Atomic spectrometry update. Elemental speciation
Chris F.
Harrington
*a,
Robert
Clough
b,
Lindsay R.
Drennan-Harris
c,
Steve J.
Hill
d and
Julian F.
Tyson
c
aSupra-regional Assay Service, Trace Element Laboratory, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, UK GU2 7XH. E-mail: chris.harrington1@nhs.net
bScience Research Innovation Centre, University of Plymouth, Plymouth, UK
cDepartment of Chemistry, University of Massachusetts Amherst, 710 North Pleasant Street, Amherst, MA 01003, USA
dSpeciation and Environmental Analysis Research Group, University of Plymouth, Plymouth, UK
First published on 13th July 2011
Abstract
This is the third Atomic Spectrometry Update (ASU) to focus specifically on developments in elemental speciation. The International Union for Pure and Applied Chemistry (IUPAC) have 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”. Within this review period an IUPAC Technical Report describing the guidelines for the terminology and critical evaluation of analytical chemistry approaches related to the area of metallomics, has been published. The working group define the metallome as the “entirety of metal- and metalloid species present in a biological system, defined as to their identity and/or quantity”. Metallomics itself is defined as the “study of the metallome, interactions and functional connections of metal ions and other metal species with gene, proteins, metabolites and other biomolecules in biological systems”. This review will therefore deal 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 will not specifically deal with operationally defined speciation, but will highlight other reviews which cover the work in this area. As with all ASU reviews, 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, we will incorporate material that is not strictly “atomic spectrometry”. For the most part, such procedures are those in which some form of molecular MS is the measurement technique. There is a growing role for this kind of MS either as the sole instrumental technique or in parallel with an elemental detector. As the contents of this Update show, there is considerable activity in the development and application of methods of elemental speciation analysis, which for some elements and combinations of techniques is a mature field as shown by the extent to which relevant topics have been the subject of review articles.
1 Topical reviews
As part of the celebrations of 25 years of activity by the Journal of Analytical Atomic Spectrometry, ASU topic group co-ordinators produced a 25-year retrospective in a review of reviews,1 which included a section on speciation. Significant developments in instrumental techniques and applications have been reviewed in the most recent of the Atomic Spectrometry Updates,2–6 including aspects of speciation analysis. Although no books devoted to elemental speciation analysis have appeared during the current review period, a large number of review articles have. The topics, as will be discussed below, range from an entire sub-discipline of speciation to the determination of the forms of one element in one type of matrix.The topic of chemical measurement in metallomics (the study of the molecular mechanisms of metal-associated life processes) has been surveyed from slightly different perspectives in three review articles. A broad review7 discusses the relevant analytical methods and instrumental techniques, particularly those methods that involve a combination of a high-resolution separation technique with a sensitive detection technique. A more focused review deals with the interaction of metals with proteins8 and covers much the same territory in terms of analytical methodology. The third review9 concentrates on the role of ICP-MS, covering detection for GE as well as for HPLC and methodology in which metal labels are attached to biomolecules. To help harmonize terminology, Murray10 has compiled a glossary of terms for separations (GC, HPLC and SFC) coupled with organic MS.
A number of metallomics reviews have concerned specific analytes and sample matrices. The “toxicometallomics” of the “exotic metalloids” Sb and Te in the blood and urine of rats and humans has been reviewed.11 It appears as though the major metabolic pathway for Sb is oxidation; although methylation is also a minor human metabolic pathway. In contrast to the metabolism of Se, which is excreted mainly as selenosugars, Te is excreted in urine as trimethyltelluronium (TMTe); whereas dimethylated Te is found in rat red blood cells. Metal speciation analysis of CSF has been reviewed12 with particular reference to Alzheimer's and Parkinson's patients, for whom recent studies indicate that metals are involved in the particular neurodegenerative process of disturbed homeostasis in the brain. Essential metals such as, Cu, Fe, Mn, and Zn have been studied to a far greater extent than the well-known neurotoxic metals such Hg and Pb. The key issues for understanding metal-induced neurotoxic effects are the transport processes across the neural barriers, the metal species, and their interactions with neuronal structures. Speciation information for Al, As, Ca, Cd, Cu, Fe, Mg, Mn, Hg, Pb, Se, and Zn in CSF is summarized together with their total concentrations.
Three reviews have dealt with protein quantification by ICP-MS.13–15 Each of these reviews discuss the complementary nature of ICP-MS and molecular MS and the power of labeling with isotopes of elements detectable by ICP-MS. Two reviews15,16 cover the chemical derivatization of protein functional groups with tags containing an element that can be determined by ICP-MS, such as Fe, Hg, I, and La. The particular case of metallothioneins, proteins that are central to the homeostatis of metals, but which are difficult to detect and quantify because of their unique primary structure and relatively low molecular weight has been reviewed.17
Speciation by non-chromatographic methods have been reviewed.18 Methods that featured electrochemical and molecular spectrometric detection were covered as well as those with atomic spectrometry detection. Aspects of LLE have attracted a number of reviewers. Miniaturization (so-called microextraction), especially in the “dispersive” mode brings some advantages in terms of speed19 and efficiency,20–22 molten salts (ionic liquids) have a number of attractive properties,23,24 including very low vapour pressure, adjustable viscosity and miscibility in aqueous phases that can be exploited for effective, environmentally friendly, separation schemes. Ionic liquids can also be immobilized on surfaces23 and in membranes, thereby facilitating flow-based separations. The entire range of possibilities for liquid membrane technologies in chemical analysis have also been reviewed,25 as has the methodology in which a single drop is the acceptor26 for extractions from either a liquid or a vapour. Pyrzynska has reviewed27 SPE with carbon-based nanomaterials, including carbon nanotubes, metal oxide carbon nanotubes, nanocomposites and carbon-encapsulated magnetic nanoparticles, for enrichment, separation and speciation of metal ions.
In the latest in a series of reviews of inorganic species analysis by CE28 advances in CE methodology designed to achieve higher detection sensitivity and greater preconcentration factors, both in the capillary and microchip formats, are emphasized. The review covers applications of CE with ICP-MS detection for a range of small and large molecules. As has been noted in previous reviews of CE developments, the majority of publications are concerned with quantification of different element species. Instrumentation for CE has also been reviewed.29
Several reviews of separation techniques have appeared. The particular combination of FFF and ICP-MS is now sufficiently mature to warrant a review30 of both instrumentation and applications. The capability of FFF techniques to resolve species based on size at sub-μm values opens up possibilities for the identification of the presence of nanoparticulate metal-containing species. The characterization of nanoparticles is clearly emerging as a frontier for speciation analysis, even though nanoparticulate forms of an element may not fall within the IUPAC definition of an individual chemical species. The importance of ICP-MS is underlined by two further reviews: one concerned with GC with ICP-MS detection,31 and one with GC and LC.32 In the first, the power of SPME is emphasized, together with the determination of metals, and in the second, applications to environmental samples are stressed. Both reviews divide the world of elements into two categories: traditional or classic (As, Hg, and Sn) and nontraditional (such as Br, I, P, and S). The success of IDA, and in particular ssIDA, is highlighted. Halko et al.33 focused on the interface between HPLC separation and atomic spectrometry detection. The list of methods covered included vapour generation with applications to the determination of As, Hg, Se, and Sb. Two reviews have dealt exclusively with the speciation analysis of these elements by the HPLC-AFS combination.34,35 The issues of post-column conversion of analytes to suitable precursors for formation of a vapour are discussed.
A large number of reviews are devoted to the determination of the species of one element in a particular matrix. These are listed here in alphabetical order of the element, starting with Al in environmental samples.36 The two As reviews37,38 are somewhat related: the first deals with the biological effects and metabolism of seafood compounds, and the second concentrates on the subset of arsenosugars, which are well known to be present in a variety of seafoods. Methods for the extraction of As species from marine algae have been summarized and discussed.39 Methods for the preservation of As species in water have been surveyed.40 This topic is discussed in more detail in Section 2.1 below. Regulations that govern the concentrations of CrVI in the environment and protect workers and end-users of Cr-containing products drive the continuous development of methodology capable of determining CrVI in a variety of solid materials (such as soil, sludge, sediment, waste materials, cement, packaging materials, and corrosion-protection layers) as well as in workplace atmospheres. These developments have been reviewed41 with particular attention to interconversions between CrIII and CrVI and their correction by ssIDA. Mercury compounds are widely distributed in the hydrosphere, and the many studies associated with gaining a fuller understanding of the biogeochemistry of this global pollutant produces continuous developments in analytical measurement technology.42 Iodine, on the other hand, is a relative newcomer to the list of elements for which information about speciation in waters is needed.43 Interest in the elucidation of the mechanism of metallodrugs has produced two reviews44,45 of the analytical methodology associated with the study of Pt-containing drugs. Antimony may be emerging as the next group 5A pollutant and the growing literature on Sb in soils has been examined46 with particular reference to a comparison with As chemistry. The importance of Se biochemistry has produced two reviews of the determination of relevant compounds by chromatography with ESD.47,48 Finally, the chemical methodology that underpins the on-going monitoring of the environment for OTCs, particularly TBT and TPhT, has been reviewed49 with particular attention to extraction, derivatization and cleanup
2 Sample preparation
2.1 Analyte stability
It has been pointed out50 that the literature concerned with the preservation of arsenic species in environmental water samples is confusing and contradictory by reviewers who consider that microbially mediated reactions and those with the FeII/FeIII redox system are not properly controlled. The reviewers conclude that filtration (as soon as possible), refrigeration and storage in the dark are prerequisites for the stabilization of AsIII and AsV, as these processes remove suspended matter and microbes, suppress biotic and abiotic reactions, and prevent photochemical reactions, respectively. They also provide guidelines for the addition of preservatives for samples other than seawater and sulfidic waters, for which there appears to be no satisfactory procedures at present. They suggest adding various combinations of EDTA, acetic, and/or hydrochloric acids, depending on the pH of the original sample. Dahl et al.51 investigated the stability of arsenic compounds in seafood samples during processing in frozen storage. They found that total As concentrations in fresh Atlantic cod and Atlantic salmon did not change on frozen storage; however, the concentration in blue mussel (the only food to contain iAs), decreased significantly. Blue mussel was also the only food to exhibit a decrease in AB content after storing frozen. Initial low concentrations of tetramethylarsonium (TeMAs+) ion, in both frozen and fresh samples, increased significantly on frying.The photochemical and chemical stabilities of trimethylselenonium (TMSe+) have been studied52 as part of a larger project concerning the synthesis, identification and chemical properties of high-purity trimethylselenonium iodide. After 2.5 h or irradiation at 300 nm, no decomposition was observed. On the other hand, the compound was not completely resistant to oxidative degradation under varying microwave digestion conditions (three different reagent mixtures and two different vessel sizes). Only SeIVand SeVI were formed, and no Se was lost. In 100 mL vessels, the extent of decomposition, even with the same reagents, varied by large amounts: for example, a mixture of concentrated HNO3 and 30% H2O2 produced anything from 13 to 52% conversion to SeIV, and, at the same time, between 5 and 10% conversion to SeVI. However in 6 mL, vessels the conversion to SeIV ranged from 63–92%, and the conversion to SeVI was between 2 and 10%. The researchers attribute these results to heterogeneity in the microwave field. They also investigated the effect of adding bromate/bromide to the HNO3 digestion, but did not find any significant improvement in the conversion to SeIV, as has been previously reported.
In a study of the stability of the Se species in plant extracts, the effects of the most common phenolic substances in plants, tannin and rutin (a flavonoid), on SeMet, SeCys2, SeMeSeCys, SeVI and SeIV during sample preparation (24 h incubation at 37 °C) and storage (4 d at 4 °C) were investigated.53 For buckwheat seeds, containing a ratio of rutin to tannin of 1![[thin space (1/6-em)]](https://www.rsc.org/images/entities/char_2009.gif)
:100 (w/w), the response for SeIV decreased during the extraction: only 40% remained after extraction with water, and 80% remained after protease extraction; however, no other Se compounds were detected by HPLC-ICP-MS. The extracts were unstable with respect to the SeIV content during refrigeration, whereas other Se species were stable. The results for other buckwheat parts (leaves, stems and sprouts) indicated no instablities. It was concluded that reactions in the extraction and storage processes may result in misidentifications and inaccurate values in Se speciation studies. One possible loss mechanism is the formation of volatile derivatives, for which a trapping method that preserves the speciation of methylated compounds has been developed.54 The researchers investigated both concentrated HNO3 and H2O2 as trapping agents and compared the results by analysis by HPLC-ICP-MS and HPLC-HG-AFS: nitric acid was the better trapping agent. Dimethylselenoxide and methylseleninic acid were identified, by HPLC-ESI-MS, as the trapped products of DMSe and DMDSe, with recoveries of 65 ± 2% and 81 ± 4%, respectively.
2.2 Extraction methods
A number of researchers have concentrated on the optimization of the extraction step. A variety of methods for the extraction of Se species from buckwheat sprouts have been evaluated.55 The extractants were water, phosphate buffer, 0.1, 0.2, 0.3 M HCl, the enzyme protease XIV on its own or in combination with cellulase, amylase or lipase. The best extractants were found to be hydrolysis with 0.3 M HCl or protease; however, only approximately one-quarter to one-half of the relevant species were accounted for, although the species in the extracts were stable for 30 d. The UAE of Br and I species from atmospheric aerosols has been optimized.56 Of all the solvents studied, pure water turned out to be the best. However, for the extraction of PBDEs and polybrominated diphenyls from a variety of polymer materials, UAE with toluene gave almost 100% efficiencies.57 Free metal ions have been separated58 from humic colloids by a tangential flow ultrafiltration device equipped with a 3 kDa filtration membrane. To selectively extract CrVI from bread, an alkaline extraction has been developed and validated.59The sample pretreatment processes for the determination of MeHg in biological materials (hair and fish) have been investigated.60 A validated method was developed in which the analyte was extracted with NaOH at 70 °C followed by separation of fat components by extraction into chloroform and hexane. Derivatization with HBr yielded MeHgBr that was extracted into toluene followed by back extraction as the L-cysteine complex into aqueous solution. For the rapid UAE extraction of MeHg from single-digit mg amounts of marine biota,61 samples were first treated with 7 M HCl followed by enzymatic hydrolysis with protease XIV in the presence of 2-mercaptoethanol. Extraction efficiencies of 90 ± 10% were obtained and the method was validated by the analysis of BCR CRM 463 (tuna fish).
Considerable effort has been devoted to the development of extractions of As species from biological and environmental samples. For the quantification of water-soluble As compounds in four freshwater fish, samples were extracted with a 1:1 (v/v) methanol : water mixture;62 under optimized conditions, efficiencies ranged from 64% for carp, to 89% for chub. Extraction of lipid-rich eel with n-hexane recovered an additional 3% of total As. The researchers concluded that the As speciation and the chemical composition of fish muscle affected the extraction efficiency. The abilities of various water : methanol mixtures with sonication, a microwave-assisted system, and ultrasonic processor to extract “As species” from salt water fish (BCR 627 Tuna) have been compared.63 Only results for AB are presented, for which the extraction efficiency ranged from 81 to 87%, with the highest efficiency obtained for 60 min ultrasound irradiation of 100 mg of sample in 20 mL of a 3:1 methanol : water mixture. Following centrifugation, the residue was re-extracted with 10 mL of the methanol-water mix. The whole procedure was repeated, the three supernatants combined, the methanol evaporated at 40 °C, and the resulting solution diluted to 100 mL and filtered (0.45 μm). The literature since 2000 on the extraction from marine algae and aquatic plants has been surveyed.39 An 8-page table summarizes the procedures described in 42 publications, from which it is clear that the solvents most often used are water and water + methanol; however, there have been a number of efforts to mobilize lipid-soluble species with chloroform + methanol. The writers conclude that there is a need to organize inter-laboratory trials on As speciation in algae. Jedynak et al.64 in a study of As speciation in white mustard (Sinapis alba) investigated the influence of sample pretreatment and extractions on the recovery and reliability of speciation analyses. They divided plants into roots, stems and leaves and extracted As species from fresh, frozen (−18 °C for a week) and dried (60 °C milled and sieved) material by two procedures: sonication in water for 60 min either with or without liquid nitrogen. Details of how the liquid nitrogen was used are not given, but as they conclude that the method involving drying followed by extraction without the liquid nitrogen was best, it doesn't really matter. About 90% of the As in the roots and stems, but only 50% of that in the leaves was extracted. Four procedures for the removal of AsIII and AsV from contaminated soils have been compared65 on the basis of preservation of the oxidation states. The extractants were (a) 10 M HCl, (b) 15% (v/v) H3PO4, (c) 10 mM phosphate + 0.5% (w/v) NaDDC, and (d) 1 M H3PO4 + 0.5 M ascorbic acid. The researchers found that MAE with the mixture of phosphoric and ascorbic acids was the best strategy. They also noted that high manganese oxide content in the soil promoted oxidation during extraction. Three research groups have developed three different extractions for As species in rice prior to determination by HPLC-ICP-MS. In the first,66 the researchers obtained 100% extraction (at least from the NIST rice flour CRM) with a 1 + 1 methanol water mixture. In the second,67 the researchers report on a comprehensive study of the effects of solvent composition, heating method, temperature, time, ratio of rice to solvent, and particle size. They conclude that heating for 2 h at 90 °C in a heating block at a rice (particle size < 150 μm) to water mass ratio of 1:10 extracts all the AsIII, AsV, and DMAV. The method was validated by the analysis of CRM NMIJ 7405a (rice flour), certified for these As-containing species. Finally, Huang et al.68 have shown that as a result of a study of the effects of slightly fewer variables, the best extraction is with 0.28 M HNO3 at 95 °C for 90 min. Samples (1.5 g), either obtained as flours or ground (no information on particle size), were mixed with 15 mL of solvent. They consider that the rice contains thiolate compounds that create a reducing environment in the extract that is counteracted by the mild oxidative power of the nitric acid, so that the relative amounts of AsIII and AsV are not changed. The method was validated by spike recoveries of the iAs species together with DMAV and MMAV.
Three research groups have come up with DLLME for inorganic speciation. Lithuanian researchers76 extracted the CrVI chelate with APDC into methanol (1 mL containing 50 μL of carbon tetrachloride) with subsequent quantification by laser-ablation of dried 7 μL droplets into a plasma source mass spectrometer. One group of Iranian workers77 extracted SbIII as the complex with N-benzoyl-N-phenylhydroxylamine into ethanol with chloroform as the disperser solvent, while another group78 extracted TeIV as the APDC complex into ethanol with carbon tetrachloride as the disperser solvent. Both methods were applied to the analysis of natural waters by ETAAS and the latter was validated by the analysis of NIST SRM 1643e (trace elements in water), which contains 1 μg L−1 Te.
As is evident from the number of review articles cited earlier, interest may be growing in the possibilities for LLE with ionic liquids. For the determination of As species AsV, was extracted79 as the ion-pair between the heteropoly molybdate and tetradecyl(trihexyl) phosphonium ion, added as the chloride ionic liquid CYPHOS®IL 101, into chloroform. Only AsV reacts, so speciation is achieved by the selective conversion of AsIII to AsV and of organic As compounds to AsV by reaction with persulfate immediately at room temperature and on boiling for 30 min, respectively. In contrast to the findings of most studies of this reaction as the basis of a spectrophotometric determination, the formation of the heteropoly complex was rapid (1 min). Because the extracted As was determined by ETAAS, other heteropoly forming species would only interfere if they influenced the extent of formation and extraction of the As complex, which did not appear to be the case as phosphate could be tolerated up to 4.5 mg L−1. For the selective retention of CrVI, Chen et al.80 coated PVC with a hydrophilic ionic liquid, N-methylimidazolium chloride, to form 1-chlorovinyl-3-methylimidazolium chloride that acted as an anion-exchanger. After elution with 0.2 M ammonium nitrate solution, the Cr was determined by ETAAS and ICP-MS. To ensure that CrIII did not interfere, it was removed by an upstream cation-exchange column.
A dual capillary column microextraction for As speciation has been developed81 in which the species are selectively retained and eluted from either a silica capillary coated with N-(2-aminoethyl)-3-aminopropyltrimethoxysilane or with 3-mercaptopropyl trimethoxysilane. At pH 9, AsV and MMAV were retained on the first column and AsIII was retained by the second, allowing DMAV to be determined in the eluent, although in the procedure developed, DMAV was determined as the difference between total As and the sum of the other species. The retained species were sequentially eluted, via column switching, by 0.01 M HNO3 (for AsV), 0.1 M HNO3 (for MMAV), and 0.2 M HNO3 + 3% (m/v) thiourea (for AsIII). Ghasemi et al.82 preconcentrated Se and Te species by hollow-fibre, liquid-phase microextraction in which SeIV and TeIV were selectively extracted as the APDC complexes into 10 μL of toluene.
Two applications of selective retention on polyaniline have been described by Chandrasekaran and coworkers. For the speciation of iAs in natural waters,89 AsV was selectively retained without any pH adjustment. After the AsIII had been washed through the column, AsV was eluted with 6% (v/v) HNO3. The determination was completed by HG-ICP-MS giving LODs of <100 ng L−1. For the determination of iHg and MeHg in waters and fish tissues,90 two procedures were developed.; at pH <3, only iHg was retained, whereas at pH 7 both MeHg and iHg species were retained. For this method, the species were eluted sequentially with 2% HCl (MeHg) and a mixture of 2% HCl and 0.02% thiourea (iHg). The first method was applied to the analysis of fish tissues that were extracted with 5 M HCl. Several tuna fish CRMs were accurately analysed. The second method was applied to a number of water samples. For the arsenic work, quantification was by HG-ICP-MS, producing LODs for both species of about 30 ng L−1.
Tsoi et al.94 developed an SPME-LC ICP-MS method for the determination of methylated and ethylated Hg in urine. The analytes were collected for 45 min on a polydimethylsiloxane-DVB fibre above the 15 mL of sample at 75 °C to which was salted out with NaCl. The fibre was then inserted in a 60 μL desorption chamber filled with mobile phase that replaced the sample loop of the LC injection valve. After 5 min desorption, the valve was switched to the inject position. The LOD was 60 ng L−1 for both species, which were detected in the urine of patients suspected of suffering from Hg poisoning.
Mercury vapour has been collected in a single drop of ionic liquid following CV generation.95 Speciation was achieved by the selective generation of Hg0 from iHg in a 10 mL sample in the presence of organoHg compounds by reaction with SnCl2. The addition of KMnO4 to the tetradecyl(trihexyl)phosphonium chloride drop more than doubled the collection efficiency as evidenced by the sensitivity of the ETAAS calibration. The 6 μL drop was directly injected into the furnace, and 10 μg of Pd was added prior to determination with a programme that included not only two drying stages at 110 and 130 °C, but also a pyrolysis step at 400 °C. The LOD was 10 ng L−1.
3 Instrumental techniques and developments
3.1 Developments in species separation
The interest in the analytical capabilities of miniaturised separation techniques such as capillary LC, nano-LC or CE that arises from the need to determine proteins and peptides in small volumes of biological fluids has been reviewed.96 Such methods have additional attractive characteristics such as the consumption of small reagent volumes. The complementary information that can be obtained from the combination of separations with ESD and from molecule-specific detection techniques, such as ESI or MALDI MS, for the fast screening of complex samples for biomolecules that contain heteroatoms, as well as for their accurate quantification represents a particularly powerful development in chemical measurement capability.Separation methods based on the size of the analyte species are applicable to biomolecular systems and to the characterization of nanoparticles. In a study of the relationships between trace metals and nanoparticles in contaminated sediment, flow FFF separation coupled with multi-angle laser light scattering and high-resolution ICP-MS showed that the toxic metals As, Cu, Pb, and Zn, were in the nano-sized fraction of the sediment and that their size distributions correlated best with those for Fe and Ti.97 Particles were also characterized by TEM. Asymmetrical flow FFF has been coupled with UV spectrophotometry and ICP-MS98 for the characterization of the extracellular polymeric substances, associated with Ca, Cd and Pb, excreted by the bacterium Sinorhizobium meliloti. The results provided information about molar-mass distributions, number-and weight-average molar masses, and polydispersity indices. In a study of the transport of metal species across neural barriers, i.e. from serum into CSF and brain, the possibilities of ultrafiltration, as an alternative to SEC, have been investigated.99 The researchers concluded that ultrafiltration has several advantages over SEC-ICP-MS in regard to sample throughput, contamination control, species stability, and availability of the undiluted low molecular mass fraction for further characterization. A combination of microdialysis and HPLC with HG-AFS detection100 has been developed for the separation of human serum albumin (HSA), free arsenicals and As-HSA complexes following the incubation of iAs and HSA. It was found that that AsIII reacted more readily than AsV, which provides a chemical basis for arsenic toxicity, and the stability constant for the AsIII-HSA species was estimated.
There is no doubt that the vast majority of methods that are developed for elemental speciation involve separation by HPLC followed by ESD. However, discerning just exactly what is novel in any given publication is challenging, as often the work described involves multiple stages, any or several of which might be modifications to previously published procedures. Researchers, are not always as clear as they might be about what features of the work are indeed new and it is not uncommon, for example, to find that the reasons for selecting a particular stationary phase are not clearly articulated. As more articles appear, not all of which are describing applications to new analytes, it may be concluded that there are still problems with HPLC separations for analytes with which the community is already familiar. Some of these problems are inherent in the separation mode itself. It is slow and analyte concentrations emerging from the column are diluted considerably from those that are injected. Others are a function of the chemical properties of the target analytes that may make it difficult to devise a combination of stationary phase and mobile phase(s) that are capable of achieving the desired separation. And yet another set of challenges arises from interfacing the separation with the detector of choice. It would seem that if costs are not an issue, ICP-MS is the best detector; however, limitations on the chemical composition of the mobile phase that can be tolerated may set the boundary conditions for the chromatographic separation. Given all these issues, it is disappointing that few researchers report quantifiable parameters, such as resolution and plate numbers, that would allow the community to decide whether a separation did indeed represent an improvement. Many researchers also do not report the extent to which analytes are retained (i.e. lost) on the column, and it is often not clear whether the detector is manifesting compound-dependent responses, as relevant data, such as slopes of calibration functions are not reported.
Three papers report on the simultaneous speciation of more than one element. For a study of the interaction between Hg and Se101 a procedure was devised for the determination of SeMeSeCys, SeIV, SeVI, L-SeMet, D-SeMet, MeHg and iHg by ICP-MS following separation on two RP columns, one of which was chiral. Following the elution of SeMeSeCys and SeIV, the eluent (0.075% v/v tetraethylammonium chloride, pH 4.5) was switched to load the SeMet species onto the chiral column, after about 2 min the eluent was switched back so that the eluting SeVI could be determined, the eluent was switched back so that the D- and L-SeMet species were eluted. Finally the eluent was switched back and the mobile phase changed to 0.1% v/v 2-mercaptoethanol, 0.06 M ammonium acetate, 5% v/v methanol, so that the Hg species were eluted. The total elution time was 27 min, but no information on re-equilibration time was given. The chromatogram of the organoSe compounds shows evidence of compound-dependent responses, but this was not discussed. Problems with serum samples were encountered: SeCys2 and selenocystamine did not elute from the column when spiked into serum samples, and SeIV and SeVI eluted in the dead volume. This latter problem was overcome by 10-fold dilution. An anion-exchange separation of the oxo-anions of As, Cr, Mo, Sb, and V102 has been developed with isocratic elution with an alkaline mobile phase of 20 mM ammonium nitrate, 50 mM ammonium tartrate at pH 9.5 that also contained 1% methanol. The analysis time was just under 6 min. Tsoi and Leung103 separated AsIII, AsV, MMAV, CrIII, CrVI, SeIV and SeVI, by AEC with gradient (no details given in the paper) elution with pH 6 mobile phases containing (a) 10 mM ammonium nitrate and 0.5 mM ammonium dihydrogenphosphate, and (b) 30 mM ammonium nitrate. The Cr species were stabilized by the addition of EDTA, which also produced an anionic species with CrIII. Interference from ArCl+ (on the AsV peak) was removed by reaction with methane in a DRC. The chromatographic run time was just under 10 min.
For the determination of As species in urine, a problem with the permanent retention of AsIII on the RP column when proteins were present in the sample by the addition of mercuric chloride solution.104 The researchers also reported that their results for the determination of AB in a new CRM (SRM 2669 arsenic species in frozen human urine) were not in agreement with the certificate value for the level 1 material (12.4 μg L−1) and they suggest that there “could be additional sources of uncertainty associated with AB measurement in SRM 2669 that are unaccounted for at this time.” A possible coelution of TMAO was discounted. Chen et al.105 investigated three modes of separation: ion-pairing, cation-exchange, and AEC. They found that the ion-pair mode was unable to differentiate AsIII from AB, which could be isolated and quantified by cation-exchange chromatography. However, all of the species were separated by AEC. Even so, they found a potential inaccurate quantification problem with urine samples containing high concentrations of AB, which eluted immediately after AsIII in their anion-exchange or ion-pairing separations. They solved this problem by incorporating a post-column HG system; AB is not hydride active, so any remaining peak must be due to AsIII.
There are multiple roles for the mobile phase in RP ion-pair chromatography. A method in which the zwitterionic bile acid derivative, 3-{(3-cholamidopropyl)-dimethylammonio}-1-propanesulfonate dynamically coated the C18 column has been developed,106 for the simultaneous quantification of both Al-protein compounds (e.g. Al-transferrin) and small-molecule Al-complex compounds (e.g. Al-Cit) in healthy human serum allowing separation in 4 min. Nygren and Bjorn107 evaluated four organic solvents: dimethylformamide, 1,4-dioxane, n-propanol and ethanol, as alternatives to acetonitrile as organic modifiers for HPLC separations that will be followed by both ICP-MS and ESI-MS detection. Gradients up to 60% modifer were run at 0.15 mL min−1 and the eluent was mixed with water flowing at 0.1 mL min−1 before introduction to the nebulizer. For acetonitrile as organic modifier, 10 mL min−1 of oxygen was added to the aerosol transport gas. The model analytes included cisplatin, cytochrome C, leucine–enkephaline and bacitracine. Oxygen was also added to the DRC so that S could be monitored as 32S16O+, allowing detection of those analytes not containing a metal. They concluded that n-propanol gave the best overall performance, especially if the column was heated to 40 °C, whereupon plate numbers and resolution increased, retention times decreased and requilibration time decreased from 30 to 10 min.
The possible benefits of separations on microbore columns have been studied by several research groups. For the determination of arsenosugars in algal extracts108 by AEC-ICP-MS, the column was 100 mm × 2.10 mm ID and it was packed with 5 μm particles. The eluent flow rate was varied from 60 to 200 μL min−1 and the results clearly showed the trade-off between analysis time and chromatographic performance. With the fastest flow rate, adequate separation in 5 min was achieved. The LOD based on peak height was 0.2 μg L−1. The researchers concluded that there was less than 10% variation in sensitivity between different species, but it is not clear what the basis for this conclusion was; As-sugars are not on the list of standards. The results of the analysis of a “reference algal extract” are presented, but it is not clear why this material is described as a “reference”, other than indicating that results from another laboratory were reported in 2000. A method based on separation on narrow-bore columns has been developed109 and applied to the determination of iAs, MMAV, DMAV, and AB in ground water. Both anion-exchange and mixed mode columns were evaluated. The flow rate was 0.3 mL min−1 and the eluent was ammonium nitrate adjusted to the desired pH (results for several pH values are presented, and those at pH 8.6 appeared suitable). It was pointed out that a unique feature of this eluent is that it can adopt any separation pH without compromising eluent strength. Chloride and methanol were found not to affect the chromatography (the Cl peak was resolved from that of any As-containing compound). Partial retention of AsV on deposits on the guard column was noted. The As could be released by the injection of phosphate but had been reduced to AsIII. This problem was overcome by the addition of phosphate to the injected samples so that the phosphate concentration was at least 2 μM.
There is interest in the development and application of multidimensional chromatography featuring SEC. A two-dimensional general method for screening the interaction of metallodrugs with proteins in biological samples has been developed in which the intact proteins were separated in the first dimension by a size-selective separation, and in the second by IEC on monolithic discs.110 Two such discs were mounted in parallel so that while one was being eluted, the other was being loaded from the size exclusion column. Gradient elution was used in both dimensions, but re-equilibration of the columns and discs was rapid and did not significantly contribute to the overall analysis time of about 6 min. Protein in the eluent was monitored as SO+ formed in a DRC by reaction with oxygen. To demonstrate the potential of the method for preclinical studies of candidate drugs, the interactions of cisplatin in serum samples were followed. Finally SEC columns have been calibrated by monitoring of the S in the standard proteins by ICP-MS.111 The addition of oxygen to the collision/reacation cell in the spectrometer allows sufficient formation of SO+ that nM concentrations of proteins can be detected, thus allowing the SEC to be calibrated without the need for a separate experiment with UV detection. Copper-binding proteins, transcuprein, caeruloplasmin, and albumin were measured in the plasma of healthy subjects and an untreated Wilson disease patients. In the plasma of the Wilson disease patient, the amount of Cu bound to caeruloplasmin was lower than that of the healthy subjects, the exchangeable Cu was increased significantly.
A method for the separation and identification of oligonucleotides by HILIC-ICP-MS has been developed112 in which the separated analytes were detected at m/z 47, corresponding to 31P16O+, formed by reaction with oxygen in the collision/reaction cell, thereby eliminating the interferences for phosphorus normally seen at m/z 31. The mobile phase was predominantly acetonitrile, flowing at 0.1 mL min−1, and plasma conditions were adjusted accordingly (high power and added oxygen). The columns were a Luna HILIC (Phenomenex) and a TSKgel Amide-80 column (TOSOH Bioscience). The technique has also been applied to the separation of OTC with ESI-MS detection.113
Three applications of short RP-HPLC columns have been described. In the first114 DMSe and DMDSe were determined based on separation on a 50 mm × 1 mm id C18 column. The mobile phase, which contained a high concentration (40%) of methanol, was flowing at 0.1 mL min−1 and the injection volumes were 1–10 μL. Methane was added to the DRC and Se was monitored at m/z 80. In the second method115 iodide and iodate in edible salts and human urine were separated on a 50 mm × 4 mm id anion-exchange column with 8 mM ammonium carbonate solution as the mobile phase. For the maximum flow rate that could be delivered of 1.5 mL min−1, the separation time was only 170 s. Although no chromatograms were shown, calibration equations are provided showing that on the basis of peak area the response to iodide was only 59% of response to iodate. For the determination of Lewisite metabolites in human urine in response to a gas attack, a rapid method was developed116 in which analytes were separated on a “RP-amide polar embedded” 50 mm × 4.6 mm id RP column packed with 3 μm particles. The column, held at 30 °C, was eluted isocratically with a mobile phase consisting of 2% (v/v) 2-propanol, 11.6 mM TBAH, 5.0 mM succinic acid at pH 5.5. The target analytes, chlorovinylarsonous acid and the corresponding AsV compound, chlorovinylarsonic acid, were well resolved from other As species, all of which (including AsV) were eluted in under 4 min at a flow rate of 1 mL min−1. The researchers specifically looked for compound-dependent responses and found none. To speed up the separation even more, ultra-high pressure LC can be applied to the problem. For the determination of oxaliplatin in human urine117 a pump capable of working up to 600 bar delivered mobile phase at up to 0.9 mL min−1 through a column (50 mm × 2.1 mm id) containing 1.8 μm particles. A gradient elution profile was applied that lasted 14 min, even though the analyte eluted in under 2 min at the lowest flow rate (of 0.3 mL min−1) and in under 1 min at the highest flow rate.
:1 metal–ligand complexes to successive complexation reactions for the uranyl- and lanthanum-oxalate systems. They report evidence for a new La-oxalate complex: La(Ox)33−. Detection of separated As and Se compounds down to 0.5 μg L−1 in extracts of coal fly ash, dogfish liver and dietary supplements by CE-ICP-MS has been achieved.126 The potentially interfering argon dimers at the Se m/z values of 78 and 80 were decreased in intensity by approximately three orders of magnitude by reaction with methane in a DRC. Arsenic was determined as 75As12C+ at m/z 87. A method for the determination of trialkyl OTCs extracted from aquatic organisms has been developed127 that does not require derivatization or preconcentration. The complexation of metals by phytosiderophores has been investigated.128 Six free metals (Co, Cu, Fe, Mn, Ni and Zn) and their complexes with three phytosiderophores (mugineic acid, epi-mugineic acid and deoxymugineic acid) were studied for hydroponically grown spring barley. Going up again in analyte size, the interactions between the metallodrugs Zn-maltolato, Zn-2-picolinato and Zn-2,6-dipicolinato complexes (candidate insulin enhancers) with human serum proteins have been studied by CE-ICP-MS.129 The protein of preference was albumin. A method for its detection in urine based derivatization with Au nanoparticles (NPs) has been devised130 in which the excess NPs and the AuNP-albumin adduct were separated by CE with ICP-MS detection. It was estimated that each albumin molecule was tagged with 2000 gold atoms on average, giving rise to an LOD of 0.5 pM for a 280 nL injection volume. The researchers concluded that their method “has potential for simultaneous determination of low-abundance multiple biomarkers of interest via multiple nanomaterials tags.” In the only study in which ICP-MS was not used, Deng et al.131 identified and determined Ca species in human red blood cells by CE with ICP-OES detection. They found eight primary calcium-containing species in human red blood cell cytoplasm and that the free Ca2+ concentration was about 4.5 μg L−1 (112 nmol L−1). The species with the greatest mobility was not free Ca2+, but a calcium-erythrocyte membrane species of MW 50–100 kDa. What was claimed to be the first report of the coupling of micellar electrokinetic chromatography with ICP-MS detection132 contained the minimum of information about how this was done: “utilizing a CETAC CEI-100 microconcentric nebulizer”. The system was applied to the determination of Pt anticancer drugs and drug candidates. Compared with UV-vis absorption, ICP-MS was considered to have better detection capability.
The combination of GE separation with synchrotron XANES imaging of the metals in the zones was applied to the study of two metal protein binding systems.133 In one, the in vitro binding of exogenous Cr to blood serum proteins, of relevance to the biochemistry of Cr dietary supplements, was found to be influenced markedly by both the oxidation state of Cr and the treatment conditions. In the second case, in vivo changes in endogenous metal speciation were examined to probe the influence of oxygen depletion on Fe speciation in Shewanella oneidensis. The combination has also been applied to a study of the liver of Nile tilapia (Oreochromis niloticus),134 when Ca, Fe, S, P, and Zn were identified in the protein spots obtained after two-dimensional PAGE. Additional quantification was made by FAAS after microwave-assisted acid mineralization of the proteins spots.
On-line coupling of SDS-PAGE with ICP-MS has been achieved135 by casting the gel into a 7.3-cm-long tube and appropriate modification of the standard electroelution apparatus. The mono-and tetraphosphopeptides in a digest of β-casein were identified by comparison with peptide standards.
The more usual GE-ICP-MS interface is LA. Castillo and coworkers applied this interface in the characterization of metal-humic acid complexes136 by one-dimensional PAGE, isoelectric focusing (IEF), and two-dimensional PAGE, in compost samples. They also used high-performance SEC, cross-flow field flow fractionation, and ultrafiltration. They also applied PAGE-LA-ICP-MS to a study of metal losses from protein binding during separations in proteomic studies.137 The systems under investigation were superoxide dismutase, containing Cu and Zn, and alcohol dehydrogenase, containing Zn. They found that the metal-protein binding depended, not only on the form of the electrophoretic process (denaturing or non-denaturing) and post-separation gel treatment, but also on the trailing ion chosen and current applied.
3.2 Chemical vapour generation
Interest in speciation based on different HG conditions is decreasing, with only a handful of publications in this review period. A method for the AFS determination of iAs has been developed138 based on control of the efficiency of electrochemical HG, which was decreased to almost zero for AsV in the presence of zinc; whereas, that of AsIII was increased. The addition of formic acid also improved the analytical performance, with LODs below 0.1 μg L−1. For the speciation of As by HG-AAS, selective HG was achieved by control of the acidity.139 Under suitably low acid conditions only AsIII was borohydride active. Total As was determined after reduction with potassium iodide and ascorbic acid. The LOD was 1 μg L−1. A procedure for the determination of four Hg species has been devised140 for the analysis of Antarctic marine biota and validated by the analysis of CRM IRMM 813 (scallop Adamussium colbecki) and MURST-ISS-A2. The basis was the successive, selective conversion of species to iHg followed by CVG with SnII. Thus by addition of 2-mercaptoethanol to stabilize MeHg, irradiation with UV light converted PhHg to iHg. Oxidation with persulfate converted MeHg and PhHg to iHg. Finally dimethylHg was added to the list of analytes by the use of borohydride to generate the volatile derivatives. The LODs ranged from 0.001 to 0.1 μg L−13.3 Developments in instrumentation
The combination of elemental and molecular mass spectrometries is a powerful tool for the elucidation of the nature of elemental species. Further information about the performance of the instrument constructed in the Hieftje lab has appeared. This TOF instrument has dual sources: an ICP141 and an ES.142 To characterize the ES channel, hexadecyltrimethylammonium, tetrahexylammonium, tetraoctylammonium, myoglobin, insulin, cyanocobalamin, leucine enkephalin, and alcohol dehydrogenase were introduced. To characterize the ICP source141 figures of merit for the determination of As, Ag, Bi, Cr, Cs, Co, Ga, Ho, Hg, and Tl were obtained, and to demonstrate the simultaneous operation of both sources cyanocobalamin, myoglobin, and superoxide dismutase were introduced. For the metals, LODs of between 30 and 100 ng L−1 were obtained. Several research groups have connected the two mass spectrometries in parallel by splitting the eluent from the HPLC column. This does create issues with the selection of the mobile phase, which now must be compatible with two quite different ionization techniques. Casal et al.143 separated Se-containing metabolomes in Se-rich yeast by cation-exchange HPLC of fractions already separated by SEC. A total of 27 compounds were identified by an instrument containing a hybrid linear ion trap and a quadrupole orbital trap. The mobile phase was an ammonium formate gradient in 20% methanol. A number of Br-containing compounds have been identified in tap water144 following separation by IC including bromochloroacetic acid, dibromoacetic acid, and bromodichloroacetic acid, as well as bromide and bromate. As part of an investigation into the extent of As biovolatilization, the species found in atmospheric particulate matter have been characterized.145 The oxidation products, of methylated arsines i.e. methylarsonate, dimethylarsinate and trimethylarsine oxide were identified in more than 90% of the 49 PM-10 (particulate matter of diameter 10 μm or less) samples taken from 8 sampling points at two geographically different locations in Argentina. For the determination of As species in heavily polluted ground water, an HPLC separation was combined with ESI MS/MS detection and ICP-MS detection.146 High concentrations of MMAIII were found as well as AsIII, AsV, MMAV and DMAV. The monitoring of AsO+ at m/z 91 and SO+ at m/z 48 by ICP-MS operating in DRC mode ruled out any possible misidentifications arising from the presence of sulfur-containing As compounds.A dielectric barrier discharge atomizer has been developed and applied to the non-chromatographic speciation of Hg compounds by AAS.147 A low concentration of borohydride (0.01% (m/v)) produced elemental Hg from iHg, and MeHg formed volatile methylmercury hydride (CH3HgH). With the low temperature atomizer both species are determined, whereas only iHg was detected with the discharge off, as CH3HgH was not atomized and does not absorb the at the Hg atom wavelength.
Also of interest in this section are reports of elemental speciation by molecular MS. A simple and fast (<5 s) method for speciation of As compounds on solid surfaces has been developed148 based on desorption electrospray ionization-tandem MS. Animal feed and plant tissues were directly monitored and MMAV, DMAV, AB, AC, 4-arsanilic acid, 4-hydroxyphenylarsonic acid, Nitarsone, and Roxarsone were detected by their characteristic m/z patterns and collision induced dissociation (CID) behaviours. A method based on extractive ESI with MS/MS has been developed for the rapid detection of radioactive inorganic species in natural water samples.149 Negatively charged uranyl acetate undergoes characteristic fragmentation in the gas phase, allowing detection of uranyl species in complex matrixes without sample pretreatment. The LOD of a few pg L−1 is comparable to that obtainable by ICP-MS.
3.4 X-ray methods
There appears to be a growing number of reports of elemental speciation studies in which the relevant information has been provided by one or several X-ray spectrometry techniques. This increase reflects, perhaps, greater access to synchrotron sources by researchers with biochemical or environmental interests. As these techniques can provide information about the immediate chemical environments of the atoms of the elements of interest at very high spatial resolution in solid samples, they are clearly complementary to those in which a relatively large amount of analyte material is extracted from the sample followed by separation and quantification. In this section, the work selected for inclusion is arranged according to sample type rather than by element or by technique.For the characterization of metals in protein spots separated by 1- or 2-D PAGE, both XRF134 and XRF plus XANES150 have been used. The metals examined were Ca, Fe and Zn in the former study, and Cr and Fe in the latter. The techniques are also able to detect non-metals P and S, which are characteristic of the presence of protein.
Several reports of studies of the uptake and distributions of elements in plant tissues have appeared. The changes in speciation of As during uptake, translocation, and storage by the As hyperaccumulating fern Pityrogramma calomelanos var. austroamericana (Pteridaceae) were examined with ICP-AES and synchrotron-μ-XANES and μ-XRF spectrometries.151 It was discovered that over 60% of the AsV absorbed in the roots was reduced to AsIII, prior to transport through vascular tissues and that AsIII was the predominant species in pinnules. The presence of AsIII coordinated with S (as sulfide) throughout the plant was interpreted as strong evidence for the involvement of sulfide functional groups in the biological reduction of AsV. Organic arsenicals and thiol-rich compounds were not detected. In a study152 of the role of iron coating on the roots of rice plants in preventing uptake of As by rice, XRF imaging, μ-XANES, transmission X-ray microscopy, and tomography were used to determine the extent of co-location of As and Fe in roots with variable Fe coatings. The researchers concluded (a) that As was not exclusively associated with Fe plaque, (b) Fe plaque does not coat many of the young roots or the younger portion of mature roots, and (c) Fe plaque does not directly restrict As uptake by rice roots; it acts as a bulk scavenger of the As that is near the root base. Carey et al.153 investigated how As species are “unloaded” into grain rice by excising the panicles (branched cluster of flowers) and then exposed them hydroponically to AsIII, AsV, glutathione-complexed As, or DMAV. Synchrotron XRF mapping and fluorescence microtomography, together with XANES (for the As speciation in the fresh grain) revealed that that DMAV is translocated to the rice grain with over an order magnitude greater efficiency than inorganic species and is more mobile than AsIII in both the phloem and the xylem. The Cd speciation in seeds and vegetative tissues of the Cd hyperaccumulator Thlaspi praecox was studied by EXAFS.154 It was found that, in intact seeds and isolated embryos, almost two thirds of the Cd ligands were thiol groups, and that there was coordination to phosphate groups via bridging oxygen's. In roots and shoots up to 80% of the Cd was bound to oxygen. The researchers concluded that vacuolar compartmentalization and binding to the cell walls were the main detoxification mechanisms in leaf tissues. In a study of the root-to-shoot transfer, localization, and chemical speciation of Co in wheat and tomato grown hydroponically in the presence of CoII, XAS measurements were used to identify the chemical environments of Co within the plants and μ-PIXE provided information on the Co distribution in the leaves.155 It was found that although the root-to-shoot transport was higher for tomato plants both plants are excluders. Cobalt was found mainly in the vascular system of both plant species complexed with carboxylate-containing organic acids. Tan et al.156 studied the accumulation of Pb in Sedum affredii (Crassulaceae), a species native to China, with synchrotron μXRF and EXAFS spectroscopies. They found that Pb was distributed mainly within the vascular bundles of leaves and stems, and a positive correlation between the distribution patterns of S and Pb was observed. The dominant chemical form of Pb (>60%) was similar to that of prepared Pb-cell wall compounds. In a study157 of the influence of water management and Se fertilization on the uptake and speciation of Se in rice, enzymatic hydrolysis followed by HPLC-ICP-MS, together with XANES were used to characterize Se in rice grain. It was found that SeVI addition to aerobic soil was the most effective way to increase Se concentration in rice grain, which was present mainly as SeMet.
Two research groups have been studying the microdistribution and speciation of elements in human nails. In a study158 of the As in toenail clippings of children living in a historic gold mining area in Victoria, Australia, synchrotron XRF and XANES were used. Experiments were conducted to distinguish between As in the nails as a result of ingestion and As that might have diffused from surface contamination. Two distinct arsenic species were found: a lower oxidation state species, possibly with mixed sulfur and methyl coordination and a higher oxidation state species, probably AsV. Depth profiling, layer structure and correlation between nail and soil concentrations, led the researchers to conclude that As in soil contributes to some systemic absorption associated with periodic exposures. Katsikini and coworkers have studied Ca, Fe, S, and Zn159 with XRF and EXAFS. They found that Ca, S, and Zn were distributed homogeneously, whereas Fe was found in clusters, of diameter 15–30 μm, in which the concentration was 10 times higher than that in the matrix. They found that Zn, in the nails from healthy donors and from patients suffering from lung diseases, was four-fold coordinated with N and S. In another study, they just focused on the Fe.160 The clusters contained both FeIII and FeII distorted octahedral environments. They were unable to distinguish between the nails of healthy individuals and those of patients with lung cancer.
A number of research groups have studied elemental speciation in soils and sediments. In a study of a freshwater lake system in the Athabasca Basin, northern Saskatchewan, Canada that receives treated process effluent containing elevated concentrations of Se, sediment, pore water, surface water, and chironomid larvae were examined by EXAFS.161 All sediments contained approximately 50% of elemental Se, which is not bioavailable, as well as inorganic metal selenides. Dissolved SeIV concentrations in pore water were correlated with SeIV provided that the sediments were classified on the basis of sand content. For the speciation of Hg in soils,162 μ-XRF,μ-XRD and μ-XANES showed that the principal Hg-species found in the soil samples were metacinnabar (β-HgS), cinnabar (α-HgS), corderoite (Hg3S2Cl2), and an amorphous phase containing Hg bound to chlorine and sulfur. No interaction among Hg-species and soil components was observed, and it was concluded that the Hg-species originated from the slow weathering of an “inert” inorganic Hg-containing waste material that included residues from incineration, roasting and retorting (U.S. EPA waste code K106) which had been dumped several years ago. Van Damme et al.163 looked at Zn speciation in mining and smelter contaminated overbank sediments, from the Geul river in Belgium, with EXAFS spectroscopy. These sediments contain 4–69 g kg−1 Zn as a result of mining and smelting activities, principally during the 19th century. The three Zn species identified were smithsonite (ZnCO3), tetrahedrally coordinated sorbed Zn, and Zn-containing trioctahedral. The researchers pointed out that the presence of the phyllosilicate phase was “the first evidence for pervasive sequestration of Zn into this newly formed precipitate at the field scale.” In a study of the Al and S speciation in hardened cement paste, μ-XRF and μ-XANES showed that Ettringite and calcium monosulfoaluminate were the main S-containing species.164 Chemometric analysis of the spectra suggested that two Al-containing clinker phases (aluminate, ferrite) and secondary phases of the hydrate assemblage (ettringite, hydrated monosulfate phases, hydrotalcite) contributed to the set of components that made up the experimental spectra. Chrysochoou et al.165 studied the speciation of CrVI in chromite ore processing residue by means of bulk XRD, and a combination of μ-XRF,μ -XAS and μ-XRD. They found that calcium aluminum chromium oxide hydrates, accounted for 60% of the total CrVI and that hydrogarnets and hydrotalcites, two mineral groups that can host CrVI in their structure by substitution, were indeed CrVI sinks. Chromatite (CaCrO4) was also identified by μ-XRD, at concentrations that were too low for detection by bulk methods.
Two research groups have reported applications to speciation in solution. Synchrotron radiation XRF was used to characterize As speciation within natural fluid inclusions from three deposits with different hydrogeochemical and geological settings. Spectra were obtained from fluid inclusions at temperatures ranging from 25 to 200 °C, and compared with spectra of aqueous AsIII and AsV solutions and minerals. It was found from the XANES studies that the fluid inclusions from all three regions contain some As in reduced form such as AsIII, As-sulfide or possibly elemental As, that was readily oxidized by the beam to AsV. Analysis of EXAFS data showed that AsIII was indeed the dominant arsenic aqueous species in the inclusions at one of the site. Interestingly, the photooxidation problem was not encountered for species produced in autoclave experiments and it was concluded that interactions between redox-sensitive complexes in solution and the products of water radiolysis generated by the beam stabilized the lower oxidation states. A method for the determination of CrIII and CrVI in aqueous solution by the chemometric analysis of the spectra from a standard laboratory energy dispersive XRF spectrometer has been devised.166 Spectra were processed (2047 variables) as follows: (1) a data matrix was constructed, (2) spectra data were mean centred, (3) principal components analysis was implemented, and finally (4) partial least squares was applied. Commercial software, Pirouette™ version 3.11 from Infometrix, was used. The LODs for both species were 17 mg kg−1.
3.5 Separation-detection interfaces
Many laboratories interested in providing information about elemental speciation do not have the unlimited financial resources that would lead them to install and operate ICP-MS and (MS)n spectrometers. So, in the real world, laboratories are faced with the challenge of delivering cost-effective measurements that provide the information needed. In turn this drives research efforts that are devoted to interfacing LC and other separations with other atomic spectrometry detectors. Modifications to conventional HPLC mean that adaptations to the interface with the detector are needed, and several of the articles that were discussed above in section 3.1.1 contained not only descriptions of novel LC separation techniques but also of modified interface devices to accommodate the changes in normal operating conditions. A similar comment can be made about research involving the use of GC with ESD that was discussed above in section 3.1.2. The availability of low-flow nebulizers and temperature controlled spray chambers means that entirely organic chromatographic mobile phases can be directly introduced to ICP-MS instruments, though it would be necessary to add oxygen to the plasma gas. These nebulizers are typically total consumption, so that even if the flow rate is significantly lower than the usual 1 mL min−1, sensitivities are not compromised, though flows may have to be split prior to introduction.Several research groups have devised interfaces for normally predominantly organic mobile phases. In a study167 of Si in organic solutions, normal phase HPLC-ICP-MS and SEC-ICP-MS were used to gain an insight into the purity of the Si standard compounds, their reactivity with different petroleum-related matrices and speciation of silicon. Sample introduction was via micro-flow injection total consumption. It was found that matrix effects and compound-dependent sensitivities could be alleviated by heating the spray chamber and sample dilution. The researchers also applied these techniques to the determination of Ni and V species in crude oil.168 In a study107 of mobile phase selection for the combined use of ICP and ESI mass spectrometries, for introduction to the plasma spectrometer, the eluent flow at 150 μL min−1 was merged with a continuous aqueous make-up flow of 100 μL min−1 and delivered to a Mira Mist enhanced parallel-path nebulizer (Burgener Research) in a PerkinElmer PC3 introduction system consisting of a Peltier cooled cyclone spray chamber at 4 °C. To overcome the compound-dependent responses caused by variations in the carbon in the plasma due to changes in the organic solvent component in a gradient elution procedure a volatile organic solvent was introduced169 directly into the spray chamber so that the sample introduction system was saturated with organic vapours; no change in sensitivity was observed during a methanol gradient separation of As species. A nebulizer for the coupling with very low flow rates of 50–4000 nL min−1 has been described.170 The very thin polyimide-coated nebulizer capillary (id 20 μm and od 90 μm) was less prone to clogging and did not create high back pressure. Different nebulizer and single-pass spray chamber geometries were evaluated, as was the addition of a make-up gas, though this was found not to be necessary as, at best, the improvement in sensitivity was about 5%. The device was used for sheath and sheathless flow nano-HPLC-ICP-MS, including the post-column addition of internal standard.
For high-temperature HPLC with an aqueous mobile phase, the end of the connecting tubing was introduced directly into the spray chamber of the ICP-AES instrument and an aerosol was generated by the thermospray effect. Although the column was maintained at 150 °C, it was necessary to provide additional heating to raise the temperature of the tubing to 220 °C. Three designs of spray chamber were evaluated and a home-made cyclonic design selected. A separate flow of argon was introduced to flush the aerosol through the chamber to the plasma. The system was used for the separation of sugars with detection via the C emission at 193.09 nm.
To interface TLC separations with ICP-MS, a device first described by Luftmann in 2004171 for coupling TLC with ESI-MS was applied for the extraction of iodinated X-ray contrast agents that had been separated on RP TLC plates.172 The spot was isolated under a solvent delivery head by clamping the head down on the plate, and solvent flushed through at a flow rate that was selected to get adequate extraction without excessive dilution.
In an overview35 of the interfacing of HPLC with AFS, the advantages for the speciation of hydride-forming elements were pointed out, but other types of interface, such as on-line UV photo-oxidation, pyrolysis or microwave assisted digestion for non-borohydride-active compounds, were also discussed. The specific cases of VG for the determination of compounds of As, Hg, Se, and Sb have been thoroughly and critically evaluated, including the problems associated with the oxidation of organic compounds and the pre-reduction to a suitable oxidation state.34 For the determination of Hg species, the addition of L-cysteine to the mobile phase degraded the alkylHg compounds sufficiently to allow cold vapour generation without the need for any postcolumn treatment.173 The LODs for iHg, MeHg and EtHg were 0.1, 0.05, and 0.07 μg L−1, respectively. For the determination of iHg and MeHg, Liu174 added L-cysteine and formic acid to the mobile phase but then irradiated the eluent with UV light to generate the Hg vapour. The LODs were 0.1 and 0.08 μg L−1 for iHg and MeHg, respectively. For the detection175 of five As compounds, AsIII, AsV, MMAV, DMAV and Roxarsone, by RP ion-pair chromatography, post column reaction with 2% (m/V) K2S2O8 (plus UV light), 7% (v/v) HCl as carrying solution and 2% (m/V) KBH4 solution as reducing agent produced the necessary volatile derivatives. The LODs were about 10 μg L−1.
Three interfaces for GC and ICP-MS with particular reference to the determination of the ∂Br-81 values for brominated diphenyl ethers has been comprehensive evaluated.122 The issues appear to be the heating of the portion of the fused silica column that extends from the chromatography oven through the torch to the base of the plasma, the heating of the sheath/make-up argon gas and the dissipation of static electricity associated with the stainless steel tubing surrounding the silica tube. The researchers conclude that their study has highlighted the challenge of designing a transfer line between the gas chromatograph and the ICP-MS instrument that allows for efficient conduction of brominated diaromatic compounds while avoiding excessive heating of the argon gas feeding the plasma. They also reported that the variation in sensitivity of the instrument was “typically ±50% during the course of the day for unknown reasons,” which must also have been a challenge.
3.6 Detection
The application of a particle beam/hollow cathode-optical emission spectrometer for simultaneous multi-element detection for chromatographic separations has been described.176 A comprehensive optimization of relevant operating conditions was made with nitrate salts of Ag, Ni, and Pb. The researchers found that monitoring multiple elements simultaneously revealed inter-element matrix effects that had not been observed previously for hollow cathode sources. They also demonstrated the capability of system to monitor both metals and non-metals, a promising development if it is, as the researchers propose, to be used as a tool for metallomic studies.4 Metrology
Whilst two of the papers cited in this section do not cover the topic of elemental speciation they are included here as the principles are applicable to speciation studies and are a good starting point for those readers new to the field of chemical metrology. The analytical approaches to IDMS measurements made at BAM, the Federal Institute for Materials Research and Testing in Germany, have been published in a review paper.177 The paper covers single, double and triple spiking procedures for IDMS, different types of instrumentation in current use, TIMS and quadrupole and MC ICP-MS, and gives examples for total elemental determinations in different matrices. The origins and principles of IDMS, along with the derivation of IDMS equations and approaches to traceability are also discussed. A second paper on this topic, by researchers at LGC, provides a tutorial review covering metrological traceability in chemical and bioanalytical measurements.178 The paper discusses the concept of metrology, the traceability requirements for both ISO 17025 and ISO 15189, and provides a scheme for analysts to meet the requirements of these two standards. A third review paper in this area covers online IDMS for both total elemental and speciation measurements.179 The article covers the history, principles and recent applications of online IDMS. The use of ‘gas phase’ generation systems, LA, ETV and HG as well as liquid phase systems, HPLC and CE and different types of ICP-MS, e.g. SF and quadrupole, instruments were also covered. References to a wide variety of elements including: Br, Cd, Cu, Fe, Hg, Se, Sb, Pt and Zn, were covered.5 Elemental speciation analysis
5.1 Aluminium
Studies of Al speciation continue to use models to calculate equilibrium composition within aqueous systems many using computer programmes to determine the thermodynamically most stable forms by considering all of the equilibrium constants. A method for the speciation of AlF3 forms in the water soluble fraction of soil samples using HPLC-FAAS has been proposed.180 The approach was based on a chromatographic separation of Al species and then examining the data using the Mineql package. The separation of Al species with nominal charge of +1. +2 and +3 required a run time of less than 4 min during a single analysis. Similarly, HPLC-FAAS has also been used in conjunction with the Mineql program for the determination of aluminium fluoride complexes,181 also with soil samples. An ETAAS method has been developed for Al in groundwater samples.182 In addition to the use of the established PHREEQC computer program for speciation calculations, other ionic associations that Al formed with Si species, SO42−, F−, Na+ and K+ ions were also considered. The distribution of the water saturation index values calculated on this basis suggested that some mineral species of the red clay layers tend to become soluble when the groundwater is mixed with the surface water.The use of ESI-MS is very often employed to study metal/ligand equilibria in aqueous solution. However, the ionization process can introduce perturbations which affect the speciation results in an unpredictable way. These effects have been studied by Di Marco et al.183 Aqueous solutions of AlII-1,6-dimethy1-4-hydroxy-3-pyridinecarboxylate at various pH were analysed and speciation results were compared with those obtained by equilibrium techniques. The differences observed were both qualitative and quantitative. The ESI-MS spectral changes were due to different instrumental settings such as the solution flow rate, the nebulizer gas flow rate, the potential applied at the entrance capillary, and the temperature of the drying gas. The effects produced by the solution flow rate and the potential applied at the entrance capillary on the spectra, strongly suggest the key role of surface activity in determining the relative fraction of the ions reaching the detector.
A C18 column coated with a zwitterionic bile acid derivative, 3-((3-cholamidopropyl)-dimethylammonio)-1-propanesulfonate (CHAPS), was used for the speciation of Al in human serum by HPLC-UV-ICP-MS.106 Small-molecule Al-complex compounds of Al-citrate (Al-Cit) and large-molecule Al-protein compounds of Al-transferrin (Al-Tf) were chosen as the model species and their retention behaviours on CHAPS modified C18 column were studied. Under the optimal conditions, large-molecule Al-protein compounds and small-molecule Al-complex compounds could be separated in 4 min. The LODs were 0.74 and 0.83 ng mL−1 with RSDs of 2.8% and 3.0% (n = 7) for Al-Tf and Al-Cit, respectively. The method was applied to the speciation of Al in healthy human serum and chronic haemodialysis patient serum, and the researchers were able to report the simultaneous quantification of both Al-protein and small molecule Al-complex compounds in healthy human serum at low concentrations.
