Atomic spectrometry update. Elemental speciation
Robert
Clough
*a,
Lindsay R.
Drennan-Harris
b,
Chris F.
Harrington
c,
Steve J.
Hill
d and
Julian F.
Tyson
b
aBiogeochemistry and Environmental Analytical Chemistry Research Group, University of Plymouth, Plymouth, UK. E-mail: rclough@plymouth.ac.uk
bDepartment of Chemistry, University of Massachusetts Amherst, 710 North Pleasant Street, Amherst, MA 01003, USA
cSupra-regional Assay Service, Trace Element Laboratory, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, UK GU2 7XH
dSpeciation and Environmental Analysis Research Group, University of Plymouth, Plymouth, UK
First published on 2nd July 2012
Abstract
This is the fourth Atomic Spectrometry Update (ASU) to focus specifically on developments in elemental speciation and covers a period of approximately 12 months from January 2011. The International Union for Pure and Applied Chemistry (IUPAC) has evaluated speciation and provided a definition as follows: “speciation analysis is the analytical activity of identifying and/or measuring the quantities of one or more individual chemical species in a sample; the chemical species are specific forms of an element defined as to isotopic composition, electronic or oxidation state, and/or complex or molecular structure; the speciation of an element is the distribution of an element amongst defined chemical species in a system.” This review therefore deals with all aspects of the analytical 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. The review does not cover operationally defined ‘speciation’, which is correctly termed fractionation. As with all ASU reviews1–6 the coverage of the topic is confined to those methods that incorporate atomic spectrometry as the measurement technique. However, in the spirit of meeting the needs of the subject, material is incorporated that is not strictly “atomic spectrometry” but the separation or sample introduction technique used could also be coupled with an atomic spectrometry detector. For the most part, such procedures are those in which some form of molecular MS is used for speciation measurements. As the content of this Update shows, the field is now maturing as evidenced by the extent to which the speciation of particular elements or technique combinations have been the subject of review articles. However, it is becoming increasingly difficult to ascertain the analytical details of the methodologies applied in speciation analysis, particularly where the paper is published in an ‘application’ based journal.
1. Topical reviews
Although no books devoted specifically to the topic of elemental speciation analysis have appeared during this recent review period, there are one or two that contain reviews of the relevant literature and these are referred to at the appropriate point in the Update. Review articles continue to appear and can be classified broadly into two groups: those whose focus is on a particular technique, and those whose focus is on the analytes and/or sample type. The entire January 2011 issue of the Journal of Analytical Atomic Spectrometry, volume 26 issue 1, was devoted to the theme of speciation. The issue included three critical reviews (all mentioned below), 20 papers and 1 technical note. Five of the papers concerned As and five concerned Se; Hg was featured in three, and Al, Cr, P, Pt and Si were each featured once. The other articles dealt with techniques or particular matrices.The application of mass spectrometry to speciation analysis has been the subject of a number of reviews. The last two years' literature on ES-MS for speciation analysis was briefly surveyed7 with particular reference to compounds of As and Se that had been studied by the combination of molecular and elemental MS. Engelhard8 has looked back over 30 years of ICP-MS development with special attention to instrumental development and emerging applications. It was pointed out that instruments with focal-plane-camera array detectors are able to record the full m/z range simultaneously and continuously, allowing fast transient signals to be reliably monitored. Such detectors also allow isotope ratios to be measured with analytically useful precision. Developments that allow the direct analysis of microparticles and single cells are also highlighted. Sanz-Medel and co-workers9 have reviewed the analytical applications of GD-MS including the possibilities for the simultaneous production of elemental, structural and molecular information from organic compounds in the gas phase. A number of references are cited in which the use of GD-MS as a GC detector is described. This group has also surveyed the applications of plasma-based mass spectrometry for the simultaneous acquisition of elemental and molecular information.10 The latter article covers a lot of the same material as the GD-MS review but does contain a section devoted to measurements made with parallel MS instruments and dual source instruments. Three other reviews cover the topic of elemental imaging with MS.11–13 The spatial location of a compound containing an element of interest with respect to the rest of the matrix is not technically “speciation” according to the IUPAC definition cited above. However, there is undoubtedly a growing interest in making such spatially resolved measurements an integral part of the study of the interaction of species of interest with biomolecules in biological systems (metallomics).
Three reviews of HPLC coupled with MS cover somewhat different topics. Gomez-Ariza et al.14 discuss approaches to probe health–relevant interactions between xenobiotics and mammalian organisms, with particular reference to the processes responsible for the transport of essential elements from the gastrointestinal tract to organs. Michalski et al.15 focus on the application of IC with ICP-MS or MS detection to the determination of selected halides, metal and metalloids species, pointing out that the toxicity of an “element” is often related to the presence of ionic species. The reviewers assess the application areas of environmental protection, biochemistry, geology, medicine, pharmaceuticals, and food production quality control. Arslan et al.16 highlight the fact that the detection capability of ICP-MS as an element-specific detector for HPLC can be enhanced, for the relevant elements, with CVG as the interface.
The application of X-ray spectrometry to elemental speciation analysis has been covered from several different angles. Spatially resolved information is available,17 even with laboratory based instruments, though synchrotron radiation is still needed to get to sub-μm resolution. Often XRF data are combined with those from other techniques, such as diffraction and absorption. There is a growing interest in the application of XAS techniques to the direct speciation analysis of inorganic elements in single cells.18 Focussing of beams allows both XANES and EXAFS to be applied to new applications in metal-based neuro-degeneration, cellular pharmacology, trace element physiology and metal toxicology. A variety of techniques have been applied to the in situ characterization of metals and metalloid species in plants.19 These include histochemical analysis, autoradiography, LA-ICP-MS, secondary ion MS, SEM including energy dispersive XRF, PIXE, and the synchrotron-based methods of XRF, XAS, and differential and fluorescence tomography. The review covers the advantages and disadvantages of various techniques. Potential artefacts, induced by either the analysis or sample preparation, are highlighted with the reviewers pointing out that an understanding of these artefacts is critical. It has been noted in a recent review20 that characterizing the molecular-scale chemical and biological processes involving contaminants at solid, liquid, and gaseous interfaces is fundamental to an understanding of anthropogenic environmental impacts. It was argued that in situ spectroscopic approaches have a number of beneficial features, and methods based on synchrotron techniques were discussed in detail. It was predicted that the need to investigate biogeochemical processes under environmentally relevant conditions and the need for risk assessment of new materials (such as nanoparticles) will drive further applications of these techniques.
The speciation of trace elements in biological systems has been covered from several different perspectives. Husted et al.21 highlight the important role for atomic spectrometry techniques in supporting advances in plant science. Case studies were presented that demonstrate how analytical developments have helped unravel the functional roles of several elements, particularly As and Se. It was concluded that ICP-MS is an essential technology in plant metallomics. A review in Chemical Speciation and Bioavailability22 focuses on applications for the characterisation of biota from marine ecosystems. The advantages and limitations of commonly used techniques were considered and it was concluded, unsurprisingly, that the extraction, separation and detection conditions of the metal species of interest in various samples are all different. The focus of the review is on elements that are essential but toxic at moderately higher concentrations, such as Cr and Se, and what were classified as non-essential elements such as As, Cd, Hg, Pb and Sn. The latest “omics” area, metallometabolomics (defined as the comprehensive determination of metabolites of a metallodrug in a biologically relevant sample) has been the subject of a recent review.23 In addition to covering high performance separations with ESD, molecular MS and various “nuclear” techniques (X-ray spectrometry and magnetic resonance techniques) were also assessed.
A review has been published on microwave assisted extraction (MAE) for Hg speciation.24 In addition to surveying methods (mostly for the analysis of biological and environmental materials), the reviewers provide a tutorial background on the basic theory and principles of microwave heating. There is also great interest currently in the determination of As species in waters.25 A review of HPLC-ICP-MS25 methods that have been developed in the last 10 years opens with the surprising, but unsupported, claim that As is an essential element for the human body. Some 45 articles are summarized, but there is little, if any, critical evaluation of the quality of the separations, and the topic of compound-dependent responses is, disappointingly, not mentioned. The authors conclude, not surprisingly, that procedures with better precision have lower detection limits, but there is a useful section on isobaric overlap correction and the use of collision/reaction cells. In reviewing the determination of organochlorine pesticide residues in fatty foods, Chung and Chen26 suggest that the only atomic spectrometry technique of relevance is GC-MIP-AES, but indicate that its performance is inferior to that of MS based detection. Compound-dependent responses are mentioned as one of the problems. The reviewers indicate that they will provide answers to the questions (1) is there a method available that can determine traditional organochlorine pesticides, together with their metabolites and derivatives in fatty foods, and (2) is there a suitable method that can monitor organochlorine pesticides and the recently added (by the Stockholm convention) persistent organic pollutants, including chlordecone, in fatty foods “together in a pot”? To which the answers would appear to be “no” and “maybe”, highlighting the need for further method development in this field. Gammelgaard et al.27 review present knowledge of (a) which Se species are available to the general population in the forms of food and supplements, and (b) how these species are metabolized in mammals. The review also covers new and improved sample preparation, separation and detection methods. These are evaluated from an analytical chemistry perspective to assess progress in what the authors call “horizontal” speciation (the identification of new metabolites) in yeasts and foods, whereas the analytical methods for “vertical” speciation (studies of conversions within cells, tissues or whole organisms) and the interpretation of the results, are evaluated from a biological perspective as well. In a wide-ranging review28 of spectroscopic approaches for P speciation in soils and other environmental systems, the authors compared XANES with NMR, FT-IR, and Raman spectrometries. The most common P species in soils and sediments, as indicated by the techniques, were found to be hydroxyapatite, octacalcium phosphate minerals, phosphate adsorbed on iron- and aluminium-oxides, pyrophosphates and polyphosphates, phosphate mono-and di-esters, and phosphonates. Sanz-Medel and co-workers29 have reviewed the determination of the most important platinum–DNA adducts by immunochemical assays, 32P post-labelling and MS based methods. They point out that as Pt-containing anti-cancer drugs exert biological effects by forming DNA adducts, quantitative methods are needed to (a) correlate the dose with the effects of treatment, and (b) to help understand drug resistance.
Due to its widespread use in industry, in particular the many consumer and personal care products containing silicon and the entry of silicones into the environment, a timely review30 of silicon speciation in environmental, biological and industrial matrices has been published. In particular the challenges to the determination, identification and quantification of Si containing compounds due to contamination and the low concentration of silicon compounds are covered. The principal species of concern include siloxanes, silanols, silanediols and silanes. State-of-the-art analytical methods for total silicon determination and silicon speciation included atomic spectroscopic methods to measure total Si at trace concentration levels and hyphenated techniques for Si speciation. Particular attention was paid to chromatographic methods coupled to sensitive and selective detectors such as MS, AED and ICP. Liquid and gas chromatography emerge as the most widespread separation techniques but other procedures such as MS, NMR, IR and XRF are used to enable a better understanding of these species. The potential and limitations of hyphenated techniques were highlighted, particularly concerning sensitivity and selectivity. Most importantly for this element, potential sources of contamination and analytical artefacts in the methods of determination were considered.
As is typical for any 12-month period, a number of books on topics more or less related to elemental speciation analysis have appeared. Probably containing more material of relevance to this Update than most is “Analysis of Chemical Warfare Degradation Products” by Caruso and co-workers31 which covers history and toxicity, as well as comparisons between different sample preparation methods, separation techniques, and detection methods. Quevauviller et al.32 have published an article on “Chemical Marine Monitoring: Policy Framework and Analytical Trends” that also contains a considerable discussion of speciation analysis. The text deals directly with the needs of chemical monitoring programmes such as those required by European Union policies including the Water Framework Directive (2000/60/EC) and the new EU Marine Strategy Framework Directive (MSFD).
2. Sample preparation
Almost all elemental speciation procedures involve sample preparation in order to transfer the species of interest, without species interconversion, into a matrix that is compatible with measurement by the chosen instrumental technique. Those reports for which the novelty of the work concerns some aspect of the sample preparation procedures will be discussed in the appropriate subsection below whilst articles whose contents are mainly concerned with the application of speciation will be mentioned in the appropriate subsection of Sections 5 or 6.2.1 Analyte stability
As with the work reported in previous ASUs, it is the stability of As species that is of concern. It was reported that EDTA preserved the AsIII and AsV species for several days in application to the analysis of river waters.33 It was shown that AsIII was leached more rapidly that AsV from contaminated sediments. Wallschlager and co-workers have demonstrated34 that thioarsenates typically found in sulfidic, low-iron waters can also be found in iron-rich groundwaters, and suggested that all preservation methods, such as acidification, flash-freezing, or EDTA addition, are ineffective. It was reported that in pure water, thioarsenates were stable for 21 days when flash-frozen and cryo-stored with minimal, oxygen-free headspace. The deleterious effects of oxygen could be offset by the addition of NaOH (0.1 M) or EtOH (1% v/v). The effects of FeII were eliminated by the addition of neutral EDTA solution, which preserved the speciation of the mono- and dithioarsenate species for 11 days. Tri- and tetrathioarsenate species were not stabilized, but were not found in the iron-rich groundwaters studied.Sanz-Medel and co-workers35 reported that to prevent the oxidative degradation of selenoproteins, during the extraction of Se species from rat colon tissues, it was necessary to bubble nitrogen continuously through the extraction mixture.
2.2 Extraction methods
As many speciation analysis methods involve separation of species in the liquid phase followed by ESD, the initial solubilisation of the target species is a crucial step. The development of MAE and UAE procedures continues to be reported as a consequence.Both As and Se species were quantitatively extracted (up to 97%) from rice and wheat flour by microwave-assisted enzymatic hydrolysis with a mixture of protease XIV and α-amylase.36 Approximately 0.5 g of sample was transferred into a 15 mL centrifuge tube to which was added 10 mg of amylase and 50 mg of protease XIV and 10 mL of 15 mM ammonium citrate. The tube was then placed in a 1 L beaker containing 450 mL of water and heated to 70 °C for 30 min. It is not clear whether (a) the tube was capped, (b) more than one tube was heated at a time, or (c) the sample was dried (though a correction for moisture content was made in the case of the CRMs analysed). After extraction, the samples were cooled, centrifuged (1870 g for 10 min) and filtered through 0.22 μm polyvinylidene fluoride (PVDF) filters. The filtrate was injected (200 μL) directly into the IC system with ICP-MS detection. The researchers claim that the accuracy of the procedure has been verified by comparing the sum of the concentrations of the elements in the individual species with the total concentration of the elements, but unfortunately this tells us nothing about the extent of species interconversion; on the other hand, recoveries of the individual species, spiked into the flour and dried to give concentrations ranging from 10 to 200 μg kg−1, ranged from 94 to 105%. Results were presented for NIST SRMs 1568a (rice flour) and 1567a (wheat flour). There were problems with the determination of Se in the rice flour as the “total Se” concentration (determined by spraying the extract directly into the instrument) was only 72% of the certified value. The “the sum of species” was only 59% of the certified value. The researchers suggest that the low Se values could be due to incomplete extraction. A similar extraction procedure was considered by other researchers37 for the solubilisation of As species in wheat and wheat products. However, the latter was rejected in favour of a MAE nitric acid procedure in which 0.35 g of material was heated (eventually to 95 °C) in a 6-stage procedure in a high pressure system in the presence of 10 mL of 0.16 M nitric acid. After cooling to 4 °C, the extracts were centrifuged (8000 rpm for 10 min) and filtered (0.22 μm). It was considered that two UAE procedures (with and without methanol) were unsuitable, and they also determined the speciation in NIST 1568a (rice flour), obtaining results that were significantly different from those discussed above,36 even though the “sum of species” was not significantly different from the certified value of 290 μg kg−1.
An MAE of inorganic As species from soil has been developed38 in which the extractant was a mixture of phosphoric and ascorbic acids. Interestingly enough, given the reducing nature of ascorbate, no conversion of AsV to AsIII was observed, but oxidation of AsIII to AsV occurred if ascorbate was absent. The method was evaluated by the analysis of two soil CRMs (ESS-2 and ESS-3 from the National Research Centre for Certified Reference Materials in Beijing (NRCCRM)) for which recoveries of total As of 90% were obtained. Recoveries of AsIII and AsV spikes (no information was provided as to how this was done) ranged from 85 to 104%. Nor was there any interference, in the subsequent HG determination, from any co-extracted humic materials. For the extraction of OTC from sediments39 focused MAE with tartaric acid and methanol eliminated interferences due to sulfur. The extracted butyltin species were ethylated and determined by GC-MS. Accurate analysis of a CRM (PACS-2 marine sediment, NRCC) was reported. For the determination of As species in oil,40 0.5 g was weighed into 15 mL polyethylene centrifuge tubes to which was added 10 mL of 0.5% v/v nitric acid in 80% v/v methanol. The two phases (oil was lower) were heated in a microwave system programmed to maintain the temperature at 80 °C for 120 min. After cooling and centrifuging at 0 °C for 10 min at 4000 g, the supernatant was decanted and dried at 70 °C by rotary evaporator for 20 min to remove most of the methanol. The extracts were then diluted to 5 mL with water and analysed by IC-ICP-MS. The extraction efficiency was 92% and the recoveries from spiked samples ranged from 90–105%.
Several research groups have developed UAE procedures for As species, including one for the extraction from foods by enzymatic hydrolysis with protease XIV and α-amylase in only 5 min.41 The extract was centrifuged at 3500 rpm for 10 min and filtered through a 0.22 μm filter, made up to volume and then diluted such that the final solution contained 1.6% v/v isopropanol to decrease the enzyme activity and “buffer the carbon effect.” The method was applied to a number of food CRMs including NIST SRM 1568a (rice flour), SRM 2976 (mussel tissue), IRMM 804 (rice flour), and BCR CRM-627 (tuna fish). Although the researchers give the impression that species are extracted without conversion, it appears that the inorganic species cannot be distinguished as all the results are presented for total iAs. Thus it seems likely that AsIII is oxidized to AsV. No information is provided about grinding and or drying, but as only 200 mg of dry sample is taken, the samples must be ground. Quite obvious compound-dependent responses were indicated by the slopes of the calibration plots, with relative responses ranging from 72% (AB) to 100% (AsIII). Extensive statistical evaluation of the results were presented from which it was concluded that the method was fit-for-purpose according to the relevant ISO and International Electrotechnical Commission guidelines. A phosphoric acid extraction procedure for solubilisation of As species (methylated and iAs) in atmospheric particles prior to determination by HPLC-HG-AFS has been described.42 The procedure was validated by the analysis of CRM GBW 08302 (Tibet soil, NRCCRM). In another sample preparation study,43 six As species were extracted from seaweed with nitric acid and methanol. However, before determination by HPLC-ICP-MS, a further clean-up step involving passage through an anion-exchange cartridge was needed. Spike recoveries between 81 and 120% were obtained except for AB and AC. The procedure was applied to the analysis of wakame (Undaria pinnatifida), kombu (Laminaria japonica) and laver (Porphyra tenera).
More aggressive UAE procedures for Hg species have also been developed. For the extraction of MeHg and EtHg from sewage sludge,44 samples were sonicated in TMAH, to which copper powder had been added, at 70 °C for 30 min. The analysis was completed by HPLC-CV-AFS. For the extraction of Hg species from 0.5 g of fish tissues45 in 10 mL of 6 M hydrochloric acid, both an ultrasonic bath (40 kHz and 160 W) and probe (20 kHz at 80 W) were applied simultaneously. Complete extraction was achieved in 60 s at 50 °C. Prior to determination by CV-AFS, the extract was centrifuged at 3000 rpm for 3 min. A single-cycle univariate search of the relevant parameter space was performed for which the figure of merit was maximum signal. The researchers varied, time, temperature, frequency (bath only) and power (for the bath only, as the probe output was fixed) and reagents (various combinations of methanol, formic acid, hydrochloric acid and sodium chloride). Apparent low “extraction efficiencies” at high values of relevant parameters were interpreted as the formation and loss of elemental mercury. The method was validated by spike (pre-sonication) recoveries for some real samples and by the analysis of DORM-2 dogfish muscle CRM (NRCC), which is certified for total Hg and MeHg. Although the extraction method shows promise for the determination of total Hg, there is some confusion over the accuracy of the speciation. The value obtained for iHg did not match the difference between the total and MeHg values of the CRM (and the researchers indicate that the material is certified for iHg, at a concentration of 0.710 μg g−1, which is incorrect.
An UAE procedure for the determination of CrVI in ambient air particles has been developed.46 Ambient particles were collected on 47 mm mixed cellulose ester filters that had been cleaned with nitric acid (10% v/v) and pre-treated with sodium bicarbonate (0.2% m/v). Filters were subsequently sonicated in 5 mL of nitric acid solution (pH 4) at 60 °C for 40 min. Recovery and inter-conversion between Cr species during sampling, sample storage and chemical analysis was investigated by the addition of enriched isotopes (53CrVI and 50CrIII) to the filter either before or after sampling. In addition the CRM NIST1648, urban particulate matter, was analysed. The pre-sampling spikes showed lower recoveries and higher inter-conversions than did the post-sampling spikes. Even so, recoveries of 91% for CrVI were obtained.
For the extraction of inorganic species of Sb and Te from cereals,47 UAE with various mineral acids was investigated. Extraction with aqua Regia showed clear interconversion of the species, whereas 1 M sulfuric acid extracted greater than 90% of the species with no changes. The procedure was validated by spike recovery measurements with quantification by HG-AFS.
An UAE procedure for organophosphorus pesticide residues in tomatoes has been developed48 for which acetone was the solvent. Following preconcentration by DLLME, the analytes were determined by GC with FPD.
Only one report of elemental speciation analysis featuring supercritical fluid extraction was received in the period under review. The OTC in clams49 were extracted at 45 °C and 30 MPa with carbon dioxide modified with 5% (mol/mol) methanol at 1.2 L min−1 (as carbon dioxide at ambient pressure) for 15 min. The extract was ethylated with 2% (m/v) sodium tetraethylborate in pH 4 buffer and the ethylated analytes adsorbed on a PDMS-DVB fibre at 50 °C for 30 min, followed by determination by GC-MS. The entire method took 1 h, and consumed less than 10 mL of solvent.
For the extraction of Se species from rat colon tissues,35 samples were first ground under liquid nitrogen. Then, 100 mg was transferred to a 1.5 mL sample tube and 0.4 g of extraction buffer added at 4 °C. The extractant was 50 mM tris hydrochloride at pH 7.4 to which a protease inhibitor cocktail containing 4-(2-aminoethyl)benzenesulfonyl fluoride, E-64 (trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane), pepstatin, bestatin, leupeptin, and aprotinin had been added. Four different procedures were evaluated in terms of recovery and species preservation: stirring in ice, ultrasonic bath in 4 °C water, ultrasonic probe in ice and an homogeniser (Ultra-Turrax) in ice. Different operating conditions for each device were investigated, as was the effect of the addition of a surfactant. It was concluded that the procedure with the Ultra-Turrax homogeniser was best, even though only 43% of the total selenium was extracted. The procedure (10 s on, 50 s off) was repeated 4 times with a fresh 0.4 g portion of buffer. The extracts were centrifuged at 10![[thin space (1/6-em)]](https://www.rsc.org/images/entities/char_2009.gif)
000g, and 4 °C for 30 min and the supernatant was transferred to a clean sample tube for immediate storage at −20 °C until analysis.
An acidic thiourea solution leached Hg species from infant hair50 as the first stage of a method for the determination of iHg, MeHg and EtHg. The EtHg was formed by the degradation of thimerosal (also known as thiomersal) from vaccines, to which it is added as a preservative. Prior to determination by CV-AFS, the analytes were pre-concentrated by SPE. The breastfed infants in question, from riverine Amazonian communities, were exposed to mercury in breast milk (from fish consumption and dental amalgam) and the method was able to detect and quantify acute EtHg exposure after injection of thimerosal-containing vaccines.
For the extraction of arsenolipids from fish meal Feldmann and co-workers51 mixed 50 g of fish meal with 400 mL of hexane to extract the nonpolar fraction. The mixture was shaken overnight and the supernatant (hexane plus arsenolipids) was evaporated on a rotary evaporator to 5 mL, which was then evaporated to dryness under nitrogen. The residue was further extracted with 400 mL of methanol/dichloromethane (1:2 v/v) following the same procedure to recover the polar fraction of the arsenolipids. The solvents were evaporated and the residue stored in the refrigerator until analysis (see Section 3.1.1 below).
:1, m/m) gave results identical to those from the more traditional high-volume method. The extraction efficiency from the CRM SELM-1 (selenium-enriched yeast, NRCC) was 93%. An article in Japanese53 contains a report of the enzyme-assisted extraction of As species from a CRM (NIES 27, typical Japanese diet) by pepsin and 0.07 M hydrochloric acid. From results presented in one of the tables, it may be deduced that only 77% of the total arsenic was extracted in the first extraction and that no species was extracted completely until a second extraction was performed. For one unknown compound, three extractions were needed.
Both MAE and UAE enzymatic procedures have been reported. A MAE method for the extraction of As and Se species in various cereal flours,36 which was discussed above, extracted the compounds quantitatively, according to the authors, with protease XIV and α-amylase solution in a microwave field at 70 °C in 30 min. However the sum of the Se species extracted from NIST SRM 1568a (rice flour) was only 59% of the certificate value for total Se, though the species extracted from NIST 1567a (wheat flour) totalled 90% of the certificate value. The researchers investigated the effects of temperature and time over the range 37–70 °C, and 10–30 min, respectively. Despite reporting that the efficiency of the procedure did not improve after 10 min, the researchers still selected 30 min as the optimum time for extraction. Dufailly et al.41 found that these enzymes needed to be purified before using for As-species extraction as they contain up to 2000 mg kg−1 of AsV. The mixture of enzymes was purified by passage of 2.5 mL though each of six Amicon Ultra-15 centrifuge filters at 6500 rpm for 1 h. The residue of each filter was dissolved in 1 mL of water and the procedure repeated (twice). Blank determinations indicated that the As content had been reduced to below the LOD for the various species. A number of parameters relating to the extraction were investigated, including volume and concentration of enzyme solution. The optimum conditions were 3 mL of 10 mg mL−1 of protease XIV. Further details of this work are discussed above in Section 2.2. The extraction of Se species from various organs of the African catfish (Clarias gariepinus) has been investigated.54 Several proteolytic digestion procedures were applied. The extraction efficiency of the “classical” protease XIV procedure was improved together with the preservation of SeCys integrity, by a method based on the initial addition of urea (7 M), reduction of Se–S and Se–Se bridges, by dithiothreitol, and alkylation with iodoacetamide. The urea concentration was diluted to 1 M before incubation with both protease and lipase.
Two conventional LLE extraction procedures have been reported. For the selective extraction of CrIII from water,55 a ternary solvent system consisting of water, tetrabutylammonium ion and chloroform was applied, in which phase separation occurred by ion-pairing between tetrabutylammonium and perchlorate. A small volume of the chloroform layer was extracted with a syringe, diluted 1 + 9 with ethanol and aspirated into a FAAS instrument. ChromiumVI, which remained in the aqueous phase, was subsequently reduced by sulfite. Preconcentration factors of up to 100 were obtained, allowing application to natural water samples. A method based on ion-pair solvent extraction for the speciation of Cr in various natural waters, soil, sediment and hair samples has been described.56 A β-diketone ligand, 2-(4-methoxybenzoyl)-N′-benzylidene-3-(4-methoxyphenyl)-3-oxo-N-phenyl-propono hydrazide in methyl isobutyl ketone (MIBK), was developed in which CrIII in a 200 mL sample was separated from CrVI as CrIII complex ion-paired with perchlorate. After back extraction with nitric acid, the solution was evaporated to near dryness and finally diluted to 2 mL for measurement by FAAS. Total Cr was determined after reduction of CrVI with hydroxylamine hydrochloride. The LOD was 0.3 μg L−1. The procedure was validated, at least as far as total Cr was concerned, by the analysis of two SRMs: LGC 6019 river water and RTC CRM044 soil (from which the Cr was extracted with a mixture of nitric acid and peroxide). Both species were found in process water from a tannery.
An organotin derivatisation-extraction procedure has been developed for the determination of 12 OTC in natural water samples.57 Analytes were ethylated with tetraethylborate after adjusting to pH 6 with tris–citrate buffer and extracted into hexane. The effects of several parameters, including nature of the buffer and extraction solvent, were evaluated. The optimized GC-ICP-MS procedure was applied to the analysis of water from the Northern Adriatic Sea, which contains mainly butyl and methyl-tin species.
Zeng et al. devised58 a supported liquid membrane procedure for the selective extraction of SbIII, as the DDC complex, into 1-octanol immobilized in the pores of a polypropylene hollow fibre. The organic phase was introduced by thermospray to a FF-AAS instrument. Total Sb was determined after reduction of SbV with L-cysteine. The optimized enrichment factor of 160 produced a LOD of 0.8 μg L−1.
A solidified floating organic drop micro-extraction procedure59 has been applied to the selective determination of CrIII. The analyte was extracted from 10 mL of sample solution, to which 0.2 mL of 1 M NaF and 1 mL of phosphate buffer solution (pH = 3.5) had been added, as the complex with thenoyltrifluoroacetone into 30 μL of 1-undecanol. The solution was stirred by a magnetic follower for 30 min and then cooled in an ice bath to solidify the undecanol, which was removed and placed in a vial at room temperature. A 10 μL subsample was transferred to the atomizer of a GF-AAS instrument. Total Cr was determined following addition of 1 ml of 0.05 M hydroxylamine hydrochloride solution to reduce CrVI to CrIII. The LOD was 6 ng L−1.
Three groups have developed cloud point extraction methods. One research group claims that AsV can be extracted as an ion-pair with Pyronine B in the presence of cetyl pyridinium chloride (CPC) at pH 8.0 into the non-ionic surfactant Triton X-114 for subsequent determination by FAAS.60 In another article61 a rather similar procedure that is specific for AsIII was described. The arsenic was extracted as an ion-pairing complex with Pyronine B in presence of SDS at pH 10.0 into Triton X-114. Pyronine B is 3,6-bis(diethylamino)-xanthylium chloride complexed 1:1 with ferric chloride, and a proposed structure of the compound formed with AsIII is shown. The accompanying discussion of the basis of the selectivity for the two As species is difficult to follow. The As present is eventually determined by HG-AAS. Total As was determined following reduction with thiosulfate. In the case of the selective AsV extraction,60 AsIII was oxidized with permanganate. The procedures were validated by the analysis of NIST SRM 1643e (simulated fresh water) and a quality control standard from Environment Canada, TMDA 53.3 (acidified, fortified, diluted Lake Ontario water). Other researchers separated62 the inorganic As species in water samples by retention on alumina, followed by elution with 0.2 M HCl. The AsIII or AsV in the eluent were then extracted as the complex with APDC or molybdate respectively into Triton X-114. The determination was completed by ETV-AAS giving LODs of 0.04 and 0.2 μg L−1 for AsIII and AsV, respectively. For the determination of Cr species in waters by FAAS,63 CrIII was extracted as the DDC complex into Triton X-100 (also a non-ionic detergent). Total Cr was determined directly. An enrichment factor of 98 produced a LOD for CrIII of 0.08 μg L−1. However, CrIII was only detected in an industrial waste water which contained 16 μg L−1 CrIII. Although CrVI was spiked into the samples at concentrations up to 15 μg L−1, it was not detected in all cases. However, it was reported that CrIII recoveries were not increased, suggesting that CrVI had not been reduced.
Two research groups have developed DLLME methods. The first of these concerns a method for the determination of trace organoarsenic compounds (DMA, MMA and Roxarsone) in edible oil.64 A 3.0 g sample was placed in a 15 mL screw cap PTFE test tube with a conical bottom to which a 1.25 mL portion of hexane (disperser solvent) containing 50 μL of pH 7 ammonium formate buffer solution (extraction solvent) was injected rapidly using a 5 mL syringe. The mixed solution was sonicated (ultrasonic bath power not given) for 5 min when the organoarsenicals were extracted into the fine droplets of the pH 7 buffer. The tube was then centrifuged at 10000 rpm for 5 min, causing the fine droplets to coalesce and deposit at the bottom of conical test tube, and 5 μL portions were injected into the LC-MS system for analysis. The overall analysis time was 30 min including the chromatographic separation. The LOD ranged from 1 to 6 μg kg−1, which was sufficient to detect DMA in frying oils. The determination of some organophosphorus pesticide residues in tomatoes has also been described48 in which a DLLME was applied to the sample after UAE with acetone. The extracting solvent was chlorobenzene, to which triphenylphosphate had been added as an internal standard, for the subsequent GC-FPD determination. The LODs ranged from 0.1 to 0.5 μg kg−1.
Two research groups have devised procedures in which the extractant was an ionic liquid. For Hg speciation in liquid cosmetics,65 the APDC complexes of HgII, MeHg and EtHg were extracted into 1-hexyl-3-methylimidazolium hexafluorophosphate. Enrichment factors ranged from 115 (MeHg) to 760 (HgII) for 5 mL of sample leading to detection limits of between 1 and 7 ng L−1. The analysis was completed by HPLC-ICP-MS, and for the four samples examined, HgII was the predominant species. Wuilloud and co-workers66 have adapted a method reported last year, for the determination of As, to the determination of Se. The APDC complex with SeIV was extracted into the ionic liquid tetradecyl(trihexyl)phosphonium chloride (CYPHOS® IL) and the ionic liquid phase was retained and separated on a microcolumn packed with Florisil®. The procedure was applied to the analysis of water and garlic samples, from which the Se species were extracted by UAE with sulfuric acid. In the preconcentration step, 4 mL of sample solution, 30 μL of 0.01 M APDC solution, 40 μL of 5% (w/v) Triton X-114 and 250 μL of 24% (w/v) NaClO4 were placed in a centrifuge tube and 50 mg of ionic liquid solubilised in 100 μL of methanol was injected. The mixture was vortexed for 3 s before loading onto the column, mounted in a six-port rotary valve in a FI system, at a flow rate of 0.5 mL min−1. After loading, washing with 0.1 M HCl + 0.025% (m/v) Triton X-114 solution removed any sample still in the lines and in the column. The retained ionic liquid phase was eluted with 200 μL of methanol acidified to 10% (v/v) HNO3, of which 40 μL was injected into the Pd-modified graphite furnace for ETV-AAS analysis. After reduction of SeVI with HCl, total inorganic Se was determined and hence SeVI by difference. The LOD of 15 ng L−1 was sufficient to detect SeIV in both garlic and some water samples (but not tap water); SeVI was detected in one garlic sample and in the river water samples.
Two research groups have developed ETV-AAS methods based on complexation with N-benzoyl-N-phenylhydroxylamine. In the first,67 SbIII was selectively extracted into chloroform with the aid of methanol as the disperser. Under the optimized experimental conditions, an enrichment factor of 110 produced a LOD of 0.03 μg L−1 for SbIII. The method was applied to the determination of SbIII and total Sb in natural waters. In the second method,68 CrIII was selectively extracted at pH 9.0 as the N-benzoyl-N-phenylhydroxylamine complex into carbon tetrachloride dispersed with the help of ethanol. For the determination of total Cr, CrVI was reduced to CrIII with hydrogen peroxide. An enrichment factor of 96 produced a LOD for CrIII of 0.01 μg L−1. The procedure was validated by the accurate analysis of two simulated water CRMs from NRCCRM, GBW08607 and GBW08608.
Three groups have reported methods involving magnetic nanoparticles, two of which were based on retention of the target analyte on silica-coated Fe3O4 magnetic nanoparticles modified with N-(2-aminoethyl)-3-amino-propyltrimethoxy-silane. In the first of these procedures,69 CrIII was selectively retained over the pH range 5.0 to 9.0 from environmental water and human serum samples. Following dispersion in the samples, the particles were magnetically retrieved and the CrIII eluted with 1.0 mL of 1.0 M HNO3. The enrichment factor was 100, producing a FAAS LOD of 0.66 μg L−1. Total Cr was determined after reduction of CrVI to CrIII by ascorbic acid. In the second procedure,70 AsV was selectively retained over the pH range from 3 to 8, with elution also by 1.0 M HNO3. Total inorganic As was extracted after oxidation of AsIII with permanganate. Under the optimized conditions, the LOD was 0.21 ng L−1. The method was applied to the speciation of inorganic arsenic in environmental waters from a tobacco growing area. The third method was applied to the determination of MeHg in seawater.71 The researchers synthesized a core–shell magnetic SPE nanoparticle composite material consisting of Fe3O4–polyaniline, which was characterized by SEM, EDX and FTIR spectroscopy. The composites showed a fibrous structure with diameters between 50 and 100 nm. The analytical procedure involved dispersion of the nanoparticles in water samples with sonication, followed by magnetic retrieval, then extraction into hexane for determination by GC-MS. An enrichment factor of 91 produced a LOD of 0.1 μg L−1.
A number of Hg speciation procedures have been reported, in which sulfur-containing reagents feature prominently. A further modification of the thiourea-based chromatography method, originally developed for MeHg and iHg, was adapted to separate MeHg, EtHg, HgII in infants' hair.50 Following extraction with acidified thiourea, iodide was added to form neutral ion-pair complexes that could be pre-concentrated by SPE on a column of hydrophobic polydivinylbenzene resin mounted in the loop of the HPLC injection valve. After HPLC separation, the Hg in the eluted compounds was converted to HgII and then to Hg0 by a post-column reaction system that was interfaced with the AFS detector. The LODs were between 0.050, and 0.10 μg kg−1 for a 20 mg sample. The use of thiourea complexes in the determination of the time-averaged HgII and MeHg concentrations in oxic waters has been described.72 The Hg species accumulated, after diffusion through an agarose layer, in a thiol-functionalised resin layer that was coated on a sample probe. Following extraction with acidic thiourea solution, the stable thiourea Hg complexes were separated and detected by IC-ICP-MS. The LODs were 0.7 and 0.1 ng L−1 for HgII and MeHg, respectively. Chen et al.73 retained HgII and MeHg on L-cysteine functionalized cellulose fibres packed in a small column in a sequential injection system. Following elution of both species with a mixture of nitric acid and L-cysteine, the species were selectively detected by CV-AFS. In the cold atomization mode, only HgII was detected, whereas in the flame atomization mode, both species were detected. The procedure was validated by the analysis of CRM BCR176 (trace elements in city waste incineration ash) and a cosmetic sample, both of which were dissolved in acid in a microwave system. The procedure was applied to a series of water samples and the extract of a seaweed sample, in all of which both species were found at concentrations above the LOD. When 1000 mL of sample solution was processed followed by elution with 100 mL of eluent, LODs of 1 ng L−1 for HgII and of 3 ng L−1 for MeHg were obtained. The determination of HgII and MeHg in seawater and estuarine water has been reported.74 The selective retention of HgII on poly(acrylamide) grafted onto cross-linked poly(4-vinyl pyridine) was achieved over a pH range of 1–2. A batch extraction procedure was used in which 50 mg of the resin was shaken with 10 mL of sample in a 100 mL polyethylene bottle. Retained HgII was only released by 20 min treatment with concentrated nitric acid, which rendered the resin useless. After filtering, the nitric acid was evaporated and the residue dissolved in water. The unretained MeHg was determined after oxidation with bromide/bromate solution. The AFS LOD, including the preconcentration step, was 2 ng L−1. The details of the procedure are difficult to find in the article, and it is not clear whether the SPE material was synthesised in house or obtained from the workers who first synthesized the material.
Both SeVI and SeIV have been pre-concentrated by retention on a column of activated carbon modified with tetrabutylammonium hydroxide.75 The researchers propose that the hydrophobic surface of the carbon is modified by the tetrabutyl functionality allowing the quaternary nitrogen, bearing a positive charge, to interact with the negatively charged Se species, which were loaded at pH 6–8. The samples, 200 mL of river or surface water, were loaded at 2 mL min−1 and eluted in 5 mL of 0.5 M nitric acid. The Se species were separated by IC with detection by ICP-MS. The enrichment factors were 76 and 93, for SeIV and SeVI respectively, leading to LODs of about 2 ng L−1. Although the method was applied successfully to a polluted river water sample, no systematic investigation of the possible interference from other anions was reported. The introduction to the paper contains the erroneous statement that borohydride can reduce SeVI to SeIV.
Several research groups have devised SPE procedures for Cr speciation. Most of these are confined to the determination of the inorganic species CrIII and CrVI. All possible SPE strategies including selective retention of CrIII, selective retention of CrVI, and selective retention of both species have been reported. This area of research does not seem to be converging, as in each of the three previous speciation ASUs several papers describing one or other of these approaches were included. For selective retention of CrIII, the following materials have been found suitable: montmorillonite saturated with potassium ions,76 4-(2-pyridylazo)resorcinol adsorbed on a cross-linked polymer modified with β-cyclodextrin77 (the ETV-AAS LOD was 0.06 μg L−1 for a preconcentration factor of 25), and cross-linked carboxymethyl starch.78 In this later procedure a slurry of the material prepared in an 0.1% agar suspension was directly injected into the graphite furnace for determination by ETV-AAS. Total chromium was determined after the reduction of CrVI to CrIII by 1% hydroxylamine hydrochloride. The LOD was 0.04 μg L−1 for a preconcentration factor of 50. For the selective retention of CrVI, modified polyurethane foams have been prepared by two research groups. For one, the modifier was simply HCl79 with elution by 3 M nitric acid; and for the other tetraphenylphosphonium bromide80 created a material that was capable of removing CrVI from aqueous HCl media. The retained species were recovered with 0.1 M NaOH and determined by FAAS. After oxidation with hydrogen peroxide in 0.1 M aqueous KOH, total Cr was determined. The LOD was 0.04 μg L−1, the enhancement factor and sensitivity increase factor were 80.0 and 30.0, respectively, suggesting that the FAA instrument in question has an LOD for Cr of about 1 μg L−1. Two research groups have devised procedures in which both species were retained. For determination by XRF spectrometry,81 the species, from water samples adjusted to pH 3, were pre-concentrated on ion-exchange resin disks: anionic CrVI was preconcentrated on the upper anion-exchange resin disk, whereas cationic CrIII was pre-concentrated on the lower cation-exchange resin disk. The disks were dried at 100 °C for 30 min and coated with a commercially available laminate film. The LODs were 0.2 μg. The sample loading rate was only 1 mL min−1, so relatively long times would be needed to achieve sub μg L−1 LODs. A further problem with mineral water was encountered: inaccurate recoveries because of overlap between the vanadium K-II and Cr K-II lines. A sequential elution procedure has been devised for the determination of Cr by FAAS.82 Both Cr species were retained on the same material, chloromethylated polystyrene functionalized with N,N-bis(naphthylideneimino)diethylenetriamine, at pH 6.7. The CrIII was eluted with HCl, and the CrVI was eluted with ammonia/ammonium nitrate buffer. The preconcentration factors were 70 and 30, and the LODs were 0.6 μg L−1 and 2.5 μg L−1 for CrIII and CrVI respectively.
There is continued interest in As speciation and SPE. For the determination of AsIII and AsV in waters,83 AsV was selectively retained in a cartridge containing a strong anion exchanger in the chloride form, allowing AsIII to be determined in the filtrate by HG-AAS. Total inorganic As was determined after reduction with 5% (m/v) KI + 5% (m/v) ascorbic acid. The LOD was 0.5 μg L−1 with a throughput of 60 h−1. Samples were filtered in the field and fractioned by passage through the anion-exchanger in a specially constructed sampling device that minimized interaction between the sample and any metallic components and atmospheric oxygen. Boyaci et al.84 synthesised an extractant containing both amine and mercapto functionalities, by modification of silica gel with 3-(triethoxysilyl)propylamine and (3-mercaptopropyl)trimethoxysilane, which they used to extract AsIII only at one pH (3 was selected) and to extract both AsIII and AsV at another pH (1 or anything higher than 4). It was suggested that the material was superior to a mixture of the individually functionalised silica gels. The retained species could be selectively eluted: 2.0 M HCl + 0.05 M KIO3 removed AsIII, and 1% (m/v) L-cysteine removed AsV. Alternately, 0.5 M NaOH removed both species, but also stripped the functional groups from the silica. Data on the performance of the material for the analysis of real samples was limited to the determination of total arsenic in one CRM (NIST SRM 1640, natural water) and the recovery of spikes into three relatively high purity waters. For the retention of four As species85 (AsIII, AsV, MMA and DMA), a combination of SPE columns was used. The columns contained immobilized macrocyclic materials AnaLig TE-01, AnaLig AN-01 Si, and AnaLig As-01 PA (supplied by IBC Advanced Technologies Inc.). Quantitative separation of AsIII, AsV, MMA and DMA was achieved based on the differences in extraction and recovery behaviour (as a function of pH) of the species on the different SPE columns. The procedure consisted of loading the sample (4 mL at 0.2 mL min−1) buffered at pH 5 onto a TE-01 column (which retained AsV and MMA) and the effluent and washings (with water) were collected, adjusted to pH 9 and loaded onto an As-01 column, which retained the AsIII allowing the DMA to pass through. The AsV and MMA were eluted together with nitric acid and the effluent divided into two portions, one of which was adjusted to pH 5 and the other to pH 8. Each was then washed into separate AN-01 columns; at pH 5 MMA was retained and AsV appears in the effluent, whereas at pH 8 AsV was retained and MMA appeared in the effluent. The AsIII retained in the As-01 column was removed with 0.1 M nitric acid. The As species in the various effluents and eluents were determined by ETV-AAS. Detection limits were not reported, but the procedure was able to detect As species in river water down to 0.1 μg L−1. The columns were robust, allowing over 100 loading and elution cycles before noticeable degradation in performance. For the collection of the same four species prior to HPLC separation a MnO2 mini-column was used.86 As might be expected, AsIII was oxidized to AsV, but the other species were unchanged. The species were eluted with TMAH and separated by HPLC with detection by HG-AAS. In a separate experiment, AsIII was selectively retained as the APDC complex on cellulose fibres, and was determined, after elution, by ETV-AAS, thereby allowing the determination of AsV by difference. The preconcentration was carried out in a sequential injection system linked to the HPLC injection valve via a homemade 0.45 μm mixed cellulose ester membrane filter. For a sample volume of 2.0 mL, enrichment factors of between 14 and 19 for the various species were obtained, with LODs of between 0.02 (AsIII), and 0.6 (DMA) μg L−1. Interestingly, the order of elution from the C30 column (Develosil RPAQUEOUS-AR-5) was AsV first (whereas usually this species is the last to elute), followed by MMA, then DMA. The method was applied to the analysis of snow water (which only contained AsV) and two samples of hijiki (Sargassum fusiforme), in one of which all species except AsIII were found.
A method has been proposed for the determination of SbIIIand SbVin seawater.87 Both species were retained on separate columns in a FI system. The SbIII was retained by [1,5-bis(2-pyridyl)-3-sulfophenyl methylene] thiocarbonohydrazide immobilised on aminopropyl-controlled pore glass, and the SbV was retained on an anion exchanger (Amberlite IRA-910). The sample pH was adjusted to 8.4 (borax–boric acid buffer) and the columns, mounted in tandem, were loaded at 1.1 mL min−1. Both species were eluted with 5% HNO3 containing 0.04% thiourea and determined by ICP-MS following HG. The sensitivity for SbIII was about 3 times that of SbV, indicating that either less efficient retention and elution, or less efficient HG, or both was in effect for SbV. The enrichment factors were 5.5 and 3.9, and the LODs were 13 and 21 ng L−1 for SbIII and SbV, respectively. The method was validated by the spike recovery analysis of two NRCC CRMs: SLRS-5 (river water) and TMDA-54.4 (fortified lake water).
An indirect method has been proposed for the determination of chlorophenols in water.88 A 10 mL filtered sample was loaded on to a small column of Amberchrom 161C resin (a polystyrene–divinylbenzene copolymer). After drying with a stream of argon, the analytes were eluted with 5 mL of dichloromethane. The analytes were then derivatised by esterification with ferrocenecarboxylic acid in the presence of 4-(dimethylamino)pyridine as a catalyst. The excess derivatisation reagents were retained on an alumina column while the derivatives were washed off with dichloromethane, which was then evaporated under nitrogen at 40 °C. The remaining material was dissolved in 100 μL of hexane and analysed by GC with AED at the iron wavelength. The LODs ranged from 2 to 4 ng L−1 allowing measurement of several compounds in lake water.
3. Instrumental techniques and developments
3.1 Developments in species separation
For an immunodetermination of carcinoembryonic antigen in human serum samples,90 a procedure was developed in which Hg was used as a label for the second antibody in a sandwich assay. As the first antibody was immobilized on a magnetic nanoparticle, the final separation stage of the unbound labelled antibody was aided by the magnetic immobilization of the sandwich complex. The Hg incorporated into the bound sandwich was released by reaction with nitric acid and detected by ICP-MS. For the consumption of only 50 μL of sample solution, the response was linear over the range 0.1 to 50 μg L−1, and the LOD was 0.01 μg L−1. The results for the analysis of three different human serum samples were in good agreement with that obtained by the commonly used clinical method of chemiluminescent immunoassay. The researchers claimed that the method was versatile and could be extended easily to the quantitative determination of other proteins, especially those used in for specific biomarker determinations.A popular chromatographic combination is IC with ammonium carbonate. A procedure for the determination of six As species in edible oil by IC-ICP-MS has been published.40 Modifications were made to the chromatographic conditions used in previous research by the same group in 2007.91 The column used was a Hamilton PRPX-100 and the mobile phase was ammonium carbonate at pH 8.5 in 1% v/v methanol. The ammonium carbonate concentration was stepped from 0.5 mM to 50 mM after 0.1 min. The total time for the separation was 7 minutes but no information about re-equilibration of the column was provided. Contrary to what the authors have written in the abstract of the paper, As species were not detected in the one fresh oil sample studied, though the used oil samples contained measurable amounts of all species that totalled up to about 20 μg kg−1. The relative amounts depended on the use to which the oil had been put; that from a seafood restaurant was relatively rich in AC. A separation of five compounds (AsIII, AsV, MMA, DMA and AB) in urine in under 3 min has been achieved92 by researchers, who also employed ammonium carbonate as the mobile phase, together with a fast gradient elution, and a collision reaction cell in the ICP-MS instrument. Species were eluted from an IC-PAK HR anion-exchange column (4.6 mm × 75 mm with 7 μm particles) with a mobile phase of ammonium carbonate (12 mM at pH 9.8, also containing 0.01% phosphoric acid and 0.5% acetonitrile) for 0.5 min followed by ammonium carbonate buffer (25 mM at pH 7.5, also containing 0.005% phosphoric acid and 0.5% acetonitrile and a small amount of nitric acid). Under these conditions, chloride co-eluted with MMAV and AsV, but the interference was eliminated by adding hydrogen to the reaction cell. They also presented some preliminary results for separation of the five compounds in less than 2 min on a C18 UPLC column containing 1.7 μm particles with a quaternary ammonium alkyl compound in the mobile phase.
An article in Japanese53 reports on the determination of inorganic As in a total diet CRM in which three different LC conditions, with or without a HG system, were evaluated. Detection in all cases was by ICP-MS. The article contains a table listing the various chromatographic conditions from which it may be deduced that two of the procedures are IC and the other is a RP procedure. The HG interface may be located by inspection of the chromatograms: one shown for one of the IC procedures contains two intense peaks for the inorganic As species (high HG efficiency) and a minor peak for MMA, low HG efficiency. Only one chromatogram of standards is shown, which shows significant compound-dependent responses. A Tosoh OA-pak A column (7.8 mm × 300 mm) was used for the separation with 0.27 mM sodium sulfate at pH 3.7 as the mobile phase. Under these conditions, TMAO did not elute until 43 min. The species present in highest concentrations were those derived from seafood, a common component of the Japanese diet. Wuilloud and co-workers devised93 a RP ion-pair procedure that did not involve an organic solvent. Organic As species were converted by post-column oxidation to inorganic As, so that HG-based detection could be used (in this case AFS). They found that a much better S/N was obtained without an organic solvent, such as methanol, in the mobile phase. The mobile phase selected after appropriate optimization was 20 mM citric acid and 5 mM sodium hexanesulfonate at pH 2.0, and the column was a C-18 Zorbax SB-AQ (4.6 mm × 150 mm). Other ion-pair agents, tetrabutylammonium bisulfate and perfluoroheptanoic acid, were evaluated and the pH was optimized. Compound-dependent responses were obtained, reflecting the variable efficiency of the post-column reactor, which involved merging a stream of persulfate followed by UV irradiation. Detection limits were 0.2, 0.5, 0.6, 1.6, and 1.6 μg L−1 for MMA, DMA, 4-aminophenylarsonic acid, Roxarsone (3-nitro, 4-hydroxyphenylarsonic acid), and 4-nitrophenylarsonic acid, respectively.
A separation in which AsVeluted first, but not until about 7.3 min, has been developed.86 Following preconcentration on a manganese dioxide column (discussed above in Section 2.2.3), As species were separated by ion-pairing on a C30 Develosil RPAQUEOUS-AR-5 column with a mobile phase containing, possibly, sodium 1-butanesulfonate, malonic acid, tetramethylammonium, hydroxide, ammonium tartrate and methanol. No details of the mobile phase composition or gradient were given or even cited, but can be found in Table S2 of the ESI accompanying an earlier paper.86 The mobile phase, in fact, consists of 5 mM sodium 1-butanesulfonate, 4 mM malonic acid, 4 mM TMAH, 10 mMol ammonium tartrate and 0.1% methanol at pH 2. There is no discussion in this or the earlier paper of the elution order, but a possible problem may be envisaged if the sample material contained a much higher concentration of AsV than of MMA (the next eluting peak) as the tail of the AsV peak would overlap the peak for MMA. This is not a problem when AsV elutes last, as is normally observed, other than lengthening the time needed before the next sample can be injected. A good example of which is shown in the report94 of an analytical procedure for multiple standard additions of As species by sequential injection analysis for their quantification in seafood extracts by HPLC-HG-AFS. To quantify AsIII, MMA, DMA in shark muscle, the sample extract was only diluted 10 times, producing an off-scale, tailing peak for AsV with a basewidth of about 4 min. After a further 10 times dilution, AsV was measured. As the method involved runs with 3 or 4 additions of the standards, the total chromatography time for all four analytes was almost 2 h.
Fish meal arsenolipids were separated by RP HPLC on an ACE-C18 column (4.6 mm × 150 mm).51 The analytes were dissolved in organic solvents as a result of the vacuum LC separation that was first applied to the hexane or methanol-dichloromethane extracts, and were separated with a gradient of water and methanol, both containing 0.1% formic acid, that started with water and transitioned linearly to 100% methanol at 4% min−1 followed by 10 min at 100% methanol, for a total time of 35 min. The column eluent was split 25% to a high resolution ICP MS instrument (platinum cones, 20% oxygen, microconcentric nebulizer) and 75% to the ES-MS instrument (LTQ Orbitrap Discovery; Thermo Scientific). The researchers discuss the problem of compound-dependent responses arising from the methanol gradient, which is nicely illustrated in one of the figures. The arsenic response changes by a factor of 4 as the methanol content changes from 20 to 80% (v/v). No arsenolipid standards are available and the researchers point out the problems of the compensatory gradient method (an additional HPLC pump is needed and the analytes are further diluted), and instead calculate the response factors as a function of retention time, averaging these over the basewidth of the eluting peaks.
There is still considerable interest in devising an HPLC procedure that will separate inorganic SeIV and SeVI and the commonly encountered selenoamino acids in reasonable time (say less than 10 min), preferably under isocratic conditions. The possibilities offered by room temperature ionic liquids (RTIL) as mobile phase additives for RP HPLC have been extensively investigated.95 These compounds have the interesting property that both cationic and anionic parts can affect the separation (both positively and negatively, so to speak). The anion could be adsorbed on the C18 stationary phase converting it to an anion-exchanger that interacts with cationic solute in the mobile phase. The hydrophobic imidazolium cation could interact with the C18 stationary phase to decrease its affinity for cationic solutes and enhance the exchange kinetics. When RTILs are involved, retention mechanisms involve a mixture of ion-pairing, ion-exchange, and hydrophobic partitioning. Four different RTILs were evaluated, together with pH, methanol addition, column temperature and mobile phase flow rate. The optimum isocratic conditions were a mobile phase consisting of 0.4% (v/v) 1-butyl-3-methylimidazolium chloride and 0.4% (v/v) 1-butyl-2,3-dimethylimidazolium tetrafluoroborate in 99.2% deionised water at pH 6 at room temperature and a flow rate of 1.0 mL min−1. Six target Se species including SeIV, SeVI, SeCys2, SeMet, SeMeSeCys, seleno-D,L-methionine were separated in 8 min. Considerable variations in the slopes of the calibrations were reported, but the issue of compound-dependent responses could not be evaluated as the researchers did not specify whether the mass/volume concentration units were referenced to the element or to the molecular species. The introduction to this paper includes a useful survey to the use of RTIL in analytical chemistry.
Other researchers have found benefits in increasing the column temperature. For the separation of halogen-containing drug metabolites,96 the temperature of a Waters Acquity UPLC BEH C18 column, mounted inside an oven with an aluminium holder, made in-house, for optimal heat transfer was ramped up to a maximum of 200 °C. A variety of mobile phases, consisting of formic acid and methanol or acetonitrile, and temperature gradients were used, depending on the analytes. Oxygen (5%) was added to the plasma gas. Analytes containing Br, Cl, and I were separated and quantified by ICP-MS and characterized by ES-MS. The two mass spectrometers were employed separately as detectors for the UPLC separation. The researchers point out that with temperature gradients, the separations can be manipulated without the changes in the mobile phase composition that cause problems with the corresponding changes in response factors.
Several HILIC columns have been evaluated for the separation of iron–citrate and copper–histidine species extracted from plants.97 The researchers found that three types of column were suitable for separations at pH 5.5: a crosslinked diol phase, a zwitterionic phase, and an amide phase. Detection and unambiguous identification of the different species was by ES-MS in the negative ionization mode.
Five HPLC columns have been assessed for the speciation of iodine in seawater.98 These included two anion-exchange columns (ICS-A2H and Excel-Pak ICS-A23), two cation-exchange columns (IonPac CG5A and TCC2 cation concentrator), a multi-mode size-exclusion column (AsahiPak GS-220 7C), and two combinations of cation exchange and anion-exchange columns (IonPac CG5A + ICSA2H, and TCC2 + ICS-A2H). The best performance (based on the resolving power and tolerance of the seawater matrix) was obtained with the multi-mode size exclusion column with a mobile phase of 50 mM malonic acid and 37.5 mM TMAH (pH 6.9) with isocratic elution at a flow rate of 1.0 mL min−1. The material is described as based upon a backbone of vinyl alcohol copolymers with an exclusion limit of 3000 RMM units, and the mechanism for the separation of iodate and iodide is by ion pair–hydrophobic interaction. The ICP-MS LODs were 20 and 30 ng L−1 for iodate and iodide respectively.
An automated system for the determination of MeHg analysis by purge and trap GC has been evaluated.99 The system (MERX-M from Brooks Rand Labs, Seattle, WA), consists of an autosampler, a purge and trap unit, a packed column GC/pyrolysis unit, and an atomic fluorescence spectrometer. The first step in the procedure is ethylation of the Hg compounds in the sample. Different instrument configurations, including chromatography columns (packed column or capillary), detector (AFS or ICP-MS, either quadrupole or sector field instruments) were compared. The MeHg LOD for capillary GC (Agilent HP-1 megabore column 0.53 mm × 15 m) with ICP-MS detection was 0.06 pg (equivalent to 1.5 pg L−1), though slightly lower values were obtained with the automated device connected directly to a detector (via the packed column, whose packing was not identified), in which case the LODs were 0.04 pg and 0.03 pg for AFS and ICP-MS (sector field) respectively. The automated system had a high throughput of 72 samples in 8 h, requiring considerably less analyst time than that needed for the manual US EPA 1630 method.
A GC-ICP-MS method has been developed for the bromine isotope analysis of methyl bromide.100 The 81Br:79Br isotope ratio is potentially a powerful tool to obtain information about sources, transport and reactive fate of the relevant compounds. Analytes were separated on a megabore column 0.53 mm × 30 m with 0.5 μm film thickness (Factor Four VF-5ms from Varian). With a multi-collector MS instrument (manufacturer not given), sample amounts of about 40 ng could be measured with a precision of 0.1 parts per thousand and with long-term reproducibility, as shown for 36 analyses acquired over 3 months, of a standard deviation better than 0.4 parts per thousand. The researchers report that the procedure is three orders of magnitude more sensitive than previous isotope ratio MS methods for Br in organobromines, thus allowing monitoring of ambient atmospheric samples.
A GC-ICP-MS method for the monitoring of 12 organotin compounds in natural waters has been developed.57 Following ethylation and extraction into hexane the analytes were separated on a 15 m DB5-MS capillary column (0.25 mm i.d. with 0.25 μm film thickness coated with 5% phenyl-methylpolysiloxane), which was chosen rather than a 30 m column, as the analytical run time was decreased from about 19 min to just under 13 min with no loss of resolution. The LODs ranged from 0.05–0.06 ng L−1 for methyltins, 0.1–0.4 ng L−1 for butyl-tins, 0.1–0.2 ng L−1 for phenyl-tins, and 0.07–0.1 ng L−1 for octyl-tins.
Chlorinated and brominated hydrocarbons have been detected using optical emission spectrometry.101 A miniaturized MIP emission spectrometer was used for measurement of chlorinated and brominated species following separation by capillary GC (Agilent Technologies DB-5 capillary column 0.32 mm × 30 m with 0.25 μm film thickness). The plasma was a helium microstrip device that was operated at less than 40 W and less than 250 mL min−1 of gas. An extensive optimization of all relevant parameters was undertaken followed by measurement of rotational and excitation temperatures of 1250–1650 K (OH bands emission), and 6100–6900 K (Fe emission from ferrocene vapour) respectively. For quantitative analysis, the C I 247.856 nm line was chosen because of the low spectral background intensity. For this line, the LODs ranged from 10 to 2000 μL L−1 for 7 organochlorine and 7 organobromine (both alkyl and aryl) compounds. Although iodoheptane was included in the compounds introduced as part of the temperature characterization study, iodo compounds were not included in the list of analytes. A method for the determination of several chlorophenols in water samples has been developed.88 Following preconcentration by SPE of the derivatives with ferrocenecarboxylic acid, the analytes were separated on a DB-5ms column (0.25 mm × 30 m with 0.25 μm film thickness) in an Agilent 6890N gas chromatograph, and detected via the iron emission at 302 nm in an Agilent G2350A atomic emission detector. For 10 mL samples, the LOD for mono- to trichlorophenols ranged from 2 to 4 ng L−1.
A method to improve the LOD for the determination of methylated Hg species in seawater using AFS has been described.102 An extensive report of the method development and optimization is given. Dimethyl Hg was purged directly from an unaltered 2 L sample following acidification to 1% with sulfuric acid, whereas MeHg was ethylated before purging and trapping on Tenax with subsequent thermal desorption. The analytes were separated on a 30 cm × 3.2 mm i.d. isothermal column at 60 °C packed with 60/80 mesh Chromosorb WAW-DMSC coated with 15% OV-3 connected to an AFS detector. There is no information at all about this device in the paper but readers are directed to an 2004 article by the same research group. The LOD was “about 2 fM” (about 0.2 parts per quadrillion) for a 2 L sample, which is a 10-fold improvement over current approaches for this analysis.
A method has been published for the determination of organophosphorus pesticides residues in tomatoes.48 The analytes were separated on a ZB-35 fused silica capillary column (0.25 mm × 30 m with 0.15 μm film thickness of 65% methyl-35% diphenylpolysiloxane copolymer) followed by FPD. Analytes were pre-concentrated after UAE by DLLME to give LODs that ranged from 0.1 to 0.5 μg kg−1 for the 13 pesticides studied, phorate, diazinon, disolfotane, methyl parathion, sumithion, chloropyrifos, malathion, fenthion, profenphose, ethion, phosalone, azinphose-methyl and co-ral.
Under appropriate conditions, preconcentration of analytes during injection may be achieved. The mechanism of enrichment for pressure-assisted electrokinetic injection of inorganic As, Se, and bromate has been investigated.105 It was found that analytes accumulated at the sample-buffer boundary and proposed the formation of a counter-ion layer at the separation buffer side of the boundary. Based on the analyte diffusion behaviour in the sample plugs, it was also proposed that this cationic layer helped to suppress diffusion. Detection was by ES-MS and the LODs were ranged between 1 and 3 μg L−1.
One-dimensional GE with LA-ICP-MS detection was used for the characterization of zinc-containing proteins extracted from slug tissues.106 By far the largest fraction of protein-bound Zn was contained in three sharp and prominent bands at 75, 100 and 150 kDa. However, analysis of the tryptic digests of the bands by MALDI-TOF-MS and public databases failed to identify the proteins within the Zn bands. On the other hand, three proteins not containing Zn were identified. The distribution of total Zn in a section of a slug sample was mapped by LA-ICP-MS. It was pointed out that an advantage of the LA-ICP-MS detection of separated proteins derives from the high sensitivity that allows detection of low abundance proteins, which are invisible after Coomassie Blue staining. In a study107 of the identification and quantification of human plasma selenoprotein P (SelP) and glutathione peroxidase 3 in NIST SRM 1950 (metabolites in human plasma), several separation techniques were used, including GE. The detection capability of the subsequent LA-ICP-MS measurement was improved by first transferring the separated bands onto PVDF membranes by electroblotting and then introducing an organic solvent (1% butanol) into the plasma along with the ablated material, so as to take advantage of the enhanced ionization in the presence of carbon in the plasma. Once the presence of Se was confirmed in a band, the gel was treated to remove the protein for identification by tryptic digestion and HPLC-MS. Sanz-Medel and co-workers108 studied the oxidative damage to DNA produced by FeII and hydrogen peroxide. After optimization of the continuous elution GE system for large DNA fragments, the GE separation of the different oxidation products was followed by monitoring 31P+ with ICP-MS. They point out that the main advantage of the method is that each oxidation product could be quantified with just inorganic phosphate as an internal standard, and also that the method functions as a diagnostic tool for monitoring enzymatic reactions, such as measuring the activity of DNA restriction enzymes. A multi-institutional study of Se speciation in different organs of African catfish (Clarias gariepinus) on a Se-enriched garlic based diet has been published.54 The work focused on the selenium distribution in the soluble protein fraction and the detection of selenoaminoacids in several organs and tissues, including the liver, gills, kidney, muscle and gastrointestinal tract. Two procedures were used, one of which involved LA-ICP-MS after separation by SDS-PAGE and electroblotting onto nitrocellulose membranes.
3.2 Chemical vapour generation
In this section, only methods in which some aspect of the vapour generation chemistry is exploited to achieve selective detection of the target analytes are reviewed. For example, post-column HG of arsenic species will allow detection of only those species that are borohydride active. In the case of As, these are often the species of interest as they are by far the most toxic; whereas, the detection of AB and AC which are non-toxic and borohydride inactive, may be less important.An IUPAC technical report109 entitled “Mechanisms of chemical generation of volatile hydrides for trace element determination”, was co-authored by many of the world's HG experts. The report might well be the last word on the rationalization and clarification of fundamental aspects of chemical HG including: (i) mechanism of hydrolysis of borane complexes; (ii) mechanism of hydrogen transfer from the borane complex to the analytical substrate; (iii) mechanisms by which the different chemical reaction conditions control the chemical HG process; and (iv) mechanisms of action of chemical additives and foreign species. The authors consider that analytical chemical HG is still dominated by “erroneous concepts which have been disseminated and consolidated within the analytical scientific community over the course of many years. The overall approach to HG has thus remained completely empirical, which has hindered possibilities for further development”.
There are two reports that describe speciation based on selective HG of one species followed by the conversion of all other species to the hydride-forming species and a second determination. A significant difference between the two values is interpreted as the presence of other species of the element of interest. Inorganic Sb and Te species in cereals have been determined by HG-AFS.47 The HG conditions were adjusted so that only the lower oxidation state species was borohydride active. A second portion of the extract was analysed after treatment with a reagent under conditions known to reduce the higher oxidation species: potassium iodide in the case of SbV and hot hydrochloric acid in the case of TeVI. The LODs ranged from 0.1 to 0.5 μg kg−1, which were good enough to detect the species in some real samples. The speciation of inorganic Sb in airborne particulate matter by slurry sampling and HG-AAS with quartz tube atomization has been described.110 In this approach, the SbIII was determined after the addition of citrate (which masks SbV), whereas total Sb was determined after reaction with potassium iodide and ascorbic acid in the presence of hydrochloric acid. The LODs were 0.1 and 0.06 μg L−1 for total Sb and SbIII, respectively, low enough to detect the species in four real samples collected in Bananeira Village, Bahia State, Brazil. It was calculated that total Sb concentrations varied from 4.3 to 4.6 ng m−3, and SbIII concentrations varied from 0.33 to 0.67 ng m−3.
A method for the determination of trivalent methylated arsenicals in mouse liver111 has been developed in which the analytes were derivatised by reaction of slurried samples with BH. Following phase separation, the hydrides were cryotrapped and sequentially released according to their boiling points. The LODs were about 6 μg kg−1. All of the analytical information is in the supplementary information that is available free from the journal website. The procedure used was published in 2008.
There are several reports of speciation procedures based on the judicious chemical manipulations inside electrothermal atomiser or vaporiser devices. For the determination of some As species in fish-based baby foods,112 10% (m/v) slurry samples were prepared in 0.01 M TMAH and analysed in the presence of three different chemical modifiers: palladium, cerium and zirconium. Using a palladium salt as the chemical modifier, the signal obtained corresponded to total As and when cerium was used as a modifier the signal was due to both inorganic As and MMA. If no signal was obtained (i.e. no As or MMA present), then a zirconium-coated atomizer was used to specifically determine DMA only. The concentration of AB could be obtained by the difference between this signal and that for the total arsenic. The underlying mechanism for the selectivity observed was not explained, but it is implied the effect is due to differential stabilization during the thermal pre-treatment (ashing) stage rather than selective atomization. The introduction to the paper includes reference to several publications in which the phenomena have been reported, some of which are more than 20 years old. The LODs were 15, 25 and 50 μg kg−1 for AB, DMA and (inorganic As + MA), respectively. When applied to the analysis of seven foodstuffs, only the first two species were detected, though for four of them the DMA concentration was below the LOD. Calibration was by standard additions. It has been proposed that an ETV-AAS speciation method could be developed by utilising the very great difference in sensitivities for Sb species obtained in the presence of complexing reagents (DDC, APDC and thenoyltrifluoroacetone).113 The basis of the method is the loss of one species during the ashing stage. This concept has been exploited for Cr speciation by ICP-MS with sample introduction by ETV.114 The volatility and stability of the CrIII/8-hydroxyquinoline chelate is such that it could be quantitatively vaporized and transported from the graphite furnace to the ICP at the relatively low temperature of 1100 °C. The LOD was 13 ng L−1 with a linear calibration over 3 orders of magnitude. The method was applied to the determination of CrIII and CrVI in water samples. In a slightly later paper,115 the same researchers describe a modification to improve sensitivity. Replacing 8-hydroxyquinoline with thenoyltrifluoroacetone improved the LOD to 8 ng L−1. This also had the effect of reducing the vaporization temperature required for the analysis to 900 °C. The CrVI was reduced to CrIII by ethanol prior to detection. The procedure was applied to several water samples in which both species were detected at single digit μg L−1 concentrations.
Although there is nothing really new in the idea of selective HG based on the reaction conditions, three articles describe specific procedures in which this concept is the basis of the speciation. For the determination of AsIII and AsV in mining river waters and leached from contaminated sediment33 selective HG was controlled by the acidity of the generating reaction. In neutral solution only AsIII reacts; whereas, in acid solution both As species react. Other researchers38 measured the species in water and soil extracts by HG-AFS by control of both the acidity and KBH4 concentration: in 2% HCl and with 1.5% KBH4, only AsIII was BH active; in 8% HCl and with 6.0% KBH4, both AsIII and AsV reacted to form arsine. No interferences from other matrix components of the seawater or soil samples (such as humic materials) were observed. A method based on slurry sampling has been proposed for the inorganic As speciation analysis of dietary supplements.116 The AsIII was determined in 1% HCl in the presence of 8-hydroxyquinoline, which was found to inhibit the HG from AsV while enhancing that of AsIII. However, when the acidity was increased to 5% HCl, 8-hydroxyquinoline enhanced the sensitivity of both species. The authors do not cite any earlier references to the effect of 8-hydroxyquinoline on arsine generation, so this may be a new phenomenon. Curiously, no signals were obtained from either DMA or MMA (deliberately added) under either reaction condition, so presumably 8-hydroxyquinoline inhibits the HG reaction from those species. The researchers suggest that this phenomenon should be investigated further. The LOD was 4–5 μg kg−1, low enough to detect the analytes in four real samples.
3.3 Developments in instrumentation
The dual source (ICP and nano-ES) mass spectrometer developed by Hieftje and co-workers, mentioned in last year's ASU,4 has been used for Cr speciation.117 Various salts, including CrIII chloride and picolinate (common dietary supplements) were introduced in a variety of solvents (water, methanol or ethanol). It was concluded that the molecular channel provided valuable information about the Cr–solvent interactions and that the information can be used to identify unresolved chromatographic peaks (should the instrument be used as an HPLC detector, which would seem to be work that is planned for the future). The ICP channel LOD was 0.03 μg L−1 and the mass accuracy of the ES channel was as good as 10 parts per million.There is continued interest in the possibilities of glow discharges in chemical measurement technology, which includes the possibilities for speciation analysis. The majority of the papers in the April 2011 issue of JAAS were devoted to GD spectroscopy, including two very substantial review articles by Jakubowski and co-workers118,119 devoted to sector field MS with either ICP or GD sources. The first of these (182 references) is a tutorial on the history, fundamentals and instrumentation, and the second (414 references) is devoted to applications. The applications review includes a substantial section devoted to speciation analysis (87 references) and in their summary and outlook, the reviewers pointing out that high mass spectral resolution brings the possibility of overcoming spectral interferences, which they characterize as the major Achilles' heel in ICP-MS.
Sanz-Medel and co-workers have reported120 on a new μs pulsed GD ion source coupled to a TOF mass spectrometer that was recently designed, constructed and evaluated for the simultaneous collection of elemental and molecular information, and as a GC detector for compounds of environmental relevance, such as volatile organic halogenated compounds. It was shown that the intensity ratios for Cl and Br could be correlated to the number of atoms in a molecule of the selected compound and, furthermore, that compound-independent calibrations were possible. When coupled with SPME introduction, the LODs were low enough to detect chloroform, at concentrations ranging from 15–36 μg L−1, in real samples of river and drinking water. Other researchers121 have determined the speciation of Fe oxides in solid samples by pulsed millisecond radio frequency GD-TOF-MS following an extensive optimization of relevant operating parameters that included sampling distance, temporal regime, discharge gas pressure, pulse frequency, and duty cycle. It was found that variations in the ratios of the Fe+ and FeOH+ signal intensities could be related to the presence of FeO, Fe2O3, and Fe3O4, and that Fe–O cluster generation correlated with oxygen abundance in the original sample.
Sanz-Medel and co-workers also reported the combination of molecular fluorescence with ICP-MS for the detection of nanoparticle-biomolecule conjugates separated by SEC.122 No details of the instrumentation are provided either in the article or the supplementary information, so it is not clear whether the eluent is split between the detectors or whether the fluorescence detector has a flow-through cell, allowing coupling of the spectrometers in series. The utility of the combined detection scheme is demonstrated by the quantitative study of the bioconjugation of quantum dots (both CdSe and CdSe/ZnS) to antibodies (monoclonal mouse anti-progesterone), a critical step for the future use of quantum dots in quantitative fluorescence immunoassays.
There is only one report of further application of dual inorganic and organic MS detection instruments. Feldmann and co-workers have used such a system for the identification and quantification of arsenolipids in fish meal.51 The analytes were separated by reverse-phase HPLC with simultaneous detection by high-resolution ICP-MS and high-resolution electrospray MS. The chromatographic eluent was split 75% to the organic mass spectrometer and 25% to the inorganic mass spectrometer. The eluent consisted of 0.1% formic acid in water at the beginning of the run and 0.1% formic acid in methanol at the end of the run. Response factors were established as a function of retention time (see also Section 3.1.1). The retention times of the arsenic species obtained from the ICP-mass spectrometer (the As-specific detector) were used to find the accurate mass from the ES-MS data (to ascertain the molecular formula) and to obtain structure information from the MS/MS fragmentation pattern. The arsenolipids, present in the highest concentration in the hexane extract of fish meal from capelin (Mallotus villosus), were three dimethylarsinoyl hydrocarbons (C23H38AsO, C17H38AsO, and C19H42AsO) with minor amounts of dimethylarsinoyl fatty acids (C17H36AsO3, C23H38AsO3, and C24H38AsO3). It was noted that an acid with an even number of carbons in the fatty acid chain, was identified for the first time. All previously identified dimethylarsinoyl fatty acids have an odd numbers of carbons.
3.4 X-ray methods
The speciation-themed January 2011 issue of JAAS contains a review of the direct speciation analysis of inorganic elements in single cells by XAS.18 It was highlighted in this review that it is the recent development of X-ray focusing optics with spatial resolution down to μm values, while maintaining high photon fluxes, that makes this application possible. However, micro-EXAFS is still more challenging to perform than micro-XANES, as the application requires good signal to noise ratios. Users are cautioned not to be complacent about the interconversion of species just because the technique gives “in situ” information and does not require any sample preparation. It is recommended that species integrity is tested systematically at all stages of an analytical procedure, including during storage and transport.In contrast to the previous ASU review period, there have been very few reports of X-ray methods applied to the speciation of elements in biological samples. Banuelos and co-workers investigated the speciation and distribution of Se in the edible parts of the cactus Opuntia ficus-indica123 with a range of techniques, including microfocused XRF, XANES, ICP-MS, and LC-MS. They were able to distinguish species in a carbon-Se-carbon environment from species in which Se was bound to oxygen, which they interpreted as selenate. It was concluded that the cactus contained mostly non-proteinaceous selenocystathionine and SeMet in the fruit and cladode (leaf-like structures), whereas the seeds contained mainly protein-bound SeMet. The molecular species were identified in Se-containing LC fractions (identified by ICP-MS) by organic MS. Other researchers89 evaluating the biotreatment of concentrated selenium wastewaters examined the solid species, formed by microbial reduction, with XANES spectroscopy at the Se K-edge. For samples incubated with the highest SeIV/SeVI concentrations, a predominance of elemental Se together with a selenide (Se-I), such as ferroselite, the thermodynamically most stable iron selenide, was found. In contrast, at the lower SeIV/SeVI concentrations, elemental Se and Se(-II) selenides were the main species detected.
X-ray microtechniques have been employed to study the speciation of Cu and Zn in size-fractionated atmospheric particulate matter by SR-TXRF-XANES.124 Sample collection was carried out in different urban and rural areas with a 7-stage May cascade impactor that had been adapted for sampling on silicon wafers. Results depended on the sample origin: the largest particles (2–4 μm) contained Cu and Zn species typical of soils, whereas the fine particles contained Cu and Zn sulfates and nitrates. It was possible to discern the speciation at concentrations as low as 140 pg m−3.
Also of interest in this section is a report of elemental speciation by a molecular spectroscopic technique. The oxidation state of several Fe samples were identified by resonance Raman spectrometry125 performed at the XRF station of the D09B-XRF beamline at the Brazilian synchrotron facility, Campinas. The samples were irradiated with monochromatic photons having energy slightly lower than the K absorption edge of Fe in order to excite the resonance Raman emission. The spectra were analysed by fitting the experimental data to theoretical expressions and extraction of the residuals on the low-energy side of the RRS peaks. The researchers found that the residuals exhibited an oscillation pattern that depended on the oxidation state of the Fe.
3.5 Separation-detection interfaces
All elemental speciation procedures that involve continuous separation of analyte species, which are sequentially delivered to an element-specific atomic spectrometric detector, require an interface between the separation device and the detector. This applies to all chromatographic (HPLC and GC) and electrophoretic (CE and GE) separations. Consequently many of the research articles discussed in this ASU contain information about the separation detection interface. Unless the researchers themselves have called attention to the novelty of the interface in question by, for example, mentioning it in the title and/or abstract of their paper, the work is not discussed in this section. The interface may be an active component of the ionization for MS detectors. It should also be borne in mind that the field of interfacing analyte separations with molecular MS is a much more active research area than the field of interfacing for elemental speciation analysis, but that advances in this area, which may well eventually lead to applications for elemental MS (or even optical spectrometries), are not covered in this Update.Marcus and co-workers126 have developed a low-power GD ionization source for the elemental MS analysis of aqueous solutions in which the surface of the liquid emerging, at atmospheric pressure, from a 75 μm i.d. glass capillary acts as the cathode of the direct current GD. The device, which operates at about 5 W, was mounted in place of the normal ES source of an Orbitrap mass spectrometer. A multi-element spectrum showed complete resolution of the lead isotopes. Although the background could be dominated by water clusters, these can be considerably decreased by changing the operating conditions, including decreasing the liquid flow to 0.1 mL min−1. Surprisingly, there would seem to be little metal oxide formation. The detection capability was illustrated with 5 μg L−1 Cs which gave a S/N of over 1000. It is pointed out that with appropriate optimisation the source may present a practical alternative to high-powered (>1 kW) ICP sources typically employed in elemental MS, particularly for those cases where integrated elemental/molecular analysis considerations are important.
A VG system involving a solution cathode GD has been developed as the interface between the HPLC separation of Hg species and AFS detection.127 In preliminary experiments with FI sample introduction, the addition of formic acid was found to be beneficial. However, for the determination of the species following HPLC separation, 2-mercaptoethanol an ion-pair reagent was found to be almost as effective at enhancing the fluorescence signal. Compared to the signals obtained in the absence of the organic modifier, formic acid increased the signals between 11 and 14 fold, whereas 2-mercaptoethanol increased the signals between 10 and 11 fold. The LOD were 0.7 μg L−1 for HgII, 0.6 μg L−1 for MeHg and 1 μg L−1 for EtHg respectively, which were sufficient to allow the analysis of CRM GBW 10029 tuna fish (NRCCRM).
Matusiewicz and Ślachciński128 developed an extremely low-flow, demountable direct injection high efficiency nebulizer as an interface between microchip-based CE separations and a MIP-OES detector. A supplementary buffer solution flow, delivered by a microsyringe pump, at the channel exit improved nebulisation efficiency, and a small evaporation chamber was incorporated into the interface to prevent losses, thereby allowing the entire aerosol sample to enter the plasma. The uptake rate was about 0.5 μL min−1 and the sample volume was 25 nL. They showed that it was possible to separate CuII and Cu(EDTA)2− in 90 s in a 26 mm separation channel etched in glass. The LODs were about 3 μg L−1.
In a review of the applications of high resolution MS with ICP and GD sources, Jakubowski et al.119 include a section relating to improved interface design for enhanced sensitivity and briefly discuss several advances in interfaces for plasma sources.
A gradient HG interface has been developed to interface HPLC separation with quartz tube AAS for As speciation.86 The hydrochloric acid concentration was stepped from the initial value of 6 M to 0.6 M to improve the sensitivity for the HG of the later eluting species. The chromatography elution order was AsV first, followed by AsIII, MMA, DMA and finally TMAO. The addition of L-cysteine to the hydrochloric acid further improved the sensitivity. The mobile phase flow rate was 1 mL min−1 and on emerging from the column, streams of hydrochloric acid (6 or 0.6 M) + L-cysteine (2% m/v) and borohydride (1% m/v in 0.5% NaOH) were merged, each flowing at 1.1 mL min−1. The combined streams passed through a 3.5 m coil heated to 60 °C (in a water bath) and then through a cooling bath (no details given) before delivery to a gas–liquid separator (GLS) that appears to have been constructed in-house and not described previously. The argon carrier gas was introduced at the GLS and not merged into the reagent flow line. A detailed drawing of the GLS is provided that would allow a glass shop to fabricate the device. Detailed drawings are also provided of three other designs that were considered inferior. Curiously, there is no discussion of the effect of flow rates of the merged reagents. In the design described, the column effluent is diluted by a factor of three, which must have been reflected in decreased sensitivities and LODs, as the signals were quantified by peak height. It is not clear why the borohydride and acid concentrations were not maximized, allowing a corresponding decrease in the flow rates and concomitant decrease in the dilution factor, which would have produced better LODs. The gradient consisted of stepping the acid concentration from 6 M to 0.6 M when the MMA peak was detected, so that the DMA and TMAO reacted at the lower acidity. The LODs, for a 100 μL injection, ranged from 0.9 to 1.6 μg L−1. When combined with preconcentration on a manganese dioxide column,86 the LODs were decreased to between 0.3 and 0.6 μg L−1 for AsV, MMA, and DMA. This aspect of the work was discussed above in Section 2.2.3, Solid-phase extraction.
4. CRMs and metrology
Two papers describing the development of speciated CRMs have been published this year. A Japanese language paper, with the abstract in English, covers the preparation and certification of a polystyrene resin containing decabrominated diphenyl ether (DBDE),129 NMIJ CRM 8108-b. The material is in the form of a disk of 30 mm diameter with a thickness of 2 mm. The Br concentrations within and between the disks were adjudged to be homogenous by XRF spectrometry. Subsequently, the Br concentration was measured by GC-MS and HPLC-UV with the found concentrations from each method agreeing within the limit of uncertainty. The concentration of DBDE in the CRM was certified as 312 mg kg−1 with an expanded uncertainty (k = 2) of 16 mg kg−1. Unfortunately, no details of the extraction procedures and analytical protocols employed are described in the abstract. The second material of interest is NIST SRM 955c, Toxic Elements in Caprine Blood, which is the first blood matrix certified for EtHg.130 The certified values and associated combined expanded uncertainty (k = 2) for MeHg, EtHg, iHg and total Hg are 4.5 ± 1.0, 5.1 ± 0.5, 9.0 ± 1.3 and 17.8 ± 1.6 mg L−1 respectively. Total Hg was measured, after spiking with 201Hg and MAE with concentrated HNO3, by CV-ID-ICP-MS. Cold vapour generation was conducted online with Sn(II)Cl2. Aliquots of the candidate CRM were spiked with IRMM-670 202Hg enriched methylmercury CRM solution, 201Hg enriched ethylmercury solution, and inorganic 198Hg solution prepared in high-purity water for the determination of Hg species by GC-ID-ICP-MS. For these analyses Hg species were solubilised with TMAH and MAE, 35 W for 4.5 minutes, followed by propylation with sodiumtetrapropylborate and sample clean up with a water-deactivated alumina SPE column. A 30 m DB-5MS + DG 250 mm i.d capillary column coated with a 0.25 mm thick film of (5%-phenyl)-methylpolysiloxane was used for GC separations and coupled to the ICP-MS by a commercially available heated transfer line. The authors noted that although EtHg decomposition was observed during the sample preparation procedure this effect was consistent and did not affect analytical performance.A new approach has been developed for the determination of the spike mass flow rate in post column species unspecific IDMS.131 Conventionally, the spike mass flow rate is determined by reverse ID-MS via the injection of a natural abundance ‘spike’ into the continuously pumped isotopically enriched spike stream. This method assumes that the spike flow rate is constant during the analytical procedure which may not be the case, for example, if a gradient HPLC elution profile is employed. The new method uses a modified ID-MS equation, in which the spike mass flow rate cancels in the final measurement calculation, and allows the analyte mass fraction to be directly calculated from isotope amount ratio chromatograms. The spike mass flow rate was also gravimetrically monitored, with data recorded every 0.1 s, to evaluate the procedure. The method was applied to the measurement of SeMet by approximate matching su-ID-HPLC-ICP-MS in the selenised yeast SRM SELM-1 (NRCC) after a two stage enzymatic digestion (Protease XIV, lipase and driselase) at 37 °C for 18 hours. An isocratic elution using a 5 μ × 3 mm i.d. × 250 mm C8 column with a mobile phase of 0.1% TFA in 2% methanol was used to separate the Se species prior to introduction to the ICP-MS instrument operated in collision/reaction mode. The found SeMet value in SELM-1 of 3.25 ± 0.11 × 103 mg kg−1 was in good agreement with the certified value of 3.389 ± 0.173 × 103 mg kg−1. The major uncertainty contributions arose from the uncertainty of the measured isotope amount ratios in the samples and spike, 36% relative for each parameter.
The determination of AB in biological tissues by ss-ID-HPLC-ES-MS and standard addition HPLC-ICP-MS has been described.132 A natural abundance AB standard, synthesised in house and characterized by quantitative 1H NMR, was used for reverse ID and standard addition HPLC-ICP-MS whilst for IDMS an in-house synthesized 13C enriched AB spike was used. Samples were extracted by sonication for 30 min with high purity deionised water. The ES-MS instrument was operated in scan mode (m/z 170–190) and the intensities of [M + H](+) ions at m/z of 179.0053 and 180.0087 were used to calculate the ion ratio for quantification of AB in fish tissues. An exact matching ID-MS approach was applied to overcome potential difficulties in mass bias correction. Concentrations of 9.65 ± 0.24 and 11.4 ± 0.39 mg kg−1 (expanded uncertainty, k = 2) for AB in two fish samples were obtained by ss-ID-HPLC-ES-MS. These results were in good agreement with those obtained by standard addition HPLC-ICP-MS, 9.56 ± 0.32 and 11.26 ± 0.44 mg kg−1 (expanded uncertainty, k = 2), respectively. A fish tissue CRM, NRCC DORM-2, was used for method validation. The results obtained, 38.7 ± 0.66 and 37.9 ± 1.8 and mg kg−1 (expanded uncertainty, k = 2) for AB by ss-ID-HPLC-ES-MS and standard addition HPLC-ICP-MS respectively, were in good agreement with the certified value of 39.0 ± 2.6 mg kg−1 (expanded uncertainty, k = 2). Detection limits were calculated to be 0.033 and 0.011 mg kg−1 AB by ss-ID-HPLC-ES-MS and standard addition HPLC-ICP-MS, respectively.
5. Elemental speciation analysis
5.1 Antimony
A new insight into poor chromatographic recoveries for antimony has been presented by Hansen et al.133 Although it is recognised that in solution Sb exists either in the pentavalent or trivalent oxidation state, most chromatographic methods used for this redox speciation analysis show a low chromatographic Sb recovery when samples of environmental or biological origin are analysed. A combination of HPLC-ICP-MS, asymmetric FFF-ICP-MS and spin-filtration was applied for analysis of model compounds and biological samples. Quantitative chromatographic Sb redox speciation analysis was possible by acidic hydrolysis of the Sb polymers prior to analysis. Sample treatment procedures were studied and the optimum solution was acidic hydrolysis by 1 M HCl in the presence of chelating ligands (EDTA, citrate), which stabilise the trivalent oxidation state of Sb. The formation of macromolecules, presumably oligomeric and polymeric SbV species, was found to be the primary cause of low chromatographic recoveries.An analytical method for the determination of SbV, SbIII and TMSbV in occupationally exposed human urine samples by HPLC-HG-AFS has been proposed by Quiroz et al..134 Studies on the stability of Sb species in urine samples as a function of preservation at 4 °C and storage at −70 °C were performed. The results revealed that Sb species are highly unstable at −70 °C, probably due to co-precipitation reactions. It is proposed that in this kind of matrix transformation during preservation may occur, such as oxidation of SbIII to SbV and transformation into species that do not elute from the column. Studies using EDTA showed that it was possible to stabilize SbIII for more than a week at 4 °C and thus avoiding co-precipitation during storage at lower temperatures. An Hamilton PRP-X100 5 μm column was used for the separation giving retention times of 0.88, 2.00 and 3.61 min for SbV, SbIII and TMSbV species respectively. The LODs were 0.18, 0.19 and 0.12 μg L−1 for the same species using a 100 μL injection loop.
Micro-grains coated with a Sb-rich layer have been characterised from soil found near a Sb mine.135 The study was performed using μ-XANES, μ-EXAFS, μ-XRD, TEM, and EPMA. The EPMA showed that soil grains locally accumulated a large amount of Sb in the secondary phases (40–61 wt% Sb2O5) with significant Fe (20–28 wt% Fe2O3). The spatial distribution of Sb in the grain was similar to that of Fe. Both Fe μ-XANES and μ-XRD of the Sb hot spots consistently showed that the secondary products were dominantly composed of ferric antimonate, tripuhyite (FeSbO4). Fits to the Sb K-edge μ-EXAFS of this phase showed second-neighbour coordination numbers similar to 30% smaller than in bulk tripuhyite, indicating that the tripuhyite included in the grain is nanoparticulate and/or has a high structural disorder. The TEM analysis suggested that the particle size of tripuhyite in the grain was around 10 nm, which is consistent with the size range indicated by μ-XRD and μ-EXAFS. This is the first report showing tripuhyite with nanocrystallinity in natural soil. The distinction between ore processing and post-depositional transformation on the speciation of Sb (and As) in mine waste and sediment has been studied.136 The study is discussed in more detail in Section 5.2 of this review.
5.2 Arsenic
The speciation of As continues to receive much attention, although increasingly the emphasis is on utilising established techniques to look at specific applications. Developments in terms of instrumental techniques which may be used to determine As species are presented in Section 3 of this review. Applications focusing on the determination of As species are presented here, although once again it should be noted that some work fails to report on the oxidation state of the compounds used. Of particular interest this year, at least in terms of the number of publications, would seem to be the determination of As in rice (Oryza sativa).The concentration of total As and the five main chemical species of As (AsIII, AsV, DMA, MMA and AB) were evaluated in 44 different rice samples (white, parboiled white, brown, parboiled brown, parboiled organic and organic white) from different Brazilian regions using HPLC-ICP-MS.137 The mean level of total arsenic was 223 ng g−1 and the daily intake of iAs from rice consumption was estimated as 10% of the Provisional Tolerable Daily Intake (PTDI) with a daily ingestion of 88 g of rice. The predominant forms in all samples were AsIII, AsV, DMA, MMA. The percentages of species were 38.7; 39.7; 3.7 and 17.8% for DMA, AsIII, MMA and AsV, respectively. However, regional differences were found, for example rice samples harvested in the state of Rio Grande do Sul had more iAs than rice in Minas Gerais or Goias, which could lead to different risks of As exposure. The Bangladeshi community living in the UK has a ca. 30-fold higher consumption of rice than white Caucasians. In order to assess the impact of this difference in rice consumption, urinary As of 49 volunteers in the UK (Bangladeshi n = 37; white Caucasians n = 12) were monitored along with dietary habits.138 Total urinary As and speciation analysis for DMA, MMA and iAs was conducted. Although no significant difference was found for total As (median: Bangladeshis 28.4 μg L−1, and white Caucasians 20.6 μg L−1), the sum of medians of DMA, MMA and iAs for the Bangladeshi group was found to be over 3-fold higher (17.9 μg L−1) than for the Caucasians (3.50 μg L−1). Urinary DMA was significantly higher (p < 0.001) in the UK Bangladeshis (median: 16.9 μg DMA L−1) than in the white Caucasians (3.16 μg DMA L−1) as well as iAs (p < 0.001) with a median of 0.630 μg iAs L−1 for Bangladeshis and 0.250 μg iAs L−1 for Caucasians. Cationic compounds were significantly lower in the Bangladeshis (2.93 μg L−1) than in Caucasians (14.9 μg L−1). The higher DMA and iAs levels in the Bangladeshis are mainly the result of higher rice consumption: As is speciated in rice as both iAs and DMA, and iAs can be metabolized, through MMA, to DMA by humans. This study shows that a higher dietary intake of DMA alters the DMA/MMA ratio in urine. Consequently, the DMA/MMA ratio as an indication of methylation capacity in populations consuming large quantities of rice should be applied with caution since variation in the quantity and type of rice eaten may alter this ratio.
The uptake of AsIIIinto rice is mainly through the silicic acid transport pathway. In a study by Moore et al.,139 the cellular and subcellular distributions of As and Si in rice roots were investigated using high-pressure freezing, SIMS and TEM. Rice plants, both the lsi2 mutant lacking the Si/AsIII efflux transporter Lsi2 and its wild-type cultivar, with or without an Fe plaque, were treated with AsV or ASIII. The formation of Fe plaque on the root surface resulted in strong accumulation of As and P on the epidermis. The lsi2 mutant showed stronger As accumulation in the endodermal vacuoles, where the Lsi2 transporter is located in the plasma membranes, than the wild-type line. The study revealed the vacuolar sequestration of As in rice roots and contrasting patterns of As and Si subcellular localization, despite both being transported across the plasma membranes by the same transporters. The spatial distribution and speciation of As (and Fe and Mn) around rice roots grown in an As-affected paddy field in Bangladesh has been investigated.140 Synchrotron μ-XRF on soil thin sections revealed that roots influence the soil distribution up to 1 mm away from the root–soil interface. Around thick roots (diameter around 500 μm), the As was closely associated with Fe concentric Fe accumulations away from the root. Near thin roots (diameter < 100 μm), in contrast, a pronounced enrichment of Fe and As was found next to the root surface. Studies with XAFS suggested that the As was mostly AsV and that the distinct enrichment patterns can be related to the extent of O2 release from primary and lateral rice roots and the thermodynamics and kinetics of Fe, Mn and As redox transformations. This suggests element accumulation and speciation in the surrounding rhizosphere soil must be taken into account when addressing the transfer of nutrients or contaminants into rice roots. In a further study by Carey et al., the retranslocation of As species from flag leaves into filling rice grain was investigated.141 Spatial distributions within grains were investigated using SR-XRF. Additionally, the effect of germanic acid (a silicic acid analogue) on grain As accumulation in AsIII treated panicles was examined. The DMA and MMA were extremely efficiently retranslocated from flag leaves to rice grain; AsV was poorly retranslocated, and was rapidly reduced to AsIII within flag leaves; AsIII displayed no retranslocation. Within grains, DMA rapidly dispersed while MMA and iAs remained close to the entry point. Germanic acid addition did not affect grain As in AsIII treated panicles. Three-dimensional SXRF microtomography gave further information on AsIII localization in the ovular vascular trace of rice grains. These results demonstrate that inorganic As is poorly remobilized, while organic species are readily remobilized, from leaves to grain. Stem translocation of inorganic As may not rely solely on silicic acid transporters. Arsenic biotransformation and volatilization in transgenic rice has also been studied by Meng et al.142 An arsM gene from the soil bacterium Rhodopseudomonas palustris was expressed in Japonica rice (Oryza sativa) cv Nipponbare, and the transgenic rice produced methylated As species, which were measured by ICP-MS and HPLC-ICP-MS. Both MMAV and DMAV were detected in the roots and shoots of the transgenic rice. After 12 days exposure to AsIII the transgenic rice gave off 10-fold greater volatile arsenicals. The study demonstrated that expression of an arsM gene in rice induces As methylation and volatilization, theoretically providing a potential stratagem for phytoremediation.
The impact of flooded paddy soils on As speciation has also been investigated using HPLC-ICP-MS and XANES.143 The flooding resulted in a decrease in Eh, a rise in pH, and an increase in the AsIII fraction in the soil solid phase of up to 80% of the total As in the soils. However, the decline in Eh did not induce the transformation of AsV to AsIII in either the soil solid or solution phases, and the dissolution of As was limited. Microbial activity was necessary for the reductive reaction of AsV to AsIII even when Eh reached the condition necessary for the dominance of AsIII. In a further study, this time in Japan, ten major cultivars (Oryza sativa L.) were grown under flooded conditions in a paddy field with low As soil levels or in pots filled with soil containing a high level of As.144 In the low As soil, the total As ranged from 0.11 to 0.17 mg kg−1, with a mean concentration of 0.14 mg kg−1, and iAs was the major species in all cultivars. There were few genotypic differences in the levels of either total As or iAs in the grain. In the high As soil, total grain As increased to a mean level of 2.4 mg kg−1 in the 10 cultivars, with markedly increased levels of DMA. The genotypic variations among cultivars in the levels of both total As and DMA were statistically significant. However, the genotypic variability of iAs levels was quite small, and these levels remained low (at about 0.2 mg kg−1) even when total As levels increased markedly. These results suggest that differences in grain As levels among Japanese cultivars may not influence dietary As exposure, because there is little genotypic difference in the accumulation of iAs.
The uptake of As into plants from different soil types has been studied. Synchrotron μXRF, μXANES and ICP-OES have been employed to study the accumulation and distribution of As145 in Parkinsonia florida plants grown in two soils spiked with As at 20 mg kg−1. Plants grown in a sandy soil accumulated at least twice more As in the roots compared to plants grown in a loamy soil. The lower As accumulation in plants grown in the loamy soil corresponded to a lower concentration of As in the water-soluble fraction of this soil. Speciation analysis by LC-ICP-MS showed only AsV in the water-soluble fraction from all treatments. In contrast, linear combination XANES speciation analysis from the root tissues showed As mainly present in the reduced AsIII form. Moreover, a fraction of the reduced As was found coordinating to S in a form consistent with As-Cys(3). The percentage of As coordinated to sulphur was smaller for plants grown in the loamy soil when compared to the sandy soil. When in contact with soil of volcanic origin and chlorine containing water, Roxarsone (4-hydroxy-3-nitrophenylarsonic acid) has been found to generate a set of organoarsenicals.146 The transformation products were identified using ICP-MS and ES-MS, ES-MS/MS and ES-TOF-MS detection after HPLC separation. The results suggested the presence of chlorinated phenylarsonic acids and a phenylarsine oxide derivative which contained As in the trivalent state. Traces of chlorine in water used for sorption experiments are suggested to be responsible for the formation of chlorinated products. After irradiation of a Roxarsone solution with visible light, different transformation products, so far not identified, were detected. In the presence of iron, dissolved organic matter (DOM) in humic material may bind considerable amounts of As, through formation of Fe-bridged As-Fe-DOM complexes and surface complexation of As on DOM-stabilised Fe-colloids (collectively referred to as As-Fe-DOM complexation). There have been few reports focusing on this complex, but Liu et al.147 have used SEC-UV-ICP-MS to investigate AsIII–Fe-DOM complexation by adding AsIII into prepared Fe-DOM. The occurrence of the complex was proposed following co-elution of As, iron, and DOM from the SEC column and co-retention of As, iron, and DOM by a centrifugal filtration membrane. The kinetic data of AsIII–Fe-DOM complexation were described by a pseudofirst order rate equation (R-2 = 0.95), with the rate constant (K′) being 0.17 ± 0.04 1/h. Stability of AsIII–iron-DOM complexation was characterized by apparent stability constants (K-s) derived from a two-site ligand binding model, with log K-s ranging from 4.4 ± 0.2 to 5.6 ± 0.4. Considering the kinetics (within hours) and stability (similar to typical metal-humates) of AsIII-iron-DOM complexation, this complexation needs to be included when evaluating As mobility in iron and DOM rich environments.
The distinction between ore processing and post-depositional transformation on the speciation of As (and Sb) in mine waste and sediment has been studied in relation to the Giant mine, Yellowknife, Canada.136 Roasting of As and Sb associated gold ore resulted in complex solid–gas phase reactions between As, Sb, and roaster-derived iron-oxides. The As and Sb, including that found in waste streams and mine-impacted sediment were characterized using EPMA, μ XRD, μXRF, bulk and μXANES. The roaster As and Sb oxides in mine waste and sediment were found in multiple oxidation states, whilst in the cyanide roaster dust AsIII and SbIII were dominant. Conversely, AsV and SbV were found in the oxidized and cyanided material collected from the roaster bed. Thus, these results have implications for predicting the fate of As and Sb in the environment, and for distinguishing between anthropogenically influenced speciation and speciation resulting from post-depositional transformations. Borate deposits in Turkey and California contain elevated concentrations of As, with adverse effects to not only local suppliers of water but also the commercial exports of boron products. Most previous studies and remediation efforts of As contamination in borate deposits have focused on sulfarsenides. However studies by Lin et al.148 using ICP-MS analysis and μ-SXRF mapping, reveal that colemanite, a major borate ore mineral, contains up to 125 mg kg−1 As. Arsenic K-edge XANES spectra suggest the presence of both AsIII and AsV species in colemanite. The data on K-edge EXAFS show preferential occupancies of AsV and AsIII at the tetrahedral B2 site and the triangular B1 site, respectively. Single-crystal electron paramagnetic resonance (EPR) spectra of gamma-ray-irradiated colemanite measured at 40 K contain an AsO32− centre, providing further support for the presence of AsV. Therefore, colemanite is a significant source of As contamination, not only in commercial B products, but also aquifers associated with borate deposits.
The microbial transformation of As species in municipal landfill leachate has also been investigated by Li et al.149 During the first 20 days of the incubation period, more than 90% arsenate AsV was found to reduce to AsIII, while AsIII was comparably stable, although a fraction of AsIII was temporarily oxidised to AsV in the first 3 days. The transformation of MMAV to DMAV was slow with only 5% MMAV methylated to DMAV after 43 days incubation. A portion of DMAV and MMAV was demonstrated to transform into thiol-organoarsenic and MMAIII, which were identified to include dimethyldithioarsinic acid (DMDTAV), dimethylmonothioarsinic acid (DMMTAV) and monomethyldithioarsonic acid (MMDTAV) by HPLC-ICP-MS and LC-ES-MS-MS. The microbial formation of DMDTAV, DMMTAV and MMDTAV is postulated to relate to hydrogen sulfide generated by bacteria. Differences in As transformation in sterilised and non-sterilised municipal landfill leachate demonstrate bacteria play a crucial role in As transformation in the landfill body.
The biomethylation of As and Te during composting in a laboratory reactor has been studied by Diaz-Bone et al.150 Time resolved investigations employing HG-GC-ICP-MS revealed a highly dynamic process during self-heated composting with markedly differing time patterns for As and Te species. Concentrations of up to 150 mg kg−1 methylated As species as well as conversion rates up to 50% for As and 5% for Te were observed. In contrast, little to no conversion was observed for Sb, Sn and Ge. In addition to experiments with metal(loid) salts, composting of As hyperaccumulating ferns Pteris vittata and P. cretica grown on As amended soils was studied. Arsenic accumulated in the fronds was efficiently methylated resulting in up to 8 mg kg−1 methylated As species. Overall, these studies indicated that metal(loid)s can undergo intensive biomethylation during composting.
Changes in As speciation and transport associated with the release of spent geothermal fluids (post production of electricity) have been studied in the Mutnovsky field, Russia.151 In the spent fluids, the As concentration reaches 9 mg L−1, while in natural hot springs expressed in the vicinity of the field, the As concentration was typically below 10 μg L−1. The aqueous phase As speciation was determined using LC-ICP-MS. The As speciation in the bottom sediments (<65 μm fraction) of the local surface waters was analysed using XAS. Arsenic in the geothermal source fluids was predominantly found as AsIII, while a mixture of AsIII–AsV is found in the water and sediment of the Falshivaia River downstream from the power plant. The extent of elevated As concentrations in water was limited by adsorption to the bottom sediment and dilution, as determined using deep well fluids. Analysis of the EXAFS spectra showed that sediment phase As is associated with both Al- and Fe-rich phases with a bi-dentate corner sharing local geometry. The geothermal waste fluids released in the surface water created a localized area of As contamination. The extent of transport of dissolved As was limited to 7 km downstream from the source, while As associated with bottom sediment travels 3 km further.
In recent years, analytical methods have been developed that have demonstrated that soluble As–S species constitute a major fraction of dissolved As in sulfidic waters. However, XAS data generated at higher (mmol L−1) concentrations suggest the presence of (oxy)thioarsenites in such waters, while LC-ICP-MS data at lower (μmol L−1 to nmol L−1) concentrations indicate the presence of (oxy)thioarsenates. Planer-Friedrich et al.152 have studied this contradiction and have shown that by XAS thioarsenites are the primary reaction products of AsIII and sulfide in geochemical model experiments in the complete absence of O2. However, thioarsenites are extremely unstable toward oxidation, and convert rapidly into thioarsenates when exposed to atmospheric O2, e.g., while waiting for analysis in an autosampler, and thus requiring a glovebox. It was shown that thioarsenites are unstable toward sample dilution, which is commonly employed prior to chromatographic analysis when sensitive detectors like ICP-MS are used. However, the problem is avoided by using undiluted samples.
Seaweeds are known As hyperaccumulators, seemingly achieving detoxification via arsenosugar biosynthesis. However, whether seaweeds synthesise phytochelatin (PC) to aid detoxification during As exposure is unknown. Hizikia fusiforme (hijiki) and Fucus spiralis were used153 as model seaweeds to evaluate this: the former is known for its high iAs concentration, whereas the latter contains mainly arsenosugars. F. spiralis was exposed to 0.1 and 10 mg L−1 AsV solutions for 24 h, whereas hijiki was analysed fresh. All samples contained AsIII, glutathione and reduced PC2, identified using HPLC-ICP-MS/ES-MS (Hamilton PRP-X100 and Eclipse XDB C18 columns). Although hijiki contained no AsIII–PC complexes, AsV exposed F. spiralis generated traces of numerous As compounds that might be AsIII GS or AsIII PC2 complexes. AsIII PC complexes seem not to be a principal storage form for long-term As storage within seaweeds. However, 40 times higher glutathione concentrations were found in hijiki than F. spiralis, which may explain how hijiki deals with its high iAs burden.
The As concentrations and species have been compared in biota from two Zostera capricorni seagrass ecosystems.154 The HPLC-ICP-MS system used one of three columns: Supelcosil PRP-SCX, HamiltonPRP-X100 or Atlantis C18 for As cations, anions and thio-As species respectively. The method was validated using a CRM (DORM-2, NRCC). Mean As concentrations were not significantly different for non-vegetative sediment, rhizosphere sediment, Z.capricorni blades, roots, rhizomes, epiphytes, amphipods, polychaetes, molluscs, crustaceans and fish, but were significantly different in detritus. Sediments and plant tissues contained mostly iAs and PO4-arsenoriboside. Detritus contained mostly PO4-arsenoriboside. Fish tissues contained predominately AB. Other animals had lower proportions of AB and variable quantities of minor As species. The proportion of AB increases through the food web was attributed to a shift from a mixed diet at lower trophic levels to animals containing mostly AB at higher trophic levels and the more efficient retention of AB compared to other arsenic species.
Time series laboratory experiments have been performed by Navratilova et al.155 to follow the degradation of As compounds naturally present in marine algae. Samples of the brown alga Ecklonia radiata, which contains three major arsenosugars, were packed into 12 tubes open to air at one end only, and allowed to naturally decompose under moist conditions. During the subsequent 25 days, single tubes were removed at intervals of 1–4 days; their contents were cut into four sections (from open to closed end) and analysed for As species by HPLC-ICP-MS following an aqueous methanol extraction. In the sections without direct contact with air, the original arsenosugars were degraded primarily to AsVvia two major intermediates, dimethylarsinoylethanol (DMAE) and DMA. The section with direct contact with air degraded more slowly and significant amounts of arsenosugars remained after 25 days. Preliminary data was also presented which suggested that the amount of non-extractable or recalcitrant As (i.e. insoluble after sequential extractions with water/methanol, acetone, and hexane) increased with time. Furthermore, treatment of the pellet with 0.1 M trifluoroacetic acid at 95 °C solubilised a significant amount of this recalcitrant As, suggesting that the As is present mainly as a cationic species of currently unknown structure.
The biomagnification profiles of As species, including total As, lipid-soluble As, eight water-soluble As compounds (AB, AC, TMA, TMAO, DMA, MMA, AsV, and AsIII), and non-extracted As have been determined in a tropical mangrove ecosystem in the Ba Ria Vung Tau, South Vietnam.156 Arsenobetaine was the predominant As species (65–96% of water-soluble As). The study utilised HPLC-ICP-MS and δ13C and δ15N analysis, and method validation using BCR 627 (tuna fish tissue) which gave recoveries of 84–90% and 92–126% for AB and DMA respectively. Linear regression slopes of log-transformed concentrations of total As, As fractions and As compounds on trophic level (as determined by δ15N) were used to determine rates of biomagnification for crustaceans, fish and total species.
Once again this year there have been a number of regional studies to investigate the spatial distribution of As in aquatic systems. Total As, AsIII and AsV in estuarine sediments from the main tributaries of Todos os Santos Bay, Brazil, was evaluated under various flow conditions.157 The concentrations of As were determined using a slurry sampling procedure with HG-AAS offering LODs of 0.1 μg L−1 for total As and 0.3 μg L−1 for AsIII. Two CRMs (MESS-2 and MESS-3, both NRCC) were also analysed. The highest concentrations were observed at estuary mouths, and exceeded lower threshold levels (7.24 mg kg−1). Due to the oxic conditions and abundance of Mn and Fe (oxyhydr)oxides in the sediments, most inorganic As in the Subae and Paraguacu estuaries was present as AsV. Nevertheless, the concentration of AsIII at several locations along the Jaguaripe River were also above the total exposure limit value, suggesting that As may be toxic to biota. In the Subae estuary, anthropogenic activities are the main source of As. At the Jaguaripe and at Paraguacu estuaries, natural sources of As need to be considered to explain the distribution patterns. Freshwater systems have also been studied. The short-term metabolic processes of AsV in a freshwater unicellular green alga, Chlamydomonas reinhardtii wild-type strain CC-125 has been studied over a 24 h period.158 The As species in the algal extracts were identified by HPLC-ICP-MS after water extraction using a sonicator. Speciation analyses showed that the levels of AsIII, AsV, and MMA in the cells rapidly increased for 30 min to 1 h, and those of DMA and oxo-arsenosugar-glycerol also tended to increase continuously for 24 h, while that of oxo-arsenosugar-phosphate was quite low and fluctuated throughout the experiment.
The identification of new biomarkers to monitor pollution and toxicological problems remains of interest this year. To this end the Iberian green frog (Rana perezi) has been investigated with respect to the accumulation of As and species distribution depending on the metamorphosis stage and level of exposure.159 LC-ICP-MS was used for the study. The TMA was the major compound detected and continuously increased in concentration during frog development. However, the results suggest that rather low As accumulation is obtained and that Rana perezi metabolised As during embryonic and larval development. The accumulation of As during the growing period (2 to 14 months) of rainbow trout (Oncorhynchus mykiss) has been monitored and shown to be significant.160 The study was performed on a farm with through-running water from the basin of water processing plant in the years 2007 and 2008. The total contents of As and AB were quantified in muscle, liver, spleen and bile separately. The total As was determined by HG-AAS and AB by HPLC combined with AFS. The concentration of As in homogenized body samples grew (P < 0.01) during the first months of life, from the 6th month of rearing no significant difference was found. The concentration of AB in homogenized bodies was low during the first months and from the 6th month the concentration stayed at an invariable level and amounted to 46 ± 6% of total arsenic. The highest concentrations of As and AB in tissues ranked in the order muscle, liver, spleen and bile. In summer months the total As in muscle was bonded in the form of AB, in winter months the content of AB dropped to 50% of total As. The content of As in liver samples remained the same during the entire monitoring, however, the AB content altered. In summer months it reached the maximum 27%, in winter months it dropped to 7% of As. The concentration of As in spleen and bile decreased in winter months, whilst AB corresponded to the value assessed in liver.
The long-term uptake and distribution of As compounds by hydroponically cultivated White mustard (Sinapis alba) has been investigated with a special emphasis on controlling the stability of the As compounds in nutrient solution during the experiment.161 Speciation of As in plant tissues and in nutrient solutions was performed by HPLC-ICP-MS. It was concluded that AsV is rapidly oxidised to AsIII during the 7 days cultivation period. The presence of plant roots increases the oxidation rate of AsIII while DMA and AsV remain stable during the exposition, and MMA is partially demethylated. When the nutrient solution containing AsIII was exchanged daily, the distribution of As was significantly different (translocation factor (TF) is 70 times higher) in comparison to the experiment without exchange of the medium. Additionally, the synthesis of phytochelatins in plants exposed to different As compounds was investigated. Phytochelatins were identified in tissues of plants exposed to AsIII and AsV, and their presence was correlated with high AsIII content. Phytochelatin synthesis was not indicated in plants grown in the presence of MMA and DMA. The results illustrate that uncontrolled conditions of hydroponic plant cultivation may be a source of data misinterpretation.
Agusa et al.162 have determined arsenic compounds in the stomach and intestine contents, bile and urine of green (Chelonia mydas) and hawksbill (Eretmochelys imbricata) turtles to understand As accumulation through intake and excretion. Stable isotopes of δ15N and δ13C were also measured by SIRMS to aid understanding the of the feeding behaviour dependent accumulation of As. The major As species in gastrointestinal contents were unknown water-soluble As, followed by unextracted As. Concentrations of AB and DMA in the tissues were higher than those in the stomach contents (prey items), indicating high bioaccumulation of these arsenicals. In green turtles, AB concentration was high in bile and increased throughout the gastrointestinal tract, suggesting significant biliary excretion of AB. Delta 15N was positively correlated with AB level in green turtles, whereas a negative relationship between residual As and δ15N was observed in hawksbill turtles. This study indicates feeding behaviour-dependent accumulation of As compounds in both turtle species.
The total As and iAs content for a range of 215 food products and drinks (seafood, fruits and vegetables, meat products, oils and fats, rice and rice products, seasonings, and alcoholic drinks) marketed in Catalonia, Spain, have been analysed using LC-ICP-MS.163 Daily As and iAs intakes for the average adult Catalan consumer were estimated at 354 and 6.1 μg per day per person, respectively, using consumption data from the Catalan Nutrition Survey (ENCAT). The highest As content was found in seafood, contributing 96% of dietary As intake, whereas rice presented the highest iAs values, corresponding to 67% of dietary iAs intake. As cooking process may affect iAs content, boiled rice was evaluated, showing an iAs reduction (up to 86%) when using higher water volumes (30:1 water:rice ratio) than those used in previous studies. This iAs exposure was slightly below the exposure risk range stated by the European Food Safety Authority (0.3–8 μg kg−1 of body weight per day), although the possibility of a risk to the population with high rice consumption cannot be excluded.
A method to determine seven As species in seafood (65 samples) has been developed and validated using IC-ICP-MS.164 A 250 mm lonPac AS7 10 μm column was used with HNO3 and 1% MeOH mobile phase. The effect of five parameters on the MAE was evaluated using CRMs. The recoveries of total As and of As species with the two best extraction media (100% H2O and 80% aqueous MeOH) were generally similar in five seafood CRMs which were also considered. However, because MeOH co-elutes with AsIII, the 100% H2O extraction conditions were selected for validation of the method. Figures of merit (linearity, LOQs (0.019–0.075 mg As kg−1), specificity, trueness (with recoveries between 82% (AsIII) and 104% (AsV) based on spikes or certified concentrations), repeatability (3–14%), and intermediate precision reproducibility (9–16%) of the proposed method were satisfactory for the determination of ASIII, MMA, DMA, ASV, AB and AC in fish and shellfish. The performance criteria for TMAO, however, were less satisfactory. The main species in all samples was AB and the percentage of iAs varied between 0.4 and 15.8% in shellfish and 0.5–1.9% in fish. The main advantage of this method was that it uses only water as an extractant and nitric acid as gradient eluent offering compatibility with the long-term stability of both IC separation and ICP-MS detection.
A study of 14 white wines produced in South America has been reported.165 Arsenic species were selectively determined by LC-ICP-MS. Separation of As species was performed using an AEC with ammonium phosphate solution (pH 6.0) as mobile phase. Samples were 10-fold diluted in the mobile phase prior to analysis. Accuracy was evaluated by recovery tests, where As species recoveries ranged from 95% to 106%. Additionally, the sum of arsenic species concentration found by LC-ICP-MS was in agreement with the total As concentration determined by ICP-MS after sample digestion. The As species detected were AsIII, AsV and DMA at levels of 2.9 to 10.3, 8.6 to 17.8, and <0.45 to 1.07 μg L−1 for AsIII, AsV and DMA, respectively.
A new method for As speciation in clinical samples has been reported by Morton and Leese.166 The method utilises μLC-ICP-MS incorporating a low-pressure delivery six-port valve with a 5 cm AEC, which allows a fully resolved separation of five As species (AB, AsIII, AsV, MMAV and DMAV) in urine in 6 min. Results are reported from 65 workers in the semiconductor industry (plus 20 controls from unexposed people), and show very low levels of As in their urine samples, similar to the levels in the controls. The authors stress that care must be taken when interpreting urinary As species results because it is not always possible to differentiate between dietary and other external sources of exposure.
The association of seafood intake with spot urine arsenic concentrations in the 2003–2006 National Health Nutrition and Examination Survey (NHANES) has been evaluated.167 The study, based on 4276 participants (>= 6 years) measured total As using ICP-MS and urine DMA and AB by HPLC-ICP-MS. Participants reporting eating seafood in the past 24 h had higher urine concentrations of total As (median 24.5 vs. 7.3 μg L−1), DMA (6.0 vs. 3.5 μg L−1), AB (10.2 vs. 0.9 μg L−1) and total As minus AB (11.0 vs. 5.5 μg L−1). Participants reporting seafood >= 2 per week vs. never during the past year had 2.3 (95% confidence interval 1.9, 2.7). 1.4 (1.2, 1.6), 6.0 (4.6, 7.8) and 1.7 (1.4, 2.0) times higher (p-trend < 0.001) concentrations of total As, DMA, AB and total As minus AB, respectively. In participants without detectable AB and in analyses adjusted for AB, seafood consumption in the past year was not associated with total As or DMA concentrations in urine. The results show that total As, DMA, the sum of iAs, DMA, and total As minus AB as markers of iAs exposure and/or metabolism is needed to address seafood intake.
Rivera-Nunez et al.168 have also utilised HPLC-ICP-MS to study As in urine. In this case six As species (AsIII, AsV, MMAIII, MMAV, DMAV and AB) were determined in urine samples from 387 individuals in southeast Michigan, USA, who were chronically exposed to slightly elevated levels of arsenic in their drinking water. The DMAV was detected in 99.2% of samples, AB in 98.2%, MMAV in 73.4%, AsIII in 45.0%, and AsV in 27.1%. No MMAIII was detected even in samples analysed within 6 hours after collection suggesting that MMAIII is not a significant metabolite in urine of people exposed to As concentrations below 20 μg L−1 in their drinking water.
A limited interlaboratory study for As speciation in whole blood has been conducted in conjunction with the New York State Department of Health's proficiency testing program.169 Four laboratories were able to report values for up to five As species (MDL 9.5 μg L−1) in blood materials representing typical environmental concentrations. For most As species good agreement was obtained among the participants and the consensus mean values obtained were assigned as target values for use as quality assurance materials in future analyses.
Geophagy or earth-eating is common amongst some Bangladeshi women, especially those who are pregnant, both in Bangladesh and in the UK. A large proportion of the population in Bangladesh is already exposed to high concentrations of As and other toxic elements from drinking contaminated groundwater. Additional exposure to As and other toxic elements from non-food sources has not been extensively studied. To address this, Al-Rmalli et al.170 have looked at As levels in baked clay (known as sikor). The samples (originating from Bangladesh) were digested and analysed for As, Cd, Fe Mn, Pb, and Zn using ICP-MS. Arsenic speciation was performed using HPLC-ICP-MS. The results revealed levels of As (3.8–13.1 mg kg−1), Cd (0.09–0.4 mg kg−1) and Pb (21–26.7 mg kg−1) present in the sikor samples. Speciation analysis revealed that sikor samples contained mainly iAs. Modest consumption of 50 g of sikor is equivalent to ingesting 370 μg of As and 1235 μg of Pb per day, based on median concentration values, and thus exceeding the permitted maximum tolerable daily intake of iAs by almost 2-fold.
Studies involving acute As poisoning in humans are obviously rare, however arsine poisoning confirmed by speciation analysis (HPLC-ICP-MS) in serum and urine following exposure in a recycling factory has been reported.171 On admission to hospital, anaemia, hematuria, and renal and liver dysfunction were observed. The patients clinical condition had improved remarkably after 5 days of transfusion and 4 units of RBC transfusion. The total As content in the serum was 244.8 μg L−1 at admission and 97.1 μg L−1 at discharge. After speciation analysis, four kinds of As compounds derived from arsine metabolism were detected in serum and urine. The concentrations of AsIII, AsV, MMA and DMA in serum at admission were 45.8, 5.2, 17.9 and 9.3 μg L−1, respectively. The concentrations of AsIII, AsV and MMA decreased with biological half-lives of 30.1, 43.0, and 96.3 h, respectively, while that of DMA was increased towards discharge. The urinary AsIII, AsV, MMA, and DMA concentrations at discharge were 384.5, 20.8, 547.4 and 1816.3 μg g−1 creatinine, respectively. These concentrations in urine subsequently decreased and their biological half-lives was 15 days. Thus, the results suggest that arsine is quickly metabolized to AsIII and then metabolized via MMA to DMA in humans. Another example of extreme As exposure has also been reported. Urinary As metabolites have been determined in subjects exposed to elevated As present in coal in Shaanxi Province, China.172 In this study, the concentrations and distributions of urinary As metabolites in 57 volunteers (36 subjects with skin lesions and 21 subjects without skin lesions) were reported. The urinary As species, including iAs, AsIII, AsV, MMAV and DMAV, were determined by HPLC-ICP-MS. The relative distributions of As species, the primary methylation index (PMI = MMAV/iAs) and the secondary methylation index (SMI = DMAV/MMAV) were calculated to assess the metabolism of As. Subjects with skin lesions had a higher concentration of urinary As and a lower As methylation capability than subjects without skin lesions. Women were found to have a significantly higher methylation capability of As than men, as defined by a higher levels of DMAV and SMI in urine among women (one possible interpretation of women with a higher concentration of urinary As but lower susceptibility to skin lesions). The findings suggested that not only the dose of As exposure but also the As methylation capability have an impact on the individual susceptibility to skin lesions induced by coal As exposure.
Arsenic plays an important role in producing oxidative stress in cultured cells. To investigate the interaction between high oxidative stress and low As methylation capacity on As carcinogenesis, a case-control study was conducted to evaluate the relationship among the indices of oxidative stress, such as urinary 8-hydroxydeoxyquanine (8-OHdG), as well as plasma micronutrients and urinary As profiles on urothelial carcinoma (UC) risk.173 Urinary 8-OHdG was measured using high-sensitivity enzyme-linked immunosorbent assay kits. The urinary As species were analysed using HPLC-HG-AAS. Plasma micronutrient levels were analysed using RP HPLC. The study showed a significant protective effect of plasma α-tocopherol on UC risk. Plasma α-tocopherol levels were significantly inversely related to urinary total As concentrations and iAs, and significantly positively related to DMA. There were no correlations between plasma micronutrients and urinary 8-OHdG. Study participants with lower α-tocopherol and higher urinary total As, higher iAs, higher MMA, and lower DMA had a higher UC risk than those with higher α-tocopherol and lower urinary total As, lower iAs, lower MMA, and higher DMA. The results suggest that plasma α-tocopherol might modify the risk of inorganic arsenic-related UC.
Genetic polymorphisms in As-metabolizing enzymes may be involved in the biotransformation of iAs and may increase the risk of developing urothelial carcinoma. A study to evaluate the roles of glutathione S-transferase omega 1 (GSTO1) and GSTO2 polymorphisms in UC carcinogenesis has been published by Chung et al.,174 following a hospital-based case-control study. Questionnaire information and biological specimens were collected from 149 UC cases and 251 healthy controls in a non-obvious iAs exposure area in Taipei, Taiwan. The urinary As profile was determined using HPLC-HG-AAS. Genotyping for GSTO1 Ala140Asp and GSTO2 Asn142Asp was conducted using PCR-restriction fragment length polymerase. GSTO1 Glu208Lys genotyping was performed using high-throughput MALDI and TIMS. A significant positive association was found between total As, iAs, MMA and UC, while DMA was significantly inversely associated with UC. The minor allele frequency of GSTO1 Ala140Asp, GSTO1 Glu208Lys and GSTO2 Asn142Asp was 18%, 1% and 26%, respectively. A significantly higher level of MMA was found in people who carried the wild type of GSTO1 140 Ala/Ala compared to those who carried the GSTO1 140 Ala/Asp and Asp/Asp genotype (p = 0.02). The homogenous variant genotype of GSTO2 142 Asp/Asp was inversely associated with UC risk (OR = 0.17; 95% Cl, 0.03–0.88; p = 0.03). Larger studies will be required to verify the association between the single nucleotide polymorphisms of As-metabolism-related enzymes and UC risk.
Among the non-cancer effects of As, cardiovascular diseases have been well documented; however, little is known about the fate of As in cardiovascular tissues. Roman et al.175 have studied the analytic bioinorganic As behaviour in cardiovascular tissues from an As exposure coronary heart disease patient group from Antofagasta, Chile, against a small unexposed As coronary heart patient group. Total As concentrations were measured in pieces of cardiovascular tissues of the As exposed and unexposed coronary heart patient groups by HG-AAS and speciation analysis by HPLC-ICP-MS. Pieces of auricle (AU), mammary artery (MAM), saphenous vein (SAP) and fat residuals (FAT) were considered in this study. The As concentrations in AU and MAM tissues were significantly different between both groups of patients and it was demonstrated that the AU was the ‘AsIII target tissue’. Linking the total concentrations of As with conditional variables and variables related to medical geology factors allowed the authors to infer that the latter were more important for the cardiovascular risk of As exposure in the Antofagasta region. Knowledge of total As and the prevalence of AsIII in the AU of patients could contribute to an understanding the effect of As on cardiovascular diseases.
A study on As speciation changes in crude and processed traditional Chinese medicines (Radix Astragali, Radix et Rhizoma Rhei, Radix Scutellariae, Radix Polygoni Multiflori and Radix Rehmanniae) and the fungi Cordyceps has also been reported.176 The HPLC-ICP-MS analysis showed that the iAs (AsIII and AsV) were the dominating species in the Chinese medicines (levels increasing after processing); MMA and DMA were not found although MMA was found in Cordyceps. The LOQ values ranged from 0.8 to 1.0 μg L−1; the reproducibility and stability had RSDs of less than 10%; and spiked recoveries ranged from 82% to 120%.
5.3 Chromium
As in previous years there have been numerous studies investigating the speciation of Cr in a range of matrices. A sequential non-chromatographic procedure has been developed by Meeravali et al.177 for selective pre-concentration and separation of CrIII and CrVI from natural and wastewaters. Triton X-114 micelle and cetylpyridinium bromide-Triton X-114 mixed-micelle were used sequentially for the extraction of hydrophobic CrIII-trifluoropentanedione and hydrophilic CrVI respectively, and ICP-AES was used for the measurements. Following optimisation, the pre-concentration factors obtained were 50 and 15 while LODs are 0.02 and 0.05 ng mL−1 for CrIII and CrVI, respectively. These LODs are better than those offered by IC-ICP-MS and comparable to those of IC-ICP-DRC-MS methods. The recoveries were in the range of 95 to 99% at 10 to 40 ng mL−1 with relative standard deviations of 2–4%. The accuracy of the procedure was validated by comparing the sum of the concentrations of each individual Cr species obtained from sequential extraction with total Cr in BCR CRM materials such as Effluent-713, Influent-714 and Industrial effluent-715. The method was then applied to a range of water samples. The speciation and determination of CrIII and CrVI in water by PIXE analysis has been reported.178 Ion exchange (Whatman cellulose P81 and DE81) was used for separation and pre-concentration of CrIII and CrVI species by filtration. The LOD reported is in the order of 1 μg L−1. However, at a very high beam current (50 nA), despite the high X-ray yield, a rapid increase of the background degraded the LOD. A CRM (NMIJ CRM 7202 ‘Trace elements in river water’ with a total Cr concentration of 4.61 ± 0.45 μg L−1) was used to obtain a relative accuracy of 96%, although it should be noted that only CrIII was found in the CRM. The RSD values for CrIII and CrVI in river water samples were 10 and 9%, respectively.The reduction of CrVI in an acidified riverine water CRM (SLRS-2, NRCC) was studied in a kinetic experiment utilising IC-ICP-MS.179 The water sample was spiked with CrVI, with or without CrIII, and the evolution of each Cr species was monitored as a function of time. This showed that the reduction of CrVI was a pseudo first order reaction. By plotting the logarithm of the peak area ratio of the instantaneous CrVI concentration over that of the original spiked concentration versus time, the reaction rate constant was obtained from the slope, and was (2.119 ± 0.040) × 10−4 s−1 in SLRS-2 at pH 1.3 and 20 °C following the addition of 20 μg L−1 CrVI. The reduction rate increased with decreasing pH and increasing temperature. The activation energy of the reaction at pH 1.3 was found to be 139.1 ± 7.0 kJ mol−1 using an Arrhenius plot. The method offered advantages in terms of small sample consumption, minimal sample manipulation, and easy data interpretation. The kinetic behaviour of the CrVI bioaccumulation process in the recently isolated bacterium, ANCR (Acinetobacter beijerinckii type) has also been studied.180 A qualitative study of the bacterium elemental profile detected by TXRF was carried out and then the Cr content was evaluated in two ways: in the cultured medium of the experiment, evaluating its decrease, and bioaccumulated in the bacteria, evaluating its increase. The results demonstrate that this new strain of Acinetobacter bacterium is able to reduce the chromium present in the culture medium with a yield of around 21%. As a consequence, it can be used as a promising microorganism for CrVI bioremediation from polluted wastewaters.
The determination of CrVIin black, green and herbal teas has been investigated.181 Samples were treated with 0.1 M Na2CO3 to determine the total CrVI and acid digestion was used to determine the total Cr concentrations by ETV-AAS. The CrVI consumed per unit cup of tea was determined by soaking the leaf or tea bag (or infusion) in 200 mL boiled water and the solution was filtered through a PVDF 0.45 μm hydrophilic filter prior analysis. Results showed that concentration of total CrVI in black teas varied between 0.03 and 3.15 μg g−1 (with an average of 1.07 ± 1.14 μg g−1), in green tea ranged between 0.03 and 0.14 μg g−1 (with an average of 0.09 ± 0.034 μg g−1) and that in herbal tea was below the LOD (0.20 μg L−1 for a 10 μL sample aliquot). It was also found that up to 17.5 μg L−1 of CrVI could be consumed per unit cup of black tea (200 mL) when a standard tea bag (2.0 g) or 2.0 g leaf was used in preparation.
The aging of artist pigments has been reported by several groups this year (see also under Zinc). Analysis by X-ray based techniques182 of the colour darkening of selected brushstrokes of the masterpiece “A Sunday on La Grande Jatte-1884” (by Georges Seurat) indicates the alteration of the chromate pigment zinc yellow. The pigment originally displays a bright greenish yellow colour but may undergo, after aging, darkening to a dull, ochre tone. Zanella et al.183 have used XANES to probe the oxidation state of Cr on paint reconstructions, and show that colour changes are associated with the reduction of CrVI to CrIII. Paint mixtures containing the pigment and linseed oil to mimic mixtures used in La Grande Jatte were subjected to artificial aging in the presence of light, SO2, and variable air humidity (50 and 90% relative humidity). High relative humidity led to the greatest degree of CrVI reduction whereas low relative humidity promoted light-induced alterations. These results are corroborated by visible reflectance measurements on the same laboratory samples.
5.4 Halogens
Two papers have been published by the same research group covering the topic of compound specific Br isotopic analysis.100,184 The first covers the isotopic composition of three different commercially available CH3Br standards by both direct aspiration and injection through a GC. A MC-ICP-MS equipped with a collision cell was used for the isotope amount ratio measurements. It is not apparent if mass bias correction was applied, or how corrections were made if mass bias was corrected for, and therefore the results could be biased. This is unfortunate given the care taken over other aspects of the measurement process. A precision of ≤0.1‰ (1σ, n = 3) was calculated for both sample introduction modes for Br amounts of ≥40 ng. Direct aspiration yielded a precision of 0.7‰ (1σ, n = 3) for sample amounts as small as 2 ng of Br. For GC injections such precision was achieved with a Br sample size of 10 ng. The second paper184 described the determination of the Br isotopic composition of six commercially available and one natural organobromine compound by GC-MC-ICP-MS. Sample were derivatised by silylation prior to separation on a 30 m mega-bore column (0.53 mm i.d, 0.5 mm film thickness, 5% phenyl-95% dimethylpolysiloxane) with a deactivated fused silica transfer line to the MC-ICP-MS. Statistical differences, assessed using Students t test, between the δ81Br value for four of the commercial organo Br compounds (2,4-dibromophenol, 1,4-dibromobenzene, 1,3,5-tribromobenzene and bromonaphthalene) and natural 2,4-dibromophenol are reported although this looks not to be the case from the data presented. Once again, no mention is made of any mass bias correction routine so the results presented may again be biased.The concentration of chloride and perchlorate ions in water samples have been determined by TXRF. Perchlorate anions were concentrated on anion-selective membranes that had been prepared on the surface of TXRF quartz reflectors which were immersed in water solutions containing ng mL−1 concentrations of perchlorate. The dried reflectors/membranes were subsequently analysed by TXRF, using a copper X-ray tube and helium flow on the target, and the possibility of discrimination between chloride and perchlorate anions was shown. Minimum detection limits <1 ng mL−1 and good linearity at the concentration range of 1–50 ng mL−1 were achieved.
Two papers described an investigation of the adsorption of iodine species onto soils by XANES and HPLC-ICP-MS for pore water samples.185,186 Iodine K-edge XANES was measured at beamline BL01B1 using a bending magnet at SPring-8 (Hyogo, Japan). In order to obtain the incident X-rays, an Si(311) double-crystal monochromator with two mirrors was used. The beam size was varied from 0.2 (vertical) × 0.3 (horizontal) to 0.2 (v) × 6 (h) mm2 depending on the iodine concentrations in the samples. XANES spectra were measured in fluorescence mode using a 19 element Ge semiconductor detector, except for reference materials that were measured in transmission mode. The I− and IO3− ions in pore waters were separated using an anion exchange column (TSK-gel Super IC-AP; 7.5 cm, Tosoh) at a constant temperature of 40 °C. The mobile phase was a mixture of 0.25% TMAH and 0.3% methanol at a flow rate of 1.0 mL min−1. Batch experiments were conducted using a locally collected gleysol packed into columns, to a 10.3 cm depth, through which solutions of either KI or KIO3 (39.4 mM) were passed. Subsequently, the material in the exposed columns was removed and separated into 1 cm sub-sections and the soil and pore water separated. The total I concentration in the sub-samples was determined, after filtration for the pore waters and extraction (5% TMAH at 70 °C for one hour) by ICP-MS. The estimated soil adsorption maxima for IO3− and I− were 25 and 4.6 mmol kg−1. It was also observed that IO3− was reduced to I− in both soil and pore water samples and the authors conclude that an improved model which accounts for this reduction needs to be developed in order to simulate IO3− soil depth profiles. In the second paper soil samples were collected to a depth of 12 cm from the Yoro area, Chiba, Japan. It was found, by HPLC-ICP-MS, that for pore waters the upper 6 cm contained organic iodine (50–60%) bound to dissolved organic matter with the remainder being in the form of I−. At a depth of 9–12 cm 98% of iodine was present as dissolved I−. For the soil samples both XANES and micro-XRF analysis showed that the iodine present was bound to organic matter.
The investigation of iodine speciation in seawater by size exclusion chromatography coupled to SF-ICP-MS has been reported which included an evaluation of five different columns. Iodine species, IO3− and I−, were separated in 400s using a Asahi Pak GS-220 multi-mode column (Showa Denko, Japan) with a mobile phase comprising 50 mM malonic acid:37.5 mM TMAH (pH 6.9) with isocratic elution at a flow rate of 1.0 mL min−1. Reported detection limits were 0.00016 mM (20 ng L−1) for iodate and 0.00024 mM (30 ng L−1) for iodide. The developed method was then used to study the vertical depth profile, of 775 m in total, of I species for seawater samples, diluted 10 fold with the mobile phase, collected in the Pacific Ocean off the coast of Aomori Prefecture, Japan. Iodate concentrations varied from 0.328 to 0.413 mM with significant variation on the upper 100 m and a gradual concentration increase with depth below this level. Iodide was found to be at the highest concentrations in surface waters (ca. 0.1 μM) with the concentration at a depth of 700 m measured as 0.009 μM. In conjunction with the vertical profile of chlorophyll a the authors conclude that the results obtained support the hypothesis that the concentration of iodide in surface waters is directly related to biotic activity.
5.5 Iron
The determination of important iron-chelating complexes using methods based on HPLC coupled to ICP-MS have been reported. The determination of hepcidin, a key polypeptide involved in iron homeostasis, by capillary HPLC-ICP-MS187 used sulphur detection to measure the peptide via the cysteine residues present in the molecule. Three different strategies to overcome the polyatomic interferences on S were evaluated: xenon as the collision cell gas; oxygen as the cell gas with detection of SO+; and use of a high resolution double-focusing mass analyser to resolve the analyte isotope. Different procedures to clean-up the urine samples were evaluated, with dialysis combined with SPE providing a 12-fold preconcentration factor. However, the use of capillary HPLC-ES-Q-TOF was found to be best suited to the determination of hepcidin in urine samples, because of the better selectivity and sensitivity it offered compared to capillary HPLC-ICP-MS. Siderophore-type complexes produced after incubation in the dark of Atlantic Ocean seawater with various ingredients, including: glucose and ammonia; glycine; or chitin and ammonium at different concentrations, were detected by HPLC-ICP-MS.188 A total of 22 different siderophore compounds were resolved and measured as gallium complexes by HPLC-ICP-MS and ten different siderophore-type complexes were identified by HPLC-ES-MS, 3 of which had not previously been identified in seawater incubations.5.6 Lead
Lead isotopes (Pb-204, Pb-206, Pb-207, Pb-208) have been utilized as a geochemical tracer of Pb origin in a variety of environmental studies, notably those involving atmospheric aerosols. Bird189 has discussed provenancing anthropogenic Pb within the fluvial environment. The paper outlines the Pb-isotope fingerprinting technique and associated analytical developments, and assesses the application of Pb isotopes in establishing the origin and dispersal mechanisms of anthropogenic and geogenic derived Pb at a range of temporal and spatial scales. The Pb isotopic signature of contemporary riverine Pb loads is shown to reflect a spatially complex influence of mineralization chemistry, anthropogenic activity as well as the hydro-morphological controls exerted upon Pb release, dispersal and storage. In relation to this, the long-term environmental legacy, and its influence upon Pb fingerprinting studies, of TEL, sourced from the combustion of leaded-petrol is also discussed.MacLean et al.190 have reported the use of XAFS, μXRF, μXRD to determine the speciation of Pb in house dust samples from four Canadian homes having elevated Pb concentrations (>1000 mg Pb kg−1). Linear combination fitting of the XAFS data, supported by μXRF and μXRD shows that Pb is complexed in a variety of molecular environments, associated with both the inorganic and organic fractions of the dust samples. The inorganic species of lead identified were: Pb metal, Pb carbonate, Pb hydroxyl carbonate, Pb oxide, and Pb adsorbed to iron oxyhydroxides. Lead carbonate and/or Pb hydroxyl carbonate occurred in all four dust samples and accounted for 28 to 75% of total Pb. Pb citrate and Pb bound to humate were the organic species identified. The impact of Pb speciation with respect to bioaccessibility and human health risk assessment for indoor environmental health was also discussed. See ‘Arsenic’ for details of Pb levels in Bangladeshi women following soil ingestion.170
In a further study on the aging of paint pigment (see also under Chromium), SR-XRF, μXRF, electron energy loss spectrometry (EELS) and μXANES have been used to investigate the degradation process of Pb chromate in yellow pigment used by artists of the end of the 19th Century and the beginning of the 20th Century.182 A darkening of the original yellow areas, painted with the chrome yellow pigment (PbCrO4, PbCrO4·xPbSO4, or PbCrO4·xPbO) is observed. The most famous of these are the various Sunflowers paintings by Vincent van Gogh. In this study, artificially aged model samples were used. The formation of CrIII compounds at the surface of the chrome yellow paint layers was observed in one sample taken from a historic paint tube (ca. 1914). About two-thirds of the Cr that was present at the surface had reduced from the hexavalent to the trivalent state. The EELS and μXANES spectra were consistent with the presence of Cr2O3·2H2O (viridian). Moreover, as demonstrated by μXANES, the presence of another CrIII compound, such as either Cr2(SO4)3·H2O or (CH3CO2)7Cr3(OH)2 is possible.
5.7 Mercury
Research into mercury species transformations, particularly Hg methylation, continues to be of interest. A study has been conducted to elucidate Hg species transformations in surface sediments that fluctuate between oxic and anoxic conditions.191 A microcosm, capable of containing a large sediment slurry volume (20 L) with a sediment (<300 μm size fraction) concentration of 93 g L−1, was designed to study methylation, demethylation and reduction and the subsequent partitioning of Hg species between the solid, aqueous and gaseous phases. Species-specific isotopically enriched tracers of iHg and MeHg, enriched in 199Hg and 201Hg respectively, were added to the microcosm to give Hg concentrations not greater than double the measured natural concentrations. Mercury species in the aqueous and sediment samples from these experiments were determined by GC-ICP-MS using previously published methods, whilst headspace gaseous Hg species were determined by CT-GC-AFS and dissolved gaseous Hg species, after purge and gold trapping by ICP-MS with detection limits of 0.15 pmol g−1 for sediments, 0.1 pmol L−1 for aqueous species and 0.01 pmol L−1 for gaseous species. The microcosm was cycled from anoxic conditions to oxic and back to anoxic over a 27 days period. Methylation of iHg, both naturally occurring and as a spike material, was observed to be greatest during the first anoxic period (1.6 and 0.9% respectively) with a lesser degree of methylation observed thereafter. Demethylation of the 201MeHg spike was found to range from 68% in the first anoxic period, at a rate of 0.02 pmol g−1 h−1, to 97% in the second anoxic period at a rate of 0.0005 pmol g−1 h−1. The reduction yields ranged from 3 × 10−5 to 2 × 10−5%. Of the gaseous species only Hg0 was observed in the reactor headspace with the sources determined as natural iHg (7–44%) and spike Hg (56–100%). A separate study focussed on the isotopic fractionation during abiotic methylation of mercury by using methylcobalamin, methyl tin, acetic acid and dimethyl sulfoxide as methyl donor compounds in the dark and under the effects of UV irradiation (325 nm @ 30 W for 15 to 45 minutes).192 Total Hg concentrations were determined using a single collector SF-ICP-MS whilst isotope amount ratios were measured using a MC-ICP-MS by monitoring 198Hg, 199Hg, 200Hg, 201Hg and 202Hg. Mass bias correction was by the use of Tl as an internal standard and an exponential model. Mercury species were separated by ion chromatography (∼1 mL of Chelex® 100, 200–400 mesh size using in house columns), based on the formation of neutral MeHgCl and charged iHg chloro complexes in dilute HCl prior to analysis by MC-ICP-MS. Subsequently, MeHg was oxidised to iHg with 3% BrCl and matrix elements removed via a second ion exchange column containing ∼0.2 mL of 100–200 mesh size AG-MP-1 M anion-exchange resin (Bio-Rad). No isotopic fractionation was observed with the use of these two ion exchange resins. It was found that mass independent fractionation occurred during experiments conducted in the dark whilst both mass independent and mass dependent fractionation occurred under UV irradiation. The former was attributed to the magnetic isotope effect operating during photo dissociation of the produced methylmercury, resulting in the selective enrichment of methylmercury with 199Hg and 201Hg isotopes. The extent of mercury species transformations, using isotopically enriched tracers of 199iHg and 201MeHg, during lyophilisation and protein extraction by ultrasonication has also been evaluated.193 Samples were spiked with the two enriched tracers prior to lyophilisation and UAE of water soluble species. White-sided dolphin (Lagenorhynchus acutus) liver homogenate, QC04LH4 supplied by NIST with a consensus mean Hg mass fraction of 3.60 μg g−1, was used in these studies as a test material. The UAE conditions were as follows; 30 s 100 W, 150 W and 200 W and 60 s at 200 W, all performed in pulse mode to avoid overheating the extraction mixture, for 0.7 g of sample suspended in 2 mL 200 mM ammonium acetate at pH 7.5. The supernatant and solid residue were separated by centrifugation with subsequent extraction of the solids with 3 mL of 4% (w/v) of SDS in water with shaking at room temperature for one hour. The SDS extractable fraction was also separated by centrifugation. Aliquots of the extracts obtained under the different sonication conditions were analysed by GC-ICP-MS preceded by digestion with 6 M HNO3, derivatisation with NaBPr4 and extraction into isooctane. No differences were observed in the iHg and MeHg concentrations (2.8 ± 0.1 μg g−1 and 0.74 ± 0.03 μg g−1) after sonication when compared to the amounts in the whole tissue. No evidence was found, by isotope pattern deconvolution, for species transformations for any of the tested extraction conditions at detection limits of 2% for methylation and 3% for demethylation.The speciation of Hg in a variety of biological samples has been covered this year. A study of MeHg metabolism and conversion into iHg in body tissues and subcellular fractions of maternal and infant rats after infant in utero and locational exposure to methylmercury (0.3 mg Hg kg−1 per day body weight) administered in drinking water from pregnancy day 0 to lactation day 20) has been reported.194 Quantification of Hg species in the cell extracts was by HPLC (C18 column, 150 × 3.9 mm, 5 μm) with a mobile phase containing 5% (v/v) methanol, 0.1% (v/v) 2-mercaptoethanol and 0.06 mol L−1 ammonium acetate at a flow rate of 1 mL min−1. The detection limits for iHg and MeHg were reported to be as low as 0.2 μg mL−1. Mercury containing proteins were isolated using a TSK-GEL G3000SWxl gel filtration column (7.8 × 300 mm i.d., TOSOH, Japan), with an eluent of 0.1 mol L−1 Tris–HCl (pH7.4) at 0.5 mL min−1, with quantitation by post column ID with a spike solution enriched in 198Hg and mass bias correction using the NIST SRM 981 common lead standard. In each case the column eluent was directly coupled to the nebuliser of a quadrupole ICP-MS. Subsequently, Hg containing proteins were identified by SDS-PAGE and MALDI-TOF-MS. The comparative distribution patterns of total mercury (T-Hg) and MeHg in the liver, kidneys, cerebrum and cerebellum between maternal and infant rats after pre-natal exposure to methylmercury showed that MeHg was the main species observed in all tissues. In maternal rats, mercury was highly accumulated in the kidneys whereas, the highest contents of T-Hg and MeHg in infant rats were in the cerebellum, more than 2 times higher than those in other organs. Three mercury containing proteins were found in the maternal and infant samples in this study. In the maternal sample, the relative molecular masses of these proteins was calibrated to be 480, 288 and 13 kDa and in the infant samples, calibrated as 100, 79 and 12.6 kDa.
Two papers cover mercury speciation in whole blood and serum/plasma by GC-ICP-MS. In the former paper195 a closed vessel MAE procedure was optimised using 0.3 mL of sample, 2 mL of 25% TMAH and 7.7 mL H2O with 10 minutes of irradiation at 180 °C. The applied microwave power level was not reported. Subsequently, the samples were derivatised with either NaBEt4 or NaBPr4 followed by GC separation in 3 minutes (30 metre, 0.53 mm id. Crossbond 100% dimethyl polysiloxane, initial column temperature 50 °C, ramped at 40 °C for at least 4 minutes with the transfer line to the ICP-MS heated to 180 °C) and detection by ICP-MS. A “three legged” torch was used to allow the GC-ICP-MS coupling conditions to be optimised by the aspiration of a 10 ng mL−1 tuning solution. The absolute LOD for the method are reported as 40 fg for iHg and MeHg after ethylation and 20 fg for iHg and 50 fg MeHg and EtHg after propylation with procedural detection limits of better than 0.5 μg L−1. The method was validated using the reference values for iHg and MeHg in NIST SRM 966 (Toxic metals in Bovine blood) and a New York Department of Health PT sample. For each material and each derivatisation procedure good agreement was obtained for the found values with the reference and target values. No EtHg was detected in either of these materials. Finally, the method was used to determine Hg species in whole blood samples, with NaBEt4 derivatisation, from a riparian population in Brazil. The MeHg concentration in these samples (n = 21) ranged from 24–109 μg L−1 and comprised over 90% of the total Hg concentration in most samples. For 11 of these samples the MeHg concentration was also determined by HPLC-ICP-MS (method details referenced within the paper) and good agreement was observed with the results obtained by GC-ICP-MS.
The second paper covers Hg speciation in serum/plasma samples by GC-ID-ICP-MS.196 Samples of plasma or serum were prepared for analysis, after spiking with 198Hg enriched (96%) MeHg, by a copper sulfate, potassium bromide, sulfuric acid, dichloromethane leaching procedure followed by back extraction into water and derivatisation with NaBEt4, collection of the ethylated species on a Tenax Tube and thermal desorbtion to the GC column via a device constructed in-house. A 15 m capillary column (0.53 mm i.d, 1.5 μm BP-1) coupled to a SF-ICP-MS instrument using an in-house constructed transfer line heated to 200 °C. The injector of the gas chromatograph was maintained at 120 °C and trapped species were desorbed for 175 s in splitless mode. The initial oven temperature was 55 °C for 2 min and then ramped to 94 °C at 49.9 °C min−1, with no hold time. The ICP-MS conditions were optimised using the 36Ar40Ar dimer and data acquisition commenced 40 s after the beginning of the thermal desorption stage. After careful optimisation, for a 2 mL sample spiked with 1.2 ng of Me198Hg the LOQ of 0.03 μg L−1 was obtained. Within and between run RSDs of <10% were estimated at concentration levels of 0.14, 0.35 and 2.8 μg L−1 MeHg. The method was validated using NIST 2977 Mussel Tissue, which, as the authors point out, is not ideal due to the different sample matrix but a plasma/serum CRM is not available, and good agreement between the found and certified values, 37.4 ± 3.8 and 36.6 ± 1.0 ng g−1 as Hg respectively, was obtained. The method was applied to MeHg determinations in 50 previously submitted plasma/serum samples which exhibited a mean value of 0.091 μg L−1 with a range of <LOQ to 0.19 μg L−1 MeHg. A common form of Hg exposure is through the use of thiomersal as a preservative in vaccines. To monitor this exposure route hair samples of breastfed infants50 have been subjected to Hg speciation analysis. Mercury species were extracted from 2 mm long hair samples by a previously validated method, involving a copper sulfate, potassium bromide, sulfuric acid and dichloromethane leaching procedure followed by back extraction into water and derivatisation with NaBPr4, collection onto Tenax followed by thermal desorbtion, isothermal GC (no conditions given), pyrolysis and detection by AFS. The LODs were estimated to be 5 and 10 pg mL−1 for MeHg and EtHg respectively for a 10 mg hair sample. The authors assume that these figures could be improved by using a larger sample size. The found concentrations in the hair samples (n = 20) ranged between 10 and 690 ng g−1 for MeHg and <LOD to 65 ng g−1 for EtHg. The median concentrations for MeHg (87 ng g−1) and EtHg (32 ng g−1) showed that a significant proportion of organomercury exposure in this sample cohort was due to intramuscular vaccines.
A number of papers cover mercury speciation in food samples and fish tissues. An enzymatic digestion procedure for a variety of food sample types has been developed.197 The samples, after defatting with acetone and toluene, were spiked with either PrHgCl or, for IDMS, 201Hg enriched MeHg followed by the addition of 3 mL of 1.5% (w/v) pancreatin and 5 mL of a 0.08 mol L−1 phosphate buffer and shaken at 37 °C overnight. To this extract 15 mL of water was added and then made up to 30 mL with concentrated HCl which was then shaken for 3 hours followed by centrifugation, pH adjustment, derivatisation with NaBPh4 and extraction into isooctane prior to analysis by GC-ICP-MS. A DB-5MS (30 m × 0.25 mm, 0.25 μm film thickness) column was used for the GC separations with the transfer line to a Q-ICP-MS heated to 280 °C. The method was validated using three CRMs, NIST SRMs 1947 (Lake Superior fish), 1566b (oyster tissue) and NRCC Tort-2 (lobster hepatopancreas) and good agreement, with recoveries ranging between 103 and 109%, was found between the certified and measured values for both conventional external calibration, with PrHgCl as an internal standard, and ID-MS. The latter method exhibited an improvement in precision (n = 3) over that obtained for the former method. Spike recoveries into a variety of foodstuffs ranged between 96 and 107% for MeHg and 88 to 117% for EtHg. The LOD for both MeHg and EtHg is reported to be 0.3 μg Hg kg−1. A second paper to report on this topic used an UAE method to extract Hg species from fish tissues.198 After lyophilisation and homogenisation by cryogenic grinding a 0.2 g sample aliquot was suspended in 10 mL of the extraction solution, 0.1% v/v HCl, 0.05% w/v L-cysteine and 0.1% v/v 2-mercaptoethanol, and sonicated for 15 minutes followed by centrifugation and passage through a 0.2 μm cellulose filter. The HPLC conditions, using a reverse phase column (C8, 3 μm, 33 × 4.6 mm), were found to be optimal with a mobile phase of 0.4% w/v L-cysteine, 0.4% v/v 2-mercaptoethanol and 0.06 M ammonium acetate flowing at 1 mL min−1 which gave a separation of iHg and MeHg in less than two minutes. The column outlet was coupled directly to the nebuliser of the ICP-MS instrument. Good agreement was found between the found and certified values for NRCC DOLT-3 (dogfish liver) and DORM-3 (dogfish muscle) for both MeHg (101 and 112% respectively) and total Hg (101 and 107% respectively) with the total Hg values obtained after a concentrated HNO3–H2O2 digestion procedure. Subsequently, the method was applied to locally bought samples of shrimp, octopus, fish, mussel, squid and canned tuna. The lowest MeHg concentrations were observed in the octopus sample (4 ng g−1) with the highest values obtained for tuna (111 ng g−1). No EtHg was found in these samples and only small amounts of iHg were detected, and good agreement between the sum of the individual species detected and the total Hg content also being reported.
A comparison between HPLC-ICP-MS and HPLC-HG-AAS has been undertaken199 using a number of different CRMs. For HPLC-ICP-MS a C8 column was used (3 μm, 33 × 3 mm) in conjunction with a mobile phase of 0.5% v/v 2-mercaptoethanol in 5% v/v methanol at pH 5.3 flowing at 1.5 mL min−1. A different column (C18, 5 μm, 250 × 4.6 mm) and mobile phase (5% v/v methanol, 0.06 M CH3COONH4 and 0.1% w/v L-cysteine at pH 6.8 flowing at 1.0 mL min−1) was used for the HPLC-HG-AAS measurements with post column hydride generation using alkaline NaBH4. Total Hg concentrations in the CRMs were determined after an MAE-concentrated HNO3 digestion procedure (600 W for two minutes, two minute hold followed by 450 W for 45 minutes) by ICP-MS. Mercury species were extracted from the biological and sediment CRMs by two methods; a MAE 2-mercaptoethanol digestion (0.2 g of sample and either 5 or 2.5 mL, fish tissue and sediments respectively, 0.5% v/v 2-mercaptoethanol at 120 °C for 15 minutes), and secondly an enzymatic digestion comprising 0.2 g of sample, Protease XIV and a phosphate buffer containing 0.05% cysteine with incubation at 37 °C for 2 hours. All supernatants from both procedures were filtered through 0.2 μm PVDF filters before analysis. Quantitative recovery of MeHg and total Hg was achieved by both extraction procedures and the results were in good agreement with the certified values for the CRMs employed (NRCC DORM-2 (dogfish muscle) and DOLT-3 (dogfish liver), NIST RM 50 (Albacore Tuna) and IRMM IMEP-20 (tuna fish) and for the MAE 2-mercaptoethanol digestion of IRMM CC580 sediment when two extractions were performed on one sediment sample). For the HPLC-ICP-MS measurements the LOD is given as 0.4 μg L−1, which corresponded to 0.01 μg g−1 in solid material. The figures of merit for HPLC-HG-AAS were, not unexpectedly, higher but broadly comparable with an LOD of 2 μg L−1, which corresponded to 0.01 μg g−1 in solid material. The calibration for each method was linear in the range of 0–100 μg L−1. No difference was observed between the results obtained by each analytical method for enzymatic digestions of the biotic CRMs or fish samples. One important point to note is that the order of eluting species changed depending on the extractant employed. With 2-mercaptoethanol MeHg elutes before iHg whilst with cysteine based extractants the iHg elutes before the MeHg which has the same retention time in each case. This effect can be useful for samples in which one species is dominant, such as iHg in sediments and MeHg in biota, and could overlap with, or cause interference via memory effects, with less abundant species of interest.
5.8 Platinum and ruthenium
Following on from the two reviews on Pt speciation in the clinical setting described in last years speciation ASU, this year another review dealing with the quantitative methods for studying DNA interactions with cisplatin has been published.29 It details some of the older analytical approaches, such as the immunochemical assays, 32P-post-labeling and some of the elemental and molecular mass spectrometry-based methods. However the period covered unfortunately seems to miss some of the more recently published methods based on the use of enzymatic digestion, SPE clean-up and HPLC-ICP-MS mentioned in last years ASU speciation review and which can be considered the current “state-of-the-art” in this area of research.Methods that can be applied to the spatial analysis of platinum-containing drugs to determine their location in tissues or cells, provide another dimension to understanding their interaction with biological systems. A LA-ICP-MS-based methodology200 for the imaging of Cu, Pt and Zn has been applied to whole kidney 3 μm sagittal sections from rats treated with pharmacological doses of cisplatin. The Pt accumulated in the kidney cortex and corticomedullary junction, corresponding to areas where the cells most sensitive to cisplatin nephrotoxicity are located. The Cu and Zn distribution maps revealed a significant displacement by Pt, as compared to control tissues. A decrease in the Pt accumulation in the cortex was observed when the nephroprotective drug cilastatin was co-administered with cisplatin. Imaging reproducibility, sensitivity (LOD, 50 fg), and resolution (down to 8 μm) were sufficient to demonstrate that LA-ICP-MS can be applied as a microscopic Pt detector at the cellular level in certain tissues. However, whilst this is an interesting model system to study, it's real world application is clearly limited, as patients undergoing cisplatin chemotherapy are screened for kidney function and hydrated extensively to reduce the known nephrotoxicity of the drug.
The cellular uptake and binding of chemotherapeutic metallodrugs has been investigated using LC or CZE coupled to ICP-MS. The quantification of intact, free cisplatin in cell models was investigated201 at a biologically relevant drug exposure concentration of 5 μM. Two complementary LC-ICP-MS methods based on pentafluorophenylpropyl siloxane (Discovery HS F5) and porous graphitised carbon (Hypercarb) were investigated. The LODs for cisplatin were 0.013 and 0.11 μg L−1 (given as total drug), respectively. Upon entering the cell, cisplatin is known to react with proteins and peptides in the cytosol forming adducts. Due to the limited selectivity of one-dimensional LC separations, protein removal was a prerequisite for accurate quantification and centrifugal filtration (10 kDa cut-off) was the method used for this. Exposure of two different cell lines to 5 μM cisplatin for 24 hours resulted in cisplatin concentration levels ranging between 0.2 and 1.5 μg g−1 protein. Despite the poor recovery of the columns regarding total Pt, the accurate cisplatin quantification was achieved via ssIDMS and standard addition. Ruthenium–indazole complexes are promising anticancer agents undergoing clinical trials. One such compound, sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP1339), which is administered intravenously (i.v.), has been investigated202 to determine its serum protein binding profile. The blood plasma from mice treated with the drug was isolated and analysed by CZE and SEC-IC both combined on-line to ICP-MS. The performance of the analytical methodology was compared and the interaction of KP1339 with mouse plasma proteins characterized in vivo. The majority of Ru compound was bound to albumin and the stoichiometry of the KP1339 protein binding was determined through the molar Ru/S ratio. In general, good agreement of the data obtained with both techniques was achieved. The SEC-IC method was found to be more sensitive as compared to the CZE-ICP-MS approach, whereas the latter benefited from shorter analysis time and lower sample consumption, which is key when dealing with such samples.
Some more studies on the analysis of oxaliplatin DNA-adducts by HPLC-ICP-MS and LC-MS/MS have been published. The use of SF-ICP-MS to investigate the fate of Pt in human cell populations following treatment with cis- or oxaliplatin and combination treatments has been reported.203 Pt–DNA adduct formation data at several time points has been determined in the leukocytes from patients undergoing Pt-based chemotherapy demonstrating significant inter-patient variability. The sensitivity and reproducibility of the technique enabled quantitation of 0.2 Pt adducts per 106 nucleotides using 10 μg of patient DNA. For in vitro studies, three cell models were used: A549 human lung adenocarcinoma epithelial cells were exposed to 50 μM cisplatin for 1 h; HCA7 human colorectal cancer cells were treated with either FOLFOX (200 μM 5-fluorouracil, 200 μM folinic acid and 50 μM oxaliplatin) or 50 μM oxaliplatin; and HT29 human colorectal cancer cells were treated with 50 μM oxaliplatin in combination with 20 μM methaneseleninic acid. The cells were harvested and either the DNA extracted and/or a commercially available kit used to fractionate the treated cells into four sub-cellular compartments. Each of the sub-cellular fractions and extracted DNA were digested separately, evaporated to dryness and reconstituted in 2% nitric acid for analysis by SF-ICP-MS. The sub-cellular Pt distribution for cisplatin treated A549 cells was shown to be as follows (all approximate values): 70% localized in the cytosol; 17% in the membrane and membrane localized fraction; 9% in the nuclear fraction; and 4% in the cytoskeletal fraction. Both FOLFOX and oxaliplatin treated HCA7 cells showed comparable sub-cellular Pt distributions, and Pt–DNA adduct formation was similar for the oxaliplatin and FOLFOX treatments with adduct yields of 5.6 and 5.5 adducts per 106 nucleotides respectively. It was found that the combination of oxaliplatin with 20 μM MSA did not change the distribution of Pt or significantly alter its accumulation in the cytosol of the HT29 cells. Mass balance experiments showed a >99% recovery of the total Pt in the sub-cellular fractions. A similar study204 investigated methods based on HPLC-ES-MS, HPLC-SF-ICP-MS and HPLC-UV detection for in vitro studies of the bifunctional adducts of oxaliplatin with mono-nucleotides, di-nucleotides and cellular DNA. Interaction of oxaliplatin with adenosine (A) and guanosine (G) mono-nucleotides resulted in the formation of five bifunctional platinum diaminocyclohexane (DACHPt) adducts. These were two isomers of the A-DACHPt-A and A-DACHPt-G adducts, and one G-DACHPt-G adduct, as confirmed by MS/MS spectra obtained by collision induced dissociation. Further, interaction of oxaliplatin with AG and GG di-nucleotides resulted in the formation of three adducts: DACHPt-GG and two isomers of the DACHPt-AG adduct, as confirmed by ES-MS and the complementary data obtained by UV and SF-ICP-MS. Finally, an HPLC-ICP-MS method for the quantification of oxaliplatin GG intra-strand adducts (DACHPt-GG) was developed and used for monitoring the in vitro formation and repair of these adducts in human colorectal cancer cells. The method detection limit was 0.14 ng mL−1 Pt which was equivalent to 0.22 Pt adduct per 106 nucleotides based on a 10 μg DNA sample.
5.9 Selenium
During the past year, two reviews have appeared. A comprehensive review of Se in food and supplements by Gammelgaard et al.27 sets out in some detail the present knowledge of Se speciation and metabolism, from both the biological and analytical viewpoints. Heras et al.205 focus on selenoproteins and the various techniques that have been employed to study them, such as multidimensional chromatography-ICP-MS, ES-MS-MS, MALDI-TOF, and SDS-PAGE-LA-ICP-MS.The determination of recently discovered metabolites has been discussed. Selenoneine (2-selenyl-N,N,N-trimethyl-L-histidine), a metabolite found by Yamashita and Yamashita in the blood of bluefin tuna and reported in the last Update, was also isolated from the livers of two species of sea turtle.206 They also identified other Se metabolites common to both marine and terrestrial animals and suggested that the turtles possess a specific metabolic mechanism by which the Se prevents the accumulation of mercury. The novel Se metabolite, Se-methylselenoneine has been identified,207 in human urine and blood after SPE by what the authors describe as “parallel coupling of RP-HILIC-ICP-MS and ES-linear ion trap quadrupole-Orbitrap-MS”. This approach offered low μg L−1 detection and the provision of information (such as the ratios of methylated to nonmethylated species in blood and urine) that could help to understand compound metabolism. However, the experimental details indicate that two chromatographic separations were performed, one for each detector, not that the eluent was split between the two. The full scan mass spectra were searched for selenocompounds at retention times determined during HPLC ICP-MS coupling. The presence of Se in the compound was confirmed by the characteristic inter-isotopic mass differences (e.g. there is a mass difference of 1.9992 AMU between 78Se and 80Se). Lastly, a study of the metabolism of selenohomolanthionine208 in rats was supported by analysis of serum and tissue supernatants, and urine, by HPLC on multimode gel filtration columns with ICP-MS detection. The compound, found in selenised Japanese pungent radish, which may be a potential new source of Se supplementation, was labelled with 77Se and injected at a dose of 25 μg per kg body weight. The researchers found that the compound differed from other Se metabolites in that it accumulated preferentially in the kidneys but produced the same urinary metabolites and was incorporated into selenoproteins.
The speciation of Se in a CRM has been determined. A LA method 80 times more sensitive than traditional LA-ICP-MS was developed to detect selenoprotein P (SelP) and GPx3 (ref. 107) at the physiological concentrations in NIST SRM 1950 (metabolites in human plasma), allowing for unambiguous identification for the first time. After separation by GE, the bands were transferred to polymer membranes by electroblotting, from which a higher LA signal was obtained than by ablation directly from the gel. The sensitivity was further increased by humidifying the plasma with a solution of an organic solvent to take advantage of the enhanced ionization efficiency for Se in the presence of carbon.
The results of a comparison study to assess the analytical capabilities of National Measurement Institutes (NMIs) to accurately quantify the mass fraction of selenomethionine (SeMet) and total Se (at low mg kg−1 levels) in a selenised wheat flour has been reported by Goenaga-Infante.209 The material used in the study, suitably prepared from a wheat grain, was provided by Nottingham University, UK. Nine results for total Se and four results for SeMet were reported. Methods were mostly MAE and ICP-MS (for total Se), and for the determination of SeMet, consisted of multiple-step enzymatic hydrolyses or hydrolysis with methanesulfonic acid followed by HPLC-ICP-MS, with ssIDMS calibration using 76Se-enriched SeMet. The total Se and speciation results presented by all participants were in “excellent agreement,” producing key comparison reference values of 17.3 mg kg−1 for total Se, and 28.3 mg kg−1 for SeMet (11.4 mg kg−1 as Se). The report also contained results for the determination of SeMet in selenised yeast tablets, which are now available as a CRM from LGC (as per CCQM-P86), by a number of “expert” laboratories, several of which are not NMIs. These results showed that accurate analyses could be made without the need for species-specific isotope dilution.
The analysis of environmental samples has been a popular topic of research in the past year. Inorganic Se speciation in groundwater210 was monitored in a suburban area of India over a three-year period to investigate the effect of seasonal variation on the species' transformations. The determination of total Se and SeIV was carried out using an HG-AAS procedure. It was found that the dominant species during the pre- and post-monsoon periods was SeVI. During the post-monsoon periods, leaching from soil was increased due to the increased oxidizing nature of the groundwater as indicated by the dissolved oxygen and redox potential (Eh) measurements. This finding has important implications for the health of people drinking groundwater from seleniferous areas. Other researchers studied the remediation of Se-contaminated waste water by anaerobic granular sludge89 with XANES, IC and SPME-GC-MS. Simmons and Wallschlager211 investigated with AEC-ICP-MS the uptake, transformation, and release of species back into the environment of three different species of inorganic Se by common green fresh water algae. The formation and the release of selenocyanate into the environment through non-industrial means was observed for the first time. It was also noted that release of the reduced metabolites coincided with increasing esterase activity in solution, indicating that cell death (lysis) was the primary emission pathway. Other workers speciated ultratrace concentrations of SeIV and SeVI in natural waters75 by HPLC-DRC-ICP-MS following preconcentration on a column containing tetrabutylammonium hydroxide-modified activated carbon. A 100-fold improvement in LODs was obtained. Potin-Gauthier and co-workers212 evaluated the distribution and mobility of Se species within soil samples with both anion-exchange and mixed-mode (anion-exchange and reversed-phase) HPLC-ICP-MS. A new method of parallel single extractions was developed in order that water-soluble Se (i.e., the most mobile Se fraction), exchangeable Se (i.e., sorbed onto soil component surface) and Se bound to soil organic matter could be determined. The LODs were between 0.1 and 10 μg kg−1. The mobility of Se within acidic environmental samples, containing high concentrations of aluminium and iron, from the Mount Etna volcano has also been reported.213 Using anion-exchange HPLC-ICP-MS; SeVI and SeVI could be separated in the rain and soil leachate samples in under 10 min with a LOD of 0.1 μg L−1. It was concluded, based on results of the interaction between synthetic acid rain (containing sulfuric acid), and soil collected from the volcano, that the Se mobility depended on competition with sulfate present in the acid rain.
Several new methods and procedures for selenium speciation have been developed in the review period. Six Se species were separated in 8 min using RP-HPLC-ICP-MS95 with the aid of room-temperature ionic liquids as mobile phase. The best separations were achieved with specifically 0.4% (v/v) 1-butyl-3-methylimidazolium chloride with 0.4% (v/v) 1-butyl-2,3-dimethylimidazolium tetrafluoroborate and 99.2% (v/v) water. This method was validated by the accurate analysis of CRM SELM-1 (selenised yeast) and applied to the analysis of selenised yeasts and clover. A new SPE material,214 prepared from chitosan cross-linked with diethylenetriamine was used to selectively adsorb SeVI from environmental water samples at pH 3.6. The retained Se was eluted with 1 M hydrochloric acid and determined by ICP-OES. Total Se was determined after oxidation with persulfate. The LODs obtained were sufficient to allow determination of both species in real samples at concentrations below 1 μg L−1. Kokarnig, et al.215 quantified Se species in human serum following protein precipitation by HPLC-ICP-MS; the cleaner matrix allowed for sub-μg L−1 detection of selenosugar 3 and of MeSeCys, which had never before been identified in serum samples. Francesconi and co-workers52 have developed a low-volume, enzymatic method for the extraction of SeMet from selenised yeast samples. In contrast to traditional methods that generally require large sample volumes consuming significant amounts of costly enzymes, the new method involved a microtitre plate that decreased the required sample volume to 1 mL per extract. Results were obtained that were identical to those by the more traditional high-volume method with a combination of protease XIV and lipase VII. Selenomethionine was determined in CRM SELM-1 (selenised yeast) by HPLC-ICP-MS and the result obtained was 93 ± 2% of the certified value.
Methods for the determination of Se in various biological media, such as food and bodily fluids, have also been developed in the period covered by this Update. Water samples and garlic extracts66 were analysed by a selective, non-chromatographic method of on-line ionic liquid dispersive microextraction with ETV-AAS detection. The LOD was 15 ng L−1. Se-enriched eggs216 were analysed for their inorganic and organic content by HG-AFS. Separation of organic and inorganic Se involved precipitation of albumen with trichloroacetic acid. The LOD was 0.07 μg L−1, allowing the researchers to demonstrate that, in these samples, the organic Se content was three to seven times that of inorganic Se. For the analysis of Se-enriched rice,104 CE-ICP-MS was used after an high efficiency enzymatic extraction. The LODs were between 0.1 and 0.9 ng mL−1, making this a simple method capable of determining SeVI, SeIV, SeCys2 and SeMet in rice directly without any derivatisation or pre-concentration within 18 min. It was reported that selenium-enriched rice contained only one species of selenium, SeMet, in the concentration range of 136–143 μg kg−1 (as Se) dry weight.
The speciation of selenium in urine by HPLC-ICP-MS has been reported in a paper in Czech.217 Measurements were made at m/z 80 and the 40Ar40Ar+ interference was removed by the addition of methane to the collision/reaction cell. Good separation of SeVI, SeMet, and selenourea was observed, but SeIV and SeCys2 were not completely resolved. The urine of “septic” patients and those of a healthy volunteer control group (both of whom were consuming Se supplements) were analysed. It was found that the Se concentration in the former group was low. The measurement of Se speciation in the liquid phase of flue gas residues after coal combustion by HG-AFS has also been reported.218 The LODs were about 0.05 μg L−1 for both inorganic and organic Se, and the method was validated by spike recovery experiments and the accurate analysis of NIST SRM 1632c (trace elements in coal).
Studies of the interconversions and transformations of Se compounds have been published in the year under review. Lunoe et al.219 observed the extent of cell death for three cancer cell lines when incubated with five different Se species. Speciation analysis was performed with RP-, AEC-, and SEC with ICP-MS detection. Selenomethionine, selenomethylselenocysteine (SeMeSeCys), methylseleninic acid, SeIV and SeVI in the concentration range 5–100 μM were incubated with cells for 24 h and the induction of cell death was measured by flow cytometry. The amounts of total Se in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Full recovery of Se in the cellular fractions was observed for all Se compounds except methylseleninic acid. In the latter case the speciation analysis showed that methylselenic acid was completely transformed during the incubations, while metabolic conversion of the other Se compounds was limited. Production of volatile dimethyldiselenide (DMDSe) was observed for methylseleninic acid and for SeMeSeCys. These compounds and SeIV showed noticeable protein binding. It was reported that there was no correlation between cell death induction and the species transformations. Goenaga-Infante et al.220 studied Se uptake and speciation in cell lysates using a high-efficiency sample introduction system, capable of achieving a 12-fold lower LOD than those obtainable with conventional nebulizers, coupled to HPLC-ICP-MS. The results, together with those from ES-MS-MS and GC-TOF-MS, indicated that exposing non-malignant and lymphoma cell lines to methylseleninic acid can lead to the formation of intracellular DMSe (not DMDSe as was found by others219) and dimethylselenidesulfide, which was identified for the first time in lymphoma cell headspace. Roman et al.,35 studied selenoprotein speciation in rat colon tissues, comparing the efficiencies of four different extraction procedures, followed by analysis by SEC- and anion-exchange-HPLC-ICP-MS with IDA. A HPLC-ICP-MS method was used to study Se speciation in aquatic plants.221 Selenium uptake was measured and the effect on the physiological characteristics were also investigated for two plant species grown in different concentrations of sodium selenate. Accumulation and distribution of Se in a species of cactus123 was identified and quantified by ICP-MS, microfocused XRF, XANES, and LC-MS. Lastly, the species distribution of Se in the organs and tissues of African catfish,54 supplemented with Se-enriched garlic, was investigated by SEC-ICP-MS and LA-ICP-MS to measure the species in the soluble protein fraction. It was indicated that the application of several sample treatments in conjunction with different chromatographic techniques (reverse phase, anion-exchange and ion exchange/size exclusion) was crucial to the unambiguous identification of Se-species.
There have been a few studies published related to the simultaneous separation and determination of multiple analytes in biological materials and food. Different forms of Se were administered to marine fish, Terapon jarbua, to study their effects on mercury uptake,222 and although a decrease in the HgII concentrations in the fish tissue was observed, all forms of Se studied were ineffective at removing or preventing the uptake of MeHg. A new method,223 combining ion-pair reversed phase and affinity chromatography with collision cell ICP-Q-MS and post-column IDA, was proposed for the quantification and distribution of Se species in human serum samples. Serum samples were collected from mercury-exposed individuals given Se supplementation in the form of Se-enriched yeast for 180 days. The results revealed that prior to supplementation, over 77% of Se was in the form of SelP, selenoalbumin (SeAlb), and GPx; whereas, after supplementation, the concentrations of SelP and SeAlb increased, concentrations of inorganic Se, SeCys2, and SeMet decreased, and concentrations of GPx remained the same. In a similar study,224 serum samples from individuals supplemented with Se-enriched yeast after long-term mercury exposure were analysed by anion-exchange HPLC-ICP-MS. The method was capable of separating four Se species in under 7.5 min, with LODs between 0.3 and 0.7 μg L−1. Lastly, the speciation of As and Se in cereal samples was investigated using IC-DRC-ICP-MS.36 The potentially interfering 38Ar40Ar+ and 40Ar40Ar+ at Se m/z of 78 and 80 were decreased in intensity by approximately 3 orders of magnitude with methane as the reactive cell gas in the DRC. This approach also allowed As to be determined as the adduct ion 75As12C1H2+ at m/z 89. The method, validated by spike recovery experiments and mass balance, had LODs between 6 and 9 ng L−1 for As and between 9 and 30 ng L−1 for Se.
5.10 Sulfur
A study has been conducted to identify, by UPLC-TOF-MS, and quantify, by UPLC-ICP-MS, glutathione adducts of clozapine,225 as part of an investigation into reactive electrophilic metabolites which have been implicated in idiosyncratic drug reactions in recent years. Conventionally, radiolabelled analogues of the compound(s) of interest are used for these studies. Human liver microsomes were incubated with clozapine, nicotinamide adenine dinucleotide phosphate-oxidase and glutathione. The glutathione-clozapine adducts were separated, after SPE clean up, using a C18 column (1.7 μm, 100 mm × 2.1 mm i.d) maintained at 65 °C with a ten minute gradient elution based on 0.1% formic acid and methanol flowing at 250 μL min−1. For the ICP-MS studies a cooled spray chamber, −5 °C, and oxygen addition to the plasma were used to overcome carbon deposition. The S containing species in the resulting chromatograms were detected at a m/z of 48 (SO+) to overcome the isobaric interferences at m/z 32 and to enhance sensitivity by up to 100 fold with a lower limit of quantitation of 0.16 μM S. Two different adducts, glutathione-clozapine and oxidised glutathione-clozapine, were identified by UPLC-TOF-MS with a concentration of 0.9 μM found for each adduct by UPLC-ICP-MS.The juice of the Noni (Morinda citrifolia) fruit is now widely sold as a dietary supplement. A study has been undertaken to identify volatile compounds in Noni juice, after extraction by steam distillation and SPME, by GC-AED and GC-MS.226 A fused silica capillary column, 30 m × 0.25 mm i.d × 0.25 μm film thickness, was used for species separations. More than 60 volatile compounds were detected with six of them, dimethyl disulfide, methanethiol, ethyl 3-methylthiopropanoate, methyl 3-methylthiopropanoate, 3-methylthiopropanoic acid and S-Methyl thioacetate, identified as sulfur species.
A non-targeted metabolomic study of sulfur and phosphorus containing metabolites in livers, plasma and urine from lean and obese Zucker rats has been reported.227 A 1.7 μm × 2.1 × 100 mm C18 column was used for the UPLC separations with gradient elution based on the following mobile phases; organic/aqueous tissue extracts and plasma extracts, solvent A was 0.1% aqueous formic acid and solvent B was methanol containing 0.1% formic acid. For bile and urine analysis solvent A was 25 mM ammonium acetate plus 0.1% formic acid whilst solvent B was methanol containing 0.1% formic acid. For tissue and plasma samples the column temperature was 50 °C with bile extract separations conducted at 65 °C and urine extracts were separated at 40 °C. Flow rates were 0.4 mL min−1 except for bile extracts for which a flow rate of 0.8 mL min−1 was used. Oxygen was added to the nebulised sample stream and was also used as a reaction gas to allow ions at m/z 47 and 48, which correspond to the phosphoxide (PO+) and sulfoxide (SO+) ions, to be monitored for quantitation. Sulfur containing metabolites were observed only in the bile extracts. Metabolites containing P were observed in the plasma and liver extract samples and the different profiles observed in the resulting ‘phosphatograms’ allowed the differentiation between lean and fat Zucker rats.
5.11 Tin
There has been a marked decrease in the number of publications reporting studies on tin speciation. A novel method using semi-permanent coated capillaries for use with CE coupled with ICP-MS for the separation of four organotin compounds has been developed by Sun et al.228 Dioctadecyl dimethyl ammonium chloride (DDAC) was investigated to enhance separation efficiency of TMT, TBT, DBT and MBT. The electrolyte composition and ionic strength were optimized and a buffer containing 0.608 mol L−1 acetic and 7 mmol L−1 sodium acetate was selected for the separation. The reported detection limits for the four organotin compounds ranged from 0.037 to 0.112 mg L−1 (as Sn). The RSD values were below 3.1% for retention time, less than 7.8% for peak height and not more than 8.4% for peak area. The developed method was successfully applied to determine the four organotin compounds in CRM BCR 477 (mussel tissue), and environmental samples including waters, wine and biological samples (Mya arenaria Linnaeus).Organotin additives are widely used as thermal and light stabilizers in PVC plastics and a study to examine the leaching rates of DMT and DBT from PVC water pipe has been conducted using a 61 m closed loop pipe system.229 Using GC coupled with a pulsed flame photometric detector (PFPD) and ICP-AES the change in concentrations of DMT and DBT in the water in the system was monitored over time and allowed to reach equilibrium. The diffusion coefficient for the OTC in the PVC pipe material was found to be 9 × 10−18 m2 s−1. This value falls within with the range of values estimated from the literature (2 × 10−18 to 2 × 10−17 m2 s−1) and is in line with predictions from a proposed mechanistic leaching rate model.
The presence of OTC in landfill leachates continues to be of interest. Most previous methods however are fairly complex (due to the presence of high levels of organic matter, proteins and fats) or suggest the use of enriched stable isotopes that are only readily available for butyltins. Vahcic et al.230 have reported a procedure for the simultaneous determination of methyl-, butyl-, phenyl- and octyl-Sn in landfill leachates using a commercially coupled GC-ICP-MS (15 m × 0.25 mm DB-5MS column coated with 5% phenyl-methylpolysiloxane). The application of methanol as co-extraction reagent and tris–citrate buffer for adjustment of pH for derivatisation of OTC in the landfill leachate was investigated as was the use of NaBEt4 and NaBPr4 as derivatisation reagents for LLE into hexane (NaBEt4 was found to be better). The reported LODs were in the order of 2 ng Sn L−1 and RSDs were mostly better than 3%. The method was applied in the analysis of OTC in landfill leachates using the standard addition calibration method.
5.12 Tungsten
Tungsten is an element that is not often the focus of speciation or metallomic studies, however the fate of the anti-diabetic agent sodium tungstate has been investigated231 in the serum of diabetic and healthy rats. It is known that oral administration of sodium tungstate preserves the pancreatic beta cell function in diabetic rats. Healthy and streptozotocin-induced diabetic rats were treated with sodium tungstate for one, three or six weeks, after which the species of W in serum, were analysed. After six weeks, the serum W concentration in diabetic rats was about 4.6 times higher than in healthy specimens. Speciation analysis of W was carried out using 2D separations, involving an immunoaffinity packed column and SEC coupled to ICP-MS. Ultrafiltration data together with the SEC-ICP-MS results showed that around 80% of the serum W was bound to proteins, mainly as a complex with albumin. An unknown protein with a molecular weight higher than 100 kDa was also found to bind a small amount of W (about 2%). Analysis of the desalted and concentrated chromatographic fractions by MALDI-TOF-MS, confirmed albumin as the main protein bound to tungstate in rat serum, while no binding to transferrin (Tf) was detected. The interaction between glutathione and W was also evaluated using standard solutions; however, the formation of complexes was not observed. The stability of the complexes between W and proteins when subjected to more stringent procedures, like those used in proteomic methodologies were also evaluated. The results indicated that the stability of the complexes between W and proteins is not high enough to remain unaltered during protein separation by SDS-PAGE in denaturing and reducing conditions. However, the procedures for in-solution tryptic digestion and for ES-MS analysis in MeOH–H2O–formic acid could be used for protein identification without large loss of binding between W and proteins.5.13 Zinc
Despite its pivotal role in determining the risks and time frames associated with contaminant release, metal speciation remains a poorly understood aspect of biosolids chemistry. Donner et al.232 have reported on the use of synchrotron-based spectroscopy techniques to investigate the speciation of Cu and Zn in a range of biosolids. High resolution element mapping using μXRF revealed considerable heterogeneity in key element associations, and a combination of both organic and inorganic Cu and Zn binding environments. Linear combination fitting of K-edge XAS indicated consistent differences in metal speciation between freshly produced and stockpiled biosolids. While sulfide minerals play a dominant role in metal binding in freshly dewatered biosolids, they were of lesser importance in dried biosolids that have been stockpiled. A degree of metal binding with Fe oxide minerals was apparent but the results did not support the hypothesis that biosolids metals are chiefly associated with Fe minerals. This work has potential implications for the long-term stability of metals in biosolids and their eventual fate following land application.The application of SR-XRF and SR-XAS has also been reported to study the distribution and speciation of Zn (Cu and Ni) in situ using hydrated roots of cowpea (Vigna unguiculata).233 The roots were exposed to 40 μM Zn for 1 to 24 h. Cortical concentrations remained comparatively low with much of the Zn accumulating in the meristematic region and moving into the stele; approximately 60% to 85% of the total Zn stored as Zn phytate within 3 h of exposure. This is reported by the authors to be the first report of the in situ distribution and speciation of Zn (and Cu and Ni) in hydrated (and fresh) plant tissues, providing valuable information on the potential mechanisms by which they are toxic.
6. Macromolecular analysis
This section details a range of diverse methods for the analysis of different biomolecules using elemental techniques.6.1 Protein and metalloprotein analysis
Investigations into the metalloprotein transferrin have focused on high accuracy analysis and its stability during the analytical protocol. The absolute quantification of the iron-containing metalloprotein Tf in harbour seals by HPLC-ICP-MS has been reported.234 The method was validated using a certified human serum reference material (ERM-DA470K/IFCC). A Tf concentration of 2.33 ± 0.03 g L−1 was found, in good agreement with the certified total concentration of 2.35 ± 0.08 g L−1. Different seal samples were analysed to demonstrate the suitability of the procedure for the quantification of Tf in real samples as well as to observe modified glycoform patterns. The stability of the metal centre in Tf under different solvent conditions, to investigate whether the metal-protein complex remains in its native state, has also been investigated.235 The analysis of metalloproteins typically involves some type of liquid chromatography or electrophoresis separation step coupled with an atomic or molecular detector. In this report UV/VIS absorbance was employed to monitor the iron content in human holo-Tf under various solvent conditions, including: changing polarity; pH; ionic strength; and the ionic and hydrophobic environment of the protein. Iron loading percentages, where 100% loading equates to a Fe3+ to Tf ratio of 2:1, were quantitatively determined to evaluate the effect of solvent composition on the retention of Fe3+ in Tf. Maximum retention was found in buffered 20 mM Tris solutions (96 ± 1%). Exposure to organic solvents and deionised water caused release of similar to 23–36% of the Fe3+ at physiological pH 7.4. Salt concentrations similar to separation conditions used for ion exchange had little to no effect on Fe3+ retention in holo-Tf. Unsurprisingly, changes in ionic strength caused by additions of guanidine HCl (0–10 M) to holo-Tf resulted in unfolding of the protein and loss of Fe3+. However, denaturing and metal loss was found not to be an instantaneous process for additions of 1 to 5 M guanidinium. In contrast, complete denaturing and loss of Fe3+ was instantaneous with >6 M additions of guanidinium. Changes to the hydrophobicity of Tf (via addition of 0 to 14 M urea) had less effect on denaturing and release of Fe3+ from the Tf compared to changes in ionic strength. A method based on the quantification of a Tf after separation by non-denaturing gel electrophoresis using LA-ICP-MS in combination with ssIDMS has been reported.236 The method used isotopically enriched 57Fe–Tf for quantitation. The stability of the metal–protein complex as well as its stoichiometry was investigated by spectrophotometry and ICP-MS, demonstrating a suitable stability over a period of one month and a molar ratio Fe:Tf of 1.94 ± 0.09, which is close to the expected value of 2. The results for the analysis of a serum CRM (ERM-DA470k/IFCC) were in good agreement with the certified value with RSDs in the range 0.9–2.7%, depending on the data treatment procedure used.
Another important metalloprotein, superoxide dismutase, a biomarker associated with oxidative cellular damage has been investigated using HPLC-ICP-MS.237 In an extension of previously published work in this area different strategies for the determination of Cu, Zn-superoxide dismutase (Cu, Zn-SOD), were evaluated. The first analytical strategy was based on the use of bovine Cu, Zn-SOD as internal standard for the quantitative analysis of human Cu, Zn-SOD. The chromatographic separation, by AEC, between both species was optimised according to their respective isoelectric point. The results revealed a faster specific degradation of the bovine standard with respect to human SOD during sample preparation. The second strategy involved the production and evaluation of an isotopically enriched metalloprotein standard to be used as tracer in ssIDMS, by measuring the Cu associated with the protein. This was achieved using HPLC-ICP-MS and applied to the quantification of SOD in bovine erythrocytes.
6.2 Tagging with metals, metal-nanoparticles and heteroatoms
Recent developments in the use of analytical methods based on elemental tagging are starting to show how this approach in combination with atomic spectroscopy detection could go some way towards the development of methods of use in real world clinical settings. The measurement of Immunoglobulin G (IgG) antibodies by immunonanogold and AAS has been reported.238 Nanogold of 10 nm size was used to label goat anti-human IgG (GIgG) to obtain an immunonanogold probe (AuGIgG) for IgG. In pH 6.8 phosphate buffer solution and the immunoprecipitator polyethylene glycol 6000 (PEG 6000), IgG reacted with the probe (AuGIgG) to form a AuGIgG-IgG-PEG immunocomplex. After centrifugation to remove the immunocomplex, AuGIgG in the supernatant was measured by AAS at 242.8 nm. The results showed that the absorption value decreased as the concentration of IgG increased, and the decreased absorption value was linearly correlated with IgG concentration in the range 0.025–0.375 μg mL−1, with a detection limit of 8 μg L−1. The assay was applied to determine IgG in human serum sample. Whilst this assay will not be replacing the current immunological methods used, there is potential for future research activity in this field. The quantitative immunoanalysis measurement of a carcinoembryonic antigen by ICP-MS90 with Hg labels also illustrates this point. A sensitive and selective method based on Hg-labelled magnetic immunoassay and ICP-MS detection was used for the quantification of carcinoembryonic antigen (CEA) in real human serum samples. Size exclusion chromatography (SEC) combined with ICP-MS was applied to the separation and detection of secondary antibody labelled with Hg and excess Hg. After complete sandwich-type immunoreactions among primary antibody, CEA and secondary antibody labelled with Hg on magnetic nanoparticles (MNPs), the concentration of CEA was determined by ICP-MS analysis of Hg released from secondary antibody with an acid-dissolution pH 1.0 step. With consumption of only 50 μL sample solution, the established method presented a limit of detection of 0.041 ng mL−1 for CEA, with a RSD of 5.2%. The response for CEA was linear over a dynamic range from 0.1 to 50 ng mL−1. The analytical results of CEA in three different human serum samples were in good agreement with that obtained by the commonly used clinical method of chemiluminescent immunoassay.The tagging methods used for the quantitation of proteins and peptides is a growing area of research activity. Protein quantitation239 using HPLC-ICP-MS with lanthanide-containing tags as metal-coded affinity reagents (MeCAT) was used to separate and quantify labelled peptides, while HPLC-ES-MS was used for identification. Synthetic-labelled peptides were used as standards to calibrate the response of the detector with compounds as close as possible to digested lysozyme and bovine serum albumin, the target species. Another method based on lanthanide labelling and ICP-MS detection240 for relative quantification of protein mixtures used the bifunctional reagent DTPAA as the labelling reagent. Two samples containing RNase A, cytochrome c, and lysozyme in different mixture ratios were labelled with the lanthanides, Ce and Sm. After separation with cation exchange chromatography, the proteins were quantified by comparison between signal intensities of Ce and Sm.
The measurement of oxidative DNA damage by gel-electrophoresis coupled to ICP-MS108 used detection of 31P as the heteroatom-tag to detect the different oxidation products. Oxidative stress was induced into plasmid DNA pBlueScript SK (2961 bp) samples by addition of Fe2+ and H2O2 through the Fenton reaction. After optimisation of the set-up for large DNA fragments, the GE separation of the different oxidation products was followed by 31P monitoring with ICP-MS and compared to conventional planar slab gels. Whilst there are a number of highly accurate and precise LC-MS/MS methods available for this application, this approach did allow for the quantification of each resulting oxidation product using inorganic phosphate as internal standard.
6.3 Metallomics and quantitative proteomics
The large datasets now available from sequencing the human genome have the potential to fuel many important research areas in the medium to long-term. Surprisingly, this has had little impact on the area of metallomics so far, but this looks soon to change, with research starting to emerge in the area of bioinformatics specifically targeting the search for new metalloproteins. Metalloproteins comprise a diverse and sizable category within the proteomes of organisms, ranging from proteins that use metals to catalyse reactions to proteins in which metals play key structural roles. Reliably predicting that a protein will contain a specific metal from its amino acid sequence is not currently possible. However, metal-directed protein purification using non-denaturing native chromatography, ICP-MS and MS/MS has been used to investigate fractions derived from the microbe Pyrococcus furiosus. A computational and statistical methodology was developed241 to aid in the identification of novel metalloproteins in order to conclusively demonstrate the extent and diversity of the uncharacterized portion of the microbial metalloproteome. The methodology enabled metal-protein interactions to be predicted, using a dataset derived from a chromatography fractionation experiment in which 870 proteins and 10 metals were measured over 2589 fractions. For each of the 10 metals, Co, Fe, Mn, Mo, Ni, Pb, U, V, W and Zn, clusters of proteins were defined in the fractionation space. The authors predicted that there are from between 5 undiscovered V- to 13 undiscovered Co-containing proteins in Pyrococcus furiosus. Molybdenum and nickel were chosen for additional assessment, producing lists of genes predicted to encode metalloproteins or metalloprotein subunits; 22 for Ni including 7 from known Ni-proteins, and 20 for Mo including two from known Mo-proteins. Another novel approach based on the use of genomics and bioinformatics to investigate metalloproteins used XRF to identify the metal centre.242 High-throughput XAS was used to measure transition metal content based on quantitative detection of XRF signals for 3879 purified proteins. Approximately 9% of the proteins analysed showed the presence of Co, Cu, Fe, Mn, Ni or Zn in stoichiometric amounts. A sequence-based de novo prediction method, MetalDetector, was used to identify Cys and His residues that bind transition metals. The XAS identifies metal type and protein binding, while the bioinformatics analysis identifies metal-binding residues, the results were combined to identify putative metal-binding sites in the proteins and their associated families. XAFS data from two of the purified Zn metalloproteins was used to validate predicted metalloprotein binding site structures. This combination of experimental and bioinformatics approaches provides comprehensive active site analysis on the genome scale for metalloproteins as a class, revealing new insights into metalloprotein structure and function.Caruso's group has embarked upon the discovery of metalloproteins in the bacteriophage lambda243 with the aim of determining their role in the phage viral coat. After large-scale preparation and purification of lambda phage according to standard protocols, state-of-the-art metallomics techniques using different combinations of chromatography and mass spectrometry were utilized for screening metal-associated molecules. The lambda phage sample was first separated using non-denaturing SEC-ICP-MS for screening associated metals and generating size distribution fractions for the various metal species, some of which include metalloproteins. Various molecular size distribution patterns were exhibited for the metals detected, Co, Cu, Fe, Mn, Ni and Zn, at different molecular weight ranges. Further identification for putative metallopeptides and metalloproteins was accomplished by collecting various metal species' fractions offline and subsequently analysing tryptically digested fractions via nanoLC-Chip-ES-MS. By searching appropriate MS databases with both Spectrum Mill and MASCOT search engines, the main capsid protein, gpE, a capsid decoration protein, gpD, and main tail component protein, gpV, were found and are known for associations with the detected metals.
6.4 Nanoparticles
The rapid increase in the use of nanoparticles in consumer and industrial products has necessitated the development of methods for the speciation of metallonanoparticles in different matrices. The speciation analysis of silver nanoparticles and ions in antibacterial products and environmental water via cloud point extraction has been reported.244 Using Triton X-114 an efficient separation was achieved for AgNPs and Ag+, which were then quantified by ICPMS after microwave digestion. The concentration of total Ag+, consisted of the adsorbed AgNP, the matrix associated AgNP, and the freely dissolved Ag, was obtained by subtracting the AgNP content from the total Ag content that was determined after digestion. The LOQs for the antibacterial products were 0.4 μg kg−1 and 0.2 μg kg−1 for AgNPs and total silver, respectively. The spike recovery of the AgNPs were in the range 70–103%, while the extraction efficiencies were in the range 1.2–10%. The possible co-extracted Ag containing NPs were distinguished by TEM, ED-SEM and UV/VIS. Real sample analysis indicated that even though the manufacturers claimed nanosilver products, AgNPs were detected only in three of the six tested antibacterial products. The interaction and stability of AgNPs in the aqueous phase245 and the influence of natural organic matter (NOM) and ionic strength on their chemical form has been investigated using asymmetrical flow field-flow fractionation (FFF) coupled with UV/VIS detection and ICP-MS. The AgNPs particles were synthesized by chemical reduction of AgNO3 with NaBH4 in the liquid phase at different NOM concentrations. The application of FFF coupled with highly sensitive detectors was a powerful approach for following the aggregation of AgNP suspensions under different physicochemical conditions and to obtain meaningful information on their chemical composition and particle size distributions. The characterisation and toxicological evaluation of AgNPs246 using ICP-MS and complementary ES-MS techniques has been developed. The HPLC-ICP-MS approach was optimised for monitoring the formation of citrate-stabilised AgNPs and the GE-ICP-MS methodology for the determination of Au/S-ratios in AgNP covered by mercaptosuccinic acid (MSA).7. Abbreviations used in this update
| 8-OHdG | 8-Hydroxydeoxyquanine |
| AAS | Atomic absorption spectrometry |
| AB | Arsenobetaine |
| AC | Arsenocholine |
| AEC | Anion exchange chromatography |
| AED | Atomic emission detection |
| AFS | Atomic fluorescence spectrometry |
| AMU | Atomic mass unit |
| APDC | Ammonium pyrrolidinedithiocarbamate |
| ASU | Atomic Spectrometry Update |
| AU | Auricle |
| BCR | Bureau Communautaire de Référence |
| BH | Borohydride |
| CCQM | Comité consultatif pour la quantité de matière – métrologie en chimie |
| CE | Capillary electrophoresis |
| CEA | Carcinoembryonic antigen |
| CPC | Cetyl pyridinium chloride |
| CRM | Certified reference material |
| CT | Cryotrapping |
| Cu, Zn-SOD | Cu, Zn-superoxide dismutase |
| CV | Cold vapour |
| CVG | Chemical vapour generation |
| CZE | Capillary zone electrophoresis |
| DACHPt | Platinum diaminocyclohexane |
| DBDE | Decabrominated diphenyl ether |
| DDAC | Dioctadecyl dimethyl ammonium chloride |
| DDC | Diethyldithiocarbamate |
| DLLME | Dispersive liquid–liquid microextraction |
| DMA | Dimethylarsenic |
| DMAE | Dimethylarsinoylethanol |
| DMAV | Dimethylarsinic acid |
| DMDSe | Dimethyldiselenide |
| DMDTAV | Dimethyldithioarsinic acid |
| DMMTAV | Dimethylmonothioarsinic acid |
| DMSe | Dimethylselenide |
| DNA | Deoxyribose nucleic acid |
| DOM | Dissolved organic matter |
| DRC | Dynamic reaction cell |
| DTPAA | Diethylenetriaminepentaacetic acid |
| DVB | Divinylbenzene |
| ED-SEM | Energy dispersive scanning electron microscopy |
| EDTA | Ethylenediaminetetraacetic acid |
| EDX | Energy dispersive X-ray analysis |
| EELS | Electron energy loss spectrometry |
| EPA | Environmental protection agency |
| EPMA | Electron probe microanalysis |
| EPR | Electron paramagnetic resonance |
| ES | Electrospray |
| ESD | Element selective detection |
| ES-MS | Electrospray mass spectrometry |
| EtHg | Ethylmercury |
| ETV | Electrothermal vaporisation |
| EU | European Union |
| EXAFS | Extended X-ray absorption fine structure |
| FAAS | Flame AAS |
| FAT | Fat residuals |
| FF | Flame furnace |
| FFF | Field flow fractionation |
| FPD | Flame photometric detector |
| FTIR | Fourier transform infrared |
| GC | Gas chromatography |
| GC-MS | Gas chromatography mass spectrometry |
| GD | Glow discharge |
| GD-MS | Glow discharge mass spectrometry |
| GE | Gel electrophoresis |
| GF | Graphite furnace |
| GLS | Gas–liquid separator |
| GPX | Glutathione peroxidase |
| GSH | Glutathione |
| GSTO1 | Glutathione S-transferase omega 1 |
| GSTO2 | Glutathione S-transferase omega 2 |
| HG | Hydride generation |
| HILIC | Hydrophilic interaction liquid chromatography |
| HPLC | High performance liquid chromatography |
| i | Inorganic |
| IC | Ion chromatography |
| ICP | Inductively coupled plasma |
| ICP-MS | Inductively coupled plasma mass spectrometry |
| ID | Isotope dilution |
| IDA | Isotope dilution analysis |
| IDMS | Isotope dilution mass spectrometry |
| IgG | Immunoglobulin G |
| IRMM | Institute for reference materials and measurements |
| IUPAC | International union for pure and applied chemistry |
| LA | Laser ablation |
| LC | Liquid chromatography |
| LLE | Liquid liquid extraction |
| LOD | Limit of detection |
| m/z | Mass to charge ratio |
| MAE | Microwave assisted extraction |
| MALDI | Matrix-assisted laser desorption ionization |
| MAM | Mammary artery |
| MC | Multi-collector |
| MeCAT | Metal-coded affinity reagents |
| MeHg | Methyl mercury |
| MeSeCys | Methylselenocysteine |
| MIBK | Methyl isobutyl ketone |
| MIP | Microwave induced plasma |
| MMAIII | Monomethylarsinous acid |
| MMAV | Monomethylarsinic acid |
| MMDTAV | Monomethyldithioarsonic acid |
| MNP | Magnetic nanoparticle |
| MS | Mass spectrometry |
| MSA | Methaneseleninic acid |
| MSA | Mercaptosuccinic acid |
| MSFD | Marine strategy framework directive |
| NIES | National institute for environmental studies |
| NIST | National institute of standards and technology |
| NMI | National measurement institute |
| NMR | Nuclear magnetic resonance |
| NOM | Natural organic matter |
| NP | Nano particle |
| NRCC | National research council canada |
| NRCCRM | National research centre for certified reference materials |
| OES | Optical emission spectrometry |
| OTC | Organotin compounds |
| PAGE | Polyacrylamide gel electrophoresis |
| PC | Phytochelatin |
| PDMS | Polydimethylsiloxane |
| PEG 6000 | Polyethylene glycol 6000 |
| PFPD | Pulsed flame photometric detector |
| PIXE | Particle induced X-ray emission |
| PMI | Primary methylation index |
| PTDI | Provisional tolerable daily intake |
| PTFE | Polytetrafluoroethylene |
| PVDF | Polyvinylidene fluoride |
| Q | Quadrupole |
| RMM | Relative molecular mass |
| RP | Reversed phase |
| RSD | Relative standard deviation |
| RTIL | Room temperature ionic liquids |
| S/N | Signal-to-noise ratio |
| SAP | Saphenous vein |
| SDS | Sodium dodecyl sulfate |
| SeAlb | Selenoalbumin |
| SEC | Size exclusion chromatography |
| SeCys | Selenocysteine |
| SeCys2 | Selenocystine |
| SelP | Selenoprotein P |
| SEM | Scanning electron microscopy |
| SeMeSeCys | Selenomethylselenocysteine |
| SeMet | Selenomethionine |
| SF | Sector field |
| SIMS | Secondary ion mass spectrometry |
| SMI | Secondary methylation index |
| SPE | Solid phase extraction |
| SPME | Solid phase micro-extraction |
| SR | Synchrotron radiation |
| SRM | Standard reference material |
| SR-XAS | Synchrotron radiation X-ray absorption spectroscopy |
| SR-XRF | Synchrotron radiation X-ray fluorescence |
| ssIDMS | Species specific isotope dilution MS |
| suIDMS | Species unspecific isotope dilution MS |
| t | Total |
| TEM | Transmission electron microscopy |
| Tf | Transferrin |
| TIMS | Thermal ionisation mass spectrometry |
| TMA | Tetramethylarsonium ion |
| TMAH | Tetramethylammonium hydroxide |
| TMAO | Trimethylarsine oxide |
| TMSbV | Tetramethyl antimony |
| TOF | Time-of-flight |
| TXRF | Total reflection X-ray fluorescence |
| UAE | Ultrasound assisted extraction |
| UC | Urothelial carcinoma |
| UPLC | Ultra performance liquid chromatography |
| UV | Ultraviolet |
| UV/VIS | Ultraviolet-visible spectrophotometry |
| VG | Vapour generation |
| XANES | X-ray absorption near edge spectroscopy |
| XAS | X-ray absorption spectroscopy |
| XRD | X-ray diffraction |
| XRF | X-ray fluorescence |
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