Spectrochimica Acta Part B 59 (2004) 135–146 Review Inductively coupled plasma mass spectrometry and electrospray mass spectrometry for speciation analysis: applications and instrumentation Amy L. Rosen, Gary M. Hieftje* Department of Chemistry, Indiana University, Bloomington, IN 47405, USA Received 15 May 2003; accepted 23 September 2003 Abstract To gain an understanding of the function, toxicity and distribution of trace elements, it is necessary to determine not only the presence and concentration of the elements of interest, but also their speciation, by identifying and characterizing the compounds within which each is present. For sensitive detection of compounds containing elements of interest, inductively coupled plasma mass spectrometry (ICP-MS) is a popular method, and for identification of compounds via determination of molecular weight, electrospray ionization mass spectrometry (ESI-MS) is gaining increasing use. ICP-MS and ESI-MS, usually coupled to a separation technique such as chromatography or capillary electrophoresis, have already been applied to a large number of research problems in such diverse fields as environmental chemistry, nutritional science, and bioinorganic chemistry, but a great deal of work remains to be completed. Current areas of research to which ICP-MS and ESI-MS have been applied are discussed, and the existing instrumentation used to solve speciation problems is described. 䊚 2003 Elsevier B.V. All rights reserved. Keywords: Speciation; Review; Inductively coupled plasma mass spectrometry (ICP-MS); Electrospray ionization mass spectrometry (ESI-MS) 1. Introduction It is a well-known fact that the toxicity, bioavailability, and transport properties of an element are highly dependent upon its chemical form. In the past, total quantification of an element was used to determine potential health hazards or benefits, but in recent years, more and more research groups have acknowledged that elemental quantification alone is not sufficient. To completely understand the ways in which particular elements will affect living organisms, it is necessary to determine the species within which the elements are found, and to quantify those species. Analysis performed to identify andyor quantify one or more distinct chemical species in a sample is known as speciation analysis w1x. Procedures for speciation analysis aimed at determining the species distribution of a particular element in a sample involve detection of compounds containing the trace *Corresponding author. Tel.: q1-812-8552189; fax: q1-81285509588. E-mail address: email@example.com (G.M. Hieftje). element of interest, preceded or followed by identification and characterization of those compounds. ‘Hyphenated’ techniques, coupling a separation method with a sensitive detection method, are often applied to obtain speciation information for trace metals and metalloids in biological and environmental systems w2–5x. It has become common practice in recent years to use inductively coupled plasma mass spectrometry (ICPMS) for ultra-sensitive detection of trace metal- and metalloid-containing compounds. For identification of those compounds that are detected by ICP-MS, electrospray ionization mass spectrometry (ESI-MS) has been gaining increasing popularity. Because traditional ESIMS reveals only the molecular weight of a compound, electrospray ionization tandem mass spectrometry (ESIMSyMS) has been increasingly used for structural characterization. ICP-MS, ESI-MS, and ESI-MSyMS provide complementary information (see Fig. 1), and when all three techniques are utilized for analysis of a sample, a complete picture of the species distribution for a given element within that sample can be obtained. 0584-8547/04/$ - see front matter 䊚 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.sab.2003.09.004 136 A.L. Rosen, G.M. Hieftje / Spectrochimica Acta Part B 59 (2004) 135–146 Fig. 1. Schematic diagram illustrating the contributions of various analytical techniques to the accomplishment of speciation analysis, as well as relationships between the techniques. Both ICP and ESI ion sources have been coupled with a number of different mass analyzers, including quadrupole, ion trap, double focusing, and time-of-flight mass spectrometers. The quadrupole mass analyzer has been the most frequently used, although each mass analyzer has its own advantages. The field of speciation analysis has come to incorporate a wide variety of analytical techniques applied to an even wider variety of biological, environmental and industrial applications. A number of excellent reviews have been written on various aspects of speciation analysis w3,5–9x, and entire symposia and journal issues have been dedicated to the subject. It is by no means the intention of the present work to provide a review of the literature from the entire diverse field. Rather, the intent is to provide a review of those problems within the field of speciation analysis that have been addressed by using ICP-MS and ESI-MS, and to provide a brief overview of ICP-MS and ESI-MS instrumentation as it is applied to speciation analysis. Although low-power and reduced-pressure ICP sources have been used by some researchers, the scope of this paper will be limited to conventional atmospheric-pressure ICP-MS. Emphasis will be placed upon the advantages of using ICP and ESI sources coupled with multidimensional separation techniques and non-scanning mass analyzers for a wide variety of applications, and upon recent trends in the field. 