5.2 Antimony
The speciation of Sb in environmental matrices using coupled techniques has been reviewed by Miravet et al.184 The review also covers the application of ESI-MS to identify individual species. A review in which the environmental chemistry of Sb in soils was compared and contrasted, with that of As has also been published.46 Knowledge gaps in environmentally relevant Sb data for soils are identified and discussed in terms of Sb mobility and bioavailability.In a study of SbIII, SbV, and Sb-containing nano-particles in urban atmospheric particulate matter,185 both SbIII and SbV forms were detected with SbIII/SbV ratios up to 1.5. These two Sb species account for 10–70% of the total extractable Sb measured directly by ICP-MS. No other soluble Sb species were detected in the samples. It was suggested that the contribution of nano-particles is responsible for the difference between ICP-MS and IC-ICP-MS data, since small size solid particles are able to pass through the sample introduction system in ICP-MS, whilst they are retained by the chromatographic column. Most traffic-related Sb air pollutants are derived from brake dust which contains Sb2S3. High-temperature oxidation products such as Sb2O3 and Sb2O4 may also be produced. Investigations have been carried out by Zih-Perenyi et al.186 to find the most selective leaching conditions for these substances. Solubility experiments were carried out and then the leaching of these compounds from artificial dusts previously spiked with these compounds at the trace level was investigated. The Sb was determined using ET-AAS and HG-ET-AAS. A 0.5 mol L−1 citric acid solution was shown to leach all of the Sb2O3 while extracting less than 10% Sb2S3 and no Sb2O4 at all. It was found that Sb2O3 and Sb2S3 traces were soluble in 6M HCI. The LODs were 1.2 and 0.3 μg g−1 for leaching by citric acid and HCI solution, respectively, which proved adequate for Sb content determination in the urban dust studied. The RSD was about 7%.
A method for the speciation of iSb by cloud point extraction combined with ETAAS has been presented.187 The method is based on the formation of a hydrophobic complex of SbIII with APDC at pH 5.0. The hydrophobic complex entered into the surfactant-rich phase, whereas SbV remained in aqueous solutions. The surfactant-rich phase containing the SbIII was analysed by ETAAS. The SbV was calculated by subtracting SbIII from the total Sb after reducing SbV to SbIII with L-cysteine. The method was used to determine the iSb speciation in leachate solutions obtained from food packaging materials. Micro-XRF analysis has been used to identify the distribution and chemical form of residual Sb used as a catalyst in the manufacture of PET bottles.188 The results are consistent with clusters of SbIII having dimensions of the order of tens of micrometers, clearly showing the ability of synchrotron radiation analyses to map elemental distribution and determine oxidation state.
A new and direct method for the simultaneous determination of SbIII and SbV in meglumine antimontate, the first-choice drug for leishmaniasis treatment, has been developed.83 Speciation was carried out using a Dowex I x 4 resin column and 1.5 M HCl solution for the separation (offline) and ICP-AES detection. The interfering effects of As, Bi, Cd, Cu, Mn, Pb and Zn were examined and only Bi was found to be a significant interferent. The liberation of SbV and SbIII from organoantimonial compounds without changing of oxidation state was carried out by means of 1 5 M HCl solution The spike recovery values obtained for SbIII in pharmaceutical sample varied from 92 to 100%, although it was not validated for Sbv.
5.3 Arsenic
The speciation of As is now well established and conducted routinely in many laboratories. However, advances continue to be made in both the development of analytical protocols and in the elucidation of chemical pathways. The most novel of these studies published within the review period are reviewed here, although it should be noted that some work fails to report the oxidation state of the compounds used. To aid validation, new certified reference materials are always welcome. The National Metrology Institute of Japan has produced a new AsV solution reference material, CRM 7912-a. The certified value of AsV in the CRM is 99.53 ± 1.67 mg kg−1. Two independent HPLC-ICP-MS methods for As species in urine have been used, with quantification by standard additions, to assess the stability of the NIST frozen human urine SRM 2669.189 Seven As species were quantified: AsIII, AsV, MMA, DMA, AB, AC, and TMAO. Analytical method LOD's for the various species in both methods ranged from 0.2 to 0.8 μg L−1 as As. The results demonstrated the stability of the As species in the CRM three years after production.The preservation of As species within the sample continues to be of interest, and has been studied for environmental water samples.50 Field filtration (0.45 μm filter), refrigeration and storage in the dark were shown to be prerequisites for stabilization of AsIII and AsV. Filtration removes suspended matter and most microbes, however the dissolved As concentration determined after 0.2 μm filtration was significantly lower than that of 0.45μm filtered samples, due to the As being sorbed onto colloidal particles sized in the range 0.2–0.45 μm. Refrigeration suppresses most biotic and abiotic reactions, and storage in the dark avoids photochemical reactions of FeIII and AsIII. Based on publications covering the period since 2000, Rubio et al.39 critically reviewed sample handling, clean up, drying and powdering of fresh samples, extraction, and analysis by HPLC-ICP-MS of As species in algae and aquatic plants. The instrumental couplings of HPLC, GC and CE with online HG and AFS detection, for the speciation of inorganic and organic compounds of As, (and Se and Sb) have also been reviewed.35 The authors stress the need for optimized coupling to realise the full analytical benefit and consider other optional intermediate steps such as online photo-oxidation (UV), pyrolysis or microwave assisted digestion for non-directly reducible compounds.
When AsIII and AsV are measured by ICP spectrometry, AsV is found to be more sensitive than AsIII (although SeIV and SeVI show the same sensitivity).190 Building on previous work, the authors have investigated the mechanism behind this using ICP-SF-MS, and it was found that the amount of hydride polyatomic species of As formed in the plasma was different between AsV and AsIII. The researchers call this dependence upon the oxidation state of the As atoms in the plasma, the incoherent molecular formation (IMF) effect. The mechanisms of the effect were further investigated and techniques for removing its influence on As determination were discussed for both ICP-MS and ICP-AES.
Quantitative analysis with HPLC-ICP-MS under gradient elution conditions with increasing content of organic solvents can be restrictive because the elemental response can vary significantly with the amount of carbon reaching the plasma. Raber et al.169 have found that by introducing a volatile organic solvent directly into the spray chamber whereby the sample introduction system is saturated with organic vapours, no change in sensitivity is observed during a methanol gradient separation. This gradient compensation method was tested for the separation of 12 As species comprising AsIII, AsV, MMA, DMA, four oxo-arsenosugars and four thio-arsenosugars within a run of 25 min using a Hamilton PRP-X100 column applying a methanol gradient up to 50% (v/v). Under these conditions 10 of the 12 species could be separated, whereas AsIII and oxo-arsenosugar glycerol co-elute near the front. The method was applied to the determination of arsenosugars in the BCR 710 oyster tissue candidate CRM (AB 32.7 ± 5.1 mg kg−1 based on the consensus mean of the final certification round).
The separation of organoarsenicals may involve hydrophilic partitioning or adsorption driven by hydrogen bonds with surface H-donor/acceptor groups of the stationary phase. To investigate the mechanism, Zwitterionic HILIC was used to study the retention behaviour of nine selected organoarsenicals.191 The results indicated that considerable electrostatic interactions occur on the column. The retention of the As species was highly dependent on water/acetonitrile ratio, pH value and salt additives. The degree of dissociation and polarity of the As species, which vary with pH, regulated the distribution of As species between the stationary and mobile phases. Increase in the ammonium acetate concentration also led to shortened or prolonged retention depending on the structure of the As species.
Narrow bore chromatography is well suited to the low flow requirements of higher efficiency nebulizers. Such a system was used with an HR-ICP-MS for As speciation.109 The AEC system takes full advantage of the detector sensitivity allowing more diluted samples (50–100 times) to be injected, delivering substantially less sample matrix to the column and a lower eluent load to the plasma. The compatibility of the NH4NO3 eluent salt used in the study to adjust the pH enabled high linear salt ramps in gradient applications, highly reproducible retention times (±1%) and LODs in the low ng L−1 range. The separation conditions were used with two different polymeric anion-exchangers: a low capacity, weakly hydrophobic material (AS11, 250 mm × 2 mm, Dionex) and a more frequently used higher capacity, higher hydrophobic material (AG7, 50 mm × 4 mm, Dionex). On both columns, AsIII, AsV, MMA, DMA, AB and Cl− were separated in less than nine minutes.
The potential of narrow bore HPLC with detection by ICP-MS for fast determination of arsenosugars in algal extracts has been explored.108 The retention behaviour of four naturally occurring dimethylarsinoylribosides on an anion-exchange microbore column was investigated, with the mobile phase flow rate ranging from 60 to 200 μL min−1. A low sample consumption system consisting of a micronebulizer and a low inner volume cyclonic spray chamber was used as the interface between the micro-column and the ICP-MS. Both the high efficiency nebulizer and the PFA micronebulizer were tested, with the former providing 20–50% greater sensitivity than PFA (depending on the liquid flow rate), but comparable LOD and slightly lower chromatographic resolution. With the setup employed and under the optimal conditions, a satisfactory separation of the arsenosugars was achieved in less than 5 min. The instrumental LOD was 0.20 μg L−1 As with an RSD of better than 3% (n = 5).
A simple efficient microextraction methodology was developed for AsV, AsIII, DMA and MMA preconcentration and determination based on the novel use of tetradecyl(trihexyl) phosphonium chloride ionic liquid as an ion-pairing reagent.79 The AsV species was selectively separated by forming AsV-molybdate heteropoly acid complex with molybdenum, followed by ion-pairing reaction with tetradecyl(trihexyl) phosphonium chloride and microextraction in chloroform. Arsenic detection was performed by ETAAS. Under optimum conditions, the analyte extraction efficiency was 99% and yielded a preconcentration factor of 125 with only 5 mL of sample. The detection limit was 0.002 μg L−1 as AsV. A dual-column capillary microextraction system consisting of N-(2-aminoethyl)-3-aminopropyltrimethoxysilane-silica coated capillary (C1) and 3-mercaptopropyl trimethoxysilane silica coated capillary (C2) was developed for sequential separation/preconcentration of AsIII, AsV, MMAV and DMAV in extracts of human hair followed by ETV-ICP-MS detection with Ir as permanent tube modifier.81 It was found that at pH 9, AsV and MMA could be quantitatively retained by C1 and only AsIII could be quantitatively retained by C2.
Work with more unusual biological column substrates has been reported. A home made PTFE micro-column loaded with polyaniline (50mg), was used for the on-line separation of trace levels of AsIII and AsV in natural waters, followed by determination by FI-HG-ICP-MS.89 Calibration was linear over the range of 0.5–50 μg L−1. The detection limits (3σ) were quoted as 0.05 and 0.09 μg L−1 for AsIII and AsV respectively and the precision at 1 μg L−1 level was found to be 2.0% for AsIII and 2.5% AsV. Method validation was carried out by analysing two BCR groundwater CRM's, BCR609 and BCR610, certified for total As. Eggshell membranes (ESM), which contain several surface functional groups such as amines, amides and carboxylic groups have been reported to have potential as SPE adsorbents192 The study focused on AsV in water samples and used HG-AFS for detection. The desorption and enrichment conditions such as pH, flow rate and the volume of sample solution, the amount of ESM and the content of NaCl were systematically optimized and the effects of other ions were also investigated. Using optimal conditions, AsV could be extracted by the ESM packed cartridge and the breakthrough adsorption capacity was 3.9 μg g−1. The LOD was 0.001 μg L−1 with an enrichment factor of 33.3, and an RSD of 2.1% for 0.6 μg L−1 As (n = 11). The speciation of AsIII and AsV by using Streptococcus pyogenes immobilized on Sepa beads SP 70 resin has been investigated using SPE with HG-AAS detection.193 The preconcentration factor was 36-fold with quantitative recoveries of AsIII of >95%. The LOD was calculated as 13 ng L−1 with a RSD of below 8%. The validation of the procedure was tested by analysis of CRM's (NIST SRM 1568a Rice floor and GBW 07605 Tea) and gave compatible results.
Single-walled carbon nanotubes have been used as an SPE adsorbent for the preconcentration and separation of Aslll and AsV prior to determination by ICP-MS.194 Experiments were performed to optimize conditions, such as pH, sample flow rate, sample volume and concentration of eluent, to achieve quantitative separation. It was found that Aslll was only sorbed on the microcolumn packed with nanotubes within a pH range of 1.0–3.0, while AsV passed through the microcolumn without retention. Under the optimized conditions, the LOD of this method for AsIII was 3.9 pg mL−1 with an enrichment factor of 50 and 0.23 ng mL−1 for AsV. The RSD for AsIII and AsV was 3.1% and 2.5% (n = 9) respectively.
A diffusive gradients in a thin film (DGT) technique, using a titanium dioxide based adsorbent (Metsorb), has been developed and evaluated for the determination of dissolved iAs and Se.195 AsIII and AsV were found to be quantitatively accumulated by the adsorbent with uptake efficiencies of 96.5–100% and eluted in 1 M NaOH with elution efficiencies of 81.2% and 75.2% respectively. Uptake occurred over the pH range 3.5–8.5, and ionic strength between 0.0001–0.75 mol L−1 NaNO3, i.e. typical of natural waters, including seawater. Reproducibility of the technique in field deployments was good (RSD <8%). Limits of detection (4 day deployments) were 0.01 μg L−1 for iAs. The performance characteristics of DGT equipped with a binding gel layer containing precipitated ferrihydrite for determining AsV, SbV,SeVI and VV has also been reported.196
Organic As intake from seafood is a major As exposure route for many people, although the As metabolism in the human body is not yet fully elucidated. The effects of genetic polymorphisms of human purine nucleoside phosphorylase (PNP), AsIII methyltransferase (As3MT) and the antibody GSTO1 on organic As metabolism has been studied following oyster ingestion.197 During a one-week dietary controlled study, fifty study subjects were provided with all their daily meals without seafood, except for two designated amounts of oyster given on the fourth day. First morning voided urine samples were provided by the study subjects for 7 consecutive days and analysed by HPLC-ICP-MS for AsIII AsV, MMA and DMA. The As3MT was suggested to be one of the major factors affecting the metabolism of dietary organic arsenic in terms of urinary DMA level. Biotransformation of iAs involving methylation catalysed by As3MT, and yielding mono-, di-, and trimethylated As, in the invertebrate chordate Ciona intestinalis has also been studied.198 Tissue levels of iAs and its methylated metabolites were determined by HG-cryotrapping-GC-AAS. The metabolic pattern is consistent with the presence of an As3MT ortholog in its genome and constitutive expression of the gene in prominent organs, making this basalchordate a useful model to examine the evolution of arsenic detoxification. ArsenicIII methyltransferase genotype affects steady-state distribution and clearance of As in Asv-treated mice has also been reported.199
Urinary As profiles have been examined using HPLC and HG-AAS.200 Folate levels were measured using a competitive immunoassay kit. Genotyping was conducted using a PCR-restriction fragment length polymorphism technique. Patients with urothelial carcinoma had higher urinary total As, iAs and MMA, and lower DMA, plasma folate and homocysteine levels than controls. The correlations between folate and DMA, and folate and homocysteine, were significant according to Pearson's correlation coefficients. Ion chromatography in combination with HG-ICP-MS was used for the separation of AsV, MMA, DMA and AB in urine.105 However, the ion-pair mode was unable to differentiate AsIII from AB: instead CEC was used for this separation. The quantification problem with urine samples containing elevated concentration of AB, which elutes immediately after AsIII in AEC or ion-pairing mode, was overcame by introducing a post-column HG derivatization step. In a study in Korea, urinary As metabolites (iAs, MMAV, DMAV) and some biological indexes such as plasma aspartate aminotransferase, erythrocyte glutathione, erythrocyte glutathione peroxidase, lipid peroxidation, and uric acid were measured in volunteer study subjects (seven males and nine females).201 Total urinary As metabolites were determined by HG-HPLC-ICP-MS. Study subjects refrained from eating seafood for 3 days prior to the first urine collection and then ingested seafood daily for 6 consecutive days. The increase in total urinary As metabolites was attributed to the increase in DMA, which is a more harmful metabolite. However, no significant change in responses of biological indexes were observed. An evaluation of As exposure in residents living in an area with a soil naturally rich in As, has been made,202 again using urinary measurements. During the summer of 2007, 322 people aged over 7 years and resident in the study area for at least 4 days prior to the investigation were recruited. The sum of urinary iAs and metabolites (iAs + MMA + DMA) were determined by ETAAS and HPLC-ICP-MS. The percent of DMA, AsIII and MMA contribution to urinary arsenic concentrations was 84.2%, 12% and 3.7% respectively. Any AsV was rarely detected and DMA was the predominant metabolite comprising the majority of measurable inorganic-related As in the urine.
A routine method was developed to quantify five As species (AsIII, AsV, MMA, DMA, and AB) in whole blood and urine using HPLC-ICP-MS.104 Whole blood was diluted 1 + 4 with a mixture of 3 mM HgCl2 and deionised water which prevents AsIII from binding to the LC column. The diluted blood was subjected to ultrafiltration to remove molecules larger than 3,000 Daltons. The urine was prepared by diluting 1 + 4 with mobile phase and deionised water. The separation was completed within 12 min, for the determination of arsenic species in whole blood, and within 11 min for urine. The method detection limit was <0.3 μg L−1 for each arsenic species. Jiang et al.100 incubated AsIII or AsV with human serum albumin at physiological conditions (pH 7.43 and 37 °C). The free As and As-HSA complexes were then separated and detected by the combined techniques of microdialysis and HPLC-HG-AFS. The authors suggest that AsIII reacted with human serum albumin more readily than AsV, which provides a chemical basis for As toxicity.
The most common routes of As exposure are reported to be ingestion and inhalation, whereas dermal uptake has been considered as a minor route based on uptake experiments with AsV. Ouypornkochagorn and Feldmann203 have studied the kinetics of AsIII, DMAV and arsenosugar penetration through full thick human skin (from one volunteer) using a Franz Cell design and comparison with AsV. The accumulation in the epidermis and dermis was demonstrated by using LA- ICP-MS as a bioimaging method, and the biotransformation reactions through the uptake experiment were monitored by hyphenated elemental MS. The penetration and accumulation of As was strongly dependent on its speciation. While arsenosugars penetrated through the unbroken skin at a similar rate as AsV, AsIII and DMAV were taken up at a much faster rate than AsV.The researchers suggest that the dermal uptake route of As has been underestimated in risk assessments where exposure to AsIII or DMAV would occur. The accumulation potential of arsenosugars and DMAV was however minimal, whereas AsIII and AsV accumulated in the epidermis and in the dermis. No significant species transformations were observed. On a similar theme, it has been suggested that children may be exposed to As during contact with structures, such as play frames, treated with chromated copper arsenate (CCA).204 Arsenic speciation was conducted using HPLC-ICP-MS to detect As species in the urine and saliva samples from children playing on CCA and non-CCA playgrounds. The results were similar in both cases with DMA and AB being the main arsenic species found in urine samples. An overview of trends in the field of instrumental analysis of arsenosugars focusing on typical experimental approaches for sample pre-treatment, extraction, separation and detection has been presented.38
Arsenic metabolism by human gut microbiota following in vitro digestion of contaminated soils has been studied.205 A high degree of methylation for colon digests both of iAs (10 μg methylarsenical g−1 biomass hr−1) and of As-contaminated soils (up to 28 μg g−1 biomass hr−1) was found. In addition to the formation of MMAV, the more toxic MMAIII was also found. The study also identified the microbiolthiolation leading to monomethylmonothioarsonic acid. This latter species, the toxicokinetic properties of which are not well known, was in many cases a major metabolite.
Arsenic species in both liver and brain of mice were measured by HG-AAS to explore the distribution of speciated As in mice exposed to AsIII at early developmental stages.206 In the liver, levels of iAs increased on postnatal day (PND) 15, and MMA increased on PND 21, however, levels of DMA in newborn mice were significantly higher than those on PND 10 and 15. In the brain, levels of iAs on PND 21 were the highest; iAs levels on PND 15 were also significantly higher than those on PND 35. The researchers suggested that transplacental transfer of arsenicals from pregnant mice into their foetus' was relatively efficient, lactational transfer from mother mice into their offspring was inefficient, and transfer of iAs from blood into brain at early developmental stages was efficient.
A comparison of four extraction procedures to assess AsIII and AsV species in contaminated soils has been reported.65 The extraction strategies were: 1) 10 M HCl, 2) 15% (v/v) H3PO4, 3) 10 mM phosphate + 0.5% (w/v) NaDDC, and, 4) 1 M H3PO4 + 0.5 M ascorbic acid. Separation and analysis of As species was performed by HPLC-ICP-MS. Oxidation of AsIII into AsV during extraction was more important in soils with high content of Mn oxides. Extraction of As from soils with 1 M H3PO4 + 0.5 M ascorbate, with microwave heating, was the best strategy to extract the majority of As while minimizing conversion of AsIII into AsV.
