ASU REVIEW
www.rsc.org/jaas | Journal of Analytical Atomic Spectrometry
Atomic Spectrometry Update. Elemental speciation Chris F. Harrington,*a Robert Clough,b Helle R. Hansen,c Steve J. Hill,d Spiros A. Pergantise and Julian F. Tysonf Received 5th June 2009, Accepted 5th June 2009 First published as an Advance Article on the web 23rd June 2009 DOI: 10.1039/b911133f
1 2 2.1 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.3 3 3.1 3.1.1 3.1.2 3.1.3 3.2 3.3 3.4 3.5 3.6 4 5 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 5.13 5.14
Literature resources and topical reviews Sample preparation Analyte stability Extraction methods Enzymatic extractions Liquid–liquid extractions Solid-phase extraction. Extraction from the vapour phase Derivatization Instrumental techniques and developments Developments in species separation Liquid chromatography Gas chromatography Capillary and gel electrophoresis Chemical vapour generation X-ray methods Separation-detection interfaces Detection Solid state speciation Metrology, CRMs and legislation Elemental speciation analysis Antimony Arsenic Cadmium Chromium Cobalt Copper Group I and II elements Halogens Lead Manganese Mercury Phosphorus Platinum Ruthenium
a Supra-regional Assay Service, Trace Element Laboratory, Centre for Clinical Science and Measurement, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, UK GU2 7XH. E-mail:
[email protected] b Science Research and Innovation Centre, University of Plymouth, Plymouth, UK c Department of Pharmaceutics and Analytical Chemistry, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark d Speciation and Environmental Analysis Research Group, University of Plymouth, Plymouth, UK e Environmental Chemical Processes Laboratory, Department of Chemistry, University of Crete, Heraklion, Greece f Department of Chemistry, University of Massachusetts Amherst, 710 North Pleasant Street, Amherst, MA, 01003, USA
This journal is ª The Royal Society of Chemistry 2009
5.15 5.16 5.17 5.18 5.19 5.20 5.21 6 6.1 6.2 6.3 7
Selenium Sulfur Tin Titanium Tungsten Vanadium Zinc Macromolecular analysis Metalloproteins Tagging with metals Quantitative proteomics Abbreviations used in this Update
This is the first Atomic Spectrometry Update (ASU) to focus specifically on developments in elemental speciation and covers a period of 10 months from January 2008. Other ASU reviews1–5 detail aspects of the research in this area, but our aim is to bring all of the work on this subject together in a single review. This new development comes as a result of the importance of the currency of the ASU reviews as a series. As a consequence of the continued and growing interest in the area of elemental speciation, which is evidenced by the volume of primary and review literature on the subject and the emergence of scientific meetings focusing on the topic, it was decided to instigate a radical change to the ASU series, culminating in the development of this new Update. Speciation has been considered by the International Union for Pure and Applied Chemistry (IUPAC) who have published guidelines6 for its definition, which are as follows: speciation analysis is the analytical activity of identifying and/or measuring the quantities of one or more individual chemical species in a sample; the chemical species are specific forms of an element defined as to isotopic composition, electronic or oxidation state, and/or complex or molecular structure; the speciation of an element is the distribution of an element amongst defined chemical species in a system. This review 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. Applications in the areas of environmental science, clinical and pharmaceutical analysis, food, industrial and related areas will be covered. 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 J. Anal. At. Spectrom., 2009, 24, 999–1025 | 999
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 quite literally, 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 reviews and book chapters.
1 Literature resources and topical reviews No books devoted exclusively to the topic of elemental speciation have been published in this review period but there have been on instrumentation used in speciation analysis. Two books devoted to inorganic MS, of which one dealt exclusively with ICP-MS, contained considerable coverage of relevant speciation topics, mostly of course, from the instrumentation point of view.7,8 Also of relevance from this perspective is a comprehensive treatise on FI and related techniques, that includes coverage of the many ways in which speciation information may be obtained by the judicious use of chemistry in flowing streams under controlled hydrodynamic conditions.9 Several reviews have highlighted developments in instrumentation relevant to speciation analysis. Most ‘‘conventional’’ GC detectors respond primarily to carbon and are poor at detecting ‘‘heteroatoms’’ such as As, Hg, and Se; whereas, AES can detect these elements with high selectivity over carbon. Advantages of GC coupled with AES or MS detection included a 3–5 orders of magnitude element:carbon selectivity, compound-independent calibration and the possibility to calculate partial molecular formulae.10 Emphasis was placed on real-life applications for non-metals such as N, P, S and the halogens and on the potential of combined AES and MS detection. Neubauer11 surveyed innovations in speciation analysis using HPLC with ICP-MS detection, while several other researchers12–15 reviewed developments in aspects of HPLC, though not exclusively confined to applications with element specific detection (ESD), that are of interest. Cecchi12 reviewed ion-pairing chromatography, Haddad et al.13 covered IC with some emphasis on metallomics and Chiaradia et al.16 discussed applications in food analysis. The coupling of CE with AAS (both quartz tube and electrothermal atomization) and AFS detection was reviewed by Li et al.14 Maxwell and Chen15 concentrated on the crucial topic of interface design, though their interest was in linking CE to ES-MS. Robb and Blades reviewed17 atmospheric pressure photoionization (APPI) for LC-MS, a combination with considerable potential for speciation analysis and concluded that ‘‘with further research and development, improvements in the performance, capabilities, and ease-of-use of APPI may reasonably be anticipated.’’ The growing use and application of stable enriched isotopes in elemental speciation is highlighted by reviews dealing specifically with research published in this area. Meija and Mester18 reviewed IDA in elemental speciation analysis and St€ urup et al.19 critically reviewed the applications of enriched stable isotopes as tracers in biological systems. They pointed out that the developments in collision/reaction cells and multicollector detection together with more advanced compartmental mathematical models for the distribution of elements in living organisms has led to a broader use of enriched stable isotopes in the biological sciences. 1000 | J. Anal. At. Spectrom., 2009, 24, 999–1025
There is growing interest in the application of element-specific detection (ESD) in proteomics and metallomics, both for the detection of metallobiomolecules and for the determination of biomolecules that can be ‘‘tagged’’ with a suitable element or that contain a heteroatom, such as S or P. Prange and Proefrock20 reviewed the three main strategies emerging for the application of ICP-MS to biomolecule quantification for research in the lifesciences. These included: the determination of covalently bound elements such as P and S; controlled labelling with elements that can be detected by ICP-MS using functional chelating agents; and the chemical labelling of biomolecules with nanoparticles containing elements detectable by ICP-MS. Shi and Chance,21 in discussing metallomics and metalloproteomics, pointed out that metals bound to proteins were mostly identified by ICP-MS, whereas the structure, dynamics and function of a metal-protein complex were elucidated with ES-MS. They also discussed the possibilities of obtaining relevant information from synchrotron radiation (SR) XAS and XRF and from bioinformatic sequence analysis. Schauml€ offel and coworkers22,23 evaluated the role of the determination of S, together with its quantification by IDA, for absolute protein quantification by ICP-MS. Sanz-Medel et al.24 covered somewhat similar topics in reviewing the last ten years of the development of analytical methods based on ESD for quantitative proteomics. The measurement by ICP-MS of heteroatom and isotope-tags with particular application to the screening and quantification of proteins and the study of posttranslational modifications was also reviewed.25 Thieme et al.26 reviewed several applications of X-ray spectromicroscopy which can provide spatially resolved determination of elemental composition as well as chemical speciation at distance scales down to 100 nm. They showed how soils and groundwater aquifer sediments have been imaged and evaluated on both the nm- and mm- scale and the effects of changing chemical conditions on the results. Clay dispersions, microhabitats and the morphological effects of biologically-induced redox changes of humic substances have been imaged tomographically, providing a detailed 3D picture of the distribution of organic and inorganic components. Spectral analysis for major chemical constituents could assess, for example, the different S species in an entire soil profile. While much of the material on the World Wide Web is of uncertain quality, the European Virtual Institute for Speciation Analysis (EVISA) (home page, http://www.speciation.net/) is an important source of information highlighting significant developments in the area and is also regularly updated. In 2008, the RSC launched the journal ‘‘Metallomics’’ (home page, http:// www.rsc.org/Publishing/Journals/MT/Index.asp) with the hope that metallomics will develop as an interdisciplinary science, complementary to genomics and proteomics, with the journal evolving into ‘‘the core publication for the emerging metallomics community as they strive to fully understand the role of metals in biological, environmental and clinical systems.’’
2
Sample preparation
2.1 Analyte stability With regard to the stability of As species, Conklin et al.27 investigated the effect of pH on the conversion of DMAV, MMAV and This journal is ª The Royal Society of Chemistry 2009
TMAOV to their respective sulfide forms in the presence of sulfide. They discovered that although there was very little conversion above pH 8, the mono- and di-thiolated forms, DMTAV and DMDTAV respectively, formed over the pH range 5–7, leaving less than 8% of the initial DMAV in solution. The MMAV was converted to MMTAV and an unidentified compound thought to be MMDTAV. At room temperature, the sulfide to oxide conversion rates were 0.3% to 2.2% per week with very little pH-induced instability for all three thio-arsenicals; whereas at elevated temperatures the rates were about 10% per week. Refrigeration reduced the conversion rate to amylase > cellulose. Most of the Se was conjugated or complexed with protein. 2.2.2 Liquid–liquid extraction. In addition to the traditional procedure of shaking of two immiscible phases, contact can be made with one phase supported as a ‘‘liquid membrane’’ in the wall of a tubular extractor. A review of the environmental and bioanalytical applications of hollow fibre membrane extraction devices emphasised the compatibility of various modes of extraction with subsequent separation by GC, HPLC or CE.54 Also discussed was the potential for increased efficiency of electromembrane extraction, a variation in which analytes are transported through the supported liquid membrane into the acceptor phase by the application of an electrical potential. Hu and coworkers55 developed hollow fibre liquid–liquid extraction (hollow fibre-LLE) procedures for the speciation of Hg. The extraction of OMCs was facilitated using toluene supported in the fibre wall and then into an HCl-thiourea solution. The inorganic species remained in the original liquid phase. For a 3.8 mL aliquot of sample solution, enrichment factors of 120, 215 and 350 were obtained for MeHg, EtHg, and PhHg, respectively. The procedure was applied to the analysis of human hair, sludge, seafood and environmental samples. For the analysis of hair and sludge, the recoveries for spiked samples ranged from 99 to 113%. The procedure was validated by the accurate analysis of NRCC CRM DORM-2 (dogfish-muscle). Jun and Bin56 compared hollow fibre liquid phase microextraction (hollow fibre-LPME) and dispersive liquid–liquid microextraction (DLLME), for the extraction of organosulfur pesticides from environmental and beverage samples. Both methods were found to be simple, fast, efficient and inexpensive. The advantages of DLLME technique were speed, extraction capacity and suitability for simultaneous batch treatment. For the analysis of complicated matrix samples such as soil and beverages, hollow fibre-LPME was more robust and thus more suitable. Both methods were applied to the determination of six organosulfur pesticides in waters, soil and beverage samples, from which the concentrations were below the LODs of 0.2–50 mg L1, depending on the species. Spike recoveries ranged from 82 to 114% for hollow fibre-LPME, and from 78 to 117% for DLLME. Berhanu et al.57 determined organophosphorus pesticides in ground and lake waters over the concentration range 25– 400 ng L1. When concentrations of 0.1 and 0.2 g L1 were spiked into the samples, the average extraction efficiency at the two concentrations was below 1%. It was therefore deduced that the extraction was non-depletive in nature, meaning that the freely dissolved and not the total concentrations were measured. Ito et al.58 determined chlorophenols in human urine by GC-EIMS following enzymatic de-conjugation with b-glucuronidase and sulfatase. The sample pre-treatment involved hollow fibreLPME, with in situ derivatization with acetic acid anydride, into toluene immobilized in the walls of a 1.1 cm long Accurel Q 3/2 polypropylene hollow-fibre membrane of 600 mm i.d., 200 mm wall thickness and 0.2 mm pore size. The LODs in the urine samples were 0.1–0.2 ng mL1. This journal is ª The Royal Society of Chemistry 2009
Two research groups have reported on single drop microextraction. Pena et al.59 extracted CrVI as the APDC complex into a single drop of organic solvent suspended in natural water samples. They described the procedure as ‘‘virtually solventfree.’’ For extraction times of 5 and 20 min, the preconcentration factors were 20 and 70, respectively. The LOD was 0.02 mg L1 for CrVI and the repeatability was 7% RSD. The technique was validated by the accurate analysis of BCR CRM-544 (lyophilized solution of CrIII and CrVI). Fan and Liu60 extracted MeHg and PhHg complexed with 1-(2-pyridylazo)-2-naphthol into 200 mL toluene with simultaneous back-extraction into a 4.0 mL microdrop containing 0.1% (w/v) L-cysteine. Enrichment factors of several hundred were obtained for 40 min extraction. There is interest in the possibilities of exploiting the high efficiencies of dispersive liquid extraction. In this procedure analytes are extracted from an aqueous phase into just a few mL of an immiscible solvent (e.g. carbon tetrachloride) which is dispersed into an emulsion by rapid introduction as a solution in a disperser solvent (e.g. ethanol) that is miscible with both phases. After only a few seconds of contact, the organic phase is collected by centrifugation. Birjandi et al.61 developed such a procedure for the speciation of OTCs in sea and river waters after derivatization with sodium tetraethylborate. The enrichment factors ranged from 825 to 1036 and the recoveries of spikes (10.0 and 100 ng L1, as Sn) from 82 to 105%. 2.2.3 Solid-phase extraction. During the present review period, about 40 publications have appeared describing the development and application of speciation procedures in which one species has been selectively retained on a solid-phase, usually in a micro column, to be followed by elution and quantification. The vast majority of the procedures are for the determination of CrIII and CrVI. In some procedures, a reagent was added to the sample to form selectively a product with one species that was then retained by the solid extractant. In other cases, the solidphase extractant was prepared in such as way that the surface binding sites were selective for one species. As a number of these procedures have been applied for the speciation in natural waters, the topic is also featured in the earlier ‘‘Environmental analysis’’ Update.5 The Cr speciation procedures can be divided into three broad categories: those in which CrIII was selectively retained, those in which CrVI was selectively retained and those in which one or the other (or both) were retained on the same material. Materials that retained CrIII included Amberlite XAD-1180 (the diphenylcarbazone complex),62 modified carbon nanofibres,63 used green tea leaves packed in a column64 and a CrIII-imprinted aminopropyl-functionalized silica gel.65 Materials that retained CrVI included anion-exchange resin (Dowex 1-X8),38 Dowex M 4195 chelating resin,66 Amberlite XAD-4 resin (as the diphenylcarbazide complex),67 alumina (as the diphenylcarbazide complex),68 a mixture of acid-activated montmorillonite and silica gel (as the diphenylcarbazide complex),69 tetraphenylarsonium chloride or tetraphenylphosphonium bromide immobilized on polyurethane foams70 and egg-shell membrane.71 For the last material, it was claimed that the retention was by a ‘‘reductive sorption process’’ but that CrIII was ‘‘virtually not retained.’’ Several procedures were reported in which both species were retained, not necessarily on the same extractant. Zou et al.72 J. Anal. At. Spectrom., 2009, 24, 999–1025 | 1003
speciated Cr in a sequential injection system incorporating dual mini-columns: CrIII was retained on a Chlorella vulgaris cell mini-column and the CrVI on a 717 anion-exchange resin. Moghimi et al.73 devised a procedure based on the selective formation of Cr diethyldithiocarbamate complexes at different pHs in the presence of Mn as an ‘‘enhancement agent of chromium signals’’, retention on octadecyl silica membrane disks and elution with organic eluents. The procedure also worked with the APDC complexes.74 Mahmoud et al. prepared alumina phases loaded with 2-pyridenecarboxyladehyde-thiosemicarbazone75 or thiosemicarbazide.76 Hassanien et al.77 prepared Duolite C20 loaded with 1-(3,4-dihydroxybenzaldehyde)-2-acetylpyridiniumchloride hydrazone and selectively retained CrIII at pH 6 and CrVI at pH 2. Several approaches to the determination of inorganic oxidation states have been devised. Jiang et al.78 determined AsIII in water following selective retention on an APDC-modified activated carbon micro-column. Xiong et al.79 preconcentrated iAs and iSe species from natural water samples with cetyltrimethylammonium bromide-modified alkyl silica microcolumn, which retained AsV and SeVI. Chen et al.80 preconcentrated MnII and MnVII on modified carbon nanofibres. The LODs for MnII and MnVI were 0.040 and 0.048 ng mL1, respectively, with an enrichment factor of 50. Carbon nanofibres were also used for Cr speciation.63 Ljubomirove and Djingova81 separated tetrachloroplatinate and hexachloroplatinate by SPE on a Dowex 1-X10 anion-exchange resin by sequential elution with the selective complexing agents 2,20 -bipyridyl or sodium-diethyldithiocarbamate. The recoveries from soil samples were 102 and 94% for PtII and PtIV, respectively, and the ICP-AES LOD for Pt was 15 ng g1. Pacheco et al.82 showed that SeVI could be preconcentrated from pH 3.0 solutions using L-methionine immobilized on controlled pore glass. Total Se content was determined directly by ETAAS without preconcentration and SeIV was calculated by difference. They devised a FI system with sample flow rate of 10 mL min1, and obtained an enhancement factor of 20 for a preconcentration time of 6 s. For an elution volume of 50 mL, the ETAAS LOD was 6 ng L1. This procedure was also applicable to the speciation of V,83 as VV was selectively retained at pH 5.0 whereas tV was quantitatively adsorbed at pH 9.0. The retained species were eluted with 10% HCl and measured with an LOD of 8 ng L1, by ultrasonic nebulisation (USN) coupled to an ICP-AES instrument. It was estimated that the SPE enrichment factor was 90. Huang and Hu84 speciated iTe species in seawater using ICP-MS following selective magnetic SPE. Within the pH range of 2 to 9, TelV could be quantitatively absorbed on g-mercaptopropyltrimethoxysilane modified silica coated magnetic nanoparticles, whilst TeVl was not retained and remained in solution. Quantitative recovery of the TelV was achieved using 2 M HCl and 0.03 M K2Cr2O7. Under optimal conditions the LOD obtained for TelV was 0.079 ng L1 with a precision of 7%. Procedures for the separation of SbIII and SbV have been reported. Wu et al.85 selectively retained the APDC complex with SbIII on the interior wall of a knotted tubular reactor at pH 1. Following elution with 1.5 M HCl, the Sb was determined by HG-AFS. For a sample volume of 12 mL, the enhancement factor was 17 and the LOD 2.3 ng L1. Retention of SbIII on Lproline immobilized on controlled-pore glass and subsequent 1004 | J. Anal. At. Spectrom., 2009, 24, 999–1025
HG-ICP-AES analysis gave a preconcentration factor of 11 and an LOD of 90 ng L1 for an 8 mL sample volume.86 The SbV passed through the column and was determined in the eluent. The method was validated by the accurate determination of total iSb in NIST SRM 1640 (river water). Recoveries from spiked river waters were between 83 and 111%. Zih-Perenyi et al.87 selectively retained SbIII from acid solution on iminodiacetic acid-ethyl cellulose and both SbIII and SbV on the chloride form of 2,20 -diaminodiethylamine-cellulose. The concentrations were determined by ETAAS. For a preconcentration factor of 25, the LODs in aqueous sample solutions and water samples were 0.18 and 0.25 mg L1 for SbIII and SbV, respectively. Marcellino et al.88 developed a method based on the selective retention of SbIII by modified Saccharomyces cerevisiae in the presence of SbV, for Sb speciation and SbIII preconcentration from water samples. Heating for 30 min at 80 C was defined as the optimal treatment whilst Sb accumulation by S.cerevisiae cells was independent of pH in the range 5 to 10 and ionic strength of 0.01 to 0.1 M. Only SbIII was retained on the column and then eluted with 40 mM thioglycolic acid at pH 10. After preconcentration, ninefold for SbIII, the LOD for SbIII and SbV were 2 and 5 ng L1 respectively using ICP-MS and 7 and 0.9 mg L1 using ICP-AES. The proposed method was successfully applied to the determination of SbIII and SbV in mineral water samples. The RSDs (n ¼ 3) were less than 10% at the lowest SbIII and SbV test concentrations of 0.1 mg L1. Corrected recoveries were in all cases close to 100%. Flow injection on-line sorption preconcentration coupled with HG-AFS has been described for natural water samples.85 With the sample kept at pH 1 only SbIII formed complexes with APDC and was retained on the walls of a knotted reactor in the presence of SbV. Following elution of the retained analyte with 1.5 M HCl, KBH4 solution was added for HG-AFS detection. A LOD of 2.3 ng L1 was obtained with a precision of 4.5% RSD for both species. A number of novel solid phases for the preconcentration of different elemental species have been reported. The approach used for Hg species included simultaneous retention of both iHg and MeHg by unmodified native sheep wool89 and extraction of Hg contaminants in water on to C-18 disks modified with 1,3bis(2-cyanobenzene)triazene.90 Huang et al.91 devised a method for direct speciation of dissolved inorganic and organic Se species in environmental and biological samples based on dual-column preconcentration/separation. The first column, containing nmsized Al2O3, selectively adsorbed iSe (SeIV, SeVI), which could be eluted with 0.2 M NaOH, but did not retain the organic Se (SeCys2 and SeMet). The second column, packed with mesoporous TiO2 chemically modified by dimercaptosuccinic acid, selectively retained SeIV and SeCys2 but not SeVI or SeMet. The detection limits obtained for SeIV, SeVI, SeMet and SeCys2 were 51, 120, 210, and 45 ng L1, with the enrichment factors of 5, 2, 1, and 5, respectively. 2.2.4 Extraction from the vapour phase. It is possible to extract volatile analytes from the vapour phase under appropriate conditions. Collection on an adsorbent-coated fibre from the headspace of a closed vessel is a well established procedure, known as SPME. Some researchers have reported on the extraction into a liquid-microdrop suspended in the headspace. For most samples, the extraction from the vapour This journal is ª The Royal Society of Chemistry 2009
phase is part of a multistage sample preparation in which the analytes are transferred to the liquid phase in an initial extraction step. There are several reports of the collection of analytes by SPME: two are highlighted here. Fidalgo-Used et al.92 determined the organochlorine pesticide bromocyclen in fish tissue by enantioselective GC-ECD and GC-ICP-MS after a clean-up/ enrichment procedure by SPME. Fish muscle tissue (10 g wet weight) was ground with 4-fold excess of activated anhydrous sodium sulfate until a fine powder was obtained. This mixture was Soxhlet extracted with 300 mL of hexane-acetone (1:1, v/v) for 16 h. The volume was decreased to 100 mL by vacuum rotatory evaporation, and 0.5 mL portions were then heated for 45 min at 80 1 C in the presence of the SPME fibre. Two different fibre types were compared: polydimethylsiloxane (PDMS, 100 mm) and polyacrylate (PA, 85 mm). They also compared immersion extraction, after evaporation to near dryness and dilution with methanol, with headspace extraction. Use of the PA fibre in the headspace mode gave the best results in terms of extraction efficiency. A 30 mm thickness PDMS fibre gave the highest extraction efficiencies of OMCs and OTCs from sediment and biota.93 The extraction of MeHg with different fibre types has been evaluated. For water samples, 100 mm diameter (PDMS) and 65 mm diameter PDMS-divinylbenzene (DVB) fibres gave the highest extraction efficiencies for the propyl derivative of MeHg.94 Although the extraction efficiency of the PDMSCarboxen (PDMS-CAR) fibre was similar to that of the other fibres, the desorption of the MeHg derivative was poor. The highest extraction efficiency (70%) for the PDMS fibre was obtained at 2 C immediately after equilibration at 30 C. However, for the PDMS-DVB and PDMS-CAR fibres there was no improvement in extraction efficiency at the lower temperatures. Recovery from a surface water sample spiked at 2 ng L1 was 85%. The procedure was applied to the analysis of a surface water sample containing 100 pg L1 MeHg. For fish samples digestion in KOH (25%, w/v) at 60 C for 180 min was suitable prior to propylation and headspace SPME.95 The procedure developed was validated with NRCC CRM DORM-2 (dogfish muscle). A PDMS-coated fibre in headspace mode for 30 min gave the best performance for the determination of OTCs in French brandies and wines.96 For wines, the LODs and LOQs ranged from 1 to 40 and 3 to 80 ng Sn L1, respectively, with recoveries ranging from 80 to 110%. Direct SPME for 40 min at 75 C with a 100 mm thick PDMS fibre and 20 mL sample volume was best in terms of linearity, precision and accuracy for the determination of organophosphorus and pyrethroid pesticides in aquaculture-seawater.97 Three research groups reported procedures involving extraction into a suspended liquid drop. Aguilera-Herrador et al.98 extracted trihalomethanes from the headspace of the water sample into a 2 mL drop of the ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate at 30 C for 30 min. The subsequent GC-EIMS determination featured an interface that prevented the ionic liquid from entering the column. The LODs ranged from 0.5 mg L1 for chloroform and bromodichloromethane to 0.9 mg L1 for dibromochloromethane. Pereira et al.99 collected methylcyclopentadienyl-manganese tricarbonyl from water samples in a 2.5 mL microdrop of octane exposed to the This journal is ª The Royal Society of Chemistry 2009
headspace of a 10 mL sample, containing 20% (w/v) NaCl, in a 15 mL vial, that was stirred for 20 min at 1250 rpm. A similar procedure was described by Campillo et al.100 for both cyclopentadienyl-manganese tricarbonyl and methylcyclopentadienylmanganese tricarbonyl. Prieto et al.101 devised a headspace stir-bar sorptive-extraction procedure for the determination of MeHg and butylSn species in surface water, sediment and biological tissue. Although the extraction device, a PDMS coated stir bar, was designed for extractions from the liquid phase, it was impossible to avoid the co-extraction of ethylboroxin, a by-product of the derivatization. A headspace version of the extraction was therefore developed. Several sizes of stir bar were evaluated and it was concluded that the largest and thickest stir bar (20 1 mm with 126 mL coating) was the most convenient size to accomplish the simultaneous preconcentration. Solid samples were first extracted with diluted HCl or KOH/methanol solutions and the organometallic compounds derivatized with NaBEt4. The extraction step was carried out for 5 h in a 0.1 M acetate buffer solution containing 0.1% (m/v) of NaBEt4 but no salting out reagent. The method was precise (4–17%) and gave recoveries of 80–120% at concentrations of ng L1 and pg g1. 2.3 Derivatization A rigorous classification of speciation procedures based on chemical derivatization is somewhat difficult. Many of the SPE extraction procedures described above could be considered to be derivatization procedures, as they are based in part, on the selective formation of a reaction product. For those elements that are capable of forming volatile derivatives (notably hydrides) there are possibilities for speciation procedures based on selective vapour generation reactions. These are discussed in more detail in section 3.2. Several of the SPME procedures described above involve derivatization (e.g. the formation of propyl derviatives of alkyltin species), though this does not necessarily confer selectivity for just one species as many of the analytes of interest undergo the reaction in question. In fact, this derviatization may be the key step in the analysis, for example the ethylation of MeHg and iHg. Speciation analysis based on this kind of derivatization is discussed in section 5. Bierla et al.102 applied carbamidomethylation derivatization to the determination of SeCys and SeMet. The derivatization reagent was iodoacetamide, an alkylating agent, which prevented the reformation of any disulfide, diselenide or S-Se linkages. The next step in the analysis was proteolytic release from blood proteins for subsequent determination by ion-pairing HPLC-ICP-MS. Using the method of standard additions, the LODs were 0.02 mg g1 (dry mass) for both amino acids. The procedure was applied to the monitoring of changes in SeCys and SeMet concentrations in lamb blood during supplementation studies with Se-rich yeast.
3
Instrumental techniques and developments
3.1 Developments in species separation 3.1.1 Liquid chromatography. It is not the focus of this ASU to analyse the literature from the perspective of the nature of the stationary phase. However, some relatively new chromatographic stationary phases are of interest. De Brabandere et al.103 J. Anal. At. Spectrom., 2009, 24, 999–1025 | 1005
separated organophosphorus compounds on a porous graphitic carbon stationary phase with a mobile phase composition that was compatible with both ICP-MS and ES-MS. Shigeta et al.104 separated the Se compounds in various mouse organs, red blood cells and plasma by microaffinity chromatography on a heparin stationary phase with ICP-MS detection. Hydrophilic interaction liquid chromatography (HILIC) can be described as ‘‘separation of polar compounds on polar stationary phases with partly aqueous eluents’’.105 Hemstr€ om and coworkers106 concluded that the best overall performance of a HILIC-ICP-MS system for the determination of cisplatin was achieved with 1,4-dioxane as eluent. Of all the high-boiling water-miscible solvents, this provided the best compromise between carbon deposition and separation efficiency. Dimethylformamide was suitable as mobile phase modifier and gave 36-times better sensitivity for the determination of cisplatin than acetonitrile.107 Furthermore, no additional O2 was needed to avoid carbon deposits on the cones. No unwanted side reaction products were formed, as happened with acetonitrile. Weber et al.108 separated small non-covalent metal species and free ligands in plant extracts on zwitterionic stationary phases. The ligands included amino acids and phytosiderophores, organic acids and synthetic chelators, such as EDTA. They compared the chromatographic behaviour of phytosiderophore complexes of Cu, Ni, and Zn on a sulfobetaine-type material with that on a phosphorylcholine-type material. The results showed a compression of the usable retention range for phytosiderophore species on the latter phase, which was attributed to the reversed charge orientation of the zwitterionic functionalities. There were considerable differences in the integrity of more labile metal chelates during separation on the two columns: in particular, Ni-malate could only be separated on the sulfobetaine phase; Cu-glutathione, Ni-aspartate, and Ni-citrate complexes were only stable on the phosphorylcholine stationary phase at pH 7.3; and Ni-histidine species were only found after lowering the pH to between 4 and 5. Dernovics and Lobinski109 separated Se-species in yeast-based dietary supplements on a normal bore (4.6 mm, i.d.) HILIC column which was coupled to a hybrid linear ion trap/orbital ion trap MS for detection of the Se-isotopic pattern. The procedure had the potential to characterize nine Se species in a single chromatographic run. 3.1.2 Gas chromatography. Although most target species are not directly amenable to separation by GC due to issues of volatility and thermal stability, there is still considerable potential for GC-based methodology. A number of research groups have developed ICP-MS detection for GC. Diaz-Bone and Hitzke110 developed a HG procedure for multi-element organometal(loid) speciation. The volatile derivatives were produced by HG under an optimized pH gradient. Methylated As Ge Hg, Sb, Sn and Te species could be determined simultaneously in soils, sediments, organic waste and compost samples with LOD in the sub- to mid- ng kg1 range, depending mainly on the blank concentration of the determinand. Epov et al.111 devised a method for the simultaneous determination of speciesspecific isotopic composition of Hg with a GC-multicollectorICP-MS instrument. They observed both mass-dependent and mass-independent fractionation in environmental samples; 1006 | J. Anal. At. Spectrom., 2009, 24, 999–1025
mass-independent fractionation of the odd isotopes, 199Hg and Hg, was measured in tuna fish. It was concluded that the methodology provided ‘‘new possibilities for the future study of species-specific stable isotope geochemistry of Hg and other trace metals.’’ Yuan et al.112 measured volatile As species released from Escherichia coli expressing the AsIII S-adenosylmethionine methyltransferase (arsM) gene, which they found was necessary for the production of arsines. The arsines were first cyrotrapped on a GC column before separation and ICP-MS detection. 201
3.1.3 Capillary and gel electrophoresis. Several research groups have described speciation procedures based on CE separation with MS detection, mostly ICP-MS. Yang et al.113 determined As species as a test of their new ICP-MS interface for coupling CE to ICP-MS which avoids laminar flow due to nebulizer suction and has a dead volume of only 5 nL. The eluent from the CE capillary is picked up by a damped persistaltic pump and delivered to a mixing T-piece, where the bulk of the liquid delivered to the nebulizer is merged. Assis et al.114 determined As species in horse urine, whole blood, plasma and mane hair following intramuscular injection of MMAV. Meermann et al.115 determined As species in fish that were separated by CE and detected by both ICP-MS and ES-TOF-MS. Cheng and Jiang measured cobalamin in nutritional supplements and chlorella foods116 and Br and I in foods;117 the interface was a microconcentric nebulizer. Michalke et al.118 studied Mn species in cerebrospinal fluid (CSF), also using SEC. Stern et al.119 investigated the complexation of REEs by humic acids. The study involved measuring the distribution of the elements between the humic acid material and a competing ligand, such as EDTA, whose binding constant is known. Pitois et al.120 separated REEs and detected the separated ions by ES-MS. A high fragmentation potential in the interface region gave spectra with mainly singly charged lanthanide oxide or metal ions. Keppler and coworkers121 studied the binding of anticancer metallodrugs to serum proteins by CZE-ICP-MS. Their work focused on Rubased drugs that are thought to be transported into the tumor in a protein-bound form.122–124 Li et al.125 measured various parameters relating to stoichiometry, thermodynamics and kinetics of the reaction between Cd and DNA by CE coupled to ETAAS. A coupling between a gel electrophoresis separation and ICP-MS detection has been developed to study oligonucleotide– cisplatin interactions.126 The electrophoresis system was equipped with an agarose gel housed in a glass column 30 2.2 mm i.d. The 2 mL samples were manually injected and a constant 300 V was applied during the electrophoretic separation. The negatively charged analytes eluted from the gel into the elution buffer at a flow rate of 100 mL min1 and then via a PTFE capillary to a Micromist nebulizer housed in a Scott spray chamber. 3.2 Chemical vapour generation In addition to speciation based on selective generation of volatile derivatives, CVG can be the interface between a separation, usually HPLC and ESD. As has been the case for a number of years now, there are many tens of papers describing HG of This journal is ª The Royal Society of Chemistry 2009
elements in groups 14 and 15 (4A and 5A) of the periodic table, most notably As, Sb and Se, and of Hg by ‘‘cold vapour’’ generation. The novelty is sometimes difficult to discern, but the interferences in the technique are such that every new combination of matrix and analyte species presents an analytical challenge. A considerable number of methods based on oxidation state selective HG have been reported. Matousek and coworkers127,128 devised procedures for the speciation of As by selective generation of arsine from AsIII or of the methylated arsines from the corresponding species in solution. No interference from dimethyldithioarsinic acid (DMDTAV), a newly described metabolite of iAs, was observed. The hydrides were cryogenically trapped and determined sequentially in order of boiling point as the trap warmed up. Thioglycolic acid was a faster reductant of pentavalent arsenicals than L-cysteine. A cryogenic trapping procedure was described by Xu et al.129 for the determination of As species in the urine of 104 workers who had ingested excessive levels of As in an accident caused by leakage of a pipeline in a copper-smelting factory. Matusiewicz and Krawczyk130 determined tSb and iSb species by HG and in-atomizer trapping FAAS, on the basis of conversion of borohydride-inactive species to borohydride reactive species by the action of Lcysteine. They investigated the performance of several atomtrapping devices including a slotted tube, a water-cooled single silica tube and an integrated atom trap. The LOD was 0.2 ng mL1 for a 120 s in situ pre-concentration time (corresponding to a sample volume of 2 mL). The sensitivity enhancement over conventional FAAS was 550 fold. The method was validated by the accurate analysis of NIST SRM 2704 (Buffalo River sediment), SRM 2710 (Montana soil), SRM 1633a (coal fly ash), SRM 1575 (pine needles), and SRM 1643e (trace elements in water). Different oxidation states of As and Sb has been determined by a novel disk electrochemical hydride generator coupled with AFS.131 Compared with the traditional thin-layer cell, the disk cell combined the advantages of quick assembly and easy operation and provided LODs for AsIII and SbIII of 0.031 mg L1 and 0.026 mg L1, respectively. Petrov et al.132 studied the influence of EDTA, carboxylic acids, amino-and hydroxocarboxylic acids, monosaccharides and humic substances on the generation of arsines. Ascorbic acid (0.02 M), EDTA (0.02 M), and glucose or fructose (0.2 M) equalized the sensitivities for AsIII, DMAV and MMAV, whereas glycine, malonic, tartaric acids, and soil humic extracts produced differences in the sensitivities. Cabral et al.133 developed a HG-ICP-AES procedure for the determination of SbIII in the presence of high concentrations of SbV. Two research groups developed photochemical VG procedures: Chandrasekaran et al.134 generated H2Se by UV irradiation of SeIII solutions in the presence of low molecular weight organic acids. The vapours were collected in a glass chamber before injection into the ICP-MS instrument in the form of a plug, which the researchers called the ‘‘collect and punch’’ method. Zheng et al.135 also used photochemical generation from SeIV solutions. Total Se could be determined by photochemically converting both SeIV and SeVI to volatile species using nano-TiO2 as a catalyst in a boiling water bath. There are a number of reports of the interfacing of chromatographic separations with ESD by HG. The advantages of using This journal is ª The Royal Society of Chemistry 2009
UV irradiation-HG, as the interface for Se speciation in waters by HPLC-ICP-MS included freedom from interference by Br136 as well as increased sensitivity. Reports in Chinese concerned a study137 of the binding between iAs species and bovine serum albumin (BSA) by dialysis and HPLC-AFS with HG as the interface and of the development138 of an IC-HG-AFS procedure for the determination of iAs and methylated species in gold mine tailings. Mazej et al.52 determined Se species in the leaves of chicory, dandelion, lamb’s lettuce and parsley. They separated the various species by a combination of anion and cation exchange chromatography in which the columns were connected on-line to a UV-HG-AFS detection system. As well as iSe species, the aminoacids SeMet, selenomethylselenocysteine and SeCys2 were quantified. A new improved system for automated HG-AAS using a coldtrap for the preconcentration and separation of arsines has been developed.127 Improved analytical performance was achieved by replacement of the conventional flame-in-tube atomizer with a recently developed multiple microflame quartz tube atomizer (multiatomizer). The system’s capacity to detect methylated AsIII and AsV species was verified using an in vitro methylation system containing recombinant rat arsenic methyltransferase and cultured rat hepatocytes treated with iAs. The automated HG-CT-AAS system provided an effective and sensitive tool for the analysis of all major human metabolites of iAs in complex biological matrices. In a similar study, selective HG, based on pre-reduction for differentiation of triand pentavalent arsenicals, was reported and applied for the oxidation state speciation of inorganic and methylated arsenicals with minimum sample pre-treatment using AAS with a multiatomizer.139 Preconcentration and separation of the produced arsines was carried out by means of CT. The study demonstrated that 2% (m/v) L-cysteine hydrochloride monohydrate currently used for off-line pre-reduction of pentavalent arsenicals can be substituted with 1% (m/v) thioglycolic acid in order to achieve much faster pre-reduction of pentavalent arsenicals at 25 C with equal sensitivities. The possibility of using single standard calibration, e.g. standards of AsIII, for the quantification of all other forms of As in urine was demonstrated. 3.3 X-ray methods A number of research groups have reported speciation studies using one or more X-ray spectroscopies. The techniques have become increasingly popular due to the minimal sample preparation needed and the resulting decreased risk of species transformation, as well as the increased availability of instrumentation. Several groups have access to synchrotron sources and are able to make absorption measurements that could not be made without such a source of high intensity variable wavelength X-rays. Such facilities can also allow XRF, XRD and even other spectrometric measurements, such as UV absorption to be made at the same time. The X-ray beam can be focused to allow spatially resolved measurements with several of the techniques. X-ray techniques are reviewed in another ASU in the series.3 There are issues related to possible sample damage induced by the X-rays. Ellis et al.140 studied the problem of X-ray-induced J. Anal. At. Spectrom., 2009, 24, 999–1025 | 1007