2. Current focal areas in speciation analysis 2.1. General fields of interest Speciation analysis is becoming increasingly important and necessary in a number of fields w4,10x (see Fig. 2). Applications within environmental chemistry include the identification of toxic metal- and metalloid-containing species in sites that have been contaminated by industrial or natural sources, as well as determination of successful clean-up strategies. Because the bioavailability and thus toxicity of elements such as arsenic and tin are highly species-dependent, total element determination is not a good indication of the threat that a contaminated site presents to the public. Identification of those chemical species that are harmful to living things is necessary to develop reasonable, chemically specific regulations for industrial waste w4x, and advances in analytical techniques are necessary to efficiently monitor this waste for contaminants. Finally, knowledge of the species present and their chemical behavior is A.L. Rosen, G.M. Hieftje / Spectrochimica Acta Part B 59 (2004) 135–146 137 Fig. 2. Fields of study within which speciation analysis has had and will continue to have significant impact. required in order to develop effective strategies to clean up contaminated sites. The fields of health and nutrition can also benefit tremendously from the information that speciation analysis provides w11x. A great number of elements are known to be essential to human nutrition and survival, including iron, copper and zinc, which are required at trace levels, as well as such elements as selenium, cobalt and manganese, which are necessary in ultra-trace amounts. Likewise, many elements, such as arsenic, mercury and lead, are known to be quite toxic to humans. However, the potential of these elements to be harmful or beneficial is highly dependent upon their speciation w11,12x. It is thus of great interest to know the species of essential and toxic elements that occur naturally in foods, as well as those that are present in vitamins and food supplements. Many current standards issued by the American Food and Drug Administration (FDA) w13x, and the National Academy of Sciences Institute of Medicine (IOM) w14x, as well as international health organizations such as the Food and Agriculture Organization and World Health Organization (FAOy WHO) w15,16x, rely only on total element concentration to determine if a particular food or beverage product is safe to consume. Identification of those particular species that are harmful to humans, as well as improvement of techniques to accurately and efficiently detect those species, will foster the development of more appropriate safety regulations, and will enable the critical evaluation of natural and synthetic supplements. Furthermore, identification of those particular chemical forms of essential elements that are easily absorbed and metabolized will aid in the establishment of more useful nutrition guidelines as well as the development of more effective supplements. Finally, advances in speciation analysis will indisputably have a tremendous impact on the field of bioinorganic chemistry w2,3,12x. Once a species enters an organism, it has the potential to undergo a great many chemical transformations, some of which may radically alter its properties. Determination of chemical species throughout the metabolic pathway and within various bodily tissues and fluids can provide information about the biological mechanisms for cellular uptake, transport, storage and excretion. Furthermore, identification of species within the body containing toxic elements can provide information about the ways in which organisms have adapted to sequester, accumulate, or detoxify otherwise harmful elements. These ideas can be extended to include therapeutic drugs as well w17x. The identification of drug metabolites is important to understand the mechanisms of medications already in use and to aid the development of more effective medications. 2.2. Essential elements Studies published in the literature regarding detection and identification of compounds containing essential elements have focused on trace essential elements, such as iron, copper and zinc, as well as ultra-trace elements such as cobalt, manganese and selenium. Major focus areas have included the identification of metalloproteins and metalloenzymes, with subsequent analysis of their structure and function w6,18x, the detection of biological 138 A.L. Rosen, G.M. Hieftje / Spectrochimica Acta Part B 59 (2004) 135–146 metal-complexing ligands w19–21x, and the differentiation between oxidation states of metals such as chromium, for which the 3q oxidation state is essential and the 6q oxidation state is toxic w11x. Common samples include foodstuffs, bodily tissues such as liver and kidney, and bodily fluids such as blood, urine, breast milk and amniotic fluid w3x. In recent years, a large number of research groups have turned their focus towards selenium, and a wealth of literature has become available on Se speciation. A review on selenium speciation by hyphenated techniques has recently been published w22x. The discovery that selenium may help prevent certain forms of cancer has prompted a pronounced interest in the mechanisms by which it functions w23–25x. Furthermore, the discovery that individuals in many countries have a diet deficient in selenium has spurred the introduction of a wide variety of selenium supplements. A number of research groups have analyzed these supplements w23,24,26–28x as well as natural sources of selenium w28–30x to determine the species of selenium present, and others have studied bodily fluids w31–33x and mammalian tissue w34x to elucidate the mechanisms by which selenium is metabolized and excreted. In addition to studies of the utilization of selenium, pathways of toxic action are of interest as well, since the margin between the nutritionally required and toxic amounts is very narrow relative to those observed for other elements w35,36x. Work performed thus far has identified the amino acids selenomethionine, selenoethionine, and selenocysteine as likely cancer-preventative species w28x, although a number of selenoamino acid derivatives, selenium-containing glutathione derivatives, and smaller organoselenium species have been detected and identified in selenium-enriched yeast w23,26,27x and in human urine w31,33x. Improvements in the detection limits of ICP-MS have resulted in the detection of over 30 distinct selenium-containing compounds in a single yeast extract. However, most of these compounds remain unidentified due to the unavailability of standards and the relatively poor detection limits of ESI-MS relative to those of ICP-MS w26x. 2.3. Toxic elements A great deal of research has been performed to identify compounds containing metals and metalloids that are toxic to humans or other organisms. Toxic elements studied include arsenic, antimony, tin, mercury, lead, aluminum, chromium, cadmium and mercury, and studies have focused both on environmental species that might be ingested, as well as species found in tissue and bodily fluids. Speciation analysis of tin, lead, mercury and antimony compounds has focused mainly on the detection and identification of small organic derivatives of these metals, as well as detection of the free metal ion w37–40x. Speciation of aluminum, chromium and cadmium has focused on biologically synthesized proteins and small molecules that act as ligands to bind and deactivate the toxic metals. Such ligands discovered so far include citrate, aconitate and malate ions, transferrin, albumin, ceruloplasmin, metallothionein, and phytochelatins w19,21,41,42x. The latter two of this list have been studied in detail due to their ability to bind and sequester a wide variety of toxic metals, and they will be discussed in a later section. Oxidation state has been of great interest as well, since the toxicity of elements such as antimony, arsenic, and chromium are highly dependent upon oxidation state w11,41x. Just as the research on essential elements has tended to focus in recent years on selenium, research on toxic elements has focused on arsenic, and a wide variety of arseniccontaining species have been discovered. Arsenic is present in a wide variety of compounds of vastly differing toxicity. Inorganic arsenic compounds (arsenite and arsenate) are known to be highly toxic, while organoarsenic compounds are generally considered to be non-toxic. Arsenic-containing organic acids such as dimethylarsinic acid and monomethylarsonic acid are believed to have intermediate toxicities w43x, and some phenylarsonic compounds have even been found to be beneficial in livestock w44x. The speciation of arsenic has been pursued by a large number of research groups owing to its disproportionately high concentration in commonly consumed seafood. The total arsenic concentration in marine plants and animals ranges from 10 to 100 mgyg, but this arsenic is distributed among a large number of compounds. About half of those arsenic compounds detected in marine animals have yet to be identified w45x. Arsenobetaine, a non-toxic organoarsenic compound, has been identified as the most abundant arsenic species in marine animals, and a number of other compounds, including simple methylated derivatives, arsenocholine, and a wide variety of arsenic-containing organic acids have been identified and well-studied w46,47x. Tentative mechanisms for the metabolism of arsenic by marine life have been postulated, but the failure to identify many of the arsenic species present has prevented a more concrete understanding w47,48x. Analysis of human urine and serum samples for determination of arsenic species has been performed as well w49–51x, but the metabolic pathway of arsenic in humans remains poorly understood. The increasing application of ESI-MS and ESI-MSyMS to identify arsenic species will reduce the current reliance on reference standards, and will aid in the identification of currently unidentified species. 2.4. Metal sequestration In studying elements that are toxic to living organisms, it is of great interest to understand the ways in A.L. Rosen, G.M. Hieftje / Spectrochimica Acta Part B 59 (2004) 135–146 which organisms are capable of protecting themselves. Plants and animals have developed a number of ways to sequester or detoxify harmful metals and metalloids. In some cases, the toxic free element is transformed enzymatically into a less harmful or completely harmless organic form which can then be stored or excreted w36,52x. In other cases, metal or metalloid ions are complexed by biologically synthesized ligands, which can be small molecules, peptides, or proteins. In both plants and animals, chemical species have been identified that are believed to have the sole purpose of binding metal ions and thus regulating the amount of free metal in the biological system. In animals, a class of proteins known collectively as metallothioneins (MTs) have been detected and characterized. MTs are non-enzymatic, low molecular-weight proteins (6–7 kDa) that are rich in cystein residues and lack disulfide bonds. They are thus capable of binding metals with sulfur affinity and have been shown to play a role in detoxification of toxic metals (Cd, Hg, Ag), as well as homeostatic control and metabolism of nontoxic metals (Cu, Zn) w18,20x. The study of MTs is complicated by the existence of a number of different isoforms. Furthermore, each MT molecule is capable of binding up to seven metal atoms. With the variety of metals that can be bound at each site, a large number of stoichiometric combinations are possible. Many subisoforms have been identified by means of ESI-MS and ESI-MSyMS, and stoichiometry has been determined for some isoforms by acidifying solutions to facilitate loss of the metals followed by comparison of metallated and demetallated masses w2,20,53,54x. However, a great deal of work remains to be done in order to isolate and detect all of the different sub-isoforms, to determine their amino-acid sequences, and to identify the metals bound and stoichiometry under various biological conditions. Improved techniques must also be developed to detect and characterize the very low concentrations present in biological organisms in situations where there is no environmental metal stress to induce increased MT production w6,19,55x. Although metallothioneins like those in animals have not been found in plants, a class of peptides known as phytochelatins (PCs) has been discovered w56x. Like MTs, PCs bind metal ions through cysteinyl sulfurs, but unlike MTs, they are known to be stress-induced and enzymatically synthesized. PCs have the general structure (g-Glu-Cys)nGly, where n ranges from 2 to 11 (known as PC2, PC3, etc.) w2x. Studies thus far indicate that PCs detoxify heavy metals and metalloids, including Cd, Cu, Pb, Zn, and As by sequestration of the PCmetal complex in intracellular vacuoles w18x, although some research has indicated a more transient role w57x. Standards exist for the most commonly found PC ligands, making their detection and determination fairly straightforward. However, PC-metal complexes are quite 139 labile and to the authors’ knowledge have not successfully been put into the gas phase intact w6x. As a result, stoichiometric determinations using mass spectrometric techniques are difficult. Furthermore, mixed-ligand complexes (e.g. PC2 and PC3 in a single complex) have been detected, but standards do not yet exist for these complexes and little is known about them w3,21x. Recent interest in phytochelatins and other metalbinding ligands in plants has centered on hyperaccumulating plants, which are able to absorb and store unusually large amounts of toxic metals w58,59x. These plants have the potential to be used in phytoremediation efforts to detoxify contaminated sites, but the mechanisms by which they are able to sequester toxic metals at such high concentrations are not well understood w52,60,61x. 2.5. Drug metabolism A number of metallocompounds and metallocomplexes have been found to have value as therapeutic drugs. Included in this category are anti-cancer drugs containing platinum-group elements, gold-containing drugs used to treat rheumatoid arthritis, lithium carbonate used to combat manic depression, and vanadium compounds used as insulin mimetics w62x. In order to fully understand how these metallodrugs behave in vivo, it is necessary to identify the products of their biotransformation and degradation. Such an analysis includes identification and characterization of chemically transformed species, as well as complexes of the drug with other species present in the biological system and with its target. In many cases, it has been found that the therapeutically active species is not the administered drug but rather a metabolite w62,63x. Such discoveries can potentially help in the development of more effective or alternative drug products. Furthermore, many metallodrugs are known to have severe side-effects, and the margin between beneficial and toxic doses can be narrow, making it necessary to understand mechanisms of toxicity as well as therapeutic action, and to identify the toxicologically active species. The platinum-containing anti-tumor drug cisplatin has been particularly well studied. The mechanism of action of cisplatin is known to include a hydration step followed by binding of the hydration product to the guanosine moieties of DNA w17x. It is believed that albumin plays a key role in the transport and activation of cisplatin, although the complex with albumin is itself inactive, and it is suspected that complexes of cisplatin with methionine are in part responsible for its toxicity w17,63,64x. Based upon work done on cisplatin, a number of related compounds have been developed that are intended to be less toxic and to be effective in cisplatinresistant tumors, although little is understood about many of them w65x. A great deal of speciation work is 140 A.L. Rosen, G.M. Hieftje / Spectrochimica Acta Part B 59 (2004) 135–146 necessary to better understand these new metallodrugs and their advantages over cisplatin. A variety of speciation techniques have been applied to elucidate the biological mechanisms of metallodrugs, as well as drugs containing non-metallic heteroatoms and drugs that do not contain a metal themselves, but which are known to bind metals in-vivo w62–67x. Most studies of metallodrug metabolism using hyphenated techniques have employed standards for compound identification, and thus the utilization of ESI-MS and ESIMSyMS to identify unknown compounds should provide new insights in the field. 3. Current instrumentation 3.1. Hyphenated techniques A wide variety of hyphenated techniques have been developed to tackle the problems of speciation analysis. Early speciation work found that complex samples could not be analyzed without the aid of a separation technique, but the most commonly used detectors for separation methods did not provide element-specific information. The interfacing of gas- and liquid-chromatography instrumentation with element-selective detectors for speciation analysis was pioneered in the late 1970s and 1980s by Van Loon w68x and Suzuki w69x. Initial configurations employed atomic absