irrigation water was 14.4 ± 0.71 μg L⁻¹ found near a metal smelting factory. The mean value for the irrigation water samples was 4.70 ± 3.3 μg L⁻¹. The second publication reported on a study to evaluate the health risk from As (and 12 other elements) in 13 types of rice consumed in Sri Lanka.⁶⁰ A C₁₈ column was used for the As speciation using HPLC-ICP-MS. The total As levels ranged from 24 to 179 μg kg⁻¹. The most predominant species detected was As^III followed by As^V, and the total iAs level ranged from 77 to 91%. Concentrations of four As species, As^III, As^V, DMA, and MMA, were successfully separated and quantified in two CRMs (SRM 1640a Trace elements in natural water, and NMIJ 7503b As compounds and trace elements in white rice flour) and in three different rice varieties collected from the Sri Lankan market. The rice samples showed multiple peaks in the chromatograms, but some of them were less than the LOQ. The LOD values of As^V, As^III, MMA, and DMA were 2.4, 5.2, 2.2, and 4.0 μg kg⁻¹, while LOQ values of the same As species were 7.3, 15.8, 6.6, and 12.2 μg kg⁻¹, respectively. The calculated Target Hazard Quotient and cancer risk values, based on total iAs, indicated potential health risks from consumption of some rice varieties in Sri Lanka.

The As speciation in fungi is also often reported. The As speciation in commonly eaten mushrooms (cultivated and wild-grown) from central Europe has been investigated by Walenta et al.⁶¹ A total of 93 mushroom samples Boletus edulis (king bolete) (n = 19), Cantharellus cibarius (chanterelle) (n = 21) and Macrolepiota procera (parasol mushroom) (n = 8). For the cultivated mushrooms Agaricus bisporus (white button (n = 10) and cremini (n = 5) mushroom), Lentinula edodes (shiitake, n = 10), Pleurotus eryngii (king oyster mushroom, n = 7) and Pleurotus ostreatus (oyster mushroom, n = 13). For analysis, around 200 mg (weighed to 0.1 mg) of each sample was mixed with 4 mL of ultrapure water and sonicated in an ultrasonic bath at room temperature for 15 min, before being centrifuged for 10 min and filtered through 0.2 μm Nylon® syringe filters. The As speciation was carried out by HPLC-ICP-MS, using both anion-exchange and cation-exchange columns. Two CRMs, were used: NIST SRM 1640a Water (n = 10) and CRM IPE-120 Agaricus bisporus (n = 6). The most commonly found As species in the mushroom fruit-bodies were DMA, MA, AB and iAs. Less frequently occurring were TMA, AC and TMAO. Some traces of trimethylarsoniopropionic acid (TMAP or AB2), dimethylarsinoylacetic acid (DMAA) and homoarsenocholine (AC2) were also detected, besides several unknown As species. The results indicated a large diversity in As speciation across the most commonly consumed mushrooms, even at trace As mass fractions. The authors highlighted the need for further elucidation of unknown As species and sometimes poor extractability (e.g. Cantharellus cibarius). The As^III induced oxidative defence and speciation changes in the wild fungi Trametes versicolor (HN01 strain) has been investigated using a multifunctional microplate reader to explore the detoxification mechanisms and HPLC-ICP-MS for As speciation.⁶² The fungi were cultivated under 40 and 80 mg L⁻¹ of As^III to induce the stress. The results indicated that T. versicolor may be one of the few mushrooms with simultaneous As accumulation and tolerance capacities and As^V was the dominant species in the hyphae of T. versicolor regardless of As related stress. The detoxification mechanisms of this strain involved alleviating the toxicity by increasing the activities of antioxidants, especially GSH, as well as by converting As^III into As^V and other less toxic As species.

The speciation of As in fish and other marine organisms has once again attracted a lot of attention. A study of As speciation in oil and in brine canned solid tuna samples using IC-ICP-MS has been reported.⁶³ The extraction of water-soluble As species (AB, DMA, MMA, As^III, and As^V) was performed with (NH₄)₂CO₃ at pH 10.5. The presence of the sample matrix showed no effect on the selectivity of the method (t-test, p < 0.05) and an external calibration method was applied. The LOQ values for the five As species were 0.59 mg per kg AB, 0.10 mg per kg DMA, 0.12 mg per kg As^III, 0.07 mg per kg MMA and 0.10 mg per kg As^V. Method accuracy was tested through the analysis of the CRM BCR-627 (tuna fish muscle tissue) and good agreement was obtained for the found and certified values. Total As in the solid canned tuna ranged from 2.65 to 5.81 mg kg⁻¹ in in-oil samples and from 2.74 to 5.70 mg kg⁻¹ in in-brine samples. These levels were higher than the limits established by the Brazilian sanitary standards (1 mg kg⁻¹) for As in fish, although the speciation showed that most As (77–85%) is non-toxic AB and the iAs species were below the LOQ. The As speciation (As^III, As^V, MMA, DMA, AB and AC) has been determined in 56 fish collagen products used in Korea.⁶⁴ The mean concentrations of total As and As species were 40 ± 81 μg kg⁻¹ and 30 ± 50 μg kg⁻¹, respectively. The mean concentration of iAs was 24 ± 32 μg kg⁻¹ and As^V was the most dominant As species. However, one sample in the study had a total As concentration of 587 μg kg⁻¹, of which 8.1 μg kg⁻¹ was iAs and 306 μg kg⁻¹ was DMA. The study demonstrated the need to monitor As levels in such products and establish standards and specifications for As in fish collagen products to assure consumer safety. Low trophic marine resources such as blue mussels and kelp are potential candidates for use as ingredients in salmon feed. However, marine biota are well known to contain relatively high levels of As, which may be present in different organic forms depending on marine biota type and trophic position. Silva et al.⁶⁵ have studied the concentrations of As species present in the raw materials, feed and farmed salmon when being fed novel low trophic marine feed resources. Atlantic salmon were fed experimental diets for 70 days. A total of 9 diets were prepared: 4 diets containing up to 4% fermented kelp, 3 diets containing up to 11% blue mussel silage, and 1 diet containing 12% blue mussel meal, in addition to a standard reference diet containing 25% fish meal. Concentrations of total As and As species in feeds, faeces, liver and fillet of Atlantic salmon were determined by ICP-MS and HPLC-ICP-MS, respectively. The use of kelp or blue mussel-based feed ingredients increased the concentration of total As, but the maximum level as defined in Directive 2002/32 EC and amendments were not exceeded. The concentrations found in the experimental feeds ranged from 3.4 mg kg⁻¹ to 4.6 mg per kg ww. Arsenic speciation in the feed varied based on the ingredient, with AB dominating in all feed samples (36–60% of the total As), while arsenosugars (5.2–8.9% of the total As) were abundant in kelp-included feed. The intestinal uptake of total As ranged from 67 to 83%, but retention in fillet only ranged from 2 to 22% and in liver from 0.3 to 0.6%, depending on the marine source used. Fish fed feeds containing blue mussel showed higher intestinal uptake of total As when compared with fish fed feeds containing fermented kelp. Fish fed fermented kelp-based feeds had higher retained concentrations of total As when comparing with fish fed feeds containing blue mussel. Overall, iAs species were not detected in salmon muscle reared on low trophic marine diets, and AB was the dominant As species.

Arsenic in seaweed and seaweed products has again been studied. Seaweed products are increasingly consumed in Europe although there is concern over potential contamination by toxic elements. A study to assess the contents and bioaccessibility of trace elements in seaweed products available in Belgium has be presented by da Silva Junior et al.⁶⁶ A total of 231 samples were collected in markets and As, Cd, Hg, I, Ni, Pb, and Se concentrations were measured. The As speciation and the effect of a washing treatment on total concentrations of trace elements were also studied. Bioaccessibility was assessed via in vitro simulation of the gastrointestinal digestion. Of the samples studies, 85%, 39% and 4% exceeded the concentration levels permitted for As, Cd and I respectively. Washing significantly reduced concentrations of all the trace elements assessed. The speciation analysis identified a high percentage of iAs in hijiki. The average bioaccessibility in the small intestine was: As (52%) > Ni (35%) > Pb (27%) > Cd (20%). The authors recommend washing of such products to reduce the concentration and bioaccessibility of trace elements in seaweeds. The temporal and intra-thallus variations in water-soluble As together with total non-polar and polar As-containing lipids have been determined in the brown macroalga Laminaria digitata in order to gain insight into the origins of arsenosugars.⁶⁷ The study also evaluated freeze-drying treatments to determine whether this changes the water soluble As speciation. Lower levels of total As (determined by ICP-MS) were detected in the samples collected in May (39.2–74.5 mg kg⁻¹) compared to those collected in February (72.6–151 mg kg⁻¹). The concentration of As^V (determined by HPLC-ICP-MS) was found to consistently increase along the thallus from the holdfast/stipe (0.78–1.82 mg kg⁻¹) to the decaying fronds (44.4–61.0 mg kg⁻¹) in both months, and AsSug-SO3 was the dominant arsenosugar in the majority of samples. The extraction efficiency was lower in fresh samples (64–77%) than in freeze-dried (95–116%) from the same month. Water-soluble, polar AsLipids, and residual As concentrations, were generally highest in February, and the non-polar AsLipids accounted for <0.42% of total As in all samples. The results suggest that the arsenosugars were not a product of As detoxification, but a by-product of normal biological activity. The authors suggest that the arsenosugars were bound to the cell membrane within the Laminaria digitata cells, and lyophilisation is required to release them quantitatively. It was not evident from the study if the lower extraction efficiency observed was due to the As being in an unextractable form, i.e. lipids, and thus not removed from cells during the water-based extractions.

A number of regional studies focusing on As speciation have been reported this year. The dumping of an estimated 57 million tons of hazardous sulfide mine waste into Portman's Bay (SE Spain) between 1957 and 1990 has caused one of the most severe cases of persistent anthropogenic impact in Europe's costal and marine environment. The resulting mine tailings deposit completely infilled Portman's Bay and extended seawards on the continental shelf, bearing high levels of metals including As. Data obtained from various sources including SR-XAS and XRF core scanner have been reported by Baza-Varas et al.⁶⁸ and revealed the presence of arsenopyrite (FeAsS), scorodite (FeAsO₄·2H₂O), orpiment (As₂S₃) and realgar (AsS) in the submarine extension of the mine tailings deposit. In addition to arsenopyrite weathering and scorodite formation, the presence of realgar and orpiment was discussed in the paper, considering both potential sourcing from the exploited ores and in situ precipitation from a combination of inorganic and biologically mediated geochemical processes. The authors hypothesise that the presence of orpiment and realgar is associated with scorodite dissolution and subsequent precipitation of these two minerals within the mine tailings deposit under moderately reducing conditions. The occurrence of organic debris and reduced organic sulfur compounds provide evidence of the activity of sulfate-reducing bacteria and provide a plausible explanation for the reactions leading to the formation of authigenic realgar and orpiment. The precipitation of these two minerals in the mine tailings has important consequences for As mobility since this process would reduce the release of As into the surrounding environment. The bioaccumulation of As and Cr species in water, soil, and vegetables collected from the Mokolo River and its vicinity and in Lephalale, Limpopo Province, South Africa has been reported.⁶⁹ Total As levels were determined by ICP-MS and the speciation by HPLC-ICP-MS. The total concentrations of As in the water samples ranged between 0.328 and 0.612 g L⁻¹, below the maximum permissible levels set by the WHO and South Africa's Department of Water Affairs and Forestry (DWAF) for drinking water. The soil quality guideline value of 12.0 μg g⁻¹ for As was surpassed at one sampling site, although the high concentrations in the soil could be linked to the use of fertilisers and atmospheric deposits introduced through coal burning by power stations. For vegetables, the observed As levels recorded in the high-flow season ranged from 1.55 to 189 ng g⁻¹, 40.1 to 153 ng g⁻¹, 9.38 to 292 ng g⁻¹ and 0.91 to 262 ng g⁻¹ for As^III, DMA, MMA and As^V, respectively. In the low-flow season, the concentration ranged from 7.03 to 52.9 ng g⁻¹, 0.338 to 30.3 ng g⁻¹, 2.48 to 15.7 ng g⁻¹ and 0.72 to 27.8 ng g⁻¹ for As^III, DMA, MMA and As^V, respectively. The study suggested that the consumption of vegetables from the area could lead to health risks in the population. Two investigations have been published for regional studies in China. The first reported on the characteristics of As speciation in cultured shellfish from Sanmen Bay, Zhejiang Province, China.⁷⁰ Sanmen Bay plays a crucial role in economic shellfish aquaculture in China, yet there are few studies on the As speciation of shellfish from this area. In the study 11 cultured shellfish species from Sanmen Bay were analysed by HPLC-ICP-MS. The results showed that organic As particularly AB, was the dominant As species, making up 21–71% of the total As. Conversely, the levels of iAs were relatively low, ranging from 0.007 to 0.093 mg kg⁻¹, and accounting for 0.2–5.7% of the total As. There was no significant correlation between iAs and total As in Sanmen Bay shellfish, and the concentration of iAs did not increase with the total As. In the second study, the As speciation in two mushrooms, Agaricus blazei Murrill and Tricholoma matsutake, cultivated in Yunnan Province, China was investigated.⁷¹ The method used both AEC and CEC chromatography coupled to ICP-MS. The total As contents ranged from 5.25 to 69.5 mg kg⁻¹. AB, iAs and MMA were the main forms of As in A. blazei samples, accounting for 42.7–88.8%, 1.59–26.2%, and 0.74–1.12% of the extractable As, respectively. The main forms of As in T. matsutake samples were AB and DMA, which accounted for 88.9–97.9%, and 1.79–3.80% of the extractable As, respectively. Some AC, TMAO, and tetramethylarsonium cation (TMA⁺) were also present, but only at trace concentrations. An estimation of health risk index based on the results indicated that there was no particular risk of iAs exposure to consumers of T. matsutake, although exposure to the higher levels of iAs in A. blazei could present a risk to health in the region.

There have been fewer reports of clinical studies based on As speciation in this review period. One of the more unusual devices described this year for As speciation was constructed using Lego™ components with application in clinical analysis. The pre-treatment device was designed for dried blood spot analysis using IC-ICP-MS.⁷² The Lego spinner, was constructed from three Lego™ components and 3D-printed tube sleeves. The device integrated magnetic mixing and centrifugal separation functions to extract and separate the As species from the dried blood spots. In the mixing mode, a magnetic stir bar was placed in the centrifuge tube with the reagents and rotated under a magnetic field to promote the dispersive extraction of As species from the dried blood spot. In the centrifugation mode of the Lego-spinner, the As extract was separated from the blood matrix for subsequent IC-ICP-MS analysis. For a dried blood spot prepared from 80 μL of whole blood, the pre-treatment operation was completed within 25 min. The LOD values of AB, As^III, DMA, MMA, and As^V in the dried blood spot were 0.09–0.15 μg L⁻¹, and precisions were <11%. The concentrations of the five As species were correlated between whole blood and the dried blood spot (r2 > 0.97), and Bland–Altman analysis indicated that the concentration difference of As species between whole blood and the dried blood spot is within ±20%. Using this approach, the As species could be preserved for at least 30 days at 4 °C. The determination of total iAs in homeopathic samples using HG-ICP-OES has been reported.⁷³ Various sample forms (sugar pellets, tablets, alcohol-based drops), containing arsenicum album or arsenicum iodatum in several potencies (C9-200, D6-10), as well as different sample preparation approaches (wet digestion, extraction, dissolution, dilution, direct analysis) were studied. The influence of the undecomposed sample matrix (mainly sugar and alcohol) on the HG process of As was also examined. Microwave-assisted wet digestion was selected for the study and under optimal conditions gave a LOD < 0.1 ng per g As, with a RSD of 0.31–7.4%, and trueness, as confirmed by recovery tests of 94.9–113%. The applicability of the developed method was demonstrated by determining As in 13 homeopathic products available on the Polish market. The fate of arsenicals in mice carrying the human AS3MT gene exposed to environmentally relevant levels of As^III in drinking water has been reported by Douillet et al.⁷⁴ Although mice are widely used to study adverse effects of iAs, the higher rates of iAs methylation in mice when compared to humans may limit their utility as a model organism. A recently created 129S6 mouse strain in which the Borcs7/As3mt locus replaces the human BORCS7/AS3MT locus and exhibits a human-like pattern of iAs metabolism was used in this study. The authors evaluated the dosage dependency of iAs metabolism in humanised (Hs) mice. They determined tissue and urinary concentrations and proportions of iAs, MMA, and DMA in male and female Hs and wild-type (WT) mice that had received 25 or 400 μg per L iAs in drinking water. At both exposure levels, Hs mice excreted less total As in urine and retain more total As in tissues than WT mice. Total As levels in tissue were higher in Hs females than in Hs males, particularly after exposure to 400 ppb iAs. Tissue and urinary fractions of total As present as iAs and MMA are significantly greater in Hs mice than in WT mice. Notably, tissue total As dosimetry in Hs mice resembles human tissue dosimetry predicted by a physiologically based pharmacokinetic model. The study employed HG-cryotrapping-ICP-MS for the As speciation, with LOD values ranging from 0.27 to 1.7 pg As. This data provides additional support for the use of Hs mice in laboratory studies examining effects of iAs exposure in target tissues or cells.

3.3. Chromium

In contrast to last year, there have been no new chromatographic separation procedures reported; the majority of the speciation methods in the most recent review period have been based on LLE or SPE.

Two variations of the same direct immersion dual-drop microextraction procedure appeared in different journals within two weeks of each other. Separation and preconcentration of Cr^III and Cr^VI were achieved in parallel by selective extraction from the same solution into two 15 μL drops of organic solvent, each of which contained an appropriate selective chelating reagent, suspended from the tips of microsyringe needles. Following extraction, the drops were diluted to 50 μL, and a 10 μL subsample taken for analysis by ETAAS. In one procedure, the researchers extracted Cr^III with thenoyltrifluoroacetone (TTFA) and Cr^VI with APDC (both in chloroform) from an aqueous solution at pH 3.5.⁷⁵ In the second procedure, Cr^III was extracted with 1-phenyl-3-methyl-4-benzoylpyrazol-5-one (PMBP) and Cr^VI was extracted with DDTC (both in 1-decanol) from an aqueous solution at pH 4.⁷⁶ The LOD values of the two methods were similar: those for the TTFA/APDC method were 3 and 4 ng L⁻¹ for Cr^III and Cr^VI, respectively, those for the PMPP/DDTC method were both 1 ng L⁻¹. The former method was validated by the analyses of CRMs GSBZ50027-94 (water), GBW10052 (tea leaves), GBW 10017 (milk powder), GBW10024 (scallop) and GBW10011 (wheat flour), and by spike recoveries (0.1–5.0 μg L⁻¹) from locally purchased (Wuhan, China), water and beverages including orange juice, well water, soda, cola, apple juice and Sprite. It was reported that Cr^VI was not detected in any beverage except well water, but was found in the water, milk powder, scallop and wheat flour CRMs. The other method (PMPP/DDTC) was validated by the analysis of CRMs GSBZ50027-94 (Cr^VI in environmental water) and GBW(E)080039 (trace elements in water) and applied to Yangtze River water, Dongxi Lake water and industrial wastewater (Wuhan, China), in all of which both species were found. Spike recoveries, measured for all samples and CRMs, ranged from 86 to 108%.

A LLME procedure in which Cr^VI was selectively extracted as an ion-pair with CTAB into CHCl₃, has been devised for the analysis of waters and industrial extracts by TXRF.⁷⁷ To a 10 mL sample adjusted to pH 0.2 were added 100 μL of 0.01 mol L⁻¹ CTAB in CHCl₃, and the mixture shaken to form a cloudy white suspension. After phase separation by centrifuging a total of 15 μL was dried in two stages on a quartz reflector. The TXRF analyser was a commercial benchtop TXRF spectrometer S2 PICOFOX (BrukerNano, Germany) containing a low-power tungsten X-ray tube (50 kV, 1 mA) and a silicon drift detector (SSD, resolution <150 eV at Mn Kα). The LOD was 0.9 μg L⁻¹, and the linear range was from 5–5000 μg L⁻¹. The procedure was applied to different types of waters (tap, well, river and sea water), a galvanic industry effluent and two different clinker aqueous extracts from a concrete manufacturer (all from the Girona region of Catalonia in northeast Spain). For the water samples Cr^VI was below the LOD whilst spike recoveries at 25 and 50 μg L⁻¹ ranged from 82 to 100%. In the effluent and extract samples, Cr^VI was found in concentrations that were in agreement with those found by a standard spectrophotometric DPC method (UNE-EN 196-10, the official Spanish version of European Standard EN 196-10:2006). The researchers confirmed that the presence of up to a five-fold excess of Cr^III did not affect the results for Cr^VI. Total Cr values were obtained by ICP-OES.

A SPE procedure in which both Cr^IIIand Cr^VIspecies were retained on a magnetic-GO-4-aminobenzenesulfonic acid composite has been applied to the analysis of sea, well and tap waters from the Malaga region of Spain.⁷⁸ The extractant material (details of whose synthesis are in a Spanish patent) was packed into two 50 cm knotted reactors that were wound round toroidal magnets and mounted in a eight-port FI valve. Sample (4 mL), adjusted to pH 3, was pumped through one reactor and the unretained matrix components washed away with water. The retained Cr^VI was selectively eluted by pumping 4 mL of 3.2% NH₄OH solution in the reverse direction and quantified by ICP-OES. At the same time, 4 mL of sample was loaded into the second reactor, which was washed and then total Cr eluted with 4.2 mL of 2.3% HNO₃ and measured by ICP-OES. During this elution step, any Cr retained in the first reactor was washed off with 2.3% nitric acid. It is not clear why the species were not determined by sequential elution from just one reactor, rather than subtracting the Cr^VI value from the total to get the Cr^III concentration. Although the volumes of sample and eluent are similar, preconcentration (enrichment factors of 15 and 23 for Cr^VI and total Cr, respectively) was achieved because the retained species would have been eluted in the leading few hundred μL of eluent. The LOD values were 0.1 and 0.08 μg L⁻¹ for Cr^VI and total Cr, respectively and the method was validated by the analysis of CRM TMDA 54.5 (fortified lake water) and SPS-SW2 (surface water), which are certified for total Cr only. However, although only Cr^III was found in TMDA 54.5, both species were found in SPS-SW2 and in all four of the real samples. Spike recoveries from all samples (50–300 μg L⁻¹) were between 91 and 112%.

A procedure in which both Cr^IIIand Cr^VIwere retained on planar SPE materials prior to quantification by LIBS has been developed.⁷⁹ The researchers fabricated an “ion enrichment chip” by 3D printing. The SPE media were inserted in the space between the top and bottom layers (length 15 mm × width 5 mm × height 0.8 mm). The first medium encountered as sample solution flowed through the device was a sheet of activated carbon fibres (length 6 mm × width 5 mm thickness 0.4 mm) and then the solution followed over an array of anion-exchange resin beads (5 rows × 6 columns, diameter 0.4 mm) that were glued to the enrichment insert (details not given). Sample solution was delivered at 21 mL min⁻¹ for 20 min (a volume of 420 mL) and then air was blown through to remove excess sample and dry the adsorbents. The media were transferred to a three-dimensional displacement platform that adjusted the position of the adsorbents under the laser beam. The researchers related the signal from the carbon fibres to the total Cr concentration, and the signal from the anion-exchange beads to the Cr^VI concentration. Presumably, the Cr^VI in the sample was somehow distributed over both extraction media, which is perhaps not surprising as the linear flow rate is about 18 cm s⁻¹, corresponding to a residence time above the carbon fibre media of about 33 ms. The LOD values were 10 and 4 μg L⁻¹ for total Cr and Cr^VI, respectively and the method was applied to a “pool water” sample from an Agricultural Science and Technology Demonstration Base. No Cr was found in this sample, but spike recoveries (0.05–4.0 mg L⁻¹) ranged from 90 to 114%. There was no pH adjustment. The method was also applied to aqueous extracts of spiked (100–1000 mg kg⁻¹) soils, though for these solutions the optimised injection speed and injection time were remarkably different: 50 μL min⁻¹ and 7 min, respectively. The only results presented for the soils were the LOD values of 13.6 mg kg⁻¹ and 3.8 mg kg⁻¹, for total Cr and Cr^VI, respectively. There is confusion over the units of these: in the abstract and table the units are mg kg⁻¹, but in the text the units are mg L⁻¹. As the retained analytes are not eluted, it is hard to understand the basis of describing this procedure as an “enrichment.”

Both nano-SIMS and micro-X-ray techniques were applied to the elucidation of Cr^IIIspeciation in rice roots with the goal of gaining a better understanding of the role of iron plaque in controlling Cr availability.⁸⁰ Both the speciation and localisation of Cr and the distribution of micro-nutrients in tip and mature regions of rice roots were examined by synchrotron-based techniques, μ-XRF and μ-XANES. Cr K-edge XANES analysis at Cr hotspots showed the dominant speciation of Cr in outer (epidermal and sub-epidermal) cell layers of the root tips and mature root were complexes with fulvic acid-like anions and with amorphous ferrihydrite, respectively. The relationship between metabolically active ¹³C-enriched root epidermal cells and metal complexes, such as CrO and FeO, in both the tip and mature root regions was mapped by NanoSIMS. The researchers concluded that the co-location of the fulvic species with signals of ⁵²Cr¹⁶O and ¹³C¹⁴N in the mature root epidermis relative to the sub-epidermis indicated the association of Cr with active root surfaces, where the dissolution of Fe plaque and release of the associated Cr is likely mediated by organic anions. Whereas the results for the root tips are indicative of the re-uptake of Cr by this region. Overall, the researchers concluded that their results indicated significant roles for iron plaque and organic anions in rice root systems on the bioavailability and dynamics of PTEs.

Separation by AF4 with quantification by ICP-MS was used in support of studies of the removal of Cr species from drinking water by various sulfonated polyethersulfone nanofiltration (NF) membranes with different molecular weight cut-offs.⁸¹ No previous work was cited for this analytical procedure nor was there discussion of any optimisation. Full details of the relevant separation parameters were given for the three-step program. During step 1 (injection and focusing), 50 μL was injected at flow rate of 0.5 mL min⁻¹ (for 2 min), with a focus flow rate of 2.0 mL min⁻¹, and a permeate flow rate of 2 mL min⁻¹. In step 2, elution was performed at a permeate flow rate of 2.0 mL min⁻¹ that decreased linearly to zero over 20 min, followed by elution for a further 12 min in the absence of permeate flow. Finally, in step 3 the system was rinsed (details not given). The total run time was 40 min. Total Cr was measured by ICP-MS, some details of which were provided in the ESI. The LOD was 0.2 μg L⁻¹. Some further information about the performance of the AF4 procedure was also given in the ESI, from which it is seen that the bottom of the working range was 5 μg L⁻¹. The researchers noted the importance of charge exclusion when Cr(OH)₄⁻ (for Cr^III) and CrO₄²⁻ (for Cr^VI) were the predominant species in the feed water. In the presence of humic acid (HA) material, Cr^III retention increased by 60%, while that of Cr^VI was unchanged. The HAs did not induce major modifications on the membrane surface charge for these membranes. Solute–solute interaction, in particular Cr^III–HA complexation, was the responsible mechanism for the increase in Cr^III retention.

A SPE-ICP-MS procedure for the speciation of inorganic Cr (and As and Se) in which the species were retained on TiO₂ NP-coated 3D-printed porous monoliths has been developed⁴⁴ and applied to the analysis of a variety of environmental waters and human urine. Although published in an analytical chemistry journal, most of the information relevant to the analysis is contained in the extensive ESI from which is seen that the extraction and elution was performed with a flow manifold, consisting of five 8-port valves, two columns and an unspecified number of peristaltic pumps, configured so that the two columns could be handled in parallel with loading of one while the other was eluted. It is also seen that the loading flow rate was 1 mL min⁻¹ for 2 min and the eluent was 3.5 mL of 0.5 mol per L HNO₃ delivered at 1 mL min⁻¹. The retention of species was based on pH control: at pH of 2.0 Cr^VI (and As^V and total Se) was retained and at pH 12.0 Cr^III (and As^III) was retained. For the speciation of Se, total Se was retained at pH 2.0, and Se^IV was retained at pH 6.0. The exact procedure is not clear, solutions at pH 2.0 were loaded onto one column and solutions of either pH 6 or 12 were loaded onto the other column. The researchers claimed that only 2 mL of sample was required, and that the throughput was 15 h⁻¹. The LOD values were 11 and 12 ng L⁻¹ for Cr^III and Cr^VI, respectively. The procedure was validated by the analysis of four CRMs NRCC CASS-4 (nearshore seawater), NRCC SLRS-5 (river water), NIST 1643f (fresh water), and Seronorm Trace Elements in Urine L-2 (human urine), in none of which were Cr^VI or Se^VI detected; As^V was not detected in CASS-4. The method was applied to the analysis of seawater, river water, agriculture waste, and human urine samples, when again Cr^VI was not detected; As was not detected in the river water, and Se^VI was detected only in the agricultural wastewater. Spike recoveries (0.5 μg L⁻¹) from these samples ranged from 96–104%.

To study Cr (and As) speciation in a water-soil-vegetable system, an HPLC-ICP-MS procedure was used.⁶⁹ All of the analytical procedures were based on previously published methods, including the HPLC separation, which was done on a Hamilton PRP-X100 column with gradient elution by a mobile phase consisting of varying proportions of NH₄NH₃ and NH₄H₂PO₄ that separated Cr^VI (and As^III, As^V, MMA and DMA). Total element determinations were validated by the analysis of three NIST CRMs SRM 1640a (trace elements in natural water), SRM 2709a (San Joaquin soil) and SRM 1573a (tomato leaves), and the speciation method was validated by spike recoveries from one soil and one vegetable sample. The procedures were applied to the analysis of 24 water samples (for which only total element concentrations were determined), 22 vegetable samples (chomolia, onion, spinach, pumpkin, and thepe) and 10 loam soil samples collected from the Mokolo River (Limpopo Province, South Africa) and nearby farms during different seasonal flows of the river. Although Cr^III had not been included in the method development and validation, the results presented for the soils and vegetables included values for Cr^III as well as for Cr^VI (and all four As species) that were well above the various LOD values. The researchers concluded that consumption of some Cr-contaminated vegetables, particularly by children, could cause serious health risks.

3.4. Copper

Two papers report on Cu speciation this year, and as they relate to Wilson's disease they are covered in Section 4 Biomolecular speciation analysis.

3.5. Gadolinium

Three different LC methods for the Gd-speciation of 7 commercially available contrast agents for MRI were compared.⁸² The systems included HPLC-ICP-MS, LC-MS/MS and IC with conductivity detection and the MRI contrast agents investigated were classified into ionic complexes with linear ligands, gadopentetate dimeglumine (Gd-DTPA), gadobenate dimeglumine (Gd-BOPTA), and gadoxetic acid disodium (Gd-EOB-DTPA), non-ionic complexes with linear ligands, gadodiamide (Gd-DTPA-BMA) and gadoversetamide (Gd-DTPA-BMEA), and the non-ionic complexes with macrocyclic ligands, gadobutrol (Gd-BT-DO3A) and gadoteridol (Gd-HP-DO3A). To separate such a diverse range of compounds using a single chromatographic system is not straight-forward, but HILIC, which has been shown to be a powerful separation technique for polar and ionic compounds, and previously used to speciate up to 6 gadolinium-based contrast agents (GBCAs) in biological and environmental samples, was developed. Separation of the contrast agents was carried out using a ZIC-pHILIC guard column (20 mm × 2.1 mm id, 5 μm, 200 Å) and a ZIC-pHILIC analytical column (150 × 2.1 mm id, 5 μm, 200 Å) at ambient temperature. Eluent A of the mobile phase was ACN, eluent B consisted of a solution of 12 mmol per L NH₄HCO₂ (pH 9) and the flow rate was 0.3 mL min⁻¹ with an injection volume of 5 μL. Using gradient elution with up to 78% acetonitrile required the use of 1.0 mm (id) quartz injector, addition of O₂ to the plasma and a cooled spray chamber (−8 °C). The LC-MS/MS method used the same or similar analytical conditions. Both the HPLC-ICP-MS and LC-MS/MS approaches were capable of speciating all 7 GBCAs, while IC was only suitable for the 3 ionic compounds (Gd-DTPA, Gd-BOPTA, and Gd-EOB-DTPA). A baseline separation of Gd-DTPA, Gd-BOPTA, Gd-EOB-DTPA, and 7 inorganic ions could be achieved using the IC approach with an AG18 guard column, an AS18 analytical column and NaOH gradient elution. However, the separation was unsatisfactory in that two peaks for Gd-EOB-DTPA appeared in the chromatogram due to dissociation of this compound in the NaOH mobile phase used. The LOD values for the 3 methods are quite informative and show the much greater sensitivity of ICP-MS as a detector and ranged from: 0.15 to 0.55 pg for HPLC-ICP-MS; 2.5 to 300 pg for LC-MS/MS: and 280 to 4500 pg for IC. The HPLC-ICP-MS method was applied to the analysis of wastewater and spiked whole blood samples. Wastewater samples were collected from influent of one of the wastewater treatment plants, which was near a large hospital in Guangzhou, China. The GBCAs were enriched prior to analysis by adding ACN (0.5 mL) to the water sample (0.5 mL) and evaporation to dryness using N₂. The samples were reconstituted in ACN : H₂O (78 : 22 v/v) a solution similar to the HPLC eluent. Spiked whole blood samples were prepared in the same way, with the addition of a protein precipitation step using acetonitrile.

3.6. Germanium

Germanium is not often featured in this elemental speciation review, however with its recognition as a Technology Critical Element and the increase in its use, and therefore presence in waste electronic and electrical equipment (WEEE), new methods for Ge species characterisation in environmental matrices are required. Thus, a method for Ge speciation in soil around a storage, processing and waste facility was developed with a focus on understanding its mobility, reactivity and transformation.⁸³ Soil sampling locations were established using in situ low-field volume magnetic susceptibility (κ) measurements. From 30 selected sampling points, two 30 cm long soil cores were collected using a Humax soil sampler. Immediately after sampling, the soil cores were placed in tubes to limit contact with atmospheric air and ore impurities. The highest extraction efficiency of Ge from the soils was achieved by using 100 mmol per L NH₄NO₃ with 1 mmol per L KNaC₄H₄O₆ as an extractant, which washed Ge from the soil within 4 h. An anion exchange IonPac AS9-HC column (250 mm × 4 mm, 9 μm) was used with a mobile phase gradient from pH 5.0 to 2.1 using a mixture of NH₄NO₃ and KNaC₄H₄O₆ at these two pH values. The samples and standard solutions were injected via a 200 μL sample loop onto the column. The Ge species including: Ge^IV, monomethylgermanium (MMGe); and dimethylgermanium (DMGe) were successfully separated after 12 min. The results showed the Ge concentration in the studied area was as high as 7.64 mg kg⁻¹, with an average concentration of 3.51 mg kg⁻¹. In most cases, the soils contained the Ge^IV form, with the methyl derivatives only found in a few soil samples.

3.7. Halogens

A review⁸⁴ of the isotopic characterisation methods for persistent halogenated organic pollutants containing C, H, Cl, Br using compound specific isotope analysis (CSIA), and based on GC and LC separations coupled to SF-ICP-MS/MS and IR-MS has covered compounds of environmental concern. A number of instrument schematics were included within the review (77 references) to show the evolution of the design of both LC and GC-combustion (C)-IRMS instrumentation. The review also describes the instrumental issues that needed to be overcome to implement the technique for the measurement of Cl and Br isotope ratios. The multi-elemental (C, H, Cl, Br) CSIA methods reviewed, have been employed in both laboratory and field experiments, but despite the instrumental advances of IRMS systems, the instrumental LOD values for GC-C-IRMS approaches are challenging, especially when applied to δ¹³C analysis. Taking into consideration the chromatographic separation required when analysing complex mixtures, the LC based methods do not provide adequate levels of resolution. The CSIA approach was extensively used to characterise the environmental processes associated with the abiotic and biotic transformation of persistent halogenated organic pollutants including those of contaminants of emerging concern. Recently, CSIA was use was expanded to larger molecules like BFRs and PCBs.

Recently developed methods have shown that the measurement of environmentally important halogen containing molecules can be achieved using GC coupled to ICP-MS and two methods have utilised this approach. The speciation of Cl in complex hydrocarbon mixtures was carried out using GC-ICP-MS/MS with a commercially available heated transfer line to interface the GC instrument to the ICP-MS/MS detector.⁸⁵ The chromatographic separation was performed on a HP-INNOWax column (60 m, 0.25 mm, 0.5 μm) using He as the carrier gas and split injection. The GC column outlet was connected via a T piece to a pre-heating line located inside the GC oven to mix the GC carrier gas flow with other gases. These gases included a mixture of Ar used to sweep the GC column effluents through the transfer line into the plasma and an Ar/O₂ mixture (80 : 20 v/v), which was used to burn off carbon deposits on the interface, resulting from the high C content of the samples. The measurement of Cl by ICP-MS is challenging because of the high first ionization potential (12.97 eV) and the existence of severe polyatomic interferences on the Cl isotopes at m/z 35 and 37 (O₂H⁺,SH⁺, ArH⁺). However, GC-ICP-MS/MS proved to be complementary to other existing Cl speciation methods such as GC-ECD, as it allies equimolar response, ease of calibration, selectivity towards Cl and good sensitivity. The investigation of new environmental marine sources of elements such as Br, I, S and Se has been undertaken using TD-GC-ICP-MS.⁸⁶ Emissions of volatile organic species from aquatic ecosystems represent an important source for the transfer of these elements into the atmosphere. The available methods to measure these species are either not sensitive enough or not automated, which limits a full understanding of species distribution and production mechanisms. A sensitive and high-throughput method for the simultaneous quantification of volatile organic species containing these elements in atmospheric and aqueous samples used a preconcentration step with sorbent tubes followed by analysis using TD-GC-ICP-MS. Selected commercially available sorbent tubes, consisting of mixed porous polymer and graphitised black carbon, offered the highest trapping capacity and lowest loss of species when stored at −20 °C for 28 days after sampling. After optimisation of the TD-GC-ICP-MS method, absolute LOD values were better than 3.8 pg, 9.1 fg, 313 fg, and 50 fg, respectively, for S, Se, Br, and I containing species. As a proof of concept, the concentrations of target species were determined in aqueous and continuously collected atmospheric samples during a cruise in the Baltic and North Seas. Unknown volatile species containing S, Br, and I were detected in both aqueous and atmospheric samples demonstrating the full potential of the method.

Iodine is an important essential element found in a range of marine algae, where it is abundantly present and nutritionally available. Speciation analysis of I in this context is therefore important, as a means to determine the relative bioavailability of each I-containing species and a method for the extraction and analysis by HPLC-ICP-MS/MS has recently been described.⁸⁷ For optimum extraction efficiency without compromising species integrity a two-step extraction method was developed using pancreatic enzymatic digestion followed by alkaline extraction with TMAH. An ion chromatography system was used with an AE separation facilitated using an IonPac AG20 guard column (2 × 50 mm) followed by an AS20 analytical column (2 × 250 mm). Elution of the species was facilitated isocratically within 10 min using a mobile phase consisting of 400 mmol per L NH₄HCO₃ with a flow rate of 0.25 mL min⁻¹. Excellent chromatographic resolution was achieved with the developed chromatography. For quantification, external calibration an internal standard was used, and calibration curves were prepared for each of the three individual I species. The method was applied to a range of marine algae including brown, green and red seaweed. In all samples, I⁻ was the major species present whereas monoiodotyrosine and diiodotyrosine were minor species found in most of the samples. Six unidentified I-containing species were also observed in varying amounts in some of the samples. The recovery of the I-containing species was determined by comparison of the sum of the species to the total I level determined using a direct ICP-MS/MS method. The highest recoveries were obtained for brown seaweed samples (66–100%) and the lowest for red seaweeds (12–45%). The recoveries for green seaweed samples were somewhere in between (41–92%). These recoveries were thought to relate to stronger binding of I in some seaweed species. The values were generally in agreement with other studies on the extraction of I-containing species from marine algae.

3.8. Iron

Analytical methods for Fe speciation continue to be developed and reported due the important biological role of this element. Maknun et al. used a combination of SEC-ES-HR-MS and SEC-ICP-MS for the quantification of Fe–siderophore complexes isolated from peat samples.⁸⁸ The siderophores were extracted from peat using water and the peat was also subjected to what is described as a biological enrichment procedure which was followed by phase separation, addition of Fe, siderophore extraction from the supernatant using SPE, elution, eluate evaporation and residue dissolution, pH adjustment (2–3) and the addition of ⁵⁸Fe and analysis. The modified isotope pattern of the Fe siderophores was used to predict the formulae of the complexes and theoretical and MS² fragmentation patterns compared to confirm these predictions. The ⁵⁸Fe : ⁵⁶Fe ratio obtained from SEC-ICP-MS analysis was used for their quantification along with LC-ES-MS isotope pattern data. In total, 19 siderophores belonging to 4 different classes were identified and quantified. The methodology can appear complex and a careful read of the paper is required for workers interested in this field of study. Four different stationary phases (C₁₈, phenyl, amide, and pentafluorophenyl propyl) have been evaluated for use in the recovery and separation of environmentally relevant trace metals (Mn, Fe, Co, Ni, Cu, Zn, Cd, and Pb) in Suwannee River Fulvic Acid and Suwannee River Natural Organic Matter. A 10 min linear gradient was from 95% H₂O, 5% MeOH to 5% H₂O, 95% MeOH, followed by 1 min of isocratic elution with 5% H₂O, flowing at 0.2 mL min⁻¹, was used for the separations. A second HPLC pump delivered a post-column compensation gradient, which was the reverse of the separation flow and mixed with the HPLC eluent, to give a constant flux of MeOH to the ICP-MS to stabilise sensitivity. This flow also included and internal standard solution (4 ng per mL In in 1% nitric acid) and O₂ prevent carbon build up on the cones. The C₁₈, amide, and phenyl phases generally yielded optimal metal recoveries (>75% for all metals except Pb), with the phenyl phase separating polar species to a greater extent than C18 or amide. A dual direct immersion single-drop microextraction (DDI-SDME) has been used for the sequential separation and preconcentration of Fe^III and Fe^II from aqueous samples, with Fe detection by GFAAS. The Fe^III in the sample selectively complexes with N-benzoyl-N-phenylhydroxylamine (BPHA) in CHCl₃ (1.0 mmol L⁻¹) at pH 1.5 leaving Fe^II in the aqueous phase. After removal of the organic phase and pH adjustment to 4.5 a second drop of the BPHA is added which complexes the Fe^II. The detection limits were 0.058 and 0.074 ng mL⁻¹ for Fe^III and Fe^II, respectively, with a precision of 5.6% or better obtainable and enrichment factors of 300-fold for both Fe species. The procedure was then applied to a milk powder CRM (GBW 10017), which was found to contain only Fe^III, and a recovery of 105% was obtained. Spike recoveries of both Fe species into the CRM at three levels gave recoveries of 94 to 105%. The method was then applied to drink samples and either one of both Fe species was detected in these. Spike recoveries, both Fe species at two levels, gave recoveries of 92 to 108%.

The use of X-ray based techniques to study Fe speciation is increasing in popularity, no doubt in part due to the wider availability of synchrotron facilities. Mijovilovich et al. investigated the modes of Fe binding and trace metal storage in the brown algae Laminaria digitata and Ectocarpus siliculosus using μXANES and XRF tomography.⁸⁹ The Fe distribution in both algae showed a higher accumulation in the cortex with symplastic subcellular localisation whilst XANES spectra indicated that Fe in L. digitata is stored in a mineral non-ferritin core, as this species lack of ferritin-encoding genes. The data also showed that the L. digitata cortex contains a ferritin-like mineral, while the meristoderm may include an additional component. The authors suggest that the formation of an amorphous mineral might be the storage strategy of non-ferritin-containing algae, thus modes of accumulation and Fe binding are different depending on tissue type. Hasegawa et al. have used XAFS to investigate Fe speciation in two fish species, Skipjack Tuna and Chub.⁹⁰ The dominant Fe species in the muscle was haem Fe (as the sum of methaemoglobin, oxyhaemoglobin, and deoxyhaemoglobin), with no detectable ferritin whilst in the liver, the ferritin contribution was 28 to 54% of the total Fe content. The authors also used MC-ICP-MS to determine the δ⁵⁶Fe values in the fish tissues with the aim of elucidating Fe metabolism. The difference in δ⁵⁶Fe values between haem Fe and ferritin ranged from 1.41‰ to 1.52‰, and the authors suggest that these results indicate that Fe storage as ferritin does not induce the lowering of δ⁵⁶Fe in the muscle, considering the low contribution of the liver Fe to the total Fe content in the body. The performance of laboratory and synchrotron based XANES measurements for examining Fe complexes with organic ligands has been compared.⁹¹ The laboratory instrument is based on a Highly Annealed Pyrolytic Graphite (HAPG) von Hamos spectrometer with the use of a cylindrically shaped crystal for combining high spectral resolution with a high solid angle of detection, modified to use a thinner HAPG crystal with a broader bending radius, which improves the spectral resolving power of the spectrometer. For validation, synchrotron XAFS measurements were performed at the μSR-XFM/μXAS beamline ID21 at the ESRF45 operating from 2.0 to 9.6 keV. Transmission mode was used for all samples except for haemoglobin, which was been measured in fluorescence mode. The compounds FeTPP(Cl), Fe(bzimpy)₂(–ClO₄)₂⁻·0.25H₂O and [Fe(bzimpy_–1H)₂]·H₂O were synthesised for the work and details of this are given or cited. The XAFS spectra of the three model/reference substances obtained with the laboratory instrument contained all of the characteristic fine structures exhibited in the results obtained by synchrotron measurements. The human haemoglobin sample analysed was also clearly identified as an Fe porphyrin system by the laboratory XAFS instrument. All three of these X-ray based papers contain a wealth of detail which will be of interest to workers in this field.

3.9. Lead

The speciation of Pb by X-ray methods has been applied to aerosol samples. Air PM_2.5 and PM₁₀ material collected on Teflon filters in an urban area in Amman, Jordan was examined⁹² for the element content by PIXE, and the speciation was determined by XANES. The measurements were made at the IAEA X-ray spectrometry endstation at the XRF beamline of Elettra-Sincrotrone Trieste, Trieste, Italy, where XANES spectra, generated with grazing incidence geometry (to maximise the footprint of the beam) at the Pb L₃-edge (13 035 eV), were collected by a Si (111) monochromator in fluorescence mode. The XANES spectra were normalised with respect to the EXAFS part of the spectrum using the ATHENA software package and then analysed through linear combination fitting to spectra of the available reference lead compounds (Pb metal, PbO, PbO₂, Pb₃O₄, PbS and PbSO₄). The researchers found that Pb concentrations increased during the working day for particle size fractions with average Pb concentration of 2.9 ± 1.7 and 3.9 ± 2.0 ng m⁻³ for PM_2.5 and PM₁₀, respectively. They found that both fractions contained Pb^II, most likely as a mixture of PbSO₄, PbS and PbO.

For the determination of Pb^II and TML (and Hg^II, MeHg and V^V) in seawater and urine, an SPE preconcentration was applied prior to HPLC-ICP-MS.⁹³ A previously synthesised, patented, magnetic nanomaterial based on a double coupling between GO and MNPs (M@GO) was further derivatised with the chelating group methyl thiosalicylate. The resulting M@GO-TS material (50 mg) was packed into a column mounted in the injection loop of a FI valve and sample, adjusted to pH 3.5, was loaded at 2.7 mL min⁻¹ for 10 min. The retained analytes were then eluted (there was, apparently, no washing step) with 7 mmol per L thiourea + 40 mmol per L H₃PO₄ at 1.5 mL min⁻¹, and 1 mL collected of which 100 μL was injected into the HPLC system for separation on a Kinetex EVO C18 column (4.6 × 250 mm, 5 μm particles). The mobile phase was a gradient of A (7 mmol per L thiourea + 40 mmol per L H₃PO₄) and B (0.16 mmol per L TBAH in H₃PO₄ at pH 4.5). The gradient elution program was 100% A, 0.0% B for 0.1 min, followed by a ramp of 1.2% s⁻¹ to 0.0% A, 100% B for 1.5 min and held for 18.4 min. The method was validated by the analysis of CRM NRCC TMDA 64.3 (fortified lake water) and by spike recoveries from two urine and one seawater samples. No Pb²⁺ was detected in any of the samples, but TML was found in one urine and the seawater. The LOD values were 5 and 20 ng L⁻¹ for Pb^II and TML, respectively and the enrichment factor for all species was 27.

3.10. Manganese

A selective DSPME procedure has been devised for the determination of Mn^II and Mn^VII in waters.⁹⁴ The extractant was multiwalled CNTs modified with N-acetylcysteine, 20 mg of which was dispersed in 150 mg of hydrophobic ionic liquid 1-hexyl-3-methyl-imidazolium-hexafluorophosphate ([HMIM] [PF₆]) and diluted with acetone (volume not given). An unspecified volume of this was injected into 50 mL of sample adjusted either to pH 6.5, for the extraction of Mn^II, or to pH 3 for the extraction of Mn^VII. Following UAE for 5 min, the phases were separated by centrifuging and the upper aqueous phase removed. Finally, Mn^II and Mn^VII were redissolved in 250 μL of HNO₃ and NaOH solutions (both 0.5 mol L⁻¹), respectively by shaking for 1 min. These solutions were then diluted to 0.5 mL with water and measured by a FAA spectrometer equipped with an integrated atom trap accessory. The LOD values were 0.12 and 0.14 μg L⁻¹ for Mn^II and Mn^VII, respectively. The method was validated by spike recovery from one well water, one drinking water and four wastewater samples. Both species were found in all samples, except from Mn^VII in the drinking water. The researchers also applied the procedure to the determination of total Mn in an extensive range of foodstuffs (almond, white rice, brown rice, spinach, apple leaves, beans, pineapple, carrot, broccoli, bran, red pepper, beetroot, radish, celery, sheep meat, fish and dark chocolate). The samples were prepared by MAE after which all Mn was considered converted to Mn^II. The method served to preconcentrate Mn with an enrichment factor of 97, and Mn was found in all samples. The method was validated by the analysis of five NIST SRMs. CRM 1640a (Mn in water), 1570a (spinach leaves), 1548a (typical diet), 1568b (rice flour) and 1515 (apple leaves).

3.11. Mercury

Four reviews covering Hg speciation have been published this year. The first of these, with 88 references, covers a key step in speciation analysis, sample preservation, with a focus on water samples and highlights the associated hazards and issues with each method cited.⁹⁵ One of these of course is the need to collect and transport large sample volumes due to the generally very low Hg species concentrations in waters. For acidic preservation of water samples in the field HCl is recommended, as the use of HNO₃ has been found to still be susceptible to large losses of Hg through both volatilisation and sorption to container walls, although H₂SO₄ is preferable if the sample is then to be subjected to a distillation and ethylation process. Solid phase extraction in the field is highlighted as a high-performance approach as species stability is often much better in the solid phase. The use of SPE in the field also reduces the amount of material that needs to be transported to the laboratory, minimises reagent transport which is becoming more expensive for acidic solutions if couriers are involved, and there are many different types of adsorbent commercially available. The review is recommended reading for those workers collecting water sample for Hg speciation studies and a similar review for As species preservation would be a welcome addition to the literature. Favilli et al. have reviewed (74 references) the use of HPLC-ICP-MS for Hg speciation.⁹⁶ The review includes sections on pre-concentration, HPLC separation conditions, ICP-MS conditions and also a considerable section on multi-element separations where Hg species are among the analytes. The application of PVG atomic spectroscopy for As, Se and Hg speciation in environmental matrices (95 references) has also been reviewed.³⁸ Relevant PVG conditions such as composition of the reaction medium, irradiation time, sensitisers or photocatalysers and different PVG system designs are described in detail. The review contains 3 tables presenting separation and detection methods used and the LOD values attained. Highlights include the section dedicated to future perspectives of the technique, including the analysis of biomolecules, the application of different nanomaterials as sensitisers (ZnO NPs, MOFs), the development of miniaturised reactors/devices and the application of PVG in visual colorimetric systems. Finally, Wang et al. have authored a review on Hg in natural gas and its condensate (245 references).⁹⁷ The coverage of the review includes; the different Hg species encountered and their adverse effects, sample pre-treatment, detection which is mainly by CV-AAS, CV-AFS and ICP-MS, the challenges involved and a large section on in situ removal systems.

As mentioned in the previous paragraph, the speciation of Hg in water samples can be challenging due to the extremely low concentrations present. This is particularly so for seawaters and it is timely that the uncertainty for the measurement of monomethyl Hg (MMHg) by SS-ID-GC-ICP-MS and dimethyl Hg (DMHg) and Hg⁰ by cryo-focussing GC-ICP-MS in this matrix has been assessed.⁹⁸ The concentration of DMHg was also measured by difference (termed calc in the subscripts used) as sample acidification converts DMHg to MMHg, which is then quantified, between the acidified and unacidified samples. The expanded uncertainty, U_ex,r (confidence interval of 95%) for cryo-focusing GC-ICP-MS was 14.4 (<50 fM) and 14.2% (>50 fM) and for SS-ID-GC-ICP-MS 5.6 (<50 fM) and 3.7% (>50 fM). For concentrations above 50 fM, U_ex,r for DMHgcalc was always lower than for direct measurements (14.2%). For MeHgcalc, on the other hand, U_ex,r was always higher for concentrations above 115 fM (range: 3.7–13.9%) than for direct measurements (3.7%). The authors state that the uncertainties achieved are considerably lower than those reported for other commonly used analytical techniques for Hg species in seawater. The paper gives a comprehensive description of how the work was carried out, including the extensive quality assurance protocols, and should be read by all workers in this field. Papers on DMHg measurements are not common, most likely due to the difficulty of the measurement process and also the high toxicity of the compound, but a second article on this topic has also been published this year. In this case Chen and co-workers developed a new DMHg generator which proved superior to the conventional approach.⁹⁹ Usually, DMHg is prepared via a reaction of Hg²⁺ with MeCo in a saline solution in a reaction bottle and this can also lead to the formation of other Hg species such as Hg⁰ and MeHg, with the latter favoured as reaction time increases. This necessitates a further purification which increases the risk of human exposure. The developed DMHg generator consisted of a 1.5 mL quartz reaction vial with a quartz circular-truncated-cone holder standing inside a 40.0 mL amber sample bottle. Both MeCo and Hg²⁺ were mixed in the reaction vial, in a molar ratio of 10 : 1, and the pH of this solution adjusted to 4 using H₂SO₄ and NaOH. The outer amber bottle contained an acetate buffer solution at a pH > 5. To produced DMHg was collected in this outer solution which was then sampled directly through the Teflon cap by purging onto an Au trap and Tenax trap in series, the former retains any Hg⁰ present while the latter retains the DMHg. The traps were then thermally desorbed onto a GC-AFS system for Hg species quantification. The method was then used to assess DMHg levels in natural water samples. No DMHg was detected in unspiked samples whilst spike recoveries, at 0.05, 0.2 and 1.0 ng L⁻¹, were all better than 93%. For the determination of Hg^II and MeHg (and Pb^II, TML and V^V) in seawater and urine, a SPE preconcentration was applied prior to HPLC-ICP-MS.⁹³ A previously synthesised, patented, magnetic nanomaterial based on a double coupling between GO and MNPs (M@GO) was further derivatised with the chelating group methyl thiosalicylate. The resulting M@GO-TS material (50 mg) was packed into a column mounted in the injection loop of a FI valve and sample, adjusted to pH 3.5, was loaded at 2.7 mL min⁻¹ for 10 min. The retained analytes were then eluted (there was, apparently, no washing step) with 7 mmol per L thiourea + 40 mmol per L H₃PO₄ at 1.5 mL min⁻¹, and 1 mL collected of which 100 μL was injected into the HPLC system for separation on a Kinetex EVO C18 column (4.6 × 250 mm, 5 μm particles). The mobile phase was a gradient of A (7 mmol per L thiourea + 40 mmol per L H₃PO₄) and B (0.16 mmol per L TBAH in H₃PO₄ at pH 4.5). The gradient elution program was 100% A, 0.0% B for 0.1 min, followed by a ramp of 1.2% s⁻¹ to 0.0% A, 100% B for 1.5 min held for 18.4 min. The method was validated by the analysis of CRM NRCC TMDA 64.3 (fortified lake water) and by spike recoveries from two urine and one seawater samples. The LOD was 0.4 ng L⁻¹ and the enrichment factor for all species was 27. No V^V was detected in either of the urine samples, only in the seawater. The use of M@GO for the non-chromatographic speciation of Hg in water samples, with ETAAS detection, has also been reported.¹⁰⁰ When dithizone is used as an auxiliary agent and the acidified to pH 5 both Hg^II and MeHg are retained on the M@GO and then eluted with HNO₃ (8% v/v) and H₂O₂ (30% v/v) and UAE at 80 °C for 10 minutes. If the sample pH is adjusted to 9, both species are again retained, but washing the collected M@GO with N-acetyl-L-cysteine removes MeHg only, and the retained Hg^II is then eluted as previously described with the MeHg content calculated by difference. The proposed procedure gave an enrichment factor of 49 and a linear range between 0.1 and 10 μg L⁻¹ as Hg. The method was validated using a suite of 9 CRMs, which included waters, fish tissues and human hair (the solid samples were first subjected to an acidic digestion procedure). For all CRMs for which Hg was certified the found and certified values were in good agreement. The method was then applied to drinking, river, and waste-water samples and each case no Hg species was detected. Spike recovery experiments on these sample gave recoveries of 90 to 104% for both Hg species studied.

Probably, most human Hg intake comes from the diet and thus Hg speciation in fish continues to be studied. A method for MeHg quantification in shark meat samples using HPLC-ICP-MS has been reported.¹⁰¹ The Hg species were extracted from dried homogenised shark meat with 0.5% L cysteine in 2% HNO₃ and UAE (50/60 Hz, 550 W) for 30 min at room temperature followed by centrifugation and filtration. A C₁₈ column (250 mm × 4.6 mm ID, 5 μm particle size, 175 Å pore size) was used for the separations with a gradient elution involving (A) 0.5% L cysteine in 2% HNO₃ and (B) 100% MeOH flowing at 1.0 mL min⁻¹. No mention is made of O₂ addition to the plasma gas but, as the chromatographic run was short with MeOH only being aspirated for half the runtime, maybe C deposition on the cones was not problematic. Under these conditions iHg eluted at 200 s and MeHg at 260 s. Method validation was by analysis of the NRCC DORM-2 CRM for which a MeHg recovery of 97% was obtained. The MeHg LOD is stated as 0.86 pg L⁻¹, which is very low and implies that there was minimal noise in the chromatographic baseline (or a misprint, given it is an order of magnitude or two lower than the LOD values cited from the literature). The MeHg mass fraction in the various shark meat samples ranged from 0.22 to 1.22 mg kg⁻¹ wet weight. A separate paper reports on the simultaneous speciation of Hg and Se in muscle from different fish species (rainbow trout, tuna, swordfish, and dogfish).¹⁰² An enzymatic extraction, protease in combination with L-cysteine and 2-mercaptoethanol and (NH₄)₃PO₄ buffer, by two protocols: incubation at 37 °C for 12 h in an oven, and enzymatic MAE (EMAE) for 45 and 60 min was used. Both methods were compared and validated by using three CRMs: ERM-CE101 (trout muscle), ERM-BB422 (fish muscle) and NMIJ7402 (cod fish tissue). The EMAE with 60 minutes irradiation provided the best extraction efficiency for total Se and Hg concentration in all the CRMs used with recovery values ranging from 90 to 116%. Reversed-phase and ion-exchange HPLC separation mechanisms coupled to ICP-MS were tested to simultaneously separate Se and Hg species from the extracts. The best results were attained using a Dionex Ion Pac S5A column (4 × 250 mm; 9 μm) and a mobile phase consisting of a mixture of 5% MeOH, 45 mmol L⁻¹ of HNO₃, 0.015% 2-mercaptoethanol and 1.5 mmol per L sodium-3-mercapto-1-propanesulfonate, at a flowrate of 0.8 mL min⁻¹. The application of these optimal conditions allowed the separation of 4 Se species (Se^IV, Se^VI, SeMet; SeCys) and 2 Hg species (Hg^II and MeHg) in less than 15 minutes. The developed method was fully validated using the accuracy profile approach. The analytical procedure offers LOD values from 0.010 to 0.013 mg kg⁻¹ for all Se species tested, except for Se^IV (0.15 mg kg⁻¹). The novel methodology was applied to different fishery products. Regarding Se species, Se^IV and SeCys levels were below LOQ in all the samples analysed whereas SeMet was quantified in all the analysed samples (0.011 to 0.054 mg kg⁻¹) as the most abundant Se species. The lowest Se species levels were found in fresh waters fish. The mean levels of MeHg varied between 0.026–1.77 mg kg⁻¹ and the lowest MeHg levels was found in the freshwater rainbow trout (0.026 mg kg⁻¹). Simultaneous speciation is a valuable tool to get a better understanding of Se and Hg antagonistic effect. Table 1 shows examples of other applications of Hg speciation presented in the literature during the time period covered by this ASU.

Table 1 Applications of speciation analysis: Hg

Analyte species	Technique	Matrix	Sample treatment	Separation	LOD	Validation	Reference
MeHg, tHg	GC-pyr-AFS, ICP-MS	Hair	HNO3 and UAE	Aqueous ethylation	Not given	None given	103
MeHg, tHg	CV-AAS	Garfish	MeHg: HCl and, toluene, UAE 30 min, centrifuged, back extraction into 1% L-cysteine HCl	Selective extraction	MeHg: 5 μg kg^−1	Tort-2, BCR-422, BCR-463, recoveries 99 to 1107%	104
					tHg: 0.5 μg kg^−1
MeHg, iHg	ID-GC-ICP-MS	Hair	25% TMAH, MAE, 75 W, 4.5 min. Aqueous ethylation	GC. Column not given. Multi-stage ramp	MeHg: 1.51 ng Hg per g^−1	IAEA-086. MeHg: 105% recovery	105
					iHg: 2.9 ng g^−1	tHg: 96% recovery
HgCl2, MeHgCl, HgS	TR-ETA-AAS	Pure compounds and mixtures	None. Fundamental study of technique	Direct analysis	0.20 ng	None used due to nature of study	106
MeHg, iHg, tHg	IR-VG-AAS	Waters	Hg species in solution. TMAH for CRM	Thermal in presence of formic acid and/or H2O2	0.41 ng g^−1	DORM-1, DOLT-3: recoveries 94 to 99%	107
MeHg, iHg	DBD-VG-AAS	Aqueous	None	None	MeHg and iHg: 100 μg L^−1	Not given	108
Protein bound Hg	SEC-UV-ICP-MS	Mushroom	UAE, Tris–HCl pH 7.5, PMFS (0.1 mmol L^−1), SDS (0.08%), SiO2 (50%)	SEC, 50 mmol per L NH4CH3COOH, 0.5% MeOH	7.4 ng g^−1	SRM 1571: 93% recovery	109
MeHg, iHg	HPLC-ICP-MS	Fish	L-Cysteine, HCl, H2O	C18 column, 0.1% L-cysteine·HCl·H2O	LOQ (mg kg^−1)	BCR710: 103% recovery spike recoveries	110
					MeHg 0.002	MeHg: 64 to 98%
					iHg 0.02	iHg: 46 to 101%

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3.12. Phosphorus

All of the recent papers of interest for P speciation analysis have involved HPLC separation coupled to ICP-MS detectors that were able to remove the considerable number of N containing interferences on ³¹P⁺, either by use of a SF mass analyser using medium resolution settings or tandem MS/MS using an appropriate collision cell gas e.g. oxygen. These relatively recently available approaches to interference removal allow for the development of new applications in the analysis of compounds containing this difficult to quantify mono-isotopic heteroatom.

Continuing on their studies of new developments in HPLC separations with ICP-MS detection, the research group of Goessler, based in Graz, has developed a simple one-step derivatisation procedure to enable the separation of the metabolite and biomarker phosphoethanolamine from the hydrophilic P-containing metabolome present in urine.¹¹¹ In particular, phosphoethanolamine has been reported as a reliable biomarker with consistently elevated urinary levels in hypophosphatasia, an inherited metabolic bone disorder. It was possible to elute the hydrophobic derivative using 1,2-hexanediol, a novel chromatographic eluent developed previously by the group and discussed in the 2023 ASU review.¹¹² The developed method features fast chromatographic separation in less than 5 min, no need for an isotopically labelled internal standard, and an instrumental LOD of 0.5 μg P per L. The method was evaluated for recovery (90–110%), repeatability (RSD ±5%), and linear correlation coefficient, R² (0.9998). The method accuracy was established by comparison with an independently developed method based on LC-ES-MS/MS without derivatisation, where agreement was found to be within ± 5 to 20%.

With the discovery of chemically reduced P-species in samples from environmental water sources, a new method has been reported for phosphate, phosphite and hypophosphite, which have now become the focus of environmental P-speciation studies. The use of a conventional IC system with a conductivity detector coupled to SF-ICP-MS in medium resolution mode has been developed,¹¹³ utilising a small clean-up column included as part of a 2 valve injection system. Commonly, IC has been used for quantifying the concentration of different species of the same element such as nitrate and nitrite, bromide and bromate, chloride and perchlorate in natural and experimental solutions. Whilst Cl ions do not interfere isobarically or by the formation of polyatomic ions with ³¹P in ICP-MS, the removal of Cl⁻ improves ionic separation and helps protect the instrument from the formation of deposits on the interface cones. Using this clean-up approach, Cl was fully removed from the samples while phosphite was retained and could be analysed by IC-ICP-MS. Concentrations of phosphite were measured down to 0.003 μmol L⁻¹ with acceptable linearity up to 1.615 μmol L⁻¹ with R² = 0.999. The clean-up column on valve 2 was an AG11-HC AE column (4 × 50 mm), and the remaining anions were separated on the IC system using an AG17-C guard column, an AS17-C AEC, an ADRS 600 2 mm suppressor and a conductivity detector. The ICP-MS instrument was coupled after the conductivity detector in the system. The eluent used was KOH, and its concentration was ramped from 1 to 40 mmol L⁻¹ over the course of each run. For the analysis of pyrophosphate the run time was 55 min. Standalone software was required for data analysis and Origin Lab software was used for smoothing the very noisy data, subtracting background levels and calculating peak areas. The method was tested with a series of standards containing 0.003 to 1.614 μmol per L phosphite (corresponding to 0.1 to 50 μg per L P). However, the clean-up system introduced a carbonate, a more significant interference than the Cl ion being removed, which also eluted much closer to the phosphite peak than the Cl ion, so the chromatograms using the chloride-removal method displayed a larger carbonate peak than those generated in normal mode. The authors indicated that this additional interference peak could be removed with further development. Organophosphorus pesticides such as glyphosate and glufosinate are now widely used, and environmental regulatory limits are being adopted in many countries due to their potential toxicity. This requires the development of robust analytical methods¹¹⁴ to determine these compounds at suitable levels in the environmental matrices of concern. A pretreatment-free analytical method was established in which these two compounds and their metabolites were separated by AE HPLC using NH₄CH₃CO₂ (70 mmol L⁻¹, pH 3.7) as eluent, and detection by ICP-MS/MS. Detection limits of 0.03–0.17 μg L⁻¹ were established by measurement of P as PO⁺ using O₂ as the collision cell reaction gas. Quantitative recovery was obtained from the spike-recovery test on river water samples containing phosphate ion as an isobaric interferent. In addition, a constant sensitivity per molar concentration was achieved regardless of the compound, due to the powerful ICP-MS ion source. This property suggests that semi-quantitative analysis of unknown P-bearing compounds is possible from a single calibration curve. This demonstrates once again the power of the new ICP-MS/MS collision cell technology for the quantification of P- and S-containing compounds.

3.13. Platinum

The chemical degradation of Pt-containing cancer treatment drugs in urine and the investigation of waste water contamination by cisplatin and carboplatin were studied using HPLC-ICP-MS.¹¹⁵ An understanding of the prevalent forms of Pt-based drug treatments is important both clinically and for developing environmental remediation strategies that reduce contamination of water systems. A prospective study was used to monitor the degradation of Pt-containing antineoplastic drugs in the urine of two patients undergoing chemotherapy. At regular intervals, urine samples were collected from cancer patients receiving treatment with cisplatin or carboplatin. The speciation of Pt-based compounds was measured in patient urine with a HILIC separation using a Discovery HS F5 column (150 mm × 2.1 μm) coupled to ICP-MS detection and elution at 250 μL min⁻¹ with 10 mmol per L NH₄CH₃CO₂ and MeOH (2% v/v). Detection was by ICP-MS by monitoring the ¹⁹⁵Pt isotope. The LOD values for each of the Pt-containing compounds measured in the study were: cisplatin 1.1 mmol per L Pt; mono aqua cisplatin 0.7 mmol per L Pt; carboplatin 0.4 mmol per L Pt; and oxaliplatin 0.2 mmol per L Pt. Following urinary excretion, oxaliplatin and cisplatin exhibit a relatively fast degradation of the original compound into several by-products, while carboplatin remained more stable in urine. As a result, the chemical in vitro stability was highest for carboplatin, while oxaliplatin and cisplatin rapidly degraded following an exponential decay curve. The kinetic parameters determined using this method could serve as input to further expand modelling databases and improve the predictive power of speciation software to estimate ecotoxicity risks. Considering the strong residual cytotoxicity of the Pt compounds studied, after renal clearance and human excretion, the contaminants are of high environmental concern, particularly carboplatin because of its greater persistence in the aquatic environment.

The formation of Pt-DNA adducts under simulated physiological conditions has been studied with the aid of CE-ICP-MS/MS.¹¹⁶ This methodology offers the added advantage over the widely used HPLC-ICP-MS approach of higher chromatographic resolution, which is useful when measuring these compounds in complex biological matrices. A drawback to using CE separations is the very small injection volume, which limits the amount of compound available for detection, but in this work it is less of an issue because of the high sensitivity of the ICP-MS detector for Pt. The selected model system for studying DNA platination and formation of DNA-adducts within this work consisted of a stable succinimide derivative of oxaliplatin and an 8-mer DNA oligonucleotide containing two potential binding sites. Both P and S were monitored at ³¹P¹⁶O⁺ and ³²S¹⁶O⁺ using O₂ as a reaction gas in the collision cell, which allowed for the investigation of drug binding to DNA and also proteins such as GSH. The drug itself was monitored on-mass at ¹⁹⁵Pt⁺. Germanium was used as an internal standard by addition to the background electrolyte, which also served as a sheath liquid. The sheath liquid flow was monitored to control the hyphenation stability and nebulisation efficiency and the Ge response was used for the normalisation of results obtained during the analysis.

3.14. Rare earth elements

A novel separation and detection system, based on HILIC chromatography and simultaneous detection by ESI-MS and ICP-MS, has been used to investigate various aspects of the interaction of actinide elements with biological and other systems. To facilitate these investigations, lanthanide elements have been used as model elements to mimic highly radioactive elements, such as Am, Cm and Np. These studies came about so that the developed analytical methods allowed the rapid determination of the selectivity and the affinity of the molecules used to isolate highly radioactive elements, while reducing the consumption of materials, effluent volumes and the time devoted to the screening experiments. This approach and analytical work flow is clearly of great importance in the nuclear reprocessing industry. To this end, a study on the binding of radionuclides with different model ligands,¹¹⁷ has produced a useful reference work, investigating a wide ranging assortment of HILIC columns (21 in total) of varying dimensions, including standard, nano and capillary (2.1, 0.3 and 0.075 mm id, respectively), with different types of mainly silica support phases for different functional stationary phases (7 in total). Specific isotope dilution was also used as a method of quantification for the determination of the affinity and selectivity of three structurally complex chelating molecules towards lanthanides used as model non-radioactive elements.

3.15. Selenium

Two reviews on Se have appeared this year. An interesting paper compiles and critically evaluates extraction protocols (aqueous, alkaline, and enzymatic) and measurement techniques (HPLC-MS/MS, HPLC-ICP-MS, GC-ICP-MS and AFS) for determining plant selenium-enriched proteins and plant selenium-enriched peptides.¹¹⁸ The paper includes tables with relevant information about different extraction protocols. Of special interest are sections devoted to bioaccessibility, bioavailability and bioactivity of Se-enriched protein/peptide extracts by means of in vitro gastrointestinal digestion procedures, cell lines and in vivo models. The paper dedicates a final section to the exploitation and utilisation of Se-enriched proteins/peptides as ingredients to prepare Se-enriched functional foods and supplements. The application of photochemical vapor generation (PVG) atomic spectroscopy for As, Se and Hg speciation in environmental matrices has been reviewed.³⁸ Relevant PVG conditions such as composition of the reaction medium, irradiation time, sensitisers or photocatalysers and different PVG system designs are described in detail. The review contains 3 tables presenting separation and detection methods used and LOD values attained for the analysis of Se, As and Hg. It is worth highlighting the section dedicated to future perspectives of the technique, including the analysis of biomolecules, the application of different nanomaterials as sensitisers (ZnO NPs, MOFs), the development of miniaturised reactors/devices and the application of PVG in visual colorimetric systems. The review includes 95 references.

The elucidation of Se metabolisms in Se-fortified plants, mushrooms and bacteria remains a topic of interest for researchers. In an interesting paper Se speciation and volatile flavour compound determination is carried out on edible flowers, stems and leaves of Cardamine violifolia, a Se-hyperaccumulating plant from China. Selenium species from C. violifolia were isolated by using UAE enzymatic hydrolysis with protease at 37 °C.¹¹⁹ Extracts were subsequently analysed by HPLC-ICP-MS by using a Zorbax SB-Ag (250 mm × 4.6 mm, 5 μm) column and a mobile phase consisted of 5 mol per L sodium 1-hexanesulfonate and 10 mol per L citric acid in 2% MeOH at pH 7.0, flowing at 1.0 mL min⁻¹. The content of Se was found to be significantly higher in flowers (543 ± 14 mg kg⁻¹) than in leaves and stems (314 ± 23 mg kg⁻¹ and 251 ± 3, respectively). Organic Se accounts for more than 98% of total Se content, primarily SeCys₂ followed by SeMet. However, the results lack a proper and unambiguous identification of Se species as Se species were identified by spiking with standards instead of using molecular mass spectrometry. A comparative study in terms of Se accumulation and speciation was performed in two types of mushrooms, Pleurotus ostreatus and Ganoderma lucidum, enriched with Se^IV and Se^VI.¹²⁰ Two sample treatment procedures were applied: (1) water extraction by mechanical shaking in a water-bath at 37 °C for 20 h and (2) enzymatic extraction with protease at pH 5.0 in a water-bath at 37 °C for 24 h. Extraction efficiency was determined with a benchtop TXRF analyser. Enzymatic hydrolysis was more effective than aqueous extraction (109 versus 79%) when applied to G. lucidum whereas no significant differences between the two sample treatments were detected for P. ostreatus. Analysis by HPLC-ICP-MS/MS using O₂ as reaction gas, a YMC Triart C18 (150 mm × 2.1 mm; 3 μm) column, and a mobile phase of 0.3% (v/v) heptafluorobutylamin acid in 3% MeOH at pH 4.0 revealed SeMet and iSe as the major Se species generated after iSe fortification in P. ostreatus and G. lucidum, respectively. Surprisingly, extracts for both mushrooms were shown to be effective in enhancing Se proteins expression (such as SelP and GPx3) in HepG 2 cells line, with the highest values when applying those extracts derived from enzymatic hydrolysis. In another study¹²¹ the effect of SO₄²⁻ on the absorption and metabolisms of Se^IV in Brassica napus was evaluated by using enzymatic hydrolysis with protease followed by separation on a Hamilton PRPX-100 (4.1 mm × 250 mm; 10 μm) column and ICP-MS detection. Low levels of SO₄²⁻ in the SeO₃²⁻ selenite containing hydroponic solution did not affect the Se absorption capacity of root system but it greatly enhanced the root-to-shoot transport efficiency of Se, leading to a substantial increase of Se concentration in shoot. Selenium speciation analysis revealed a high proportion of SeMet and Se^VI in shoot and roots whereas SeMeCys was the major Se organic species in xylem sap where only a small amount of SeMet was detected. The authors hypothesised that most SeMet accumulated in roots was transported to shoots as the easily transportable forms Se^VI and MeSeCys and then transformed, via selenide, into SeMet. Analysis by qPCR of SeO₃²⁻ absorption related genes evidenced the presence of upregulated genes with both Se^IV addition and S deficiency, supporting the idea that S-deficient conditions favours SeMet biosynthesis. Twelve extractions protocols have been evaluated for isolating Se species from Se-enriched Ganoderma Lucidum (246 ± 53 mg Se per g) before LC-UV-AFS measurements with separation using a PRP X-100 column.¹²² The sample treatments tested included the use of buffer solutions alone (deionised water, Tris–HCl buffer, pH 2.1; phosphate buffer, pH 7.5 and glycine–HCl buffer, pH 2.1) or in combination with different enzymes (trypsin, protease XIV, protease K, pepsin). The use of buffer solutions alone provided extraction efficiencies of less than 10% whereas the combination with enzymes increased extraction efficiencies up to 65%, with the highest value attained by a mixture composed of an aqueous solution of pepsin and trypsin. Speciation revealed Se^IV (63%), SeCys₂ (10%) MeSeCys (15%) and SeMet (1.5%) as the major Se species present. Although the study contains valuable information as it compares a plethora of sample treatments protocols for Se isolation and includes tables with reported data on Se analytical methodologies, the study fails when performing Se identification as ES/MS–MS is needed to correctly assign Se species structure. Selenium speciation in Se-enriched wheat supplemented with Se^IV and Se^VI and a mixture of both was carried out by using HPLC-ICP-MS and XAS techniques.¹²³ For HPLC-ICP-MS measurements, wheat samples were subjected to enzymatic hydrolysis with protease at 37 °C for 16 hours and Se-species were then separated by a PRP X-100 column (250 × 4.1 mm, 10 μm) under gradient elution between 5 to 15 mmol per L NH₄ citrate with 2% MeOH at pH 5.0. Speciation by XAS was performed without the need for any sample treatment. The authors detected 5 major Se species (SeMet, MeSeCys, SeCys, Se^IV and Se^VI) that were identified by spiking with commercial standards. The XAS measurements allowed confirmation of the presence of Se⁰ and C–Se–C and C–Se–Se–C forms. Treatment with Se^IV produced mainly C–Se–C forms while Se^VI enhanced the production of C–Se–Se–C. The novelty of the paper lies in the study of the effect of Se supplementation on the levels of phytohormones implicated in signalling pathways. Although a clear tendency could not be established, it seems to be that the symptoms of toxicity observed in the plants were due to Se exposures, excluding the effects of other stresses during plant growth. The use of soil amendments to increase adsorption of Se^IV and Se^VI by using XAFS and XRF spectroscopies has been reported on.¹²⁴ Analyses using XAFS proved particularly useful for studying sorption mechanisms of Se and for determining variation of its oxidation state. The incorporation of Ca-modified zeolite in surface soils representative of Mediterranean agrosystems notably improved Se^IV availability and favoured the retention of Se^VI by creating adsorption sites. Zhu et al.¹²⁵ evaluated the influence of a fermentation process on SeO₃²⁻ metabolisms by the probiotic Bifidobacterium logum. Parameters affecting Se incorporation, such as level of Se^IV (18–400 μg mL⁻¹), feeding time (12, 16 and 24 h) and fermentation stage are described in detail. Specific methodologies were applied for each measured parameter. Total Se content and its distribution between water soluble and water insoluble fraction was achieved by acid-wet digestion and UAE with water followed by ICP-MS measurements, respectively. Selenium species in the whole bacteria culture and water extracts were determined by applying an enzymatic protocol with protease and β-mercaptoethanol and HPLC-ICP-MS analysis by means of a PRP X-100 column under gradient elution using a mobile phase composed of triethylamine and HAc. Se-metabolites in the culture medium after fermentation were determined by SEC-ICP-MS analysis along with collection of the Se-containing peaks and analysis by HPLC-ES-Orbitrap-MS by using a C18 column with a mobile phase composed of formic acid in ACN under gradient elution. The results revealed SeMet as the most abundant species in the bacteria and γ-Glu-SeMet as the predominant Se-metabolite. A negative correlation was detected between the production of SeMet and γ-Glu-SeMet, suggesting that the synthesis of Se-containing proteins could be enhanced by suppressing the production of γ-Glu-SeMet. The greatest Se accumulation and SeMet production was obtained when fermentation conditions were set at 18 mg per mL Se^IV and 16 h of feeding time.

The metabolism of Se in animals and humans has been the subject of different papers. Two parallel studies have been carried out by Bierla et al. to evaluate Se metabolisms in rats fed with different levels of Se (0–5 μg Se per g) as SeO₃²⁻ in one paper¹²⁶ and as SeMet in the second.¹²⁷ After 28 days of feeding, rats were anesthetised and livers were removed and analysed by using HPLC-ICP-MS and molecular mass spectrometry (Orbitrap MS/MS) Depending on the information required, specific methodologies were developed: (a) ICP-MS measurements with acid digestion to determine total Se in livers; (b) SEC-ICP-MS analysis to obtain the profile of Se-containing biomolecules from aqueous liver extracts. For this purpose, an Acquity UPLS protein BEH SEC (125 A, 1.7 μm 4.6 × 150 mm) column with an exclusion range of 1000–80000 Da was employed and NH₄CH₃CO₂, pH 7.5, as the mobile phase and (c) HPLC-ICP-MS and Orbitrap MS/MS measurements to determine and identify selenoamino acids and selenometabolites in liver extracts obtained by applying enzymatic hydrolysis and a carbamidomethylation process. Analysis of the aqueous liver extracts by SEC-ICP-MS revealed the presence of identical HMM and LMM Se species profiles, regardless of either Se^IV or SeMet supplementation. Speciation analysis of LMM (<10 kDa) of the water-soluble liver extracts by HPLC-ICP-MS and Orbitrap MS/MS identified SeMet and selenosugars, such as acetyl galactosamine conjugated to GSH (GSSeGalNac) and methylacetyl galactosamine (CH₃-SeGalNac), and derivatives of the selenogluthatione (GS SeSG and CH₃SeSG) in both Se^IV and SeMet feeding rats. There was an absence of SeMet in rats supplemented with Se^IV. The amount of selenosugars and SeMet produced was dependent on the level of selenite and SeMet supplied. When SeMet was supplemented at a level below Se requirements (0 to 0.08 μg Se per g) no LMM compounds were detected, including SeMet. At Se adequate levels (0.24 μg Se per g) and above SeMet accounted for only 10% of LMM species detected whereas methyl and glutathionyl selenosugars accounted 70% of the LMM species. Above normal Se requirements, SeMet was the predominant species. In relation to Se^IV supplementation, from 0 to 0.08 μg Se per g Se seems to be incorporated into SeCys-selenomolecules, in Se adequate-rats (0.24 μg Se per g) Se was present as SeCys but 32% was in the form of selenosugars and 22% as inorganic Se bound to proteins. In liver of rats fed with 5 μg Se per g, 22% of Se was present as selenosugars and selenosugars bound to proteins but 64% was present as inorganic Se bound to proteins. The results obtained highlighted the role of selenosugars in SeO₃²⁻ toxicity due to the excessive accumulation of selenosugars linked to cysteines on proteins. The study also indicated that iSe is not metabolised to SeMet in rats. Recognition of Se species is still a problematic part of Se speciation and in a third paper Bierla et al.¹²⁸ develop a software tool to assist in isotopologue pattern based mining for Se species in combination with a HILIC-ES-HR-MS set up. This approach was applied for samples containing a high-water soluble Se content such as Torula yeast and the hyperaccumulating plant Cardamine violifolia. These organisms are of interest as they can produce important Se compounds involved in Se detoxification processes such as selenohomolanthionine (Torula) and selenolanthionine (C. violifolia) along with other minor species involved in the metabolomic pathways. The developed software was evaluated in terms of repeatability and Se isotope selection. The application of this tool enabled identification of 88% of the Se species found from yeast and plant samples. The metabolism of selenoneine, a Se metabolite connected with Hg detoxification processes, was evaluated by using mice as in vivo model.¹²⁹ Selenoneine was biosynthesised using the genetically modified fission yeast strain FY2530 cultured in the presence of ⁷⁶Se^VI. The resulting ⁷⁶Se-selenoneine at a concentration level of 100 mmol per L Se was administered to six male 5 weeks old mice via drinking water for 6 days. Pooled urine samples were collected every 24 h. After 6 days the mice were sacrificed, and blood and tissue samples collected. Selenoneine and Se-methylselenoneine were analysed for using LC-ICP-MS by means of a Ultrahydrogel-120 (7.8 × 300 mm) column and a NH₄Ac solution containing 0.1% IGEPAL™ as mobile phase flowing at 1 mL min⁻¹. Using these conditions, ⁷⁶Se-selenoneine eluted at a retention time of 9 minutes. Selenoneine and Se-methylselenoneine, were also properly identified by LC-PAD-High resolution mass spectrometry. The results showed the presence of Se-methylselenoneine in urine and selenoneine distributed in liver and kidney with Se mass fractions of 16.1 ± 3.3 and 13.3 ± 1.9 nmol g⁻¹, respectively. Se-methylselenoneine was also detected in liver and kidney but at a concentration below the LOQ value. These results seem to indicate that selenoneine is methylated in liver and kidney and then excreted through the urine via a unique metabolic pathway. Finally, the relationship between Se and cardiovascular diseases was evaluated within the context of a 10 years epidemiological prospective study (ATTICA) conducted in the Attica region (Greece) from May 2001 to December 2012.¹³⁰ Participants (400) were randomly selected to measure Se and distribution of the plasma selenoproteins (GPx3, SelAl and SelP). Plasma selenoproteins were simultaneously measured by employing a wide implemented protocol consisting of the use of 2 chromatographic affinity columns (Heparin Sepharose, HEP, and blue Sepharose; BLUE) coupled with ICP-MS. Selenium results obtained were correlated with parameters such as sex, age, obesity status, dietary habits of participants, cholesterol, and glucose levels, among others. The mean serum Se found (91 μg L⁻¹) agreed to the values found for European population (50 to 90 μg L⁻¹) but was lower than the mean reported for US population (124 μg L⁻¹). The highest concentration Se protein was SelP followed by GPx3 and SeAlb. An aspect that deserves attention from the study is the higher cardiovascular risk observed in those individuals with the high total Se but with the lowest GPx3 levels. In this sense the European Society for Clinical Nutrition and Metabolisms has recently recommended GPx3 as a marker of functional Se status. The main strength of this study is to consider variables as sex and health status, but unfortunately the prospective character of the study cannot permit the identification of proper relationships.

Several studies have reported the role of Se against Hg toxicity in mushrooms and fishery products. The antagonist effect between Se and Hg includes the formation of SeHg compounds, demethylation of methylmercury and variation of Hg distribution, among others. In this line, Pleurotus mushrooms were cultivated on Hg-contaminated substrate as Hg^II and simultaneously exposed to Se^IV and Se^VI under different levels of supplementation.¹⁰⁹ After a growth period (14–18 days), mushrooms were collected, freeze-dried and submitted to different analytical protocols: (a) acidic digestion followed by ICP-MS for total Hg and Se determination; (b) protein extraction by using a Tris–HCl solution at pH 7.5 containing PMFS (0.1 mmol L⁻¹), SDS (0.08%) and SiO₂ (50%) prior to SEC-UV-ICP-MS analysis for Se and Hg bound to proteins determination and (c) L-cysteine (0.5 mol L⁻¹) and formic acid as extractant solution with subsequent analysis by HPLC-ICP-MS for Hg^II and MeHg determination. The Hg species separation was performed on a Premier C18 (4.6 × 150 mm, 5 μm) column using 5 mol per L L-cysteine, 25 mmol per L NH₄CH₃CO₂, 5% MeOH (pH 5.0) as mobile phase flowing at 1 mL min⁻¹. The protective effect of Se was evidenced through the reduction of total Hg accumulated by the mushrooms. It was found that Se^IV was more effective than Se^VI in reducing Hg uptake, decreasing the total Hg concentration by up to 96%. The presence of Se^IV also reduced the fraction of Hg-bound to MMW compounds (17–44 kDa) by up to 80%. The results on Hg speciation also supported the antagonistic relationship between Se^IV and Hg in terms of decreasing Hg methylation, thus decreasing MeHg content in exposed mushrooms by up to almost 100%. Two papers report on Hg and Se speciation in fish and fish-derived products. Fernández-Bautista et al.¹¹⁰ determined Se and Hg contents, Se : Hg ratios, Se and Hg species and Selenium Health Benefits values (HBV_Se) in fish fillet (tuna, farmed and wild salmon), fish-derived products (fish roe such as lump fish roe, trout roe and salmon roe) and ultra-processed products made from fish (crab sticks, surimi-derived elders, cod noodles and salmon noodles) acquired in local supermarkets. Information of total Se concentration was acquired by acidic MAE followed by ICP-MS measurements whereas for Hg determination a direct Hg analyser was used without applying any sample treatment. Nowadays, is widely accepted that Se : Hg ratios and HBV_Se values provides more realistic information in terms of Hg toxicity than just presenting contents of both elements separately as HBV_Se refers to the remaining Se content available once part of it has been sequestered by Hg through Se : Hg interaction. Positive values of HBV_Se indicate that fish and fish-derived product consumption does not entail any risk for Hg toxicity. Total Se and Hg content in the samples analysed varied from 0.77 to 4.4 mg kg⁻¹ and from 0.030 to 0.75 mg kg⁻¹, respectively. Positive values of HBV_Se and Se : Hg molar ratios greater than 1 were obtained, therefore moderate consumption of these products seems not to have a negative impact on human health. The highest HBV_Se value was observed for fish roe samples as they present relatively elevated concentration of Se (21 mg kg⁻¹) but low levels of Hg (0.030–0.048 mg kg⁻¹). Conversely, tuna exhibited one of the highest Hg concentrations (0.75 mg kg⁻¹) but HBV_Se value remains positive due to its high Se content (1.6 mg kg⁻¹). Selenium species were isolated from the samples by enzymatic hydrolysis using protease and 30 mmol L⁻¹ Tris–HCl followed by incubation during 24 h at 37 °C in a thermostatic bath. Selenium speciation analysis in the extracts by HPL-ICP-MS and HPLC-ES-MS/MS (Phenomenex Kinetex EVO C18 column. 150 × 3 mm, 5 μm) revealed SeMet and MeSeCys as the major Se species present in fish and fish-derived products. Mercury speciation studies by extraction with an L-cysteine/HCl/H₂O solution followed by separation on the same column and ICP-MS detection evidenced MeHg as the major Hg form found in fish and fish-derived products. A different paper¹⁰² reports the single-run simultaneous speciation of Hg and Se in muscle from different fish species (rainbow trout, tuna, swordfish, and dogfish). Enzymatic extraction by using protease in combination with L-cysteine and 2-mercaptoethanol and (NH₄)₃PO₄ buffer was by two protocols: incubation at 37 °C for 12 h in an oven, and enzymatic MAE (EMAE) for 45 and 60 min. Both methods were compared and validated by using three CRMs: ERM-CE101 (trout muscle), ERM-BB422 (fish muscle) and NMIJ7402 (cod fish tissue). The EMAE with 60 minutes irradiation provided the best extraction efficiency for total Se and Hg concentration in all the CRMs used with recovery values ranging from 90 to 116%. Reversed-phase and ion-exchange HPLC separation mechanisms coupled to ICP-MS were tested to simultaneously separate Se and Hg species from the extracts. The best results were attained by using a Dionex Ion Pac S5A column (4 × 250 mm; 9 μm) and a mobile phase consisting of a mixture of 5% MeOH, 45 mmol L⁻¹ of HNO₃, 0.015% 2-mercaptoethanol and 1.5 mmol L⁻¹ sodium-3-mercapto-1-propanesulfonate, at a flowrate of 0.8 mL min⁻¹. The application of these optimal conditions allowed the separation of four Se species (Se^IV, Se^VI, SeMet; SeCys) and two Hg species (Hg^II and MeHg) in less than 15 minutes. The developed method was fully validated using the accuracy profile approach. The analytical procedure offers LOD values from 0.010 to 0.013 mg kg⁻¹ for all Se species tested, except for Se^IV (0.15 mg kg⁻¹). The novel methodology was applied to different fishery products. Regarding Se species, Se^IV and SeCys levels were below LOQ in all the samples analysed whereas SeMet was quantified in all the analysed samples (0.011 to 0.054 mg kg⁻¹) as the most abundant Se species. The lowest Se species levels were found in fresh waters fish. The mean levels of MeHg varied between 0.026–1.77 mg kg⁻¹ and the lowest MeHg levels was found in the freshwater rainbow trout (0.026 mg kg⁻¹). Simultaneous speciation is a valuable tool to get a better understanding of Se and Hg antagonistic effect.

A number of researchers have focussed on Se NPs, specifically those aspects referring to characterising biogenically synthesised Se NPs. The metabolic transformation of Se NPs and Se^IV by Escherichia coli and Staphylococcus aureus was evaluated by HPLC-ICP-MS, HPLC-ES-MS/MS and spICP-MS.¹³¹ Bacteria were cultured in the presence of in-house synthesised chitosan-modified Se NPs (Ch-SeNPs) and Se^IV at 0, 1, 2 mg L⁻¹ over 72 h. Special attention was paid to the isolation of Se NPs from the bacteria cell cultures. Two procedures to disrupt the bacteria cell wall before treatment with protease were employed: enzymatic disruption by employing lysozyme and TAE buffer (for E. coli as a Gram-negative bacteria) or Tris–HCl buffer (for S. aureus as a Gram-Positive bacteria), and mechanical disruption with glass beads. The former method reported the best results in terms of extraction efficiency (>96%), Se-containing protein breakdown, artifact formation and chromatographic resolution. The major Se species identified in both bacteria was SeCys, representing 80% of the total Se accumulated, regardless of the form of Se supplementation. The confirmation of the identity of SeCys species was achieved by performing a carbamidomethylation protocol prior to enzymatic hydrolysis and HPLC-ES-MS/MS measurements. Biogenic SeNPs produced when bacteria were exposed to SO₃²⁻ were characterised by sNP-ICP-MS. Mechanical disruption was used to isolate SeNPs from bacteria culture as this provided an extraction efficiency of around 80% for short treatment times. The extracts were further diluted and measured by sp-ICP-MS by monitoring the ⁸⁰Se isotope with H₂ as a reaction gas and a dwell time of 3 ms. A disagreement between TEM and sNP-ICP-MS for the size distribution results was observed. The dissimilarity was attributed to the different ionisation efficiency of dissolved and NP Se. Once the Se ionisation efficiencies were corrected for a good agreement between results from TEM and sNP-ICP-MS was obtained with a size distribution for biogenic Se NPs ranging from 150 to 350 nm in E. coli and from 140 to 300 nm in S. aureus. A useful article¹³² has critically evaluated the effectiveness of different sample treatments to determine biogenic Se NPs, produced by yeast, by sNP-ICP-MS. Enzymatic hydrolysis with driselase in a 4-step approach, mechanical cell lysis with different types of beads (glass, metal, sand and glass and sand mixture) and chemical procedures by applying basic media (NaOH, TMAH) and an anionic surfactant (SDS) were considered. The extracts obtained from each treatment were further analysed by sNP-ICP-MS at a dwell time of 100 μs and the ⁸⁰Se isotope was monitored with H₂ as a reaction gas. The transport efficiency (2.4 and 4.4%) was estimated using both the particle size and frequency methods for Se NP size and number concentration, respectively and a LOD_size of 18 nm was attained. The samples evaluated were a baker's yeast incubated in house with SO₃²⁻, a commercially available Se-enriched yeast and a commercial suspension of 150 nm Se NPs, the latter for optimisation purposes. A significant number of pulses due to Se NPs were detected in Se-enriched baker's yeast. No Se NPs were detected during analysis of the CRM SEL-M1 extracts, possibly due to any present being at a size below the LOD. The size distribution was highly affected by the sample treatment protocol applied and exposure time of the yeast to Se^IV. Mechanical lysis resulted in narrower and more symmetrical size distribution that the enzymatic treatment, possibly due to the enzymatic modification of the Se NPs. Enzymatic hydrolysis gave the highest efficiency in terms of mass recovery whereas the mechanical procedures provided a more realistic size distribution. Chemical procedures were not recommended by authors due to the partial dissolution of SeNPs. In the last study covered in this section, Se NPs synthesised by SO₃²⁻ fortified mushrooms were characterised by sNP-ICP-MS.¹³³ A mechanical lysis protocol with 500 mm diameter glass beads was selected to isolate SeNPs. The resulting fungi lysates were centrifuged, and the supernatants collected and measured by sNP-ICP-MS/MS with oxygen as a reaction gas and a dwell time of 5 ms. Transport efficiency was calculated by using AuNPs according to the particle frequency method. To ensure a complete lysis of the material, P was monitored as being considered as a constitutive element in cells. The absence of ³¹P¹⁶O⁺ in the sNP-ICP-MS particleogram was used by the authors to check for degradation of mushroom cells. Individual events on the background signal corresponding to Se NPs were detected in sNP-ICP-MS analysis of mycelium extracts whereas the fruiting bodies did not reveal any evidence of Se NPs, which is likely to be due to the high metabolic activity of this part of the mushroom. Mean particle diameters of 90–120 nm were observed with size distribution of 60–250 nm.

The speciation of Se in environmental samples has also been reported on this year. Determining the Se concentration and speciation in seawaters is challenging due to the low levels of Se (<1 ng L⁻¹) and the interferences present in ICP-MS. To solve this, the coupling GC-ICP-MS has attracted the attention of 2 reports. One reports on the development of ss-IDA and a large volume injection with a programmed temperature vaporisation (PVT)-GC-ICP-MS to determine Se^IV, Se^VI, organic Se^−II + Se⁰, and total Se.¹³⁴ The paper presents a detailed evaluation of different derivatisation reagents for Se^IV, including 3 tetralkylborate compounds (NaBET₄, NaBPr₄, Bu₄NH₄BbU₄) as well as 4 different phenylenediamine compounds, and derivatisation parameters such as pH, solvent (toluene, hexane, isooctane) and reagent to sample ratio were also considered. Derivatised Se compounds were then analysed by GC-ICP-MS. The GC was equipped with a J & W HP-5 column (30 m; id 0.32 mm, 0.25 film thickness). The GC inlet was operated with 20% O₂ in Ar to limit the introduction of the organic matrix. The best results were achieved using phenylenediamine compounds, in particular 4-chlor-1,2-phenylendiamin (4CloP, 97%) and 3,5-bis(trifluoromethyl)-1,2-phenylenediamine (3,5-TFMoP) and toluene as solvent. The use of toluene facilitates the application of higher injection temperatures, thus decreasing analysis time. Specific pre-treatment protocols were applied for the different Se chemical forms. Analysis for Se^IV was direct whilst a pre-reduction step of Se^IV to Se^VI with 6 mol per L HCl was employed to determine total iSe, and a MAE with H₂O₂ followed by reduction with 6 mol per L HCl was used to determine total Se concentration. The organic Se^−II and Se⁰ fraction was calculated as the difference between total Se concentration and iSe. This analytical approach offered LOD values of 0.1 pg for Se^IV and 0.2 pg for Se^VI and Se^VI, and a method detection limit (MDL) of 0.9 ng L⁻¹ for Se^IV, 1.9 ng L⁻¹ for Se^IV and Se^VI and 3.1 ng L⁻¹ for total Se. The authors emphasised the importance of the LOD values obtained as the lowest reported in the literature for seawater and other matrices using GC coupled to a detection system. The accuracy of the method was evaluated by using CRMs from sea and freshwater, unfortunately only the freshwater CRM NIST 1643f was certified for Se for which a recovery of 102% was obtained. The low LOD values achieved allowed quantification of Se at very low concentrations in 168 waters collected in the Baltic and North seas. The Se^IV detected in most of the samples ranged from 0.9 and 19.7 ng L⁻¹. For Se^VI 67 out of 168 samples were found below the MDL and Se^VI values ranged from 1.97 to 73.2 ng L⁻¹ in the remainder. A great variability in the reduced Se^−II and Se⁰ fraction, covering 3.1 to 164.5 ng L⁻¹, was observed. The coupling of GC to ICP-MS has been also used to determine emissions of volatile organic Br, I, S and Se compounds from aquatic systems into the atmosphere.⁸⁶ The volatile species were collected onto commercially available sorbent tubes and subsequently analysed by thermal desorption (TD) GC-ICP-MS. The trapping capacity of the sorbent tubes was first determined by directly loading standards of the targeted species (DMS, CHBr₃, CH₂Br₂, CH₃I, CS, CHBr₂, CHBrCl, C₂H₅I, C₃H₇I, CH₂ICl, CH₂I₂). Sorbent tubes containing a graphitised carbon material and Tenax provided the best trapping capacity for the volatile species analysed for. The retained volatile species were stable at −20 °C for at least 28 days, which allows for long field campaigns. Water samples were collected in 5 L Niskin bottles, which were degassed by purging with the sparge gas exiting through a PTFE tube connected to a U-shaped column immersed in ice with the sorbent tubes connected to the U column outlet. The volatile compounds were purged for 25 minutes with N₂ as a flowrate set at 400 mL min⁻¹. In the case of air samples, an automated device including a sampling pump with a variable flowrate was used to sample atmospheric volatile species. A tube desorption time of 2 min at 200 °C and a N₂ flowrate of 25 mL min⁻¹ was chosen as optimal conditions for species desorption from the sorbent tubes. The MDL ranged from 23 to 122 fmol in aqueous samples and 0.06–0.3 parts per trillion by volume in atmospheric samples. The lowest MDLs were found for Se being 8.8–167 amol and 0.2–0.4 parts per quadrillion by volume, for water and air samples, respectively. The method was applied to aqueous and atmospheric samples collected in the Baltic and North Seas and the target compounds were detected and quantified in aqueous samples from all depths. The low LOD values provided by this high throughout multi-elemental method make it useful for environmental studies.

3.16. Silver

Most papers covering Ag speciation involve the separation of ionic Ag species from Ag nanoparticles (NPs), usually involving some form of chromatography or field flow fractionation coupled with ICP-MS whilst the use of X-ray based techniques is less commonly reported. Before analysis though a sample preparation step is usually needed and Jiménez et al. have evaluated three solvents, HPW, TMAH (25%) and 10 mmol per L Na₄P₂O₇ (TSPP) for the extraction of Ag species in faeces collected pigs and chickens fed with a Ag-based nanomaterial.¹³⁵ For the TMAH extraction, cysteine and CaCl₂ were used to evaluate their stabilisation effect on the Ag forms, i.e. the prevention of the formation of particulates from any Ag⁺ present. The extracts were analysed using sNP-ICP-MS, hydrodynamic chromatography ICP-MS (HDC-ICP-MS) and AF4-ICP-MS and the total Ag content of the faeces as determined by ICP-MS after an acidic digestion method. Water extracted 3% and 9% of the Ag present for faeces of pigs and chickens, respectively with less than 10% of these amounts being in the NP form. The TMAH methods extracted about 40% of the total Ag present in the faeces samples with less than 1% of the total being Ag NPs. Both stabilisers, cysteine and CaCl₂, exhibited a similar effect on Ag NP preservation for chicken faeces, whereas cysteine-Triton X-100 was twice as efficient for pig faeces. The TSPP method, which was a multistep sequential approach, enabled the fractionation of the silver in the faeces, with Ag₂S (41%) and ionic Ag (62%) being the most abundant fractions. The paper gives a detailed account of the evaluations undertaken and the possible reasons for the differences observed. A HPLC-ICP-MS method, using a C₁₈ column (250 × 4.6 mm, 7 mm particle size, 1000 Å pore size) has been developed for the separation of Ag NPs and dissolved Ag^I in surface waters and algal cell extracts.¹³⁶ The optimal mobile phase was found to be 10 mmol per L SDS, 2 mmol per L citrate buffer, and 2 mmol per L tiopronin, flowing at 0.5 mL min⁻¹ and detection limits of 2.0 ng L⁻¹ for Ag^I, 3.1 ng L⁻¹ for 10 nm Ag NPs, and 2.2 ng L⁻¹ for 30 nm AgNPs were estimated. A linear relationship was found between the Ag NP retention time and log particle diameter, up to 40 nm, and the authors propose this can be used for Ag NP particle size determination in extracts of real samples. The method was used to study the interactions of Ag^I and citrate-stabilized Ag NPs (10 nm and 30 nm) with green microalgae, Acutodesmus obliquus, in river water and Ag biosorption was ranged from 77 to 82%. The biosynthesis of small NPs (6 nm) from Ag⁺ ions and the partial dissolution of AgNPs was also observed. The paper contains a good discussion on the choice of mobile phase components, particularly the reagents used to prevent the formation of Ag NPs from Ag⁺. The Ag biodistribution and gut toxicity of two commercially available colloidal silver products, Mesosilver™ (which is a mix of Ag NPs and Ag⁺) and AgC which is solely Ag⁺, has been evaluated after dietary exposure in male mice using μXRF μXAS. The results obtained showed that the Ag NPs and Ag⁺ ions in Mesosilver™ underwent physicochemical modifications in the gut. In the colon, most Ag atoms were oxidised and dissolved to form Ag complexes with protein and/or peptide thiol groups. A sub-chronic exposure dose led to a moderate impact on the gut barrier for both colloidal Ag products. An increase in colonic LCN-2, used to assess gut inflammation, was observed only after exposure to AgC. Exposure to Mesosilver™ led to a decrease in Ruminococcus and Anaerosporobacter in the gut whilst Intestinimonas amounts increased. Exposure to AgC resulted in an increase in Clostridium sp. ASF356 and Tyzzerella, while the relative abundance of Anaerosporobacter decreased and the Saccharomycetes fungal population increased. The paper contains a detailed discussion on the inferences that can be obtained form these gut flora increases and decreases.

3.17. Sulfur

A new analytical method for the simultaneous determination of the breakdown products from the widely used dithiocarbamate (DTC) fungicides, ethylene-thiourea (ETU) and propylene-thiourea (PTU) in fruits and vegetables has used HPLC-ICP-MS/MS.¹³⁷ The main representatives of DTCs currently in use worldwide are ziram, ferbam, thiram, maneb, zineb, nabam, metiram, mancozeb, and propineb. These compounds are highly unstable and decompose in the environment and in foodstuffs into ETU and PTU. These compounds were baseline separated in less than 5 min using a reversed-phase HPLC column and detection as the oxides of ³²S and ³⁴S using ICP-MS/MS with O₂ as the collision cell reaction gas. The two fungicide degradation products and sulfate were separated on an Aqua C18 RP column (150 mm × 2.0 mm, 3 μm 125 Å), using an eluent containing methanol (5% v/v) pumped isocratically at 0.25 mL min⁻¹. The sulfate in the chromatogram originated from (NH₄)₂SO₄ ammonium sulfate and standards of ETU (99.9%) and PTU (99%) were used as the source of S for the calibration. The performance criteria in terms of LOQ, accuracy, repeatability, and intermediate precision were assessed using the accuracy profile methodology approach, which involves the expression of the systematic error (trueness) and the random error (repeatability and intermediate precision) for a series of analyte concentrations in various matrices and within a range of concentrations called the “validity domain”. Interested readers are encouraged to access the work describing the approach more fully that are referenced in the paper. The LOQ values were 6.0 μg L⁻¹ for ETU and 10.1 μg L⁻¹ for PTU, which corresponds to 0.006 mg kg⁻¹ for ETU and 0.010 mg kg⁻¹ for PTU, taking into account the sample mass (50 g) and the extract volume (50 mL). The spike recovery ranged from 87% to 101% for ETU and from 98% to 99% for PTU. The ETU and PTU levels measured in both reagent blanks and in the unfortified real-life fruit and vegetable samples were below the method LOQ values. The method was successfully applied to the analysis of a selection of real-life foodstuffs (fruits and vegetables) hence proving its full capacity to be applied to routine determination of ETU and PTU.

3.18. Technetium

The speciation of Tc (the most important radionuclide for SPECT in nuclear medicine) has been carried out by using HPLC-ICP-MS and HPLC-ES-HR-MS.¹³⁸ A C₁₈ column (accurate 2.6 mm, 150 mm) was employed to separate most of the Tc-species in several radiopharmaceutical products. Due to the lack of Tc standards, isobaric dilution analysis (IBDA) was applied to enable calibration. This calibration technique is derived from IDA analysis but in this case IBDA uses a spike containing a chemically similar element with an isobaric isotope. Here, a Ru post-column spike was continuously added after chromatographic column generating a ratio of m/z 99 to 101 and the alteration of this ratio due to the elution of ⁹⁹Tc was used for Tc compound quantification. The differences in sensitivity of Tc and Ru were corrected for by approximating the element sensitivity of Tc in ICP-MS using the response of its neighbouring Group 4d elements. A LOD and LOQ of 0.05 ng Tc per g⁻¹ and 0.17 ng Tc per g, respectively were obtained. Two major Tc signals were detected in the ⁹⁹Tc mass flow chromatograms of two experimental radiotracers developed for the scintigraphy of tumours. The first was assigned by HPLC-ES-HR-MS to the pertechnetate anion and the second signal was assigned to the radiotracer ⁹⁹TcC₄₀H₄₇O₉N₇⁺. Pertechnetate is the most thermodynamically stable Tc species under aerobic conditions and is used as the precursor for all Tc-labelled radiotracers. The method was further applied to four different commercially available tracers: pertechnetate (for scintigraphy of thyroids), Tc-MAG3 (widely applied for scintigraphy of kidneys) and Tc-tetrofosmin and Tc-MIBI (for myocardial scintigraphy). In TcMAG3 the desired tracer was identified as ⁹⁹TcC₈H₉O₆N₃S₁ and in ⁹⁹TcMIBI as ⁹⁹TcC₃₆H₈₀O₁₀P₄ and ⁹⁹TcC₃₆H₆₆N₆O₆. Several unknown products were also detected in Tc-Tetrofosmin and Tc-MIBI which were highly time dependent and degraded within hours to pertechnetate.

3.19. Thallium

In a review of the determination of Tl in biological and environmental samples, just over 200 references from the period 2012 to 2022 were cited.¹³⁹ Most of the papers covered featured quantification by atomic spectrometry though a small number of molecular spectrometry (including fluorimetry) methods were also mentioned. The reviewers discussed sample preparation procedures, including SPE, CPE, and DLLME. Three ASU-style tables summarise the AAS, OES (mostly ICP) and the ICP-MS methods. Speciation methods for Tl^I and Tl^III are covered in three short sections entitled “redox techniques”, “chromatographic separation” and “selective extraction,” comprising approximately 10, 3 and 6 citations, respectively. The basis of the redox techniques was the combination of some selective measurement and the oxidation of Tl^I to Tl^III or reduction of Tl^III to Tl^I. The reviewers concluded that there is still a need for (a) more efficient preconcentration and matrix isolation procedures for which ion-imprinted polymers (IIPs) and metal–organic frameworks were considered promising, (b) novel optical probes for fast sensing of Tl^I and Tl^III, for which aptamer modified-quantum dots or IIPS-conjugated aggregation induced emission materials were considered promising, and (c) facile and effective speciation methods, for which no specific suggestions were made.

A version of the same direct immersion dual-drop microextraction procedure already described for Cr speciation (Section 3.3) has been developed for the determination of Tl^I and Tl^III in foods.¹⁴⁰ Samples were adjusted to pH 6 and 0.3 mL of 0.6 mmol L⁻¹ picric acid added (as counter ion for the extraction of Tl^I) and two 15 μL drops of organic solvent containing chelating/extracting agents, suspended from syringes, immersed on the solution which was heated to 35 °C and stirred for 20 min. The drop of 1-dodecanol containing 1.5 mmol L⁻¹ pyridylazo naphthol was selective for Tl^III and the other of nitrobenzene containing 1.2 mmol L⁻¹ of dicyclohexano-18-crown-6 was selective for Tl^I. After extraction each drop was diluted to 100 μL and 20 μL taken for analysis by ETAAS. The LOD values were 8.3 and 6.3 ng L⁻¹ for Tl^I and Tl^III, respectively and the method was validated by the analysis of IGGE CRM GBW 10017 (milk powder) and by spike recoveries from real samples of beverages (orange juice, soda, cola and sprite drink) purchased from local markets in Wuhan, China. All samples contained measurable concentrations of Tl^I, but Tl^III was detected in only one orange juice.

3.20. Tin

Since the IMO ban on the use of organotin based antifouling systems came into force in 2008, the number of studies reporting on Sn speciation has declined almost to zero. This year there was just one paper which reported on the determination of inorganic and organic forms of Sn in waters of different salinities.¹⁴¹ Various approaches were evaluated for the separation of the Sn species: LLE with various solvents, precipitation with halides or aqueous solutions of ammonia and Fe^III chloride, as well as SPE, although little of this was truly novel and many of the reported extraction efficiencies were very low (<15%). The best separation of the Sn species was achieved using the silica gel sorbent Diapak C18, which, under optimised conditions, selectively extracted more than 95% of the OTCs from waters with different salinities with minimal co-extraction of iSn. Quantification of the extracted Sn was by HG-ICP-OES and HG-ICP-MS with LOQ values for the iSn after SPE from OTCs of 0.03 g L⁻¹ (ICP-MS) and 0.05 g L⁻¹ (ICP-OES).

3.21. Uranium

The binding and stability of U within biological systems and with biomolecules is of interest from a biogeochemical and toxicological perspective and this area has been investigated and described in two recent papers. An analytical strategy developed¹⁴² to investigate the affinity of synthetically prepared, termed biomimetic, peptides for U was based on the simultaneous coupling of HILIC with ES-MS and ICP-MS. In this instrumental set-up the peptide complexes formed with UO₂²⁺ were structurally identified using negative ion ES-MS/MS, which showed that doubly charged [UO₂(peptide)]²⁻ complexes with 1 : 1 stoichiometry were formed. These complexes were quantified by external calibration using measurement of U by ICP-MS. Three different HILIC microbore columns with amide, diol and hybrid silica stationary phases were investigated, including: acquity BEH amide (100 × 2.1; 1.7 μm), YMC Triart Diol (100 × 2; 1.9 μm) and acquity BEH HILIC (100 × 2.1; 1.7 μm). The composition of the mobile phases was obtained by online mixing the appropriate proportions of, solvent A (60/40 ACN/H₂O v/v containing 20 mmol per L NH₄CH₃CO₂) and solvent B (80/20 ACN/H₂O v/v containing 20 mmol per L NH₄CH₃CO₂). This provided acceptable separation of the mixture of four di- and tri-phosphorylated linear and cyclic biomimetic peptides of interest. To recover UO₂²⁺ adsorbed on the column, successive injections of the tetra-phosphorylated peptide were carried out after each chromatographic run until the background ²³⁸U signal was less than 1000 cps. In order to prevent carbon deposition on the sampler cone due to the use of organic solvents, additional O₂ (8 mL min⁻¹) was introduced into the argon plasma. Platinum cones and a 1 mm inner diameter injector were also used for this purpose. It was possible to demonstrate that (i) the increasing number of phosphorylated residues promotes the affinity of the peptides for UO₂²⁺, (ii) the position of the phosphorylated site in the peptide backbone has little effect on this affinity, (iii) the cyclic structure of the peptide favours complexation in comparison with the linear structure. These results are in agreement with those previously obtained by other spectroscopic techniques, which were used to validate the method. In the second paper¹⁴³ the developed molecular and inorganic MS detection system was used to determine the effect of competing divalent metals including, Ca, Cu and Zn, on the binding of the uranyl ion UO₂²⁺ to a synthetically prepared model tetra-phosphorylated peptide. Because of the isobaric interferences on the main Ca isotope Sr was used as a substitute. The application was found to be suitable for demonstrating whether synthetically prepared peptides could be used to affect the binding of uranyl to biomolecules, in particular proteins found in biological systems.

3.22. Vanadium

For the determination of V^V (and Pb^II, TML Hg^II and MeHg) in seawater and urine, a SPE preconcentration was applied prior to HPLC-ICP-MS.⁹³ A previously synthesised, patented, magnetic nanomaterial based on a double coupling between GO and MNPs (M@GO) was further derivatised with the chelating group methyl thiosalicylate. The resulting M@GO-TS material (50 mg) was packed into a column mounted in the injection loop of a FI valve and sample, adjusted to pH 3.5, was loaded at 2.7 mL min⁻¹ for 10 min. The retained analytes were then eluted (there was, apparently, no washing step) with 7 mmol per L thiourea + 40 mmol per L H₃PO₄ at 1.5 mL min⁻¹, and 1 mL collected of which 100 μL was injected into the HPLC system for separation on a Kinetex EVO C18 column (4.6 × 250 mm, 5 μm particles). The mobile phase was a gradient of A (7 mmol per L thiourea + 40 mmol per L H₃PO₄) and B (0.16 mmol per L TBAH in H₃PO₄ at pH 4.5). The gradient elution program was 100% A, 0.0% B for 0.1 min, followed by a ramp of 1.2% s⁻¹ to 0.0% A, 100% B for 1.5 min held for 18.4 min. The method was validated by the analysis of CRM NRCC TMDA 64.3 (fortified lake water) and by spike recoveries from two urine and one seawater samples. The LOD was 0.4 ng L⁻¹ and the enrichment factor for all species was 27. No V^V was found in either of the urine samples, only in the seawater.

To determine V speciation in ancient shales, mapping by μXANES and microfocused scanning XRF microscopy were applied.¹⁴⁴ Measurements were performed at the XRF beamline at the Australian Synchrotron. Bulk characterisations were also performed by XANES as well as XRD and ICP-MS. The researchers found that V speciation was predominantly phyllosilicate-hosted V^III and that the global statistical colocalisation analyses showed strong associations between V and K, consistent with V hosted by potassium-rich clay minerals. They concluded that, although synchrotron-based X-ray methods were a powerful set of analytical tools, for V in ancient marine sediments it was unlikely that authigenic (generated where found) and detrital (composed primarily of particles or fragments detached from preexisting rocks either by erosion or by weathering) could be differentiated.