redox changes of metals in metalloprotein crystals with simultaneous macromolecular crystallography, X-ray absorption spectroscopy (XAS) and optical spectroscopy. In particular, they focused on nitrite reductase from Alcaligenes xylosoxidans, which contains both type-1 and type-2 Cu centres, and discovered that the type-1 Cu centre photoreduced rapidly, resulting in the loss of the 595 nm peak in the optical spectrum. On the other hand, the type-2 Cu centre remained in the oxidized state for much longer. They concluded that their results clearly demonstrated ‘‘the importance of using two online methods, spectroscopy and XAS, for identifying well defined redox states of metalloproteins during crystallographic data collection.’’ Several research groups have reported on the application of XANES in elemental speciation studies. Often the experiments provide spatially resolved information in conjunction with focused XRF spectroscopy. The systems that have been studied include: the oxidation states of Cr in Cr-treated wood,141 Zn in rocket plants,142 Hg and S in hair, serum and red blood cells,143 Hg migration from dental amalgams through teeth,144 As in contaminated soil,145 As in rice grains,146 As in fur and feathers,147 As in radishes,148 As in hydroponically grown cucumbers,149 or corn seedlings,150 As in iron minerals,151 As in HepG2 cells,152 Cu in contaminated agricultural soil,153 multiple elements (As, Cd, Cr, Hg, Ni and Pb) in feed coals, ashes, and stack emitted materials at a coal-burning electricity generating facility,154,155 Pb in forest floor soils,156 S in Norway spruce year rings,157 Nb in steam-corroded zirconium based alloys,158 I in soil-water systems.159 As can be seen from the comprehensive list of applications cited above, there are a number of applications of X-ray techniques to study As speciation. Meirer et al.149 used XANES to measure As species to a minimum concentration of 30 ng mL1 in cucumber xylem with no alteration of the oxidation state observed during the measurements. Analysis of xylem saps showed that AsV taken up from a nutrient solution was reduced to AsIII whilst AsIII contained in a separate nutrient solution was found to be partially oxidized to AsV. The results obtained in this study were comparable to earlier results obtained by HPLC-HRICP-MS.160 Smith et al.148 used both XANES and HPLC-ICPMS to monitor the uptake, transport and transformation of AsV in radishes (Raphanus sativus), grown in either As-contaminated mine waste or As-amended liquid cultures. The use of XAS imaging indicated that AsV, the predominant form available in the growth media, was transported from the roots to the shoots via the xylem. Subsequently, AsV was reduced by the plant and AsIII-S compound(s) accounted for the majority of As in the leaf and stem of living plants. In this study the application of synchrotron techniques permitted the identification of AsIII-S species which were ‘‘invisible’’ to conventional HPLC-ICP-MS analysis. Speciation and uptake of arsenic accumulated by corn seedlings was studied by using XAS and ICP-DRC-MS.150 Studies using XANES showed that no matter if the treatment was with SbV or SbIII, As was present as a mixture of an AsIII sulfur complex and an AsV oxygen complex. The XANES data was corroborated by the EXAFS studies showing the presence of both O and S ligands coordinated to the As atoms. A study of the interaction of As with S in biomolecules161 with HPLC-ICP-MS and XAS revealed that in the roots of mesquite (Proposis sp.) 1008 | J. Anal. At. Spectrom., 2009, 24, 999–1025
grown in soil containing 30 ppm of AsIII, only AsIII was present whereas both AsIII and AsV were found in plants grown with 50 ppm AsV. Critical complementary speciation information provided by XAS revealed that As within plants was mainly coordinated to three S atoms, with interatomic distances of 2.26 A. Other applications of X-ray spectrometry of interest have included: a study of the relative concentrations of As, Hg and Se in protein bands separated by thin-layer isoelectric focusing;162 spatially resolved measurements of Cu and Zn in leaf tissues by micro PIXE;163 the speciation of Eu on the surface of alumina by X-ray photoelectron spectroscopy, XPS;164 and the characterization, in terms of quantification of total organic and elemental C, of the surfaces of airborne particulate matter by XPS;165 the speciation of Se in river sediment cores by SR-XRF.166
3.4 Separation-detection interfaces While quite satisfactory performance by the direct coupling of the chromatographic separation to the intake of a conventional nebulizer may be obtained, this does not work for some separation techniques. This may be due to mobile phase composition or issues with flow rate incompatibility, which is particularly acute for the CE interface (papers reviewed in section 3.1.3). Several groups have investigated alternatives to conventional pneumatic nebulization. Use of USN improved the sensitivity of the determination of ultratrace Se species four-fold but gave only a two-fold improvement in LOD167 to 42 ng kg1 for the ICP-MS determination of Se in yeast. Addition of methane to the plasma gases also increased the sensitivity. Shigeta et al.104 employed a low flow total consumption nebulizer as interface between the microaffinity chromatography and ICP-MS detector. Castillo et al.168 linked micro-HPLC to an ICP mass spectrometer fitted with a micro-nebulizer for the simultaneous determination of As and Se species in environmental water samples. The LODs were in the range 0.03–0.04 mg L1 for As species and 0.35 mg L1 for Se species. Sadi et al.169 modified the interface between the HPLC and ICP-MS detector for the determination of Cd-phytochelatins in genetically modified Arabidopsis thaliana, so as to allow mobile phases with high methanol content to be used. They replaced the sample uptake and drain tubing, connectors and spray chamber O-rings with solvent resistant substitutes made of PTFE. A high-purity silica torch with a narrow injector (1.5 mm) and platinum sampler and skimmer cones were also used. The spray chamber was Peltier cooled. Chang et al.170 coupled HPLC separation using a mobile phase containing 12% methanol with ICP-MS detection by a high efficiency DIN for the speciation analysis of Hg and Pb in fish. Two research groups have reported on the development of LA applications for speciation analysis. Becker et al.171 separated metal-binding proteins in rat liver and kidney by non-denaturating gel electrophoresis and detected Cd, Cr, Cu, Fe, Ni and Pb, by scanning the gel bands using LA-ICP-MS. Lafleur and Salin172 used high performance TLC to separate Cr species that were detected by LA-ICP-MS with Si from the silica gel TLC plate acting as an internal standard. This journal is ª The Royal Society of Chemistry 2009
3.5 Detection As might be expected, there are not many reports of novel detectors. Two research groups have reported on the development of chemiluminescence (CL) detection for HPLC separations. Idowu and Dasgupta173 converted the As species in the eluent to hydrides by post-column UV irradiation followed by reaction with borohydride. Following gas-liquid separation, CL was generated by reaction with ozone in a cell mounted in front of a photomultiplier tube. For 100 mL injections, the LODs were 0.2–0.5 mg L1. Su et al.174 developed a liquid-phase CL detector based on the quenching of the luminol CL reaction by active oxygen species generated photochemically at TiO2 nanoparticles. The LODs for SeCys and SeMet were 6 and 10 mg L1, respectively. There appears to have been little reported in the recent literature about compound dependent responses of atomic spectrometry instruments, though there has been some discussion of the topic at recent conferences. Published work is often difficult to examine from this perspective, as the data for the response of the instrument to standards of the different species are often not reported, nor is it clear whether concentrations are based on the mass of the species or of the element of interest. Narukawa et al.175 found that the ETAAS sensitivity of As was 1.3-times higher for arsenobetaine than for iAs, which they attributed to differences in the mechanisms of their atomization processes, suggesting that the two species are not converted to a common form during the ashing stage and further suggesting that a chemical modifier is needed.
3.6 Solid state speciation Many of the materials that are the subject of speciation studies are solids, whereas many of the techniques that are brought to bear on the measurement challenge are only capable of accepting liquid samples. This mismatch in capability is the source of much of the development work in dissolution and extraction techniques that are reported in the literature and were reviewed in section 2.2 of this Update. There are, however, some possibilities for speciation measurements on the solid material. The various X-ray spectrometric techniques discussed in section 3.3 are all directly applicable to the solid phase and can provide useful information largely free of sample preparation artifacts, provided that the concentration of the analyte species is high enough to give an acceptable S/N. Some analytical problems are directly amenable to working with solid samples and reports on the speciation analysis of solids have been published. Shuuaeua et al.176 developed a procedure for the determination of Hg species in solids by the simple expedient of heating the sample and detecting the evolved gases with AAS. They applied their method to the analysis of lake sediments and plankton and were able to quantify HgCl2, MeHg and HgS down to LODs of about 0.2 ng. Robertson-Honecker et al.177 showed that they could speciate Cr in solids by GD-MS with a millisecond pulsed rf discharge, based on the measurement of the cluster ions specific to the various oxidation states: Cr2+ and Cr2O+ cluster ions came from CrIII in the sample whereas the CrO3+ cluster ion was due to CrVI. Malherbe et al.178 investigated the potential of rf-GD-AES for quantification of This journal is ª The Royal Society of Chemistry 2009
oxide films, with particular reference to obtaining depthresolved speciation information. They compared the preliminary results for Al and Fe with those of XRD measurements and concluded that the GD-AES technique demonstrated ‘‘excellent capabilities.’’
4
Metrology, CRMs and legislation
A major interlaboratory comparison (CCQM – P86) for the determination tSe and SeMet in pharmaceutical yeast tablets, was reported by Goenaga-Infante et al.179 Fifteen laboratories, of which ten were National Measurement Institutes, from 12 countries took part in the comparison. As no methodology was prescribed, apart from moisture content determinations, a wide variety of analytical methods were used including single-step and multiple-step enzymatic hydrolysis, enzymatic probe sonication and hydrolysis with methanesulfonic acid for the determination of SeMet. Overall, satisfactory agreement amongst participants was achieved; results averaged 337.6 9.7 mg kg1 for tSe (n ¼ 13) and 561.5 44.3 mg kg1 for SeMet (n ¼ 11). Recovery of SeMet from NRCC CRM SELM-1 (SeMet in yeast) averaged 95.0% (n ¼ 9). This year saw the release of a new CRM (CRM 7402a) by the National Metrological Institute of Japan for MeHg in Cod muscle.180 Quantification was by ssIDMS preceded by two separate extraction protocols using either acidic or alkaline methanol. Both ethylation and phenylation were used to derivatize the extracted Hg species prior to GC-ICP-MS separation of Hg species and isotope ratio measurements. The certified value given for MeHg was 0.58 0.02 mg kg1 as Hg.
5
Elemental speciation analysis
5.1 Antimony A review181 has discussed the current practice and future perspectives for Sb speciation in biological and environmental samples. Analytical methods and techniques were covered, with emphasis on identifying their main limitations, as well as highlighting advantages and recent breakthroughs. So far only inorganic SbV and SbIII, mono-, di- and tri-methylantimony species, along with four complexes of SbV with lactate and citrate ligands, and two complexes of SbIII with glutathione have been identified. The challenges identified for this element included: quantitative extraction; preservation of the analyte integrity once extracted; and low chromatographic recoveries. The authors speculated that because of the generally low extractability of Sb compounds, solid-state methods may be needed. Re-assessment of some current practises seems appropriate if further advances in Sb speciation analysis in biological samples are to be achieved. Methods for the analysis of Sb-containing drugs and drug formulations were described. Computational chemistry in combination with HPLC coupled to ICP-MS or ES-MS was applied to resolve the composition and structure of the Sb species present in dilutions of the drug Pentostam.182 By using SEC separation, it was shown that the original drug consisted of large SbV-glyconate polymers that degraded upon dilution. In dilute solution, the drug was a mixture of non-complexed SbV, large polymeric complexes, as well as several low molecular mass SbVglyconate complexes of various stoichiometry. The latter were J. Anal. At. Spectrom., 2009, 24, 999–1025 | 1009
separated on a mixed-mode chromatographic column, Obelisc N. The 1:1 complex became the most abundant low molecular mass SbV complex upon dilution. Density functional theory was applied to deduce the most likely coordination sites. Other developments included the selective determination of SbIII and tSb by HG-ICP-AES and ICP-AES for the development of formulations of meglumine antimoniate medication.133 The proposed analytical method for the determination of SbIII in the presence of large quantities of SbV provided adequate selectivity, linearity, accuracy and precision. Reflecting the growing interest in the safety issues surrounding Sb compounds in the environment a number of papers detail work in this area. The particular problems of speciation in atmospheric aerosols have been reviewed by Smichowski.183 The forms of methylated As and Sb species in pore waters were reported for the first time as part of a study on the movement of these elements across the water-sediment interface.184 An experimental approach known as the ‘sediment or fauna incubation experiment’ was used which enabled the determination of chemical speciation across redox zones in undisturbed systems. The predominant organometallic species were DMSb and DMA followed by the monoethylated species. The data indicated that methylation significantly influenced the translocation of As and Sb in sediments and that the As transformation in sediments was faster than the Sb transformation, but was more susceptible to disruption due to acidification. A recent determination of Sb species in mine waste contaminated soils using HPLC-ICP-MS reflected the difficulty of extracting Sb species from solid samples.185 Shaking with a 25 mM citric acid solution at 90 C for 15 minutes extracted 47–78% of the Sb from soils. A heated (50 C) anion-exchange column was then used with a 20 mM EDTA mobile phase at pH 4.5 and flow rate 1.5 ml min1 to separate the Sb species. Column recoveries ranged from 78 to 104%. The predominant form of Sb was SbV and little conversion of the SbV occurred during extraction. 5.2 Arsenic The study of As speciation in different human biofluids has continued to receive attention. Urine and blood samples from cancer patients being treated with high doses of arsenic trioxide were analysed using HPLC-HG-AFS and in some cases using HPLC-ICP-MS.29 The main As species found in urine were AsIII, MMAV and DMAV and in blood AsV, MMAV and DMAV. Ion pair RP HPLC-ICP-MS was used for the speciation of six As species, AsIII, AsV, DMAIII, DMAV, MMAIII and MMAV in urine by isocratic elution within less than 6 minutes.186 In addition a cation exchange column was used for separation of AB, AC, TMA+ and TMAO. The major urinary metabolites in samples taken from children living in an As-affected area in the Iron Quadrangle in Brazil, were DMAV and MMAV, whereas TMAO and AC were not detected. In most samples, MMAIII was detected up to 2.0 ng As mL1, but DMAIII was not detected in any case. Methods involving HPLC-ICP-MS were examined for the determination of phenylarsenic compounds, originating from chemical warfare agents, in the urine of patients exposed to phenylarsenic compounds via accidental pollution.187 Analysis of the urine samples from the patients indicated that the main arsenic species were DPAA and PMAA, along with some 1010 | J. Anal. At. Spectrom., 2009, 24, 999–1025
unknown arsenicals. The determination of As speciation in saliva using HPLC-ICP-MS has been reported.188 Species identities were confirmed using LC-ES-MS/MS to be AsIII, AsV and their methylation metabolites, MMAV and DMAV. These were detected in >300 saliva samples collected from people who were exposed to varying concentrations of As. The biotransformation and biochemical role of As in different biotic and abiotic models has received attention. The enhanced analytical power of using a combination of GFC and anion exchange chromatography with ICP-MS detection was demonstrated by detecting a major As-binding protein in the plasma of rats after oral administration of AsIII.189 The molecular mass was estimated to be 90 kDa based on results using GFC and the As bound to this protein was only in the form of DMAIII in the plasma. Use of SDS-PAGE and MALDI-MS, revealed that the purified protein consisted of haptoglobin (Hp) (37 kDa) distributed in three bands and the haemoglobin (Hb) alpha chain (14 kDa) found in a single band. These findings suggest that the binding-protein is a ternary DMAIII-Hb-Hp complex. Another advanced analytical approach137 applied to study the binding of AsIII and AsV to BSA involved the use of dialysis and HPLC-HGAFS at physiological pH 7.4. The speciation of As in fish samples was the focus of a study by Meermann et al.,115 using CE-ICP-MS for quantification purposes, with a spray chamber designed and built in-house and a ES-TOF-MS instrument to identify the As species AB, AsIII, AsV and DMAA. Under optimal conditions CE-ICP-MS provided higher sensitivity, typically 80 nM LOD compared with 500 nM, and better reproducibility for quantitative measurements than CE-ES-TOF-MS. Detection by SR-XRF in combination with thin-layer isoelectric focusing was developed for the study of As-containing proteins in liver tissues of carp sampled from a mercury-polluted area of Wanshan, Guizhou Province, China.162 The results showed the presence of As and Se in one of the three Hg-containing bands at pI 3.7 in bighead carp and 6.2 in grass carp. This may indicate an antagonistic function of Se against the toxicity of As and Hg. In addition, As and Hg often co-exist in the same band, suggesting that the two elements may be involved in similar metabolic processes. Recent studies have assessed As speciation in the ingredients used in East Asian and Japanese foodstuffs. Rodriguez et al.190 analysed As speciation in fish sauce samples from Thailand and Vietnam. Total As concentrations of 0.69–2.75 mg L1 were determined in six different fish sauces by ICP-MS with He as a collision gas. The AsIII or AsV concentrations were below the 0.01 mg L1 LOD of AEC-ICP-MS. The presence of AB (82 to 94% of the tAs) was revealed by CEC coupled to an ICP-MS instrument. Other species detected were AC (4.9 to 7.7%), TMAO (0.7 to 7.8%), and trimethylarsenopropionate (0.5 to 2.1%). A mass balance showed no significant difference between the total As content of the samples and the sum of the species present as detected by speciation analysis. Two fish sauce samples, which contained at least 1.5 mg L1 As, were analysed by HPLC-ES-MS to confirm the identification of As species detected by HPLC-ICP-MS. No thioAs species were detected. Although it could be concluded that the fish sauces under study pose no health risk and are suitable as a medium for iron fortification, further investigation of other sauces on the market is required. Complementary analytical techniques for As speciation This journal is ª The Royal Society of Chemistry 2009
were used by Meharg et al.146 who measured total As and individual As species in white and brown rice samples from the United States, China and Bangladesh. Analysis by SR-XRF showed that in white rice As was generally dispersed throughout the grain, whilst in brown rice As was found to be preferentially localized at the surface. The latter finding was confirmed by LA-ICP-MS. The speciation of As in the rice grains by m-XANES and AEC coupled to an ICP-MS instrument, revealed the presence of mainly iAs and DMAA. 5.3 Cadmium Speciation methods to investigate the interaction of Cd with biological macromolecules have been developed. A RP HPLCICP-MS technique was developed for the separation of Cdphytochelatin complexes (Cd-PC2, Cd-PC3, and Cd-PC4) in the plant Arapidopsis thaliana.169 After minor adjustment to the interface in order to accommodate high methanol content and to achieve optimum sensitivity for Cd detection, the following LODs were achieved: 91.8, 77.2 and 49.2 ng L1 for Cd-PC2, CdPC3, and Cd-PC4, respectively. The primary complex found in both wild-type and transgenic plants was Cd-PC2. In order to study the binding, electrophoretic behaviour, stoichiometry, thermodynamics and kinetics of the interaction of CdII with DNA, CE coupled on-line with an ETAAS instrument was developed.125 The stoichiometry for the interaction was determined to be 1:5 and two types of binding sites on DNA were observed, both, showing strong affinity of CdII to DNA. 5.4 Chromium A simple, rapid and sensitive ES-MS/MS method for the determination of CrVI in urine was developed because CrVI is a substantial component of Urine Luck, a product which is used to conceal the presence of drugs in urine.191 The method was based on the complexation of CrVI with DDC and its extraction into isoamyl alcohol in the presence of citric acid. A 1 mL aliquot of isoamyl alcohol containing Cr-DDC complex was injected into the MS-MS instrument and analysed in the FI mode. Quantification was performed using selected reaction monitoring at m/z 363.8 of the product ion CrO(DDC)2+ obtained by CID from the precursor ion CrOH(DDC)3+ at m/z 513.1. The LOD and LOQ for CrVI were 0.05 and 0.18 ng mL1 respectively, using only 10 mL of urine. Validation was carried out by analysing urine samples obtained from volunteers and CRMs such as NIST SRM 2670a (toxic elements in urine) and NIST SRM 1643e (trace elements in water). The most important advantage of this method is that it gives excellent product ion mass spectra for the identification of CrVI. 5.5 Cobalt Chen and Jiang116 used MAE and CE-ICP-MS for the determination of Co species cyanocobalamin, (CN-Cbl, vitamin B12), hydroxocobalamin (OH-Cbl), and CoII in nutritional supplements and Chlorella samples. Over 92% of the total Co species were extracted using a 5% v/v HNO3 solution within a period of 10 min with spike recovery in the range of 94 to 105% for the various Co species. The CE-ICP-MS LODs were 0.3, 0.2, and 1.7 ng mL1 (as Co) for CN-Cbl, OH-Cbl, and CoII, respectively. This journal is ª The Royal Society of Chemistry 2009
The major cobalt species in the supplements and chlorella samples was found to be cyanocobalamin. 5.6 Copper Tissue supernatants from mouse neonates bearing a mutation in the Cu-transporter gene, Atp7a, were analysed using narrow-bore HPLC-ICP-MS in a study on Cu metabolism.192 Accumulation of Cu in the intestine was found to be as the metallothioneine (MT)bound form and mRNA expression of the two MT isoforms was increased. The MT-bound Cu form was depleted in the liver and mRNA expression decreased in comparison with wild-type mice. The analyses conducted supported the suggestion that narrowbore HPLC-ICP-MS used for MT protein determination can be a complementary technique to a real time-polymerase chain reaction (PCR) used for MT mRNA determination in Cu speciation. 5.7 Group I and II elements Only one study was exclusively concerned with the metabolism of a Group I or II element. A new method for speciation analysis of Mg species occurring in rat plasma was developed using on-line coupling of CE with an ICP-AES instrument.193 Baseline separation of seven Mg-containing species was achieved by using a 120 cm (100 mm i.d.) fused-silica capillary, a 20 kV separation voltage and a solution of 50 mM acetate (pH 5.5) as electrolyte buffer. Seven Mg species were present in the rat plasma, one of which was identified as the free Mg2+ ion as confirmed by spiking experiments. One protein-bound Mg species in the rat plasma was associated with albumin and three other species were combined with globulin. 5.8 Halogens Pereira et al.194 monitored 79Br, 81Br, 35Cl and 34S in several pharmaceutical compounds by RP HPLC coupled to a multidetection system composed of UV spectroscopy, evaporative light scattering detection (ELSD) and ICP-MS. Applying the principle of post-column solvent compensation, the organic modifier content was kept constant in ELSD and ICP-MS under gradient elution. The LOQs were 80 and 2 ng mL1 for chlorpropamide and bromazepam, respectively. Shelbourn et al.195 also determined 79Br, 35Cl and 34S in pharmaceutical products using RP HPLC with both UV and ICP-MS detectors. An active pharmaceutical ingredient, containing Br, Cl and S, was hydrolysed with alkali to monitor the parent compound and two degradation products containing either Br and S, or Cl. The relative amount of each species calculated from the ICP-MS and UV chromatograms suggested that the UV method introduced a bias due to the difference in extinction coefficients for each compound. In order to maintain the conductivity of the ICP-MS plasma and a more stable system compared with use of RP HPLC, a high temperature HPLC thermal gradient method with water as the mobile phase was developed to determine the disposition and metabolic fate of 2-, 3- and 4- bromobenzoic acids following in vitro incubation with rat hepatocytes.196 The improved system stability resulted in better sensitivity, as shown by increased signal intensity for high temperature liquid chromatography compared to conventional RP separations. J. Anal. At. Spectrom., 2009, 24, 999–1025 | 1011
Cuyckens et al.197 used a multi analytical approach for studying the metabolism of a Br containing drug. A combination of ICPMS and ES-MS was deployed for the metabolite profiling and metabolite identification of a new anti-tuberculosis compound (R207910). Fluctuations in the Br sensitivity caused by the HPLC gradient were counteracted by the use of species-unspecific isotope dilution analysis (suIDA) and the results obtained were compared with those acquired via radioactivity detection of [14C]R207910. Data-dependent scanning using isotope recognition on ion trap or orbitrap MS instruments or isotope-triggered processing of Q-TOF data provided qualitative bromatograms that were similar to those obtained with ICP-MS detection. The combination of accurate mass measurements and fragmentation behaviour in the MS2 or MSn spectra allowed the structures of the main metabolites of R207910 in methanolic extracts of dog and rat faeces taken 0–24 h post-dose to be characterized. 5.9 Lead The determination of Pb in road dust has for many years attracted attention and it continues to do so, although the techniques employed have become more refined. For example, the simultaneous determination of TML and TEL in BCR CRM 605 (urban road dust) by IDA GC-ICP-MS employed 206Pb-enriched TEL and TML.198 The spike was synthesized from NIST SRM 983 a radiogenic lead isotope standard. An acetic acid-methanol extraction was used and after adjusting the pH of the extracted solution to 5, the extracted organolead compounds were derivatized using tetrabutylammonium tetrabutylborate and measured. The results obtained for TML and TEL for BCR CRM 605 were 8.22 0.04 and 1.12 0.06 mg kg1 respectively. The certified value for TML in BCR CRM 605 is 7.9 1.2 mg kg1, whereas TEL does not have a certified value. 5.10 Manganese The size distribution of Mn containing biomolecules, including proteins, peptides and carbonic acids, in serum and CSF were determined, to characterise the carriers involved in transporting Mn across the neural barrier and into the brain.118 Further identification of the Mn species in CSF was achieved successfully using CE coupled to a ICP-DRC-MS instrument. Although the size distribution of Mn-carriers in serum samples with mean Mn concentrations of 1.7 0.8 mg L1 showed a main peak in the transferrin/albumin range, a Mn peak at 700 Da, which increased with total Mn concentration, was also observed. In CSF, Mn was found to be mostly bound to low-molecular-mass Mn carriers in the range of 640–680 Da. This is similar to the low-molecular-mass compound in serum and to Mn-citrate complexes thought to be present in body fluids. The citrate concentration of 673 mM was huge compared with that of Mn. The most abundant Mn-species was Mn-citrate at a concentration of around 0.7 mg L1 (as Mn). 5.11 Mercury Several groups studied Hg speciation in various human and biological samples. Using XANES it was found that in people from China, iHg is the main species in hair (92%) while 1012 | J. Anal. At. Spectrom., 2009, 24, 999–1025
inorganic and MeHg are both about 50% of tHg in red blood cells and serum samples.143 Two methods based on hollow fibre liquid–liquid-liquid (three phase) microextraction (hollow fibreLLLME) and hollow fibre LPME (two phase) were compared for the determination of MeHg content in several matrices, amongst others in spiked human hair.55 The LODs for MeHg and Hg were 0.1 and 0.4 mg l1 respectively. Lin et al.42 studied flour samples after method validation with NIST SRM 1568a (rice flour), NIST SRM 1567a (wheat flour) and NRCC DOLT-3 (dogfish liver). Of note was the low organic content of the mobile phase. Use of a C8 column as the stationary phase and a pH 4.7 solution containing 0.5% v/v 2-mercaptoethanol and 5% v/v methanol as the mobile phase provided rapid (five minutes) chromatographic separation of iHg, MeHg and EtHg. The separated Hg compounds were converted to Hg vapours by an in situ nebulizer/VG system prior to ICP-MS detection. A MAE procedure was adopted for extraction of the Hg compounds from rice flour, wheat flour and fish samples into the mobile phase. Mercury complexes with biothiols were characterized by ES-MS equipped with an ion trap mass analyser.199 Biologically relevant thiols were separated with molecular identification of Hg and MeHg complexes derived from their reactions with cysteine (Cys) and GSH: Hg(Cys)2; Hg(GS)2; MeHgCys; and MeHgGS. Chromatographic baseline separation was performed within 10 min with formic acid as the mobile phase on a RP HPLC column and the eluent was analysed by simultaneous coupling with ES-MS and ICP-MS. When plant extracts were spiked with Hg complexes, no perturbation of the separation and detection conditions was observed, suggesting that this method is capable of detecting Hg-biothiol complexes in plants. 5.12 Phosphorus Ion pairing RP HPLC coupled to an ICP-MS instrument has been used for the determination of deoxyribonucleotides in plasmid DNA.200 An instrument with a hexapole collision-cell containing O2 was employed for the detection of 31P16O+. Optimum separation conditions for the four deoxyribonucleotides, 20 -deoxycytidine-50 -monophosphate (dCMP), 20 -deoxythymidine-50 monophosphate (dTMP), 20 -deoxyguanosine-50 -monophosphate (dGMP) and 20 -deoxyadenosine-50 -monophosphate (dAMP) were obtained using a mobile phase containing 10 mM ammonium acetate at pH 4.8 and 2.5% (v/v) methanol. The LODs were 0.211, 0.204, 0.173 and 0.225 mM for dCMP, dTMP, GMP and dAMP, respectively. The structures of organophosphorus compounds in aquatic systems are at present not well characterized although these compounds are known to be important in regulating the trophic status of aquatic systems. To identify these compounds, LC employing a porous graphite carbon stationary phase has been coupled to ICP-AES and ES-MS/MS.103 Eight different P-containing species were separated by chromatography but the MS analysis gave additional information on the structure of almost 40 different compounds, several verified as nucleotides. The characterization and quantification of organophosphorus and organonitrogen compounds in aquatic systems has recently been reviewed with emphasis placed on biogeochemical considerations and analytical challenges.201 Many aspects of analytical This journal is ª The Royal Society of Chemistry 2009
methodology were covered, including NMR and X-ray spectrometries. The detection of 12 chemical warfare agent degradation products (CWADPs) all containing P, was achieved using HPLC coupled with ICP-MS and ES-MS.202 None of the compounds were detected in natural samples. Spiking of foodstuffs with up to 10 CWADPs demonstrated LOD of 20–80 ng mL1 which were lower than or comparable to the LOD achievable with currently published LC methods. 5.13 Platinum Reviews by Bosch et al.203 and Brouwers et al.204 provided systematic surveys of the latest analytical techniques for the determination of cisplatin in biological samples and of publications describing the analysis of both Pt- and Ru- containing anticancer agents using ICP-MS detection, respectively. The majority of Pt speciation studies are directly concerned with the metabolism of Pt containing anti-cancer drugs. Other types of study included the development of a method for differentiating PtII and PtIV in soil based on SPE, sequential elution and separation by use of selective complexing agents.81 Lenz and co-workers205,206 studied the fate of cancerostatic Pt compounds in biological wastewater treatment of hospital effluents. A pilot membrane bioreactor system was installed in a hospital and fed with wastewater from the oncology ward to investigate the Pt-compound adsorption in a sewage treatment plant. Analysis using HPLC-ICP-MS showed that carboplatin was mainly present as the intact drug in both the influent and, due to low adsorption, also in the effluent of the membrane bioreactor system. Speciation studies to identify of Pt-species after incubation of a biomolecule with a metallodrug have been reported. EstebanFernandez et al.207 studied the interaction of human serum proteins, transferrin, albumin and innmunoglobulin-G and human serum with cisplatin. The detection of the proteins was conducted by AEC and simultaneous molecular detection by UV absorption (280 nm) and elemental detection (195Pt) using ICPMS. The Pt-transferrin complex was detected by simultaneous measurement of Fe, Pt and S. The nature of interactions was also investigated by ES-Q-TOF of the intact protein and a bottomup-approach was performed by analysing the Pt-protein adducts after tryptic digest by capillary HPLC-ICP-MS and ESI-Q-TOF parallel detection. Br€ uchert et al.126 presented a novel approach for the in vitro study of cisplatin interactions with 8-mer oligonucleotides. The approach was based on a recently developed coupling of continuous elution gel electrophoresis (GE) to a sector field (SF) ICP-MS instrument. The electrophoretic separation conditions allowed both detailed determination of the reaction kinetics as well as the observation of dominant intermediates. Several speciation studies concerned the metabolism of Ptdrugs. Koellensperger et al.208 used 194Pt isotopically enriched carboplatin and species-specific isotope dilution mass spectrometry (ssIDMS) for the accurate quantification of carboplatin in urine, by using an HPLC method which was compatible with both ICP-MS and ES-TOF-MS detection. The procedural LOD of HPLC-ICP-MS and HPLC-ES-TOF-MS were 0.1 ng g1 and 15 ng g1, respectively. Both methodologies were successfully applied to the quantification of carboplatin in the This journal is ª The Royal Society of Chemistry 2009
urine of a chemotherapy patient. For ICP-MS and ESI-TOFMS the total combined uncertainties were 5.7% and 23%, respectively. It was reported that cisplatin, satraplatin and its degradation product, JM118, bind irreversibly to albumin in human plasma.209 The analysis was based on total Pt measurements after different procedures of fractionation and ultrafiltration. Speciation was performed on the plasma ultrafiltrate by HPLC-ICP-MS. Sodium thiosulfate, GSH or acetylcysteine lowered the reactive Pt levels in whole blood when added, as demonstrated by reduced Pt-protein and Pt-DNA binding in the presence of the S compounds.210 Total Pt concentrations in plasma and plasma ultrafiltrate were determined by ICP-MS while the major Pt-DNA adducts were separated and measured by off-line RP HPLC with either UV or ICP-MS detection. Sar et al.211 developed a capillary RP HPLC-ICP-MS method for the detection of Pt-DNA adducts and applied it on cells of D. melanogaster flies that had been exposed in vivo to different cisplatin concentrations. Analytical performance characteristics of the capillary HPLC-ICP-MS method used to detect 31P and 195 Pt in the adduct generated by the interaction of cisplatin with a DNA oligonucleotide and after enzymatic hydrolysis was compared to those obtained using conventional HPLC-ICP-MS in a previous study. The LOD of 31P and 195Pt were in terms of absolute mass about two orders of magnitude lower for capillary HPLC (0.7 and 2.0 pg, respectively) than for conventional HPLC (2.4 and 0.1 ng, respectively). Ip et al.212 quantified the amount of oxaliplatin and its major biotransformation product Pt(R, R-diaminocyclohexane)Cl2 in the blood plasma of colorectal cancer patients given oxaliplatin and found that Pt(R, R-diaminocyclohexane)Cl2 was undetectable in patient samples, despite the fact that the RP HPLC-ICP-MS method had a low LOD of 5 nM. A separation based on HILIC was applied with ICP-MS and ES-MS detection for the determination of free intracellular cisplatin in in vitro grown T289 human malignant melanoma cells.107 5.14 Ruthenium The development of CE-ICP-MS as a tool for studying the hydrolysis of Ru anticancer drugs and their reactivity towards biomolecules was reported. Polybrene-coated fused silica capillaries were used in two different studies on Ru-containing drugs. In the first the hydrolytic stability and binding of the Ru anticancer drug candidates KP418, KP1019, and RAPTA-C to dGMP were described.123 The compound RAPTA-C was found to hydrolyse fastest and showed the highest reactivity toward the DNA model compound, whereas KP418 was the most stable in both these respects. In the second study,121 the interaction of KP1019 with human albumin and transferrin, was studied and the applicability of the method to human serum and plasma and, more importantly, to real-world patient samples was proven. A high degree of binding was noted between KP1019 and albumin in serum, plasma and the patient samples. Only minor fractions of Ru were found attached to other proteins. Polec-Pawlak et al.124 used CE-ICP-MS for evaluating and comparing the protein-reactivity patterns of the two Ru-drugs, KP1019 and KP1339. The measurements were carried out at pH 7.4 by using a separation electrolyte of 10 mM phosphate buffer and a make-up liquid of 2 mM phosphate buffer and 20 mg L1 J. Anal. At. Spectrom., 2009, 24, 999–1025 | 1013
Ge (as external correction standard). Rate constants of interaction with albumin and transferrin were determined, at pharmacologically fitting drug-to-protein ratios, as on average 0.0319 0.0021 min1 and 0.0931 0.0019 min1 (KP1019) and 0.0316 0.0018 min1 and 0.0935 0.0053 min1 (KP1339), respectively. 5.15 Selenium Several reviews on Se speciation were published in the period of this Update. Renzanka and Sigler213 provided an overview of the state-of-the-art of Se speciation in microbial cells, fungi and plants. Falandysz214 reported Se speciation in edible mushrooms, and Gammelgaard et al.215 focused their review on the complementary use of molecular and element-specific mass spectrometry for the identification of Se-containing compounds related to human metabolism. In the last of these reviews, Se compounds were only considered identified if molecular mass spectra for the authentic biological sample had been provided. Orga216 discussed the advantages and disadvantages of the coupled mass spectrometric techniques and Morales et al.217 reviewed the recent progress in the field of CE coupled techniques for the identification of Se species. Schauml€ offel23 elegantly demonstrated how ICP-MS coupled to separation techniques such as SEC and GE via LA, can be employed at different steps in the proteomic workflow for sensitive ESD (e.g. for S and Se). Emphasis was placed on the coupling of CE and nanoHPLC to ICP-MS instrumentation, by demonstrating that element-specific peptide mapping using nanoHPLC-ICP-MS is the key technique, particularly in combination with peptide sequencing via nanoHPLC-ES-MS. The measurement of Se containing macromolecules in plasma has been described. Shigeta et al.104 injected intravenously 82Seenriched SeIV into mice fed Se-adequate and deficient diets and studied the time-dependent changes in the distribution of the enriched Se in organs, red blood cells and plasma. Speciation analysis was conducted by micro-affinity chromatography (heparin) coupled with low-flow ICP-MS. The Se-containing proteins selenoalbumin and GPx peaked at 1 h and quickly decreased from 1 to 6 h after injection, whereas selenoprotein P peaked at 6 h and gradually decreased from 6 to 72 h after injection. Xu et al.218 separated five Se species (selenoprotein P, GPx, selenoalbumin, and two unknown selenospecies (U1 and U2) in a pooled plasma sample from five healthy people by using AEC and a 35 min gradient elution program. The relative proportions of the species, measured by HPLC-ICP-DRC-MS using suIDA, were 45.5%, 19.1%, 15.1%, 2.9% and 8.1% respectively. To quantify the proteins, an enriched 77Se spike solution was mixed continuously into the chromatographic effluent to change the isotope ratio of 80Se:77Se. In order to keep the flow stable and mixed sufficiently, a flow rate of 20 mL h1 was adjusted using an accurate syringe pump. Methane was employed as a reaction gas in the DRC to achieve the determination of 80Se free of spectroscopic interference from 40Ar2+ and 79 BrH+. Studies concerned with Se metabolism in cells have been reported. Cuello et al.219 investigated the potential protective effect of MeSeCys against a chemical oxidative stress induced by tert-butyl hydroperoxide in human hepatoma HepG2 cells. 1014 | J. Anal. At. Spectrom., 2009, 24, 999–1025
Speciation of Se derivatives by using RP HPLC-ICP-MS indicated MeSeCys as the only selenocompound in the cell culture. Cell viability and markers of oxidative status were evaluated and the results showed that pre-treatment of cells with MeSeCys for 20 h in the nM to mM range conferred a significant protection against this oxidative insult. Gabel-Jensen et al.220 studied the metabolism of methylseleninic acid (MSA), in the gastrointestinal tract by incubation of MSA with homogenized intestinal epithelial cells from pigs. The major Se-containing metabolite was identified by analysis of the supernatant by RP HPLC-ICPMS. The identity of the compound S-methylselenocysteine was established by LC-ES-MS/MS after purification by preparative chromatography. The usefulness of isotopically enriched Se sources in metabolic speciation studies was demonstrated. Suzuki et al.221 who gave a single oral dose of 10 mg Se kg1 body weight each to find the in vivo bioavailability and organ distribution of three isotopically enriched Se sources, 76Se-MeSeCys, 77Se-MSA and 82Se-selenite. Before the start of the experiment, the rats had been depleted of natural abundance Se by dosing with a single isotope, 78Se. Recovery of the three Se sources in the major organs/tissues/ blood was determined 3 h after the oral administration. The distribution of isotopically enriched-Se species was determined for the liver supernatant on two columns (SEC and RP) by HPLC-ICP-MS. The study revealed that MeSeCys was more efficiently distributed in organs than MSA in its intact form, suggesting that MeSeCys is the best MMSe source in most organs/tissues. The same group investigated the in vitro methylation and demethylation of glutathione-reduced forms of 76 Se-selenite, 77Se-MSA and 82Se-DMSeO, in rat organ supernatants and homogenates.222 The resulting chemically labile reaction products, selenide, MMSe and DMSe were detected indirectly by speciation analysis with HPLC-ICP-MS after oxidation with 150 mM sodium periodate in the presence of 1% (v/v) methanol. It should be noted that MMSe has never been detected directly, possibly due to its instability. The timerelated changes in Se isotope profiles showed that demethylation of MMSe to selenide was efficient but that of DMSe to MMSe was negligible. The methylation of selenide to MMSe and MMSe to DMSe was efficient but that of DMSe to TMSe occurred less efficiently. The interest in Se speciation in yeasts continues. The combination of elemental and molecular MS is at the forefront of modern Se speciation studies, as demonstrated by the studies of Dernovics and Lobinski.109,223 In the first study, six of the eight Se-derivatives of glutathione identified had not previously been reported. Species from the aqueous extract of a selenized yeast sample were subjected to SEC and the fractions selected for further analyses were, after pooling from multiple SEC runs, frozen and lyophilised. The AEC-HPLC-ICP-MS chromatogram of the most intense SEC fraction produced seven peaks containing eight Se compounds. Subsequently, the Se containing compounds were identified by HPLC coupled to a Q-TOF-MS instrument, used in either full-scan TOF mode, or in product ion MS/MS mode. The entire method was optimized under a strict mass balance control protocol. In the second study, HILIC was coupled with a hybrid linear ion trap/orbital ion trap MS. Quantitative elution recovery was verified by online ICP-MS. Confirmation that all the species present were found was This journal is ª The Royal Society of Chemistry 2009
achieved by the parallel use of AEC HPLC-ICP-MS optimized for maximum resolution. Tastet et al.34 combined 2D-GE, HPLC-ICP-MS and HPLC-ES-MS/MS to identify Se-containing proteins in an aqueous Se-rich yeast extract. An ultrasonic probe was employed for fast protein extraction avoiding sample heating and thus reducing the risk of protein degradation. The efficiencies of different extraction steps were critically evaluated by total Se analysis and SEC-ICP-MS. The subsequent analyses of selected digests by HPLC-ES-MS/MS allowed amino acid sequences to be attributed to peaks detected by ICPMS. This revealed the presence of two Se-containing proteins: stress-induced protein 18 (SIP 18) and heat-shock protein 12 (HSP 12). In a separate paper36 the same group identified a further 17 Se-containing proteins after 2D GE and analysis by LA-ICP-MS, HPLC-ICP-MS or Q-TOF-MS, operated in TOF and MS/MS modes. Calibration using IDMS techniques have been applied to assess the Se speciation in food products. Goenaga-Infante et al.167 reported the measurement of ultratrace g-glutamyl-SeMetSeCys species of relevance to cancer research in extracts of garlic by HPLC-ICP-MS. Quantitation was by ssIDMS using a 77Seenriched g-glutamyl-SeMetSeCys spike, produced in-house from an aqueous extract of 77Se-enriched yeast. The LOD for 78Se was 42 pg g1. Kirby et al.224 used a suIDMS technique and HPLCICP-MS to assess the Se speciation in food products. Enzymatic digested samples were injected onto a C8 RP column and eluted with an isocratic mobile phase consisting of 10% methanol (v/v) and 0.1% (v/v) heptafluorobutanoic acid at a flow rate of 1 mL min1 and a column temperature of 25 C. Identification of Se species occurred through retention time comparisons with available standards. An enriched 76Se spike (5 mg L1) was continually mixed at a flow rate of 0.1 mL min1 with the eluent of the HPLC column through a T-piece prior analysis. The 78/76Se isotope ratio was determined after the measurements of 78Se and 76 Se had been corrected for the interferences introduced by 77 Se1H+ and 75As1H+ by means of using specific intensity equations. The information on Se species was used to gain an understanding of the bioavailability in biofortified and processfortified wheat biscuits used in a clinical trial. The major Se species identified were SeMet and SeMet, selenoxide, respectively. The biotransformation of Se by bacteria (Lactobacillus) and yeast (Saccharomyces), in lactic fermentation process of yogurt and kefir was investigated.225 Following enzymatic extraction, Se speciation profiles were obtained by AEC and ion-pairing RP HPLC with ICP-MS detection. The SEM analysis of enriched samples revealed the presence of segregated Seo when the added concentrations were >5 mg g1. The main Se species formed depended on the type of microorganism involved in the fermentation process, SeCys2 and MeSeCys being the main species generated in yogurt and SeMet in kefir. The Se species were stable for at least 10 and 15 days in kefir and yogurt, respectively. After this period, the SeCys2 concentration decreased whereas that of MeSeCys increased significantly. An analytical method was developed by Bierla et al.226 in order to discriminate between the quantity of selenoproteins containing SeCys and other proteins containing SeMet. The method was applied to monitoring the Se speciation in tissues of chicken and lamb,226 lamb blood102 and whole milk of This journal is ª The Royal Society of Chemistry 2009
cows227 during supplementation studies with Se-enrich yeast or iSeIV. The analytical procedure was based on protein unfolding (with urea), derivatization of SeCys and SeMet by carbamidomethylation using iodoacetamide followed by quantitative proteolysis. In all studies, the mixture of the derivatized Seamino acids was purified by SEC and analysed by ion-paring RP HPLC-ICP-MS. The SeIV present in the samples was derivatized as well and was determined along with the selenoaminoacids. The origin and identity of species were identified by the co-elution with the SeIV, 77SeMet and with a synthetic standard of carbamidomethylated selenocysteine. The method of standard additions was used to quantify SeMet, SeCys and SeIV and the method was validated by the determination of the Se mass balance.
5.16 Sulfur There is interest in the determination of S, particularly with the relatively low resolution ICP-MS. In the measurement of thioarsenicals in urine by HPLC-ICP-MS, the use of Xe as a collision gas gave a much improved LOD down to about 10 ng g1 for S at m/z 32 than that obtained with He, presumably because of the greater efficiency with which 32O2+ was dissociated.228 A study on the role of S in arsenic cycling found that the oxidation of AsIII was linked to the presence of S-oxidising bacteria and that the production of AsV was greatly enhanced by the addition of sulfide or thiosulfate.229 The rate and extent of these processes was linked to the concentration of bacteria and the reducedS:AsIII ratio. Studies by IC-ICP-MS also showed that mono- and di- thioarsenate formed in lake water amended with both AsIII and sulfide. A new strategy for the determination of S drugs and biothiols used silver nanoparticles capped with different functional groups as the matrix and affinity probes, in surface-assisted laser desorption/ionization MS and atmospheric pressure MALDIMS.230 Biothiols adsorbed on the surface of silver nanoparticles through covalent bonding were subjected to UV radiation that enabled desorption and ionization. The proposed method was successfully applied to the determination of cysteine and homocysteine in human urine samples using an internal standard (penicillamine). The LOD and LOQ for cysteine and homocysteine were 7 and 22 nM. The advantages of the method over other methods for biothiol analysis were; simplicity, rapidity and sensitivity without the need for time-consuming separation and tedious preconcentration processes. Such an approach could also be used in conjunction with atomic spectrometric detection of S, potentially with excellent analytical figures of merit. The speciation of S in marine cloud droplets and particles has been studied as part of the Marine Stratus Experiment using a range of microanalysis techniques.231 These included SEM with energy dispersive analysis, TOF-SIMS and scanning transmission X-ray microscopy with near edge XAFS. Two distinct classes of S-containing particles were identified: chemically modified sea salt particles and secondary-formed sulfate particles. The results also indicated a substantial heterogeneous replacement of chloride by methanesulfonate and non-sea-salt sulfate. J. Anal. At. Spectrom., 2009, 24, 999–1025 | 1015
Volatile S species were determined in ecological samples such as guava leaves using pulsed FPD detection and GC-EIMS232 and in Norway spruce using SR-XAS.157 In the latter study, functional groups were identified using S compound spectra as fingerprints. Inorganic sulfide, organic sulfide, methylthiol and highly oxidized S were reported. The authors compared the data with total S content profiles from ICP-AES and reported better accuracy and temporal resolution using SR-XAS at the sulfur L-edge in total electron yield detection mode. The presence of hydrogen sulfide has been shown to enhance the emission signal for sulfide when determining sulfide and sulfate in natural waters.233 The generation of the hydrogen sulfide took place in a coil in which sulfide reacted with HCl. The contribution of sulfide was obtained by subtracting the emission intensity measured straight in the water sample from that measured in acid medium. Heilmann and Heumann234 developed a suIDA method based on GC-ICP-MS for the routine quantification of S species in petroleum products. A special dosing unit was designed to deliver 34S-labeled dimethyldisulfide to allow determination without any matrix effects from coeluting hydrocarbons. 5.17 Tin An evaluation of a combined fractionation and speciation approach for the study of size-based distributions of OTCs in environmental matrices associated with landfill leachate, utilized asymmetric flow field-flow fractionation with multi detection and off-line speciation using headspace SPME linked to GC with pulsed FPD.235 Two distinct colloidal populations were characterized: organic rich and inorganic colloids with gyration radii up to 120 nm. Total tin and the MBT and DBT species were distributed over the whole colloidal phase although TBT, MMT, MOctT, and DPhT were also detected. The occurrence of OTCs in leachate and biogases from landfill sites was investigated by using GC-ICP-MS to determine up to nine different organotin compounds in the leachate at concentrations in the 0.01–6.5 mg L1 range.236 Five volatile tin species, including methyltin, mixed methyl-ethyltin, and ethlytin, were found at concentrations up to 25 mg m3. Two main parameters were suggested as being important in terms of influencing the organotin composition: first, wet deposition may induce leachate dilution and alter methylation/ethylation mechanisms; second, evolution of the waste degradation stage yields different volatilization mechanisms (i.e. permethylation or perethylation). Van et al.237 investigated the transformation of phenyltin species during sample preparation of biological tissues with multi-isotope spike ssIDA and GC-ICP-MS in which three isotope-enriched phenylSn compounds were used. The degradation of phenyltin compounds was influenced by the acidity of the extractant, the presence of complexing reagents and the use of ultrasonic or microwave radiation. An integrated passive sampler consisting of a 47 mm C-18 Empore disk as the receiving phase overlaid with a thin cellulose acetate diffusion membrane has been developed for the measurement of time-weighted average water concentrations of DBT, MBT, TBT and TPhT in water.238 Once accumulated in the receiving phase the compounds were stable over prolonged periods. The device was successfully evaluated in field trials in conjunction with GC and either ICP-MS or FPD. Headspace 1016 | J. Anal. At. Spectrom., 2009, 24, 999–1025
SPME and GC ion trap MS/MS have been employed to measure OTCs in river sediments.239 The technique used a one-step procedure based on tetraethylborate in situ ethylation, although a leaching/extraction step was also required to extract the OTCs from the sediments. The LODs of 0.3, 1.0 and 0.4 pg g1 for MBT, DBT and TBT, respectively, compared favourably with those obtained by other methods. The use of GC-MS/MS to determine OTCs in water, sediment and gastropods was reported.240 The methodology and problems associated with the speciation of OTCs in marine sediments has been reviewed by Staniszewska et al.241 Following an incident in which a worker exposed to DMT compounds exhibited neurological symptoms similar to those of TMT encephalopathy, speciation analysis of the tin compounds in urine was performed by HPLC-ICP-MS.242 High concentrations of DMT and time-dependent increase in TMT concentrations were found in both mice and rats exposed only to DMT trichloride during the 4-day treatment. Both concentrations decreased gradually after the cessation of treatment. These results confirmed the production of TMT in vivo, probably through methylation of DMT. In addition, an HPLC-MS/MS analytical method for the detection of TMT in human urine was developed and applied to confirm the presence of TMT in the patient’s urine.243
5.18 Titanium Different HPLC separations followed by ICP-MS detection were used to study the speciation of Ti in human serum.244 The development of such analytical methods is extremely important for improving our understanding of the effects of Ti found in patients wearing prostheses or of the biochemical pathways of this metal when used as an anticancer drug. This work confirmed that transferrin (Tf) is the only carrier protein binding Ti in human blood serum samples. The quantitative speciation of Ti– Tf in standards and human blood serum samples was achieved using IDA. An important finding of this study was that chemical degradation of metalloproteins occurred during analytical separations thereby nullifying the quantitative speciation data. This study demonstrated for the first time the usefulness and potential of a ssIDA approach for investigating quantitatively metal-protein associations which can be dissociated under certain experimental conditions.
5.19 Tungsten The coupling of SEC to an ICP-MS instrument using 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 150 mM NaCl at pH 7.4 as the mobile phase, proved to be a suitable methodology for the screening of tungsten-protein complexes.245 Analysis of human serum incubated with tungstate resulted in three analytical peaks, one related to tungstate-albumin binding, one to free tungstate and one to an unknown protein (MW > 300 kDa). The association of tungstate with albumin and the other unknown protein was confirmed by MALDITOF-MS analysis of the tungsten-containing fractions collected from SEC-ICP-MS chromatograms, after desalting and preconcentration. This journal is ª The Royal Society of Chemistry 2009
5.20 Vanadium 246
IV
V
Aureli et al. employed HPLC-ICP-MS to measure V and V in natural mineral waters. Cationic and anionic VIV and VV species were converted into V-EDTA complexes which were separated effectively by AEC. Pre-column EDTA complexation also prevented species interconversion as a resulting from loss of CO2 from samples and their exposure to atmospheric oxygen. The method, which was tested on samples encompassing a wide range of potential interfering ions and total dissolved solids, provided accurate and reproducible results according to mass balance budgets, mean recovery of 100% (range 96–107%, RSD 4%). Recoveries from spiking experiments were in the range 98–103%. The LODs were 0.16 and 0.025 mg L1 for VIV and VV, respectively. The predominant species in PET-bottled mineral water samples was VV. Species distribution was consistent with theoretical predictions based on thermodynamic equilibria of V in water. The potential of high resolution MS for speciation analysis with no prior separation was demonstrated by Qian et al.247 who determined the elemental formulae for the entire family of vanadyl, VO and VOS porphyrins in a complicated asphaltene matrix. The instrument used APPI and FT-ICR-MS. 5.21 Zinc Laursen et al.248 used absorption (416 nm), fluorescence (excitation 420 nm fluorescence 589 nm) and XRF (40 kV, 40 mA) for the quantitation of Zn-protoporphyrin (Zn–pp), which was identified as the major pigment in certain dry-cured meat products (Parma ham, Iberian ham and dry-cured ham with added nitrite). The Zn–pp was extracted from the hams by homogenization in distilled water with subsequent centrifugation of the homogenate followed by filtration, extraction into acetone and isolation on a disposable C18 column, (Sep-Pak). The LODs for all methods were 1 mg kg1. No significant difference in Zn–pp concentrations, 15.6 and 14.3 mg kg1 for Parma and Iberian ham respectively, was found for the hams which all contained Zn–pp. This study also focused on XRF determination of Fe in the extracts and, as no detectable Fe was found in the three types of ham extracts investigated (LOD Fe 1.8 mg kg1), the authors concluded that iron containing pigments, e.g. haem, do not contribute to the noticeable red colour observed in some of the extracts.
6 Macromolecular analysis 6.1 Metalloproteins The relationship between metalloprotein distributions and cancer development in human liver tissues was investigated.249 Proteins in the cytosol and microsome of tumour and surrounding nontumour tissues from five individuals with hepatocellular carcinoma were separated with isoelectric focusing. The contents of Cu, Fe, Mn and Zn in protein bands were measured with SRXRF. In the cytosol, the metal contents in protein bands from tumour tissues were generally less than those from corresponding non-tumour tissues, especially in the Fe-containing bands with isoelectric points of 6.5 and 8.0 and the Cu, Zn-containing band of 5.5. It was possible to distinguish between tumour and non-tumour tissues on the basis of the metal distributions This journal is ª The Royal Society of Chemistry 2009
among the detected metal-containing bands separated by isoelectric focusing. The direct isotopic labelling of a metalloprotein complex in vivo was achieved in order to measure ferritin-bound Fe.250 The spike material 57Fe-Ferritin was produced by cloning and overexpressing the Phaseolus vulgaris ferritin gene pfe in Escherichia coli in the presence of 57FeCl2. Recombinant ferritin contained approximately 1,000 Fe atoms per molecule at an isotopic enrichment of more than 95% 57Fe. The high transfer efficiency (20%) of enriched iron into 57Fe-ferritin, the absence of species conversion of the isotopic label for at least 5 months of storage at 20 C, in addition to the fact that negligible amounts of nonferritin-bound iron existed in the purified 57Fe-ferritin solution made this spike suitable for quantification of ferritin-bound Fe. Such methods are essential in order to assess more accurately the Fe-storing capacity of ferritin, as opposed to estimating it by measuring the concentration of the protein shell of the molecule in fluids and tissues. The glycated form of haemoglobin could be measured by ICP-MS with LOD of 1 mg mL1 protein following separation by CEC and detection of the Fe contained within the haem group of the protein.251 Furthermore, Fe naturally present in glycated and non-glycated Hb, was determined accurately by suIDA using isotopically enriched 57Fe (92.4%) as the spike material. The proposed method was validated by determining glycated and non-glycated Hb (physiological levels about 100–130 g L1) in RMs commonly used to calibrate clinical analysers and LGC Promochen RM 405 (glycated Hb in human haemolysate). Protein-metal complexes of Cd, Cr, Cu, Fe, Ni, Pb and Zn present in rat tissues (liver and kidney) were separated in their native state in the first dimension by blue native PAGE and detected by scanning the gel bands using LA-ICP-MS with and without collision cell as a microanalytical technique.171 A Pb-containing protein band was detected at around 65 kDa by LA-ICP-MS. Several proteins were identified by using MALDITOF-MS together with a database search, but the protein binding Pb was not identified. An on-line IDA method in combination with SEC-ICP-TOF-MS was used to study the cytosolic speciation of the metals Cd, Cu, Ni, Pb and Zn, in the liver of the European eel.252 The distribution of the metals among cytosolic fractions displayed strong differences; whereas Cd, Cu and Zn were largely associated with the MT pool, Ni and Pb were not as might be expected from the liver tissue concentrations. A preliminary speciation study on metalloproteins in CSF was presented by Ellis et al.253 who used HPLC-ICP-MS as an initial screening tool for any ultimate metal species differences (Cu, Fe, Mg, Pb, and Zn) in different patient populations. Samples of CSF were pre-fractionated in three different molecular weight ranges (MW < 5 kDa, MW 5–50 kDa, MW > 50 kDa) by using spin concentrators with cutoff points of 5 and 50 kDa. Two columns (C18 and anion-exchange fast protein LC) were investigated for the separation of the metals in CSF using a capillary LC system. Fractions of interest from the HPLC-ICP-MS analysis, were further characterized by the novel application of the commercially available microfludic nanoLC-CHIP/ion trap-MS instrument and a data base system was used for identifying proteins. A possible iron associated protein, suggested as receptor activity-modifying protein, RAMP 2, was identified. J. Anal. At. Spectrom., 2009, 24, 999–1025 | 1017
6.2 Tagging with metals A number of research groups have developed speciation procedures for the indirect determination of biomolecules by forming derivatives containing readily determined metals. Ahrends et al.254 called their procedure ‘‘the metal-coded affinity tag approach’’ to quantitative proteomics. Peptides and proteins were tagged with different monoisotopic lanthanide ions by reaction with the macrocyclic metal chelate complex, 1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), linked to the cysteine residues. The macrocyclic metal chelate complex, loaded with different lanthanide (III) ions, was the essential part of the tag. The combination of DOTA, an affinity anchor for purification and a reactive group for reaction with amino acids, constituted a reagent that allowed quantification of peptides and proteins in an absolute fashion. For quantitative determination, the tagged peptides and proteins were analysed using FI-ICPMS, a technique that allowed detection of metals with high precision and sensitivity (LOD for BSA was 110 aM). This approach was used to analyse proteins of the Sus scrofa eye lens as a model system. Patel et al.255 derivatized bradykinin and substance P with cyclic diethylenetriaminepentaacetic anhydride that was subsequently labelled with Eu3+. Relative quantitation was achieved by differentially labelling two peptide sources using natural and enriched 151Eu, respectively. The 151Eu:153Eu isotope ratio was measured by HPLC-ICP-MS and used to calculate the original peptide ratio. Razumienko et al.256 devised metalloimmunoasays with element-tagged antibodies for proteins and also for the DNAbinding activity of transcription factor p53 in leukemia cell lysates through its interaction with oligonucleotides immobilized in microplate wells. The study demonstrated the applicability of ICP-MS to proteomic studies through the use of antibodies directly labelled with polymer tags bearing multiple Ln atoms, thus enabling fast and quantitative identification of multiple analytes in a single well. Guo et al.257 were able to determine sulfhydryl and disulfide bonds in peptides and proteins by reaction with the monofunctional organic mercury ion RHg+ as a MS tag, which they pointed out, had the advantages of reacting with one sulfhydryl group, thereby offering a well-defined definite mass shift and a stable characteristic isotopic distribution pattern. In the labelling of ovalbumin with p-hydroxymercuribenzoic acid, Kutscher et al.258 obtained a molar ratio of label to protein of 4:1, which would be expected on the basis of the four free sulfhydryl groups from cysteines. Jakubowski et al.259 labelled three protein, BSA, chicken egg white lysozyme and porcine gastric mucosa pepsin with 127I using a commercially available reaction kit containing the stable isotope to allow detection by LA-ICP-MS after separation by SDS-PAGE. The labelling procedure did not affect mobility because the change of the molecular weight was relatively small for the proteins investigated. Seuma et al.260 developed a LA-ICP-MS procedure for imaging cancer biomarkers in tissue sections in which the relevant proteins were reacted with Au/Ag tagged antibodies. The distribution of two breast cancer-associated proteins, MUC-1 and HER2, was studied based on multiple line rastering of tissue sections and measurement of relevant Au/Ag tagged antibodies bound to the tissue. Application to the quantitative assessment 1018 | J. Anal. At. Spectrom., 2009, 24, 999–1025
of HER2 expression in tissue microarrays was demonstrated. Roos et al.261 also used LA-ICP-MS to detect electrophoretically separated cytochromes P450 by element-labelled monoclonal antibodies. Antibodies for two enzymes were differentially labelled with Eu via a covalently linked chelator and with I. Multielement labelling strategies (multiplexing) seem to provide some promising results for ICP-MS applications in quantitative proteomics. In one application the labelling of the two different proteins, BSA and hen egg white lysozyme, with the commercially available chelating compound DOTA was investigated.262 The assay developed was optimized to detect proteins labelled with stable isotopes of Eu, Ho and Tb and separated by SDS-PAGE. Detection has been performed by LA-ICP-MS after electroblotting of the target proteins onto nitrocellulose membranes. A range of total protein amounts from 0.015 pM (BSA) to 105 pM (lysozyme) were covered. A calibration was performed for BSA in the range from 0.015 to 15 pM and a LOD of