The use of SR-XRF to characterize As speciation within natural fluid inclusions from three deposits with different hydrogeochemical and geological settings has been reported.207 The studied samples represent different compositions of Au-bearing fluids. Data obtained by XANES show that initially the fluid inclusions contained different As species, although the AsIII readily oxidized under the beam to AsV. Therefore, EXAFS spectra for the AsIII aqueous complex could only be collected on the sample containing large fluid inclusions with high As concentrations (>1000 ppm).
The rapid determination of volatile As species (AsH3, CH3AsH2, (CH3)2AsH and (CH3)3As released from lake sediment has been studied using a short packed cotton column coupled with AFS.123 Based on this, a new online system for collection and speciation of volatile arsenic species was developed. A special PTFE chamber was designed and applied for both incubation and collection of volatile arsenicals generated from the sediment. Real sample analysis and spiking experiments indicated that the system was sensitive enough for the analysis of volatile arsenicals produced in the environment. The biovolatilization of As as arsines in the form of AsH3, and mono-, di and trimethylarsine has often been determined under laboratory conditions. Although environmental point sources such as landfill sites or hot springs have been characterized, only limited knowledge is available on how widespread the formation of volatile methylated arsenic compounds are in the environment.145 The environmental concentration of the organoarsenicals in this study ranged from 4 to 60 pg m−3 As as TMAO, while the maximum concentrations for DMA and MMA were 16 and 6 pg m−3 as As, respectively. No difference in terms of the concentration or distribution of the organoarsenicals in PM10 samples was identified. These workers suggested that that biovolatilization of As as methylated arsines is a widespread phenomenon. Arsenic species in municipal landfill leachates were investigated by HPLC-DRC-ICPMS and LC-ESI-MS/MS208 and AsV, AsIII, MMAV, DMAV, as well as S-containing organoarsenic species were detected. Two S-containing As species in a municipal landfill leachate were identified as dimethyldithioarsinic acid (DMDTAV) and dimethylmonothioarsinic acid (DMMTAV) by comparing their molecular ions, fragmentation patterns and S:As ratios with those of in-house synthesised thiol-organoarsenic compounds. The findings demonstrated the potential for transformation of DMAV to DMDTAV and DMMTAV in a DMAV-spiked landfill leachate environment.
HPLC-ICP-MS with an octapole reaction system has been used to speciate As in the stream waters of a refining process.209 Four As species, AsIII, AsV, MMA, and DMA were separated in a single run by IC using gradient elution with 100 mmol L−1 NH4NO3, pH 8.5, adjusted by addition of NH3, as eluent. Repeatabilities of peak position and of peak area evaluation were better than about 3%. Detection limits (as 3σ of the baseline noise) were 22, 19, 25, and 16 ng L−1 for AsIII, AsV, MMA, and DMA, respectively. The CRM materials: BCR 714 (Waste water influent - Trace elements), BCR 1714, and BCR 715 (industrial effluent wastewater), plus two different refinery samples -inlet and outlet wastewater were also used. MMA was also detected in groundwater from a former herbicide production plant in the USA.146 The site has areas where the total As concentrations were thousands of mg L−1, representing one of the most severe cases of As contamination ever reported. Structure-specific detection of MMA, along with AsIII, AsV, MMAv, and DMAv, was achieved using HPLC-ESI-MS/MS. To enable the electrospray of MMA and AsIII, dimercaptosuccinic acid (DMSA) was used to derivatize these trivalent arsenicals online, so that their complexes with DMSA could be detected using negative ionization ESI-MS/MS. Aerobic degradation of organoarsenicals (including Roxarsone) in wastewater lagoons has been documented.210 The preliminary study suggested the direct and/or indirect association of particulate Cu in catalyzing Roxarsone degradation under aerobic conditions in samples with high % solids content. An organocopper Roxarsone metabolite was found only in the high-Cu wastewater sample. No organocopper metabolite was found in the low-Cu wastewater sample, and the Roxarsone did not undergo degradation under aerobic conditions even after 16 days.
Seasonal changes of As speciation in lake waters in relation to eutrophication has been investigated.211 Surface water samples were collected from 18 lakes (n = 1–10) in Japan during July 2007 and February 2008. The lakes were classified as mesotrophic (7 lakes) or eutrophic (11 lakes) based on the total phosphate and chlorophyll-a concentrations in the water column. Inorganic, methylated and ultraviolet-labile fractions of As were determined by HG-AAS with UV irradiation. Organoarsenicals (mainly methylated and UV-labile fractions) comprised 30–60% of the total As in most lakes during summer, although, AsIII and AsV dominates (about 60–85%) during winter. The occurrence of UV-labile fractions of As was higher in eutrophic lakes than those in mesotrophic lakes in both seasons. The concentration of DMA was high in eutrophic lakes during winter; and in mesotrophic lakes during summer. The results suggest that the conversion of AsIII and AsV to more complicated organoarsenicals occurred frequently in eutrophic lakes compared to that in mesotrophic lakes, and is likely to be due to biological activity in the water column.
Arsenic concentrations in fresh and frozen samples of raw, boiled and fried Atlantic cod (Gadhus morhua), Atlantic salmon (Salmo solar) and blue mussel (Mytilus edulis) were determined.51 The results show that the total As concentrations of the fresh Atlantic cod and Atlantic salmon samples were not different from the frozen samples within the same seafood type. For blue mussel, the total As concentration decreased significantly after storage. Inorganic As was found only in blue mussels and, importantly, no significant increase of iAs was observed after processing or after storage by freezing. The content of tetramethylarsonium ion was generally low in all samples types, but increased significantly in all fried samples of both fresh and frozen seafood. Upon storage by freezing, the AB content was reduced significantly, but only in the samples of blue mussels. Arsenolipids were also extracted from canned cod liver and purified by SPE using a silica gel column and ethyl acetate/methanol as eluent.118 The results obtained by GC-ICP-MS and GC-MIP-AES showed the existence of numerous arsenic compounds in the SPE fractions collected. Information on the molecular weights of the major arsenic species were provided by TOF-MS equipped with an CE-ESI-MS sprayer kit. A mixture of iso-propanol and water (50:50, v/v) containing 0.2% formic acid served as sheath-liquid at a flow rate of 3 μL min−1. Sample introduction was performed applying pressure to the sample vial which was connected with the coaxial sheath-liquid sprayer via a short piece of fused silica capillary (50 μm I.D., 360 μm O.D.). European whelks (Buccinum undatum) have been shown to accumulate high levels of As. Since the accumulation process is not well understood, Urgast et al.212 have investigated the geographical variability of the As concentration in them. The mean As concentrations of the whelks was shown to be site specific and to vary by a factor of 3.5 in ten different allocations in Northern Britain.
The use of pressurized conditions to assist enzymatic hydrolysis of seafood tissues for As speciation (AsIII, MMA, DMA, AsV, AB and AC) has been studied by Moreda-Pineiro et al.69 Arsenic species were released from the dried seafood tissues using pepsin as a protease and the As species were separated/quantified by anion exchange (IonPac AS7, 250 mm × 4 mm id) HPLC-ICP-MS. Total sample solubilisation was not achieved after the procedure, however the authors report that it was sufficient for breaking down the bio-molecules to release the As species. The method offered RSDs lower than 6% for AsIII, DMA and AsV and 3% for AB. The LOQs were 18.1, 36.2, 35.7, 28.6, 20.6 and 22.5 ng g−1 for AsIII, MMA, DMA, AsV, AB and AC respectively. The optimized methodology was successfully applied to different CRMs (DORM-2 Dogfish Muscle and BCR 627 Tuna fish tissue) which offer certified AB and DMA contents and also to different seafood products (molluscs, white fish, and cold water fish).
Arsenic in dry seafood products such as kelp, Sargassum fusiform, Laver, and Enteromorpha prolifera, were studied using HPLC-ICP-MS.213 It was found that, of the As species in dry seafood products, three were unknown arsenicals, speculated to be arsenosugars. Later studies using HPLC-ESI-TOF-MS identified these as dimethylarsinic (DMA)-glycerol ribose, DMA-phosphate ribose and DMA-sulfate ribose. Some seaweeds contain high amounts of iAs - in particular, Hijikia fusiforme has an As content of approximately 50%. Ichikawa et al.214 have used HPLC-ICP-MS employing an Inertsil AS column (2.1mm × 150 mm) to examine the absorption, metabolism, excretion and accumulation of As compounds in mice after the administration of Hijiki. The experiment, wherein a single dose of cooked Hijiki was administered to the mice, revealed that the urinary and faecal excretion of As compounds was highest on the first day of dosing, and that 66–92% of As was excreted within 3 days after administration of the first dose. When repeated doses of cooked or dried Hijiki were administered to the mice, As was detected in various organ tissues, but only approximately 5% of the administered dose of As was detected as residual As. These results suggest that the As present in cooked Hijiki is accumulated in very small amounts in mice.
Antarctic coastal environments offer the unique opportunity to study elemental cycling under pristine conditions. Grotti et al.215 have reported on the determination of As species in various tissues from a range of marine organisms collected from coastal environments, and compare their results with those from similar studies in temperate and tropical waters. The As species were determined in aqueous methanol extracts of tissues (including muscle, liver, gonads and spleen) by HPLC-ICP-MS. The major compounds were AB and oxo-arsenosugars, with their relative proportions depending on the position of the organism in the food chain and, for some species, on the type of tissue analysed. Several minor compounds, such as dimethylarsinate, trimethylarsine oxide, trimethylarsoniopropionate and arsenocholine were also found.
Axenic cultures of the phytoplankton Dunaliella tertiolecta were dosed with either AsV, MMA or AB at environmentally realistic concentrations (2 μg L−1) to investigate incorporation and transformation of As species.216 Total As concentrations in cultures dosed with AsV were higher than those dosed with MMA and AB (6–10 μg g−1 compared to 1–3 μg g−1). Arsenic concentrations in AsV-dosed cultures increased over time, whereas As concentrations in MMA- and AB-dosed cultures remained constant, indicating that AsV is more rapidly and continuously incorporated into the cell in comparison to MMA and AB. Small amounts of AsIII, MMA, DMA and arsenoribosides were also found in the lipid and water soluble As fractions. All cultures showed an increase in DMA and arsenoriboside species over time. The significance of these results is that while AB, the major arsenical in marine animals, is not produced or accumulated by this phytoplankton species, the possible precursors to AB formation are produced.
The transportation of As in phloem has not been fully elucidated. Ye et al.217 have quantified the chemical species of As in phloem and xylem exudates of castor bean (Ricinus communis) exposed to AsV, AsIII, MMAV, or DMA. In the AsV and AsIII exposed plants, AsV was the main species in xylem exudate (55%–83%) whereas AsIII predominated in phloem exudate (70%–94%). The ratio of As concentrations in phloem to xylem exudate varied from 0.7 to 3.9. Analyses of phloem exudate using HPLC-ICP-MS and HPLC-ESI-MS identified high concentrations of reduced and oxidized glutathione and some oxidized phytochelatin, but no AsIII -thiol complexes. It was thought that AsIII -thiol complexes would not be stable in the alkaline conditions of phloem sap. Small concentrations of oxidized glutathione and oxidized phytochelatin were found in xylem exudate, where there was also no evidence of AsIII-thiol complexes. MMAV was partially reduced to MMAIII in roots, but only MMAV was found in xylem and phloem exudate. Despite the smallest uptake among the four As species supplied to plants, DMA was most efficiently transported in both xylem and phloem, and its phloem concentration was 3.2 times that in xylem. The results indicate that free iAs, mainly AsIII, was transported in the phloem of castor bean exposed to either AsV or AsIII, and that methylated As species were more mobile than iAs in the phloem. To help elucidate the molecular structure for arsenic-thiol complexes, Park and Butcher218 have investigated the interaction between As species (AsIII, AsV MMAV, and DMAV) with biomolecules containing thiol groups (glutathione and cysteine) by ESI-MS. In each mass spectrum for solutions of As species and thiol compounds, various peaks such as protonated As-thiol complexes, protonated non-complexed thiol compounds, Na bound cluster ions, and proton bound cluster ions were observed. These As-thiol complexes produced a variety of fragment ions by cleavage of chemical bonds, and by interaction of other binding sites on thiol compounds in MS/MS experiments. The fate and speciation of As during uptake, translocation, and storage by the As hyperaccumulating fern Pityrogramma calomelanos var. austroamericana (Pteridaceae) were examined using ICP-AES and SR-XANES and μXRF.151 More than 60% of AsV absorbed was reduced to AsIII in roots, prior to transport through vascular tissues. In pinnules, AsIII was the predominant redox species (72–90%), being found as solvated, oxygen coordinated compounds. The presence of putative AsS coordination throughout the fern tissues (4–25%) suggests that S2 functional groups may contribute in the biochemical reduction of AsV to AsIII during uptake and transport at a whole-plant level.
Arsenic contamination of rice is widespread, but the rhizosphere processes influencing As attenuation remain unresolved. In particular, the formation of Fe plaque around rice roots is thought to be an important barrier to As uptake, but the relative importance of this mechanism is not well characterized. Seyfferth et al.152 have elucidated the co-localization of As species and Fe on rice roots with variable Fe coatings, using a combination of techniques: XRF, μXANES, transmission X-ray microscopy, and tomography. Two dominant As species were observed in fine roots - inorganic AsV and AsIII - with minor amounts of DMA and AsIII-tris-glutathione. To investigate how As species are unloaded into grain rice, panicles were excised during grain filling and hydroponically pulsed with AsIII, AsV, glutathione-complexed As, or DMA. Total As concentrations in flag leaf, grain, and husk, were quantified by ICP-MS and As speciation in the fresh grain was determined by XANES.153 The results demonstrate that DMA was translocated to the rice grain with over an order of magnitude greater efficiency than inorganic species and is more mobile than AsIII in both the phloem and the xylem. Phloem transport accounted for 90% of AsIII, and 55% of DMA, transport to the grain. Polished rice from various production regions of China have been analysed for total As and As species using HPLC-ICP-MS.219 Total As concentration ranged 65.3–274.2 ng g−1, with an average value of 114.4 ng g−1. Four arsenic species, including AsIII, AsV, DMA and MMA, were detected in most rice samples. A similar study also utilising HPLC-ICP-MS has been conducted for rice in Korea66 which found that in a batch of 11 samples, nearly 57% of the total As was iAs species.
5.4 Chromium
In recent years, the extensive use of Cr in industrial processes has led to the promotion of several directives and recommendations by the European Union (and elsewhere), that try to limit and regulate the presence of CrVI in the environment and to protect industrial workers using Cr and end-users of manufactured products. This area has now been reviewed41 focusing on solid matrices, and emphasising different extraction procedures.As in previous years, most methods for Cr speciation reported this year were modifications to existing approaches. Various approaches for determining CrIIIand CrVIin water samples have been reported. The immobilization of N-methylimidazolium onto a PVC surface forms bonded hydrophilic ionic liquid 1-chlorovinyl-3-methylimidazolium chloride which entailed anion exchange nature and positively charged surface, facilitating retention of CrVI with a retention capacity of 23.2 mg g−1 for CrVI at pH 5.6.80 This has been used in an SPE approach using mini columns in conjunction with either ETAAS or ICP-MS. The retained CrVI is effectively recovered with elution by 0.2 M NH4NO3 solution. A linear range of 0.01–1.0 μg l−1, LOD of 3 ng L−1 and a RSD of 2.9% at 0.5 μg L−1 for CrVI, was obtained. The procedure was used for tap water and snow water.
Solid-phase extraction combined with metal furnace atomizer AAS has also been reported for the determination of CrIII and CrVI at sub-ppb levels in water.84 A 500 mL sample was adjusted to pH 3 with nitric acid and then passed through an iminodiacetate extraction disk placed on a cation-exchange extraction disk at a flow rate of 20–40 mL min−1 for concentrating CrIII The filtrate was adjusted to pH 10 with aqueous ammonia and then passed through an anion-exchange extraction disk at a flow rate of 2 mL min−1 for concentrating CrVI. The CrIII and CrVI collected were eluted with 40 mL of 3 M nitric acid for CrIII and 40 mL of 1 g L−1 diphenylcarbazide solution for CrVI. Each eluate was diluted to 50 mL with deionized water and injected into a U-type tungsten board on the metal furnace The linear range for both CrIII and CrVI was 0.1–0.5 ng with an LOD for both species of 81 pg (3σ). A selective method for preconcentration and determination of CrVI in aqueous samples has been reported which does not require an elution step.220 After adsorption in ‘batch mode’ onto Aliquat 336-AC, determinations were made directly on the solid by XRF. A pre-concentration factor of 71-fold was achieved.
A method has been described for Cr speciation at sub-μg L−1 levels in potable water samples using IC-ICP-MS with a collision/reaction interface.221 The redox pair CrVI and CrIII was separated on an IonPac AG-7 guard column within 7.5 min using gradient elution with 0.1 M ammonium nitrate and 0.8 M nitric acid as the mobile phases. Hydrogen was used as the interface gas to eliminate Cl-based and C-based polyatomic interferences. Water samples were analysed directly, without pretreatment, to preserve the original Cr speciation.
Two different types of modification to activated carbon, i.e. treatment with concentrated solution of HNO3 and outgassing treatment at high temperature, were studied in order to obtain the most effective adsorption of CrVIions from solution.222 The reduction of CrVI to CrIII and further ion exchange mechanism of adsorption onto oxidizing activated carbon and the surface precipitation to Cr(OH)3 in case of outgassed activated carbon were found as the main adsorption mechanisms of CrVI ions onto modified activated carbons. The presence of chlorides and nitrates strongly decreased the adsorption of CrVI onto outgassing activated carbon. Following on from their work with As referenced above,194 Chan et al. have also reported the use of microcolumn packed single-walled carbon nanotubes for Cr speciation when coupled to ICP-MS for detection.223 The effects of the experimental parameters, including pH of the solution, sample flow rate, volume and concentration of eluent, sample volume and interfering ions, on the separation and determination of CrIII and CrVI were investigated. It was found that CrIII was selectively sorbed on the microcolumn packed with single-walled carbon nanotubes in the pH range from 2.0 to 4.0, while CrVI remained in solution.
A method employing ultrasonic probe-assisted LLLME combined with ETAAS has been developed for the determination of CrVI species in water samples.224 In this procedure, the hydrophobic chelate of CrVI with APDC was extracted into the fine droplets of 1-hexyl-3-methylimidazolium hexafluorophosphate, which was dispersed into the aqueous sample solution using the ultrasonic probe. The LOD of the proposed method was 0.07 ng mL−1 for CrVI, and the RSD (n = 5) was 2.0 ng mL−1 CrVI was 9.2%. A similar approach was used by Razmisleviciene et al.76 Dried microdroplets (7 μL) of the extracts were ablated from a polystyrene substrate and the CrVI quantified by LA-ICP-MS. External calibration was used in combination with 195Pt as an internal standard (no matrix-matched standards were required). The LODs were 0.11 and 0.31 μg L−1 for 52Cr and 53Cr, respectively, with precision between 4 and 8%. The method was applied to determine CrVI in tap and river samples.
Stable Cr isotope fractionation data for CrVI reduction, CrIII oxidation and isotopic exchange between soluble CrIII and CrVI in aqueous media has been collected to help elucidate the reaction kinetics and Cr fractionation behaviour during redox transition and isotope exchange.225 All Cr isotope measurements were performed by MC–ICP-MS. The Cr signals, 50Cr+, 52Cr+, 53Cr+ and 54Cr+ were detected simultaneously together with the monitor signals 48Ti+, 51V+, and 56Fe+ to correct for minor isobaric interferences on 50Cr+ and 54Cr+ when necessary. Further polyatomic interferences such as 40Ar12C+ on 52Cr+, 40Ar14N+ on 54Cr+ and 40Ar16O+ on 56Fe+ were completely resolved by operating the instrument in medium-resolution mode. The results indicated that the isotopic composition of CrVI in a natural system will not be influenced by equilibration with any CrIII and thus reveal the true extent of reduction, given that the Cr isotope composition of the source CrVI and the fractionation factor for the prevailing conditions are known.
Urinary Cr speciation analysis can provide information of the individual exposure levels to CrVI compounds. A method based on ion-pair reversed-phase HPLC combined with ICP-MS to simultaneously determine CrIII and CrVI in human urine has been developed for assessing the occupational exposure to CrVI.226 A PEEK column was used and separation of CrIII and CrVI was achieved within 4 min with an LOD of 0.03 μg L−1 at 100 μL injections. The speciation of the in vitro binding of exogenous Cr to blood serum proteins was influenced markedly by both the oxidation state of Cr exposed to the serum proteins and the treatment conditions, which is of relevance to the biochemistry of Cr dietary supplements.150
The uptake and localisation of Cr in the cell structure of the yeast Saccharomyces cerevisae was studied following uptake of CrIII-citrate and CrIII-histidine from the growing medium.227 The total cellular Cr and the distribution of Cr between the cell walls and spheroplasts were determined by AAS. Chromium accumulation was shown to depend on treatment time and metal concentration as well as the nature of the bound ligand. The pH dependence pattern of Cr accumulation was similar for both of the CrIII-organic compounds: pH 6.5 > pH 5 > pH 8. Substantial differences were found between the two CrIII organic compounds, in the total Cr accumulation as well as in the distribution in yeast cell walls and spheroplasts. A procedure for the speciation of Cr in bread samples has been described.59 The samples were prepared by a wet acid digestion procedure for total Cr followed by a selective alkaline extraction of CrVI, The Cr was measured by ETAAS. The method was applied to 152 bread samples. The calculated daily intake was up to 12.7 μg day−1 for total Cr and 1.98 μg day−1 for CrVI representing 10% of the reference daily intake of 120 μg/day.
The Cr speciation in marsh soils developed in weathering CrIII ore processing residue (COPR), was characterized using sequential extractions and synchrotron microbeam and XAS analyses.228 The extractions suggested substantial Cr associated with reducible and oxidizable soil components, and significant non-extractable residual Cr. Bulk XAS data further indicated CrIII incorporated in Fe(OH)3, and CrIII associated with organic matter. The authors concluded that the effects of anoxia on Cr speciation, and the potential for active COPR weathering releasing CrVI deeper in the soil profile require further study.
5.5 Gadolinium
The use of Gd based MRI contrast agents continue to provide interest for speciation analysis. The Gd species present in rat kidney arising from the MRI contrast agent Gadoversetamide, Gd-DTPA-BMEA, have been studied by both HPLC-ICP-OES and HPLC-ESI-MS.229 Three different RP C18 columns were used in this study, two of 250 × 4.6 mm and one 50 mm by 4.6 mm. The mobile phase for each was 10 mM ammonium acetate in water, with the addition of acetonitrile (5%) when the 50 mm column was used. A water based UAE procedure was used to solubilise the GD species from freeze tried tissue samples with varying recovery factors. The authors conclude that further work involving enzymatic UAE procedures needs to be conducted to improve extraction efficiencies. The LOD for Gd-DTPA-BMEA in the tissue samples was calculated to be 0.5 mg kg−1. The imaging of gadolinium (Gd) spatial distribution in a mouse tumour model postadministration of PEGylated Gd liposomal nanoparticles has been studied by LA-ICP-MS.230 At 2 h postinjection the presence of Gd within tumour tissue was confirmed and, when correlated to histology, was found to be prevalent in regions of higher vascularity. Gadolinium was also found in the kidneys.5.6 Halogens
Interest in the detection of brominated compounds by ICP-MS continues to grow which may be due to the recent legislative requirements (e.g. EU Directive 2008/105/EC). The isotopic composition of PBDEs by GC-MC-ICP-MS, has been studied as a potential tool for degradation monitoring and source identification of organobromines in the environment.122 Separation by GC was performed on a 30 m megabore column, 5% phenyl–95% dimethylpolysiloxane, 0.53 mm i.d., 0.5 mm film thickness, with a fused silica capillary transfer line to the ICP-MS. Three different approaches to heating the transfer line were evaluated. In two cases the fused silica capillary from the GC passed through electrically heated stainless steel tubing, of 1/8′′ and 0.53 mm i.ds., whilst the third transfer line, was heated by both hot argon and electrical methods. δ81Br values for a commercially available flame-retardant mixture were determined with previously characterised monobromobenzene used as the isotopic reference standard. It is not clear how, or even if, mass bias correction was performed on the measured Br isotopic ratios. The precision of the results obtained was poor and it is also not clear how precision values were calculated from the data presented. If no mass bias correction was applied it is possible that this may account for the poor precision values. A mixture of tri-, tetra-, penta-, and hexabrominated 81Br enriched PBDEs has been synthesised for use as spike material for the determination of PDBEs by IDMS.231 The isotopic enrichment was measured by GC-MC-ICP-MS and was found to be 99.53% 81Br.The concentrations of three PDBE congeners, 28, 47, and 99, in the synthetic mixture was measured by reverse IDMS, using a certified natural isotopic abundance PDBE mixture, by both GC-ICP-MS and GC-EI-MS. Subsequently, using GC-EI-MS and an isotope pattern deconvolution procedure, detection limits of less than 0.5 ng L−1 were obtained for congeners 28, 47 and 99 in natural waters by IDMS. This was achieved using 100 mL of sample and an isooctane LL extraction procedure. Mass bias correction for GC-ICP-MS isotope ratio measurements was performed using a mixture of natural isotopic composition PDBE congeners. A method for the rapid screening of 9 PDBEs, (congeners 47, 85, 100, 138, 155, 201, 206, 207, 208 and 209) in biological samples by RP-HPLC-ICP-MS has also been developed.232 The PDBE congener separation was achieved using a C18 column and a gradient elution of between 30:70 and 5:95% water:acetonitrile at a flow rate of 1.5 ml min−1 in twelve minutes. A high RF forward power, oxygen addition at 8% to negate carbon deposits and He2 as a collision cell gas to minimise ArH+ polyatomic interferences allowed detection limits of 17 ± 1 ng ml−1. The method was applied to the determination of decabromodiphenylether in rat liver and faecal extracts and gave reproducibilities of 6.8 and 6.9% respectively for three measurements. The method was validated by comparison with measurements of 14C labelled decabromodiphenylether in the same samples with the results obtained by both methods being in good agreement.
In response to the need to detect brominated flame retardants (BFRs) in waste electrical and electronic equipment an HPLC-ICP-MS method has been developed for the determination of selected PBDEs and polybrominated biphenyl (PBB) in four polymers: high-density polyethylene (HDPE), polystyrene (PS), acrylonitrile–butadiene–styrene copolymer (ABS), and polypropylene (PP).57 The PBDEs and PBB in the polymers were extracted with toluene, using UAE for 30 min at ambient temperature after dissolution of the polymer samples. The BFRs in the extracts were then determined by HPLC–ICP–MS, using a single point external calibration. Chromatographic separations were performed using 250 × 4.6 mm C18 columns with ternary mobile phases, consisting of methanol, acetonitrile and water, and differing gradient elution profiles. These mobile phases necessitated the addition of oxygen to the Ar carrier gas at 20% of this gas flow rate. Extraction efficiencies of the various BFRs from four different reference materials were always close to 100% and the results obtained were also in good agreement with Comite consultatif pour la quantite de matiere metrologie en chimie (CCQM) study P114. The overall LOD for BRFs in the solid polymers ranged from 6 to 8 mg kg−1 which, although higher than GC-ICP-MS based methods, are still low enough to satisfy the Restriction of Hazardous Substances (RoHS) requirement of 0.1% for BRFs in polymers.
A rapid method using IC-ICP-MS has been employed for iodine speciation in edible salts and human urine.115 A 50 × 4 mm anion exchange column, with a mobile phase of 8 mmol L−1 of (NH4)2CO3 at a flow rate of 1.5 ml min−1, was used to separate IO3− and I−, with detection limits of 15 and 81 ng L−1 respectively, in under three minutes. The method was validated with three different IO3− CRMs and good agreement was obtained with the certified values. A mass balance approach was adopted for the edible salt and human urine samples and the sum of the found concentrations were also in good agreement with the total I content as determined by ICP-MS.
A different and somewhat unusual approach was undertaken to determine iodine containing X-ray contrast agents by coupling TLC with ICP-MS.172 The device was developed to allow on-site sampling analyte separation, which may reduce the potential for sample contamination and/or species transformations. Three X-ray contrast agents Biliscopin, Imagopaque and Peritrast, the latter was used as an internal standard, were separated on silica gel RP18 TLC plates using a mobile phase containing methanol and 0.2% aqueous formic acid (4:6, v/v). The separation conditions were optimised to give a spot size of 4 mm diameter or less. Subsequently, a previously developed device for extraction of the TLC spots was directly coupled to the ICP-MS nebuliser and the transient signals arising from the eluted spots of spiked urine samples monitored. The mobile phase for spot elution was methanol:2.5 mM ammonium acetate buffer (pH 7.4, 8:2, v/v) with oxygen addition to the plasma to minimise carbon build up. Good agreement between the gravimetric values and found values for spiked urine samples was obtained with RSDs ranging from 0.1 to 1.9% whilst detection limits of less than 50 nmol L−1 were obtained.
5.7 Lead
The accumulation and transport of Pb in Brassica juncea and Sesuvium portulacastrum plants and the possible formation of complexes of this element with bioligands, such as phytochelatins, in the roots and shoots of plants exposed to different amounts of Pb(NO3)2 were studied.233 Speciation studies on the plant extracts were conducted using SEC and ion pair HPLC coupled to both UV and ICP-MS, to monitor Pb and S. Identification of the species separated by chromatography was performed by MALDI-TOF-MS. In both types of exposed plants it was possible to identify the presence of the phytochelatin isoform PC3. The results obtained suggest that both types of plants can be useful in studies of phytoremediation, but the ability of S. portulacastrum to accumulate and redistribute Pb from root to shoot is more effective than that of B. juncea.The spatial distribution and speciation of Pb in tissues of the accumulator plant Sedum alfredii (Crassulaceae), was investigated using SR-μXRF and powder EXAFS spectroscopy.156 Lead was predominantly restricted to the vascular bundles of both leaf and stem of the plant. Micro-XRF analysis revealed that the Pb distributed predominantly within the vascular bundles, and a positive correlation between the distribution patterns of Pb and S was observed.
5.8 Mercury
Analytical methods for the speciation analysis of Hg compounds in natural waters have been reviewed.42 Differing approaches currently in use for sample collection and storage, pre-concentration, separation, and detection are critically compared. The paper discusses chromatographic and non-chromatographic Hg speciation methods as well as derivatisation methods and the coupling of pre-concentration and/or separation methods to detection systems. The authors conclude that, due to the ubiquity of Hg in natural waters, albeit usually at very or ultra low concentrations, there is a need to develop easy to use, in situ, reagent-free pre-concentration devices, and ultimately in situ quantification methods.The methods for the measurement of isotopic ratios in individual Hg species for identifying fractionation and for IDMS continues to advance. A new approach for calculating Hg isotope ratios after measurements by GC-MC-ICP-MS has been described.234 The method, which is based on calculation from the slope of a linear regression of transient signal intensities, gave improved precision compared with other methods of calculation in use with values of between 0.003 and 0.02% RSD, for injections of as low as 90 pg of Hg species, which is close to the theoretical values from counting statistics. A comparison has been made between GC-MS and GC-ICP-MS for the determination of Hg species by multiple spiking ss-IDMS.235 The method was based on a previously reported isotope pattern deconvolution procedure which identified interconversion reactions for Hg species, confirmed by GC-ICP-MS analyses, during open focussed microwave extractions from DOLT-4 Dogfish liver CRM. No species interconversion reactions were observed for the same extraction protocol with BCR 464 Tuna fish CRM.
Acid-leaching and distillation-derivatisation are two common sample preparation procedures for HG speciation. A combination of these two, described as acid leaching-ion exchange-thiosulfate extraction (TSE), followed by aqueous phase ethylation, have been used to measure MeHg by ID GC-ICP-MS.119 Two sediment CRMs, BCR 580 and IAEA 405, were used to evaluate the accuracy of the TSE procedure. Recoveries of 94 to 109% were obtained for BCR 580 and 94 to 105% for IAEA 405 for which recoveries using an acid leaching procedure were poorer at 78 to 96%. No artefact formation, evaluated using sediments spiked with inorganic 200Hg2+, was observed with the TSE procedure.
Two papers cover non-chromatographic Hg speciation.140,147 In the former paper Hg species were extracted from biological materials using a combination of 0.1% (v/v) HCl, 0.1% (v/v) 2-mercapoethanol and 0.15% (m/v) KCl. Subsequently, the Hg species were converted to Hg0 for AFS detection, using a two valve two pump sample introduction manifold, with SnCl2 for Hg2+, SnCl2 and UV irradiation for PhHg+ with the addition of 2-mercaptoethaol to prevent MeHg+ decomposition, using K2S2O8 and UV irradiation the combined total from Hg2+, PhHg+ and MeHg+ was given whilst NaBH4 was used to determine the combined concentration of all four Hg species under study (Hg2+, PhHg+, MeHg+ and Me2Hg). The concentrations of individual Hg species were then calculated by subtraction. The LODs obtained were 1, 40 68 and 99 ng L−1 for Hg2+, PhHg+, MeHg+ and Me2Hg respectively. The method was evaluated using two CRMs, IRMM 811 Admussium colbecki and MURSST-ISS-A2 Antarctic Krill. For IRMM 811, which at the time of the research was not certified for Hg, the total Hg content found by summing the individual species determined by CV-AFS was in good agreement with the total found by a microwave digestion procedure whilst for MURSST-ISS-A2 the total Hg content found by species summing was in excellent agreement with the certified value. No PhHg+ or Me2Hg was detected in either CRM. The second paper in this field used a dielectric barrier discharge (DBD) atomiser and NaBH4 to generate Hg vapour with detection by AAS. Inorganic Hg was detected without the use of the DBD plasma whilst total Hg was determined with the DBD device in operation. The MeHg content was then determined by difference with the assumption being that this value was entirely due to MeHg and not other organoHg species. A tuna fish CRM, GBW10029, was used to evaluate the device and the found values for MeHg and total Hg were in very good agreement with the certified value for the CRM. Found LODs were 35 and 54 ng L−1 for Hg2+ and MeHg respectively.
The simplification of the apparatus and reagents for Hg speciation continues to provoke interest, often due to financial restrictions. The separation and detection of Hg species by HPLC-CV-AFS using only aqueous L-cysteine (1 g L−1) and ammonium acetate (0.06 mol L−1) as the mobile phase and a 150 × 6 mm RP C18 column has been reported.173 The CV reagents were 10% HCl v/v and KBH4 0.5% w/v in 0.2% w/v KOH with no UV assistance. The reported LODs with this system were 100 ng l−1 or better for inorganic Hg, MeHg and EtHg. Two CRMs were used for method validation and the results obtained for both DORM-2 Dogfish Muscle, using an alkaline leaching procedure, and TORT-2 Lobster Hepatopancreas, using and acidic leaching procedure, were in good agreement with the certified values. A mini-column, of 30 mm length with an i.d. of either 2, 3 or 4 mm, inserted in place of the conventional sample loop, with polyaniline as the stationary phase has been used as a pre-concentration and separation device prior to CV-ICP-MS detection.90 Under optimal conditions LODs of 25 ng L−1 and 32 ng L−1 were obtained for iHg and MeHg respectively. The results obtained for three Tuna CRMs, ERM-CE463, ERM-CE464 and IAEA-350, after extraction of Hg species by UAE with 5M HCl, were in good agreement with the certified values, with recoveries of 95%. A UAE method which can be used for small sample masses has recently been developed and validated.61 Focussed ultrasound, using a probe with a 2mm tip diameter, in conjunction with either acid leaching (HCl 7M) or enzymatic extraction (15mg protease type XIV in 2.5% (v/v) 2-mercaptoethanol) was used for Hg species extraction from freeze dried tissue samples of 2 to 50 mg. Recoveries of MeHg from BCR CRM-463 were 99 ± 3% and 93 ± 1% respectively for the two extraction protocols. Mercury species separation was performed using a C18 column, 150 × 3.9mm, with a mobile phase consisting of 0.1% (v/v) formic acid, 0.1% (v/v) heptafluorobutyric acid, 2% (v/v) methanol, and 0.02% (w/v) mM l-cysteine at pH 2.1 and detection by ICP-MS.
5.9 Phosphorus
Most of the papers which utilise phosphorus detection are covered in the ‘macromolecules’ section of this review. However, two papers come outside of the scope of that section and are covered here. In the first paper236 two compounds which are thought to be inhibitors of renal stone formation, pyrophosphate (PPi) and phytic acid (IP6), were selectively extracted from small volume urine samples by AEC SPE after sample dilution and using HCl of differing molarities. The LOD for PPi was 7 μg L−1 and recoveries from spiked samples were of the order of 100% for PPi. No LOD for IP6 could be found in the paper by this reviewer, it is also difficult to ascertain the optimal SPE elution profile. The second paper covers the extraction of organophosphorus pesticides from natural waters by TiO2 nanotubes with subsequent separation and detection by GC-FPD. For optimal extraction efficiency 50 ml of sample, adjusted to pH 6 was passed through 6 ml PP SPE cartridge containing 0.2 g of the in house fabricated TiO2 nanotubes. The analytes of interest were then eluted in hexane, which was evaporated off and redissolved in methanol prior to GC-FPD analysis. The linear range of the method was 0.1 to 40 μg L−1 and LODs of 0.11, 0.014, and 0.0025 mg L−1, and LOQs of 0.37, 0.047, and 0.0083 mg L−1 for chlorpyrifos, phorate, and methyl parathion respectively were obtained. The method was validated with real environmental water samples and the spiked recoveries were in the range of 86.5–115.1%.5.10 Platinum
Two reviews relating to the speciation of platinum have been published in this ASU period. Unsurprisingly, both relate to the use of platinum chemotherapy agents, which remains the main area of interest in relation to the speciation of Pt. The first45 describes in some detail the overarching research that has been carried out looking at the Pt-drugs, the formation of their active derivatives, how these interact with DNA to cause apoptosis, their limitations due to side-effects and resistance, and how interaction of the drugs with different cytosolic proteins reduces their effectiveness. The speciation methods covered include: chromatography and capillary electrophoresis coupled to ICP-MS or ESI-MS. The review also deals with the analysis of human samples such as serum and the excretion products found in urine and how this can be used to assess the in vivo metabolism of the drugs. The second review considers the analytical methodologies used for metallomic based studies and covers the most common sample preparation methods that have been developed with this goal in mind, as well as molecular and elemental techniques. A range of methods are described for relevant sample matrices including: blood, urine, cell cytosol, DNA, and the drugs (cisplatin, carboplatin and oxaliplatin) and their active derivatives. The majority of studies are still based on in vitro experimentation, or incubation of the standards with synthetic DNA, which can lead to results dependent on the experimental conditions used. The review makes a pertinent point in describing the necessary in vivo studies as challenging, due to the complexity of multicellular systems and the low adduct concentrations found in human samples. From a clinical perspective, the measurement of cisplatin DNA-adducts in patient samples must be undertaken to get an understanding of the real interactions and this is only now being reported, as will be seen in the studies described below.Two methods for the analysis of in vivo human samples and of relevance to the measurement of cisplatin in the clinical setting have been reported. Determination of the cisplatin 1,2-intrastrand guanine-guanine (GG) DNA adducts, which has been reported,74 can give an indication of a patients response to chemotherapy, because cellular levels of these specific adducts can indicate cellular sensitivity. Measurement of the DNA adducts at the low concentrations present in the limited sample (<10 mL intravenous blood) volumes available from patients, requires highly specific and sensitive analytical methods. This was achieved in the study by using a highly specific enzyme-based adduct isolation method with a sensitive detection system based on HPLC-ICP-MS, using a C8 column. The values for the LOD and LOQ for the 1,2-GG adduct were 0.21 and 0.67 fmol Pt mg DNA−1. When applied to a cell-line system the method showed a statistically significant higher concentration of adduct in the sensitive cells compared to the resistant ones. When applied to human samples, the cisplatin adduct was found to be present in all patient samples taken one hour after infusion with cisplatin and ranged from 113 to 1245 fmol Pt μg DNA−1. Another method237 with potential applications in the clinical setting, used a microflow-injection chemiluminescence system with a spiral microchannel for the determination of cisplatin in human serum samples. The configuration used, enhanced the sensitivity because of the more efficient mixing of the analyte with the reagent solutions. A masking agent (1,10-phenanthroline) removed the interference of common ions found in human serum on the measurement of cisplatin via detection of PtII. The reported detection limit was 1.24 nmol L−1 as cisplatin, with a sample consumption of only 2 μL. This type of analytical approach could make possible the analysis of cisplatin on the autoanalysers commonly found in clinical laboratories.
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. Bouslimani et al.238 have reported the use of MALDI-TOF-MS for the imaging of oxaliplatin derivatives in treated rat kidney sections. The MALDI method allowed for the detection and localization of oxaliplatin and its monocysteine and monomethionine complexes in thin sections of the treated rat kidney and showed that they were located in the cortex, indicating poor drug penetration into the organ. Micro-PIXE has been used239 to examine the intracellular localization of cisplatin in esophageal cancer cell lines. The intracellular and intranuclear location of Pt was measured in cell-lines with differing cisplatin sensitivity and the results showed that the most sensitive cells had the highest intracellular Pt concentrations.
5.11 Selenium
Several reviews of methodologies and of specific applications, have appeared during the period covered by this Update. The developments made over the last decade in organoselenium speciation in biological liquids were discussed,48 focusing on sample pre-treatment procedures and separation with atomic or mass spectrometric detection. The current knowledge and future directions240 of Se bioavailability research have also been reviewed, suggesting that efforts should be made to improve techniques for species quantification, and that more reliable and sensitive biomarkers for Se status are needed, in order to assess bioavailability more accurately. The review also stresses the need for better understanding of how genotype can affect bioavailability in order to make better-informed dietary recommendations. Recent trends47 in Se speciation in biological sample matrices were discussed in a comprehensive review, covering topics from sample preparation and method development to interference removal and specific applications, focusing predominantly on IC-HPLC-MS.Discovery of a new compound, as well as a novel way to prepare a known compound for purposes of standardisation, has been reported. A new Se species was isolated and characterized from the blood and tissues of bluefin tuna via MS, NMR, and LC-ICP-MS.241 The compound, having the molecular formula C18H29N6O4Se2, has been named selenoneine after its sulfur analog ergothioneine, and is the most predominant organic form of Se in tuna tissues. Also within the timeframe of this Update, a comprehensive study52 of the chemical properties of high-purity trimethylselenonium iodide (TMSeI) has been made for the first time utilizing various techniques, including NMR spectrometry, FT-IR absorption spectrometry, ESI-MS and differential scanning calorimetry. The TMSeI was synthesized from DMSe and methyl iodide, with a purity of 99.8 ± 1.1%. The synthesized compound may be used as a new standard for analyses.
New methods for the determination of volatile species have been discussed. A method for the determination of DMSe and DMDSe in milk and milk by-products was proposed, utilizing SPME followed by GC-MIP-AED with standard additions calibration to compensate for matrix effects.242 The SPME was optimized for volume or mass of sample, ionic strength, adsorption and desorption times, and temperature. Depending on the sample, the LODs were 70–110 pg mL−1 and 80–400 pg mL−1 for DMSe and DMDSe, respectively; although none of the 23 samples evaluated had concentrations above those limits. Winkel et al.54 successfully applied HNO3 as a trapping liquid for preserving volatile Se species, followed by separation and detection by HPLC-ICP-MS and HPLC-HG-AFS. Recoveries were 65.2 ± 1.9% and 81.3 ± 3.9% for DMSe and DMDSe, respectively. Dimethyl selenoxide (DMSeO) was identified by HPLC-ESI-MS as the trapped product of DMSe, whereas MSA was the only product of DMDSe. The method proved to be simple, reproducible, robust, and widely applicable. For a number of in vitro metabolism model reactions, Gabel-Jensen et al.243 monitored the formation of methylselenol (MeSeH), DMeSe, and DMeDSe from a variety of Se compounds by direct headspace GC-MS, eliminating the loss of volatile species. Results suggested that DMeDSe may be a marker for the production of MeSeH in in vitro models, but that its volatilization may be suppressed in plasma, and so care must be taken when using the results as a model for in vivo systems. A fast and simple method114 for the separation and quantification of DMSe and DMDSe in biological samples by means of HPLC-ICP-DRC-MS was also proposed. A short RP column was used with an eluent of 40% methanol, allowing a LOD of 8 nM for both species and a separation time of under 10 min. Cancer cells were incubated with MSA, SeMet, SeMeSeCys, and sodium selenite, and results showed that DMDSe was present in some of those cells. The researchers suggest that this method is a viable alternative to analysis by GC-ICP-MS.
The characterization of Se-enriched yeast and other dietary supplements continues to be a topic of much research. Twenty seven water-extracted, Se-containing metabolites in Se-enriched yeast were separated by strong cation-exchange HPLC-ICP-MS and identified with ESI-orbital trap-MS.143 Orbital trap (Orbitrap) is a modified ion trap that uses only electrostatic fields—no RF or magnetic fields—to confine sample ions. Gradient elution by ammonium formate in 20% methanol was optimized for ICP-MS sensitivity as well as ESI compatibility. This method allowed for the first time the ability to compare Se-enriched yeast from different manufacturers in terms of these Se metabolites. Vacchina et al.244 speciated SeMet and a new SeMet precursor, 2-hydroxy-4-methylselenobutanoic acid, via ion-pairing-RP-HPLC-ICP-MS, resulting in a LOD of 1 ng mL−1. The results were validated by the analysis of CRM SELM-1 (selenium-enriched yeast), calibration by standard additions, and calculation of the mass balance. The method was applied to yeast samples, for which the conversion of 2-hydroxy-4-methylselenobutanoic acid into SeMet was monitored. Far et al.245 employed both normal phase-HPLC and HILIC to separate fifteen selenometabolites in the SEC fraction of the extract of Se-rich yeast, that has previously eluded speciation due to the coelution of salt and sulfur analogues. Optimal separation was achieved using HILIC-ICP-MS with isocratic elution and low concentration ammonium acetate buffer in 80% acetonitrile. Twelve of the 15 peaks were identified by ESI-QTOF-MS/MS. In a separate study, the safety of antioxidant food supplements was called into question with the analysis of three supplements.246 With the use of HG-AFS and ion-exchange HPLC-ICP-MS, the study concluded that the supplements were misrepresented in terms of expected Se concentration as well as the major Se species used for supplementation. Similar incongruities were found in another study71 employing a new, rapid methodology for screening Se in Se-enriched food supplements by UAE digestion-HPLC-ETAAS. Only 7 out of 10 total Se values found were in agreement with the supplement's expected value. Selenite, SeVI, SeMet, SeMeSeCys, and SeCys2 were studied in this work, and the results showed that SeMet was only detected in 3 out of 10 samples, SeIV was the major species in 2 of the 10, and 5 of the 10 samples contained other species not included in the study. The researchers suggest this method can be extended to include other Se species as well as to a wider range of Se-enriched foods and supplements for routine screening. They also suggest that stricter regulation is needed to control the quantity and chemical form of Se in these supplements.
The analysis of environmental water and soil samples has been discussed in the past year. Whole sediment samples from a freshwater lake exposed to mining and milling wastes were analysed by XAS and μXRF.161 High concentrations of elemental Se and inorganic metal selenides were found in the sediment samples. Najafi et al.247 examined environmental water and agricultural soil samples using a simple method involving electrodeposition-ETAAS, based on the selective reduction of water-soluble SeIV and SeCys2 on a mecury-coated electrode. Under acidic conditions, only SeIV and SeCys2 deposited on the electrode surface. Spike recoveries were between 91–99%, and a LOD of 1 μg L−1 was obtained. In another study, water samples and sediments from the hyporheic zone, the region of streams where ground- and surface waters meet, were analysed.248In situ geochemical measurements of surface and pore water showed that Se uptake was occurring, while μ-synchrotron-XRF of the sediment samples identified reduced elemental Se, selenides, and selenite. The results support the researcher's hypothesis that reduction in the hyporheic zone promotes sequestration of surface water Se. Liu249 extracted iSe species onto a nano-Al2O3 solid phase column for subsequent quantification by ICP-MS. The optimized eluent used was 100 mmol L−1 NaOH solution at pH 7.0. Detection limits of 6 and 11 ng L−1 for SeIV and SeVI, respectively, were obtained, and spike recoveries from real environmental water samples ranged from 80–98%.
New methods for the determination of Se in food and beverages have emerged. Total and iSe in wines were determined by anion-exchange HPLC-ICP-MS.250 Different clean-ups were investigated to remove matrix interferences. The concentration ranges of SeIV and SeVI were 1.7–8.5 ng mL−1 and 1.8–14.7 ng mL−1, respectively, representing a potential health risk. In another study, SeMet, SeCys, and SeIV were determined in chicken eggs by SEC followed by RP-HPLC-ICP-MS.251 Carbamidomethylation (i.e. alkylation of cysteine residues by reaction with iodoacetamide) of SeCys was implemented to stabilize the Se compound and prevent its loss during the sample preparation. The end products of the carbamidomethylation were identified for the first time by ES-QTOF-MS after 2D HPLC purification. Additionally, insight into the metabolism and bioavailability of these compounds was modeled by digesting the eggs with simulated gastric and gastrointestinal juices. Cubadda et al.252 identified and quantified Se compounds in wheat grain samples from India by HPLC-ICP-DRC-MS, using RP, cation-exchange, and anion-exchange columns after ultrasound-assisted enzymatic extraction. Results were validated by the analysis of wheat-based CRMs, NIST1567a wheat flour and NIST8436 durum wheat flower, and demonstrated that the samples, collected from the Nawanshahr-Hoshiarpur Region of India, contained 29–185 μg g−1, the highest Se concentration ever recorded in cereal grains.
Several studies on the conversions and transformations of Se in plant materials were published during the time period of this Update. The effect of the presence of phenolic antioxidants on the concentration and stability of Se species in plants during sample preparation and storage was investigated.53 Speciation was performed by on-line coupling of ion-exchange HPLC-ICP-MS following water and enzymatic hydrolysis, and was applied to a real buckwheat seed sample. Results from the in vitro experiments and the real sample suggest that sample preparation and storage may give rise to speciation changes and consequently inaccurate quantification. Similarly, a method was developed55 for studying the transformation of Se in buckwheat sprouts grown from seeds soaked in either SeMet, SeIV, or SeVI. The optimized procedure involved extraction by hydrolysis with 0.3 M HCl and protease. All three Se compounds were detected in the extracts, regardless of the soaking solution, but the concentrations in the extracts depended on the both the original concentration solution and the extractant. Chan et al.253 created a comprehensive Se profile, detailing total Se and both high and low MW compounds in each of the compartments of soybean plants grown in soil supplemented with sodium selenite. The Se species were separated by SEC-ICP-MS, indicating that the high MW species accounted for 82% of the total Se in the bean compartment, whereas low MW species were primarily accumulated in the pod, leaf, and root of the plant. The use of ion-pairing-RP-HPLC-ICP-MS and ESI-ion trap-MS for the determination of the low MW compounds demonstrated that the major Se species in the bean were SeMet and SeCys2, whereas the other compartments were comprised of mostly inorganic species. In a separate study, the effects of soil or foliar Se application on the accumulation of Se in Brassica species were investigated254 using a new method of HPLC-ICP-MS. High accumulation within the seeds and meal was shown, and biotransformation studies using enzymatic hydrolysis followed by HPLC-ICP-MS showed that up to 85% of the total Se was present as SeMet. Li et al.157 investigated Se uptake and speciation in rice, comparing the difference between growing under flooded versus aerobic conditions. The plants were grown in soil spiked with SeIV or SeVI, or in the absence of Se. Selenium was extracted from the grains via enzymatic hydrolysis and separated and analysed by HPLC-ICP-MS and XANES. The addition of SeIV to aerobic soil was the most effective way to enrich the rice grains with Se, SeMet being the predominant species.
Studies of the simultaneous separation and determination of multiple analytes have been reported. A new method for the simultaneous speciation of Se and Hg was developed for HPLC-ICP-MS and applied to urine and umbilical cord blood samples.101 Column switching was employed, using two different mobile phases, successfully separating SeIV, SeVI, L-SeMet, D-SeMet, SeMeSeCys, MeHg, and iHg in under 27 min. Hsieh et al.126 simultaneously speciated As and Se compounds with CE-ICP-DRC-MS. The method employed a fused-silica capillary column, with 25 mM 3-(cyclohexylamino)-1-propanesulfonic acid and 0.5 mM SDS buffer at pH 9.5. Selenite, SeVI, SeCys, SeMet, SeMeSeCys, and five As species were separated by this method, which was applied to the analysis of NIST SRM 1633a coal fly ash, NRCC DOLT-3 dogfish liver, and a Se dietary supplement. The LOD for Se was 0.5–1.4 μg L−1, and spike recoveries ranged between 91–103%. In a separate study, quantitative speciation of several As and Se species from petroleum refinery wastewaters was obtained by HPLC-ICP-MS with an octapole reaction system.209 Simultaneous separation of SeIV, SeVI, selenocyanate (SeCN−), and four As species was achieved using IC with gradient elution of 100 mmol L−1 NH4NO3 at pH 8.5, adjusted with NH3. The LODs for SeIV, SeVI, and SeCN− were 81, 56, and 75 ng L−1, respectively, while even lower limits were obtained for the As species. The method was validated by the analysis of wastewater CRMs BCR 714, 1714, and 715.
The biotransformation and metabolism of Se compounds provides important clues into the prevention and development of disease. A comparison of the toxicity, distribution, and metabolism between selenohomolanthionine (SeHLan), a newly identified selenoamino acid, and SeMet was made, using rats as a model.255 While SeMet accumulated in the pancreas, SeHLan tended to accumulate in the kidney. Urinary metabolites, on the other hand, were comparable regardless of the injected selenoamino acid, suggesting similar metabolic efficiencies and offering a potential alternative to supplementation with SeMet. Stanczyk et al.256 studied the effect of Selol 2%, a biologically active product containing organoselenium compounds in the +4 oxidation state, on the oxidation-reduction potential of lung tissue from healthy rats, comparing the changes in concentrations of thiol compounds relevant to the metabolism of glutathione as a function of the adsorbed Se. The researchers used a new method of RP-HPLC with molecular fluorescence detection to separate the 5 relevant thiol compounds (glutathione, cysteine, homocysteine, gamma-glutamylcysteine, and cysteinylglycine), while total Se in the lung tissue was determined by ICP-MS. The initial oxidative stress observed with the administration of the product was soon overcome after 4 h by anti-oxidant activity, leaving the cells at a healthier, lower potential than they had been prior to the addition, suggesting that the compound does not have a negative effect on healthy cells.
The determination of macromolecules such as DNA fragments and selenoproteins has been helped by recent advances in technology and analytical methods. A 15 kD selenoprotein was quantified in human prostate carcinoma cells and rat prostate cells of various Se concentrations using HPLC-ICP-MS.257 Rat prostate cells deficient in Se contained 1.02 ng μL−1 and human prostate carcinoma cells contained 2.07 ng μL−1 of the selenoprotein, while the prostate cells of rats with adequate Se contained 4.88 ng μL−1, suggesting a potential link between the 15 kD selenoprotein and cancer development. Jitaru et al.258 validated a clean-up for a more accurate determination of glutathione peroxidase (GPx), selenoprotein P (SelP), and selenoalbumin (SeAlb) in human serum by anion-exchange SPE followed by affinity-HPLC-ICP-MS. The identity and purity of the Se species were confirmed with MALDI-TOF-MS. In a related study, two methods72 for the determination of Se in human serum selenoalbumin were compared, the results of which quantified for the first time the concentration of Se in SeAlb, GPx and SelP within three commercially available sera. The first method, using ssIDA with RP-HPLC-collision/reaction cell-ICP-QMS following enzymatic hydrolysis, quantified the resulting SeMet, whereas the second method, using affinity-HPLC-collision/reaction cell-ICP-QMS, determined GPx and SelP. The researchers believe these sera may be used as a reference for future validation studies. Additionally, some of the same researchers259 quantified selenoproteins for the first time in the commercially available human serum BCR-637 CRM. The concentration of Se present as GPx, SelP, and SeAlb was obtained from 13 different analytical methods with affinity HPLC-ICP-MS and can be used for validation of these proteins in human serum. Letsiou et al.260 determined Se in human serum samples from healthy individuals by dual-column affinity chromatography with ID–ICP-QMS. The values obtained for Se in SelP, GPx, and SeAlb were comparable to literature values and may help to serve as a baseline for comparison for individuals with Se-related health issues.
5.12 Sulfur
Sulfur isotope ratio measurements by MC-ICP-MS are slowly becoming more routine as the traditional IRMS community becomes more aware of the capabilities and ease of use of the technique, particularly in terms of sample preparation. In a comprehensive paper demonstrating this Amrani et al.121 describe the measurement of δ34S for S containing compounds by GC-MC-ICP-MS. The combination of a six port valve and an additional make up line into the GC oven post column allowed a stream of SF6 in He to be directed to the plasma in either continuous fashion or in a timed pulsed mode. The former flow was used for instrumental tuning and optimisation whilst the pulsed mode was used to introduce transient signals for either mass bias correction or as a δ34S reference standard. Instrumental mass bias was concluded to be sufficiently stable, with a difference of 0.12‰ between SF6 peaks (n = 10), that mass bias correction standards could be introduced at intervals of greater than 20 min allowing 30 min GC runs to be used. Peak integrations used only the central part of the transient signal to improve the precision of the δ34S measurements which approached 0.1‰ for samples containing >40 pmol S and was closer to 0.5‰ for 6 pmol S which is comparable to compound specific δ34S, δ13S and δ15N measurements by IRMS. A comparison of data provided by the GC-ICP-MS method with that from LA-ICP-MS for samples of nine different organosulfur compounds exhibited excellent agreement between the results obtained by the two methods. To demonstrate the capabilities of the developed method samples of a tenfold diluted low S crude oil, a highly complex matrix which contains many co-eluting compounds, were analysed. Values for δ34S for identified compounds ranged from 1 to 24‰ with precisions in the range of 0.2 to 1.0‰, close to that obtained for pure standards. In another comprehensive paper three different analytical strategies, compound independent calibration (CIC), post-column IDMS and post-column Isotope Pattern Deconvolution (IPD) were evaluated for the determination of S-containing biomolecules.261 Four different ICP-MS instruments, two quadrupole (one with a collision cell) and two SF (one a multicollector) were used in this study. The aim of the work was to develop analytical methodologies for the characterisation of an in house prepared 34S-labelled yeast, 93% 34S, with regards to its sulfur-containing biomolecule composition. Yeast proteins were extracted with water and then hydrolysed with 3% v/v HCl at 120 °C for 10 min. The solid residue remaining after the protein extraction was also hydrolysed in the same manner. Isocratic chromatographic separations of sulfate, cysteine, glutathione and methionine were by a 150 × 2.1 mm C18 column with a mobile phase consisting of 0.3% aqueous heptafluorobutyric acid and 2% methanol adjusted to pH 2.5. A gradient HPLC method was also developed using the isocratic method as the starting conditions. For CIC sulfur detection by the quadrupole instruments was performed at mass 48 (32S16O+) whilst mass 32 was used with the SF instruments at medium resolution. For each instrument it was found that, when isocratic HPLC separations were undertaken, the instrumental response was independent of the analyte molecule and depended only upon the amount of S present. The lowest detection limit, 8 ng g−1 was obtained for the single collector sector field instrument. The collision cell ICP-Q-MS and the multicollector SF gave LODs of 21 and 25 ng g−1 respectively whilst the ICP-Q-MS gave an LOD of 42 ng g−1. However, not all of the S containing compounds eluted under these HPLC conditions so a gradient elution profile was subsequently employed rendering CIC inapplicable for further quantification. Further quantifications were performed using post column IDMS in conjunction with IPD using 33S and 34S spike solutions with a natural abundance S standard used for mass bias correction. These measurements were undertaken using a single collector SF-ICP-MS. The total S recovery for the hydrolysed protein extract was low compared with the total S content of the yeast but the work demonstrated the presence of S species highly enriched in 34S and that analytical methodologies could quantify these compounds when extracted from the yeast.5.13 Tin
The environmental distribution, fate and chemical speciation of OTC in the environment has recently been reviewed.49 However, the speciation of organotins would seem to have reached a level of maturity with few novel reports being published over the last year, perhaps also indicating that many Sn species of interest can now be determined routinely.Recently solid phase methods have been developed. A rapid and sensitive method based on SPME followed by GC-ICP-MS has been developed and validated for the determination of DMT, DBT and TBT in water samples.91 A divinylbenzene/carboxen/polidimethylsiloxane fibre was used for the SPME sample treatment. Coupling of the ICP-MS with GC was accomplished with an in-house interface. Limits of detection of 0.04 to 5 ng L−1 were achieved with linearity over two orders of magnitude for all the investigated compounds.
A method for selective determination of TBT in the presence of SnIV by combining HG-ICP-AES and SPE employing baker's yeast, Saccharomyces cerevisiae, has been proposed.262 The procedure is based on selective retention of TBT by the yeast at pH 6. A detection limit of 1.9 μg L−1 and a quantification limit of 6.3 μg L−1 were obtained. Tributyl Sn and SnIV were determined in the range of 0–25 μg L−1 and the proposed approach was applied to river water, sea water and biological extracts, with recoveries of 114, 101 and 86%, respectively.
A range of tri-organotins (TMT, TET, TPT and TBT) have been extracted from aquatic organisms using microwave-assisted extraction and CE-ICP-MS.127 Extraction was achieved within several min and LODs without any derivatization and preconcentration in the range of 0.2–0.7 μg L−1 Sn were reported. The organotins were successfully determined in dried samples of the softshell clam Mya arenaria Linnaeus and Corbicula fluminea within 17 min with a recovery of 93–104% and an RSD (n = 6) of 2–5% for spiked samples. It was suggested that dried Mya arenaria Linnaeus may be a good biomarker for organotin pollution. Ultrasonically-assisted extraction into the micelles of both non-ionic and anionic surfactants has been applied as a methodology for the isolation of TBT from fish and mussel tissue prior to determination with ETAAS.263
The use of HILIC-ESI-MS has been evaluated for the determination of TBT and TPT in water samples.113 Separation was performed in isocratic mode on an Atlantis HILIC silica (2.1 mm × 150 mm, 5 μm) column with a mobile phase of acetonitrile-0.1% aqueous HCOOH (86:14, v/v) at a flow rate of 0.2 mL min−1. Under optimum conditions, the LOD for TBT and TPT were 10 and 20 pg (injected onto the column), respectively.
5.14 Zinc
Development of ZnII compounds with insulin-enhancing activity is underway for potential use to treat diabetes. These may prove to be alternatives to well-established V compounds that can have undesirable side effects. Studies using CE-ICP-MS have been used to investigate the interaction of Zn(II)-maltolato, -2-picolinato and -2,6-dipicolinato complexes with human serum proteins.129The Zn-binding plant metallothionein E–C from wheat has been used to evaluate intermediary metallospecies that evolve during the course of metal transfer to the chelator EDTA, as a model reaction to mimic the biological function of the protein as a zinc donor.264 Zinc release from the two-domain protein E–C appears to be extremely rapid and non-cooperative, and progresses with loss of one Zn ion from the fully loaded Zn-6 species, and a transient build-up of Zn-5 and Zn-4 species, which further react to give species with 0–3 zinc ions. Additional proton NMR data provided further insights into the different behaviour of the two domains upon metal depletion.
Sediments contaminated with metalliferous minerals can be a major source of toxic metals. Zinc species in the overbank sediments from the River Geul in Belgium, which contain 4000–69,000 mg kg−1 Zn as a result of mining and smelting activities, have been determined by Van Damme et al.163 Three main Zn species were identified by powder Zn K-edge EXAFS: smithsonite (ZnCO3), tetrahedrally coordinated sorbed Zn (sorbed ZnIV) and Zn-containing trioctahedralphyllosilicate. Smithsonite is a primary mineral, which accounts for approximately 20–60% of the Zn in sediments affected by mining and smelting of oxidized Zn ores (mostly carbonates and silicates). This species is almost absent in sediments affected by mining and smelting of both sulfidic (ZnS, PbS) and oxidized ores, presumably because of acidic dissolution associated with the oxidation of sulfides. Thus, sulfide minerals in sediment deposits can act as a secondary source of dissolved metals by a chemical process analogous to acid mine drainage.
6 Macromolecular analysis
This section details a range of diverse methods for the analysis of different biomolecules using elemental techniques, with the majority focusing on ICP-MS. Easter et al.112 have developed a method for the separation and detection of oligonucleotides using hydrophilic interation liquid chromatography (HILIC) coupled to ICP-MS. Polythymidylic acids of various lengths (10–30 nucleotdes) were separated using gradient elution and detected using ICP-MS by monitoring the m/z 47 signal, due to PO+. The PO+ species were generated by using oxygen in the reaction cell and improved the sensitivity of the method by overcoming the interferences on P at m/z 31. The LODs were determined to be between 0.55–1.69 pmol for the 10 to 30 mer model oligonucleotides analysed. An on-line column switching method110 coupled to ICP-MS detection has been developed to investigate the interaction of proteins with metallodrugs. The approach combines an initial separation on a SEC column, followed by IC separation of the eluting fractions using two monolithic disks in parallel. The elution time from these disks using gradient elution was 3 min, which allowed for one disk to be eluted, whilst the second was being loaded from the SEC separation. Proteins were detected by ICP-MS using S as a hetero-atom tag, which was monitored as SO+ using oxygen in the reaction. Using this approach LODs were in the pmol range. The system was developed to monitor any metallodrug-biomolecular interaction and the feasibility was tested by looking at the interaction of cisplatin with serum samples prepared by in vitro incubation.6.1 Protein and metalloprotein analysis
Studies looking at the influence of the analytical conditions on the stability of metalloproteins during separation by gel-electrophoresis or SEC and the selection of mobile phase for combined use of HPLC coupled to elemental or MS detection, have been investigated. Jimenez et al.137 studied the effect of electrophoresis conditions on the determination of SOD and alcohol dehydrogenase by LA-ICP-MS. In metal-binding proteins with weak metal-protein affinity, metal loss can occur during the electrophoretic separation, or the post-separation gel treatment (eg blotting), but is also dependent upon the trailing ion used and the current applied. Non-denaturing approaches were best for analysis of alcohol dehydrogenase, with tricine as the trailing ion, whereas for SOD either denaturing or non-denaturing conditions could be used. The use of SEC-ICP-AES265 to assess the effect of different buffers on the Cu, Fe and Zn containing metalloproteins present in rabbit plasma, has been reported. Phosphate buffered saline (PBS) (0.15 M, pH 7.4), Tris-buffer (0.1 and 0.05 M, pH 7.4), Hepes-buffer (0.1 M, pH 7.4) and Mops-buffer (0.1 M, pH 7.4) were evaluated to determine whether redistribution of the metal ions was occurring during the separation. The Tris, Hepes and Mops buffers caused redistribution of Zn from proteins <100 kDa to the fraction >100–600 kDa. For Fe, a redistribution from the <100 kDa to the >600 kDa fraction occurred with the Hepes and Mops buffers. The PBS-buffer gave results for all three types of metalloproteins which were in agreement with other literature data for proteins in rabbit plasma and the study recommended its use for metallomic investigations on mammalian plasma. A study107 looking at four different organic modifiers (dimethylformamide, 1,4-dioxane, n-propanol and ethanol) to establish common sets of chromatographic conditions that could be used for HPLC hyphenation to ICP-MS as well as ESI-MS, has been reported. The evaluation criteria were in comparison to acetonitrile and included low solvent vapour loading, maximum analyte sensitivity and minimised carbon depositions, for ICP-MS, whilst maintaining chromatographic and ESI-MS performance. For the combined use of HPLC-ICP-MS and HPLC-ESI-MS with a common set of chromatographic conditions, n-propanol gave the best overall performance compared to acetonitrile.The analysis of superoxide dismutase (SOD) has been the focus of attention for a number of research groups, each of whom has used a different style of chromatography prior to inorganic or organic MS detection. The use of HPLC-ICP-MS266 with post column IDA based on Cu was used to quantify SOD in red blood cells. The performance of the method was compared to the enzyme activity, as determined by the spectrophotometric pyrogallol autoxidation method, and showed a good correlation between activity and protein concentration determined by IDA. Protein identification in the red blood cells was confirmed by MALDI-TOF-MS and by use of an appropriate matrix the metal-protein interactions were preserved. Deitrich et al.267 have used non-denaturing planar gel-electrophoresis LA-ICP-MS for the absolute quantitation of SOD by species-specific IDA. The stability of the metal-protein complex under non-denaturing conditions during 1-D PAGE was confirmed and the performance of the method was evaluated, showing that 4 to 64 μg SOD was quantified with a recovery rate between 82 to 110%. The extent of orthogonal diffusion in the gel was also evaluated using the isotopically enriched spike solution. Capillary electrophoresis coupled to ESI-MS or UV spectroscopy and MALDI-TOF-MS were used to study268 the different structures of bovine SOD. In all cases the conditions used lead to the loss of metal from SOD and the apo-SOD spectra were obtained.
An important clinical paper269 describes the use of LC-ID-MS for the measurement of haemoglobin A(1c) which is an important maker of glycaemic control in diabetic patients. The method used enzymatic cleavage based on a reference method and spiking with D7-labelled glycated and non-glycated hexapeptides as internal isotopic standards. The developed method was in excellent agreement with the reference method and contributes to the continued quality improvement of this important measurement area.
6.2 Tagging with metals or metal-containing labels
Biomolecular quantification strategies based on labelling with metals and subsequent analysis by ICP-MS, with parallel characterisation by molecular MS has reached a sufficient stage of maturity for the research in this area to be reviewed. The studies covered15 include: the chemical derivatization of protein functional groups with lanthanide chelates, ferrocene, organomercury compounds and iodine. Using these labelling methods detection limits for the proteins were in the attomole level and calibration was possible using simple metal standards or stable metal isotopes.Methods involving the detection of biomolecules by the use of metal-containing labels are gaining prominence, with a number of key papers published in this review period. A method270 for labelling methylcytidine and other ribonucleosides with Os was developed to measure RNA methylation. The procedure involves labelling ribose residues with Os by formation of a ternary complex between cis-diol ribose groups, hexavalent Os and tetramethylethylenediamine. The separation of Os-labelled cytidine, uridine, 5-methylcytidine and guanosine was achieved using a C18 reversed-phase column with detection of 189Os by ICP-MS. The LODs for the different labelled species ranged from 21 to 38 pmol L−1. For the detection of peptides and proteins by ICP-MS and ESI-MS271 the amino groups were labelled with succinimidylferrocenyl propionate (SFP) to give derivatives that had considerably lower polarities that those of the native analytes and could be well separated by means of RP-HPLC. The success of the procedure was demonstrated by the determination of both basic and acidic model proteins, including lysozyme, b-lactoglobulin A and insulin. The researchers also devised a dual-labelling strategy, in which the thiol groups were also labelled with a ferrocene-based reagent: in this case ferrocenecarboxylic acid(2-maleimidoyl) ethylamide.
Labelling of intact proteins with isotopically enriched metals so that high accuracy calibration using IDA and ICP-MS has been reported. Kutscher and Bettmer272 have used p-hydroxymercuribenzoic acid containing an enriched isotope, 199Hg, to label cysteine-containing proteins. The method was tested on the measurement of insulin with detection by MALDI-MS or ICP-MS. Relative and absolute quantification of the protein were described, either by using a differential labelling procedure applied to the comparative study of two different samples or by the use of species-specific IDMS. The absolute quantitation of lysozyme, insulin and ribonuclease A was demonstrated75 using 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-10-maleimidoethylacetamide (MMA-DOTA) loaded with an enriched isotope of europium, 153Eu. Quantification was achieved by HPLC-ICP-MS with species-unspecific ID ICP-MS. The labelling conditions were optimized using ESI-MS and varying reagents, pH, temperature, time and the ratio of Eu to MMA-DOPA. A comparison was made between analysis of the intact labelled proteins and the use of trypsin to digest them prior to separation and detection. Analysis of the intact proteins was found to achieve the best results, because 100% digestion efficiency was not achievable and the peptides required a higher degree of chromatographic resolution. The LODs for the three intact proteins ranged from 0.819 to 1.638 fmol.
The use of tags containing nanoparticles or quantum dots has continued. Li et al.273 have used gold-nanoparticles to label Escherichia coli 0157:H7 prior to detection by ICP-MS. This novel labelling application used antibody affinity binding gold-nanoparticle labels to rapidly detect as few as 500 E. coli cells in 1 mL of sample. The assay was highly specific for this particular pathogen due to the use of the anti-body label, provided results in 40 min. and further development would allow its use to detect a variety of other bacterial pathogens. Quantum dot (QD) labelling274 has been used in combination with immunoaffinity monolithic capillary microextraction and ICP-MS to determine human IgG. An aminopropyltriethoxysilane-silica hybrid monolithic capillary was prepared using an ionic liquid sol–gel method and onto which was immobilized a goat anti-human IgG antibody. The human IgG was isolated from human serum samples by a sandwich immunoreaction involving the immobilized antibody, followed by reaction with a second antibody labelled with CdSe QDs. The Cd and Se were released from the captured QDs by an acid (pH 2) dissolution step and the concentration of IgG determined by measurement of the eluted Cd and Se using ICP-MS with a microconcentric nebuliser. Only 50 μL serum was required for analysis giving an LOD of 0.058 μg L−1 using the Cd signal and 0.097 μg L−1 using Se. The results for some real human samples were in agreement with the more commonly used clinical method for IgG based on immunoturbidimetry.
6.3 Metallomics and quantitative proteomics
An IUPAC report275 has been published dealing with the definition of the terms “metallome” and “metallomics”. The report also covers the associated analytical techniques and methods for the systematic study of metal content, speciation and biological activity. The working group define the metallome as the “entirety of metal- and metalloid species present in a biological system, defined as to their identity and/or quantity”. Metallomics itself is defined as the “study of the metallome, interactions and functional connections of metal ions and other metal species with gene, proteins, metabolites and other biomolecules in biological systems”. The guidelines go on to discuss the analytical techniques and methods for the systematic study of the metallome, and interested readers are directed to this paper for a fuller discussion of this important area.The impact of metallomics as a subject area on its own has reached such a level that a number of reviews have been published dealing with various activities associated with the measurement of metal-containing compounds and the metallome. The use of high resolution separation methods such as capillary electrophoresis and 2-D HPLC, in combination with detection by ICP-MS, ESI-MS or MALDI-TOF-MS has been the subject of two reviews.7,9 Calibration strategies including the use of ICP-MS for absolute protein quantitation13 and the application276 of IDA with ICP-MS for quantitation, have stressed the potential for the use of elemental mass spectrometry in advancing metallomic and proteomic studies. The possibility for multiplex absolute protein quantitation277 by the measurement of heteroatom-tags, using complementary technique such as ICP-MS, MALDI or ESI-MS has also been the subject of review. The capability of using the multi-elemental features of ICP-MS was stressed, with the potential to apply this to multiplex protein determinations, with either liquid sample introduction or laser direct sampling from a solid being discussed. Clearly there is a need for multi-biomarker strategies to be developed, which can be applied to clinical, medical and pharmaceutical studies and no doubt this new area will be the subject of future work involving elemental speciation and metallomic approaches.
The application of metallomics to human health studies have started to address real clinical situations and shows how the advances in this area can be realised in the real world. The use of metallomics for the discovery of new biomarkers associated with neuroscience have been reported. Susulini et al.278 have studied human serum from patients being treated with Li for bipolar disorder, using MALDI-TOF-MS/MS and LA-ICP-MS in combination. The samples were analysed using 2-D PAGE and the metals bound to the protein spots were identified by LA-ICP-MS and the metalloproteins characterised by MALDI-TOF-MS/MS. Differentiation of the three patient groups studied was possible based on the different metalloproteins present and the report highlights the role this combination of techniques could make in biomarker discovery. The search279 for biomarkers to predict cerebral vasospasm after a subarachnoid haemorrhage has involved the use of SEC-ICP-MS followed by ESI-MS. After SEC separation, a reversed-phase chip based separation was used in combination with ESI detection to identify metal-containing species, that could be used as biomarkers. Six protein families with possible biomarker properties to indicate the on-set of vasospasm were identified from a data base search engine.
A significant paper280 describing the metalloproteome of micro-organisms makes the point that the individual metalloproteins are largely uncharacterised. A robust, metal-based approach was used to determine all the metals an organism assimilates and identify them on a genome-wide scale. This was based on high-throughput MS/MS and ICP-MS analysis to characterise the cytoplasmic metalloproteins from Pyrococcus furiosus. Of 343 metal-containing peaks in isolated chromatography fractions, 158 did not match any predicted metalloprotein. Unassigned peaks included metals known to be used, Co, Fe, Ni, W and Zn, plus metals the organism was not thought to assimilate, Pb, Mn, Mo, U and V. Further purification of 8 of the 158 unexpected metal-containing peaks yielded 4 novel Ni and Mo containing proteins, whereas the other 4 contained mis-incorporated Pb and U. It bis clear from this work that the metallome is much more diverse and extensive than previously thought.
7 Abbreviations used in this Update
| AAS | Atomic absorption spectrometry |
| AB | Arsenobetaine |
| ABS | Acrylonitrile–butadiene–styrene copolymer |
| AC | Arsenocholine |
| AEC | Anion exchange chromatography |
| AED | Atomic emission detection |
| AES | Atomic emission spectroscopy |
| AFS | Atomic fluorescence spectrometry |
| Al-Tf | Al-transferrin |
| APDC | Ammonium pyrrolidinedithiocarbamate |
| ASE | Accelerated solvent extraction |
| AsMT | Asmethyltransferase |
| BCR | Bureau Communautaire de Référence |
| BDE | Brominated diphenyl ethers |
| BFR | Brominated flame retardants |
| BH | Borohydride |
| CCA | Chromated copper arsenate |
| CE | Capillary electrophoresis |
| CEC | Cation exchange chromatography |
| CHAPS | 3-((3-cholamidopropyl)-dimethylammonio)-1-propanesulfonate |
| CIC | Compound independent calibration |
| COPR | CrIII ore processing residue |
| CRM | Certified reference material |
| CSF | Cerebrospinal fluid |
| CV | Cold vapour |
| CVG | Chemical vapour generation |
| CZE | Capillary zone electrophoresis |
| DBT | Dibutyltin |
| DDC | Diethyldithiocarbamate |
| DGT | Diffusive gradients in a thin film |
| DLLME | Dispersive liquid–liquid microextraction |
| DMA | Dimethylarsenic |
| DMDSe | Dimethyldiselenide |
| DMDTA | Dimethyldithioarsinic acid |
| DMeDSe | Dimethyldiselenide |
| DMMTA | Dimethylmonothioarsinic acid |
| DMSe | Dimethylselenide |
| DMSeO | Dimethyl selenoxide |
| DRC | Dynamic reaction cell |
| DVB | Divinylbenzene |
| EDTA | Ethylenediaminetetraacetic acid |
| EI | Electron impact |
| EPA | Environmental Protection Agency |
| ES | Electrospray |
| ESD | Element elective detection |
| ESI | Electrospray ionisation |
| ESM | Eggshell membranes |
| ET | Electrothermal |
| ETAAS | Electrothermal atomic absorption spectrometry |
| ETV | Electrothermal vaporisation |
| EXAFS | Extended X-ray absorption fine structure |
| FAAS | Flame AAS |
| FFF | Field flow fractionation |
| FI | Flow injection |
| FPD | Flame photometric detection |
| FT | Fourier transform |
| GC | Gas chromatography |
| Gd-DTPA-BMEA | Gadoversetamide |
| GE | Gel electrophoresis |
| GPx | Glutathione peroxidase |
| HDPE | High-density polyethylene |
| HG | Hydride generation |
| HILIC | Hydrophilic interaction liquid chromatography |
| HPLC | High performance liquid chromatography |
| HSA | Human serum albumin |
| i | Inorganic |
| IC | Ion chromatography |
| ICP | Inductively coupled plasma |
| IDA | isotope dilution analysis |
| IDMS | Isotope dilution mass spectrometry |
| IEF | Isoelectric focusing |
| IP6 | Phytic acid |
| IPD | Isotope Pattern Deconvolution |
| IR | Infra red |
| IRMS | Isotope ratio mass spectrometry |
| ISO | International Organisation for Standardisation |
| LA | Laser ablation |
| LC | Liquid chromatography |
| LL | Liquid liquid |
| LLE | Liquid liquid extraction |
| LLLME | Liquid liquid liquid micro extraction |
| LOD | Limit of detection |
| MAE | Microwave assisted extraction |
| MALDI | Matrix-assisted laser desorption ionization |
| MC | Multi-collector |
| MeHg | Methyl mercury |
| MeSeH | Methylselenol |
| MIP | Microwave induced plasma |
| MMA | Monomethylarsinous acid |
| MRI | Magnetic resonance imaging |
| MS | Mass spectrometry |
| MSA | Methylseleninic acid |
| MW | Molecular weight |
| NaDDC | Na diethyldithiocarbamate |
| NIST | National Institute of Standards and Technology |
| NMIJ | National Metrology Institute of Japan |
| NMR | Nuclear magnetic resonance |
| NP | Nanoparticles |
| OTC | Organotin compounds |
| PAGE | Polyacrylamide gel electrophoresis |
| PBB | Polybrominated biphenyl |
| PBDE | Polybrominated diphenyl ethers |
| PCR | Polymerase chain reaction |
| PEEK | Polyethyletherketone |
| PET | Polyethylene terephthalate |
| PIXE | Particle induced X-ray emission |
| PNP | Purine nucleoside phosphorylase |
| PP | Polypropylene |
| Ppi | Pyrophosphate |
| PS | Polystyrene |
| PTFE | Polytetrafluoroethylene |
| Q | Quadrupole |
| RoHS | Restriction of Hazardous Substances |
| RP | Reversed phase |
| RSD | Relative standard deviation |
| SDS | Sodium dodecyl sulfate |
| SeAlb | Selenoalbumin |
| SEC | Size exclusion chromatography |
| SeCys2 | Selenocystine |
| SeHLan | Selenohomolanthionine |
| SelP | Selenoprotein P |
| SeMeSeCys | Selenomethylselenocysteine |
| SeMet | Selenomethionine |
| SF | Sector field |
| SFC | Supercritical fluid chromatography |
| SPE | Solid phase extraction |
| SPME | Solid phase micro-extraction |
| SR | Synchrotron radiation |
| SRM | Standard reference material |
| ssIDA | Species specific isotope dilution analysis |
| TBAH | Tetrabutylammonium hydroxide |
| TBT | Tributyltin |
| TEM | Transmission electron microscopy |
| TeMAs | Tetramethylarsonium |
| TET | Tetraethyllead |
| TIMS | Thermal ionisation mass spectrometry |
| TLC | Thin layer chromatography |
| TMAO | Trimethylarsine oxide |
| TMSe | Trimethylselenonium |
| TMSeI | Trimethylselenonium iodide |
| TMT | Tri methyl tin |
| TMTe | Trimethyltelluronium |
| TOF | Time-of-flight |
| TPhT | Triphenyltin |
| TSE | Thiosulfate extraction |
| UAE | Ultrasound assisted extraction |
| UV | Ultraviolet |
| XANES | X-ray absorption near edge spectroscopy |
| XAS | X-ray absorption spectroscopy |
| XRD | X-ray diffraction |
| XRF | X-ray fluorescence |
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