3.23. Zinc

A detailed study on the speciation of Zn, Pb, Cu and As in Pb/Zn smelter slags, a hazardous industrial waste from the Imperial smelting process, was carried out by Chen et al.²¹ The results are presented in the context of a review where the state of the art of a variety of techniques (XAS, XRF and SEM among others) is presented along with tables containing information about the mineralogical composition of smelter slags. The XRF measurements indicated the presence of around 8.4% of Zn in the slag but no crystalline phase was detected. The use of XAS provided authors information on the chemical form of the elements at very low concentration levels, irrespective of crystallinity. The XAS measurements detected the presence of Zn as ZnS and in solid solutions with Fe oxides such as spinel, Fe₃O₄. In a second paper¹⁴⁵ XRF and XANES techniques were also employed to perform Zn speciation and desorption kinetics studies in mining soils amended with phosphate (added as NH₄H₂PO₄). Analysis by XAS evidenced a decrease of recalcitrant forms of Zn as gahnite and montmorillonites species. The origin of the Zn dissolution process observed was attributed to the decrease of soil pH when phosphate was added, from 8.3 to 6.3. Consequently, phosphate amendment seems to be not suitable for remediation of Zn in the soil due to increased Zn mobilisation in the soil. The characterisation of ZnO NPs in cosmetic and pharmaceutical products was evaluated by using sNP-ICP-MS and high-resolution continuum source (HRCS) GFAAS.¹⁴⁶ Ten samples of cosmetic and personal care products were studied, including 5 contact powdered eyeshadows of different colours, 3 baby skincare creams and 2 selfcare lotions. Triton X-100 was used to prepare suspensions from the selected products and its concentration was optimised by using a composite design. Based on the results obtained, 0.55% TritonX-100 solution and 3 minutes sonication and 1% TritonX-100 and 70 minutes sonication were selected as the best conditions for HRCS-GFAAS and sNP-ICP-MS, respectively. It seems to be that sonication time was highly dependent on the technique selected for performing analysis. Suspension dilution and sample volume were also optimised. By using 0.1% Triton X-100 and a dilution not higher than 1 : 1000 two atomisation peaks were detected by HRCS-GFAAS, the first peak was associated with the presence of ionic Zn and the second one to the NP form. The peak associated with the NPs was deconvoluted to avoid small contributions of the ionic Zn near to the ZnO NP region. In addition, the use of Triton X-100 improved separation of the atomisation peaks favouring the speciation analysis of the samples. Sizing by HRCS-GFAAS was performed by using a size calibration procedure with ZnO NP standards. The ZnO NP size distributions in different commercial products were found to be within the 18 to 500 nm range and agreed with values provided by TEM analysis. Analysis of the samples by sNP-ICP-MS gave no NP signals due to the high ionic Zn background levels present in the extracts. Thus, a separation technique is required to resolve this interference. Lastly the behaviour of nano/microplastics as a vector of organic and metal contaminants is an active area of work as micro/nanoplastics can adsorb chemical compounds in their heterogeneous surface. In this line, Gomez-Gonzalez et al.¹⁴⁷ evaluated the adsorption of ZnO NPs to microplastics under different environmental conditions (tap water, sea water and artificial seawater without DOM). The procedure was applied not only to suspensions of ZnO NPs (80–200 nm) and polystyrene microplastics standards (200 nm) but also to commercial sunscreen containing ZnO and an exfoliating cleanser containing microbeads. All ZnO NP containing samples and microplastic containing samples were subjected to an aging procedure through continuous stirring for 7 days. The aged ZnO NP and microplastic suspensions were then incubated together for 24 h under stirring. Analysis by SEM revealed the presence of a significant amount of Zn adsorbed over the microplastic surfaces as nano–micro Zn aggregates which ranged from individual NPs to 2–3 mm aggregates, the total amount of Zn adsorbed on the micro/nanoplastics was quantified by ICP-OES and results evidence the poor dissolution of Zn nanoparticles as less than 75 μg L⁻¹ was detected in the water samples after 7 days of aging. XANES analysis allowed the authors to perform Zn speciation on the adsorbed particles, reporting a significant ZnO transformation towards Zn-sulfide and Zn-phosphate compounds in both standard solutions and ZnO based sunscreen and exfoliating mixture solutions. This type of study is of relevance from the environmental point of view due to the persistent nature of these compounds that pose a threat to microorganisms and biota.

4. Biomolecular speciation analysis

The advances in the accuracy and traceability of metalloprotein measurements by ID-MS calibration since 2006, when the first description of on-line ssID-MS analysis method for the accurate measurement of Cu-containing proteins was reported have been reviewed.¹⁴⁸ Covering 124 papers it summarises both species-specific and species-unspecific IDMS methods for the measurement of intact proteins containing metals and non-metals. The review was divided into sections dealing with; classic proteomics using IDMS using protein standards isotopically labelled with ²H, ¹³C or ¹⁵N; S-based IDMS using post-separation addition of ³⁴S; Fe-containing proteins including transferrin, ferritin and haemoglobin; Cu and Zn containing proteins; selenoproteins; and protein-bound xenobiotic metal elements, such as Pt. The reviewed focused on the most important metal containing proteins from a clinical aspect, so as to illustrate how the attributes of chromatography coupled to inorganic MS can be used for the direct measurement of metalloproteins.

The direct speciation methods for the measurement of Cu-containing biomolecules in the investigation of Wilson disease is the focus of attention due to the increasing number of clinical trials in progress for the treatment of this disease. As part of a special themed issue of the medical journal Diagnostics detailing Advances in Wilson Disease and Other Neurodegenerations with Brain Metal Accumulations, a review describing the current methods for “Monitoring of Copper in Wilson Disease”¹⁴⁹ has included a small number of papers detailing the measurement of Cu-containing biomolecules in patient samples. The aim of the review was to present the current state of knowledge on the biochemical tests of Cu metabolism in the diagnosis and monitoring of Wilson disease and it used a number of terms and keywords to search several library databases e.g. PubMed. This has focused on the separation and detection of Cu-containing metalloproteins such as ceruloplasmin, using speciation methods based on HPLC-ICP-MS. The review was limited to describing the advantages and disadvantages of the few pieces of work that have developed methods in this area as well as providing patient results. Interested readers are referred to papers in that review, which have also been discussed in recent previous ASU elemental speciation reviews. Non-ceruloplasmin-bound Cu (NCBC) and Cu speciation in serum with extraction using functionalized dendritic silica spheres followed by ICP-MS detection has been developed to understand the correlation of Cu with various physiological processes and diseases.¹⁵⁰ The dendritic spherical silica particles were functionalized with EDTA, by the hydrolysis of tetraethyl orthosilicate with the aid of structure-directing agents. The functionalised silica formed serves as an adsorbent with abundant binding sites to facilitate efficient extraction of NCBC. The retained NCBC on the absorbent was recovered by stripping with HNO₃. By hyphenating with ICP-MS detection, it provided an LOD of 1.3 pmol for NCBC. The degradation of ceruloplasmin, the major Cu-containing protein in blood, with 200 mmol per L H₂O₂ released the bound Cu as NCBC to distribute among other ligands, which may be efficiently retained by the adsorbent and facilitated the determination of the total Cu. The linear ranges for NCBC and total Cu were 0.21–10 μmol L⁻¹ and 0.42–30 μmol L⁻¹. The recovery rates for spiked NCBC or total Cu in serum were shown to be 97–108% and 94–102%, respectively. The analysis of serum for a healthy subject resulted in 1.8 μmol per L NCBC and 9.5 μmol L⁻¹ for total Cu, which is in agreement with published reference ranges for these characteristics. In addition, the proportions of 8.5–12% for NCBC were derived from the serum of healthy adults, while those for the patients with lung, hepatocellular and oesophageal carcinoma were found to be 10–12%, illustrating no obvious difference to the normal group.

The use of elemental standards, in particular inorganic S, is probably one of the most important applications of using inorganic MS as a method for the measurement of proteins and metalloproteins. The group at the University of Oviedo continues to explore this fertile area of study with excellent papers, the most recent of which uses S for the quantification of human cytokines, an important biomarker of inflammation and other cellular responses.¹⁵¹ With the use of molecular MS approaches such as LC-MS/MS for the measurement of protein concentration, protein standards are required as calibration references. This requires there to be suitable standards for each target protein species, which must be appropriately purified, characterised, and whose concentration and mass purity must be accurately assessed. This is not straightforward, requires specialist expertise, and is time consuming and expensive to do. In this work, an absolute quantitative analytical strategy was developed, based on the detection of S by ICP-MS/MS that uses sulfate as generic standard to quantify and certify the mass purity of protein standards. Standards of Recombinant Human Fms-related tyrosine kinase 3 ligand in 2 batches (Flt3L-A) and (Flt3L-B) as well as Recombinant Human Gamma-Interferon Inducible Protein 10 (IP-10) were used to illustrate the methodology. The system comprised a capHPLC separation coupled to ICP-MS/MS via a commercially available total consumption nebuliser. A reversed phase capHPLC BIOShell™ A400C4, 3.4 μm, 150 mm × 0.3 mm, column was used at a controlled temperature of 80 °C. The chromatographic mobile phases consisted of a gradient of ultrapure water (A) and acetonitrile (B) both with 0.2% of formic acid pumped at a flow rate of 4 μL min^{− 1}. This chromatographic system was also coupled to an ES-MS/MS instrument and the same eluent was used with both detectors but is very much representative of the requirements to generate multiply charged ions for ESI analysis. Molecular mass spectrometry analyses were performed with a ES-QTOF MS instrument and analytes were ionized by heated ES operating in the positive ionization mode. The advantage of ICP-MS/MS is that the elemental S response factor can be made species-independent, hence any well-characterised and certified S-containing standard can be used. Therefore, in a single chromatographic analysis, correlation of S chromatographic peak areas of analyte and standard directly results in the quantification of S concentration. The methodology is based on the requirement that the signal response factor (S signal per unit of concentration) is equal for both analyte and standard. This response factor has been shown previously to be determined by a number of factors, including: the efficiency of the transport and ionisation processes; and the chromatographic gradient used. These factors must be considered when developing a generic methodology for protein quantification and particularly protein standards certification. The paper goes into considerable detail for what is required as well as referencing previous work where these effects have been further studied and investigated. The analytical workflow used FIA to establish the S-response factor followed by the capLC separation to determine the concentration of the measurands. In this case the chromatographic recovery and mass purity values obtained for the Cytokine standards IP-10, FLT3-LG (batches A and B), and commercial BSA used as quality control, were between 96 to 103%, indicating excellent quantitative performance of the method.

One of the main issues to overcome when using speciation methods for the measurement of intact proteins by ICP-MS/MS is the chromatographic resolution step, which precedes detection. Gel-electrophoresis is a very well-established method to separate proteins, either in 1 or 2 dimensions, however coupling to ICP-MS/MS is limited when this is carried out using conventional GE, and requires the analysis of the gel plate using LA. However, new approaches using column GE offers the potential to make the hyphenation of these two methods easier to achieve and faster. However, the method of GE is slower than other chromatographic approaches, even if it does offer higher resolution. Therefore a new approach¹⁵² to speeding up column GE separations is encouraging. In this study, a GE-ICP-MS system was optimised by adjusting the analytical conditions, including the concentration and pH of the running buffer and the proportion of polyacrylamide gel used. The results showed that the migration speed of proteins in GE was enhanced by increasing the electrolyte concentration in the running buffer solution. Additionally, the ICP-MS response, which was dramatically decreased because of the change in running buffer solution, was stabilised by adjusting the pH of the running buffer. Optimisation of the polyacrylamide gel ratio allows GE-ICP-MS to maintain high resolution for proteins of similar molecular weight with increased detection speed. After increasing the concentration of running buffer by 10 times, 4 iodine labelled proteins including carbonic anhydrase, ovalbumin, bovine serum albumin and conalbumin were successfully separated at baseline by the GE-ICP-MS system at pH 8.0 in 40 min using a resolving gel (8%, 7 cm) and a stacking gel (4%, 1 cm). The developed separation was shown to be 3 times faster than the original one. Finally, the optimised method was applied to the qualitative analysis of an Ag-binding protein in rat plasma samples.

5. Abbreviations

μXRF	micro X-ray fluorescence
3D	three dimensional
AAS	atomic absorption spectrometry
AB	arsenobetaine
AC	alternating current
AC2	homoarsenocholine
ACN	acetonitrile
AE	anion-exchange
AEC	anion-exchange chromatography
AF4	asymmetric flow-field flow fractionation
AFS	atomic fluorescence spectrometry
APDC	ammonium pyrrolidine dithiocarbamate
AsSug	arsenosugar
ASU	Atomic Spectrometry Update
As^V	anodic stripping voltammetry
BCR	Community Bureau of Reference
BFR	brominated flame retardants
BPHA	N-benzoyl-N-phenylhydroxylamine
BSA	bovine serum albumin
CE	capillary electrophoresis
CEC	cation-exchange chromatography
CF	continuous flow
CNT	carbon nanotube
CPE	cloud point extraction
CRM	certified reference material
CSIA	compound specific isotope analysis
CT	computed tomography
CTAB	cetyl trimethylammonium bromide
CV	coefficient of variation OR cold vapour (but NOT in same review)
DBD	dielectric barrier detector
DDI	dual direct immersion
DDTC	diethyldithiocarbamate
DIDDME	direct immersion dual-drop microextraction
DLLME	dispersive liquid–liquid microextraction
DMA	dimethylarsenic (include oxidation state if known)
DMGe	dimethylgermanium
DMHg	dimethyl Hg
DMS	dimethylsulfide
DNA	deoxyribonucleic acid
DOC	dissolved organic carbon
DOM	dissolved organic matter
DPC	diphenylcarbazone
DSPME	dispersive solid phase microextraction
DTC	dithiocarbamate
EC	elemental carbon
ECD	electron capture detector
ED	energy dispersive
EDTA	ethylenediaminetetraacetic acid
EMAE	enzymatic microwave assisted extraction
ENM	engineered nanomaterial
EPMA	electron probe microanalysis
ERM	European reference material
ES	electrospray
ETA	electrothermal atomisation
ETAAS	electrothermal atomic absorption spectrometry
ETU	ethylene-thiourea
EXAFS	extended X-ray absorption fine structure
FAAS	flame atomic absorption spectrometry
FIA	flow injection analysis
FTIR	Fourier transform infrared
GBCA	Gd-based contrast agent
GC	gas chromatography
Gd-BOPTA	gadobenate dimeglumine
Gd-BT-DO3A	gadobutrol
Gd-DTPA	gadopentetate
Gd-DTPA-BMA	gadodiamide
Gd-DTPA-BMEA	gadoversetamide
Gd-EOB-DTPA	gadoxetic acid disodium
Gd-HP-DO3A	gadoteridol
GE	gel electrophoresis
GF	graphite furnace
γ-Glu-SeMet	γ-glutamyl-selenomethionine
GO	graphene oxide
GPx3	glutathione peroxidase 3
GSH	glutathione
HA	hydroxyapatite
HAPG	highly annealed pyrolytic graphite
HDC	hydrodynamic chromatography
HFLLME	hollow fibre liquid–liquid microextraction
HG	hydride generation
HILIC	hydrophilic interaction liquid chromatography
HMIM	1-hexyl-3-methyl-imidazolium-hexafluorophosphate
HMM	high molecular mass
HPLC	high performance liquid chromatography
HR	high resolution
HRCS	high-resolution continuum source
IAEA	International Atomic Energy Agency
iAs	inorganic arsenic
IBDA	isobaric dilution analysis
IC	ion chromatography
ICP	inductively coupled plasma
Id	internal diameter
IDA	isotope dilution analysis
IDMS	isotope dilution mass spectrometry
IE	ion exchange
IEC	ion exchange chromatography
IGGE	Institute of Geophysical and Geochemical Exploration
iHg	inorganic mercury
IIP	ion-imprinted polymer
IR	infrared
IUPAC	International Union of Pure and Applied Chemistry
LA	laser ablation
LC	liquid chromatography
LLE	liquid–liquid extraction
LLME	liquid–liquid microextraction
LMM	low molecular mass
LOD	limit of detection
LOQ	limit of quantification
LPME	liquid phase microextraction
MAE	microwave-assisted extraction
MC	multicollector
MDL	method detection limit
MeCo	methylcobalamine
MeHg	methyl mercury
MeOH	methanol
MeSeCys	methylselenocysteine
MIL	magnetic ionic liquid
MMA	monomethylarsenic
MMGe	monomethylgermanium
MMHg	monomethylmercury
MMM	medium molecular mass
MMMTA	monomethylmonothioarsonic acid
MOF	metal–organic framework
MP	microwave plasma
MRI	magnetic resonance imaging
MS	mass spectrometry
MSIS	multi-mode sample introduction system
MSPE	magnetic solid-phase extraction
NCBC	non-ceruloplasmin-bound Cu
NIST	National Institute of Standards and Technology
NMIJ	National Measurement Institute of Japan
NP	nanoparticle
NRCC	National Research Council of Canada
ODS	octadecylsilane
OES	optical emission spectrometry
OTC	organotin compounds
PAD	pulsed amperometric detection
PCB	polychlorinated biphenyl
PCR	polymerase chain reaction
PIXE	particle-induced X-ray emission
PM10	particulate matter (with an aerodynamic diameter of up to 10 μm)
PM2.5	particulate matter (with an aerodynamic diameter of up to 2.5 μm)
PMBP	1-phenyl-3-methyl-4-benzoyl-5-pyrazone
PMFS	phenylmethylsulfonyl fluoride
PTE	potentially toxic element
PTU	propylene-thiourea
PVG	photochemical vapour generation
Pyr	pyrolysis
Q	quadrupole
REE	rare earth element
ROX	roxarsone
RP	reversed phase
RSD	relative standard deviation
SAX	strong anion exchange
SDME	single-drop microextraction
SDS	sodium dodecylsulfate
SeAlb	selenoalbumin
SEC	size exclusion chromatography
SeCys	selenocysteine
SeCys2	selenocystine
SelP	selenoprotein
SEM	scanning electron microscopy
SeMeCys	selenomethylcysteine
SeMet	selenomethionine
SF	sector field
SI	Système International d'unités
SIMS	secondary ion mass spectrometry
sNP	single nanoparticle
SPE	solid phase extraction
SPECT	single-photon emission computed tomography
SPME	solid phase microextraction
SR	synchrotron radiation
SRM	standard reference material
SS	species specific
TBAH	tetrabutyl ammonium hydroxide
TC	total carbon
TD	thermal desorption
TEM	transmission electron microscopy
tHg	total mercury
TLC	thin layer chromatography
TMA^+	tetramethylarsonium cation
TMAH	tetramethylammonium hydroxide
TMAO	trimethylarsine oxide
TMAP	trimethylarsoniopropionate
TML	tetramethyllead
TOF	time-of-flight
TPD	targeted protein degradation
Tris	tris(hydroxymethyl)aminomethane
TTFA	thenoyltrifluoroacetone
TXRF	total reflection X-ray fluorescence
UAE	ultrasound-assisted extraction
UV	ultraviolet
WD	wavelength dispersive
WEEE	waste electrical and electronic equipment
WHO	World Health Organisation
XAFS	X-ray absorption fine structure spectroscopy
XANES	X-ray absorption near-edge structure
XAS	X-ray absorption spectroscopy
XPS	X-ray photoelectron spectroscopy
XRD	X-ray diffraction
XRF	X-ray fluorescence

Conflicts of interest

There are no conflict of interest to declare.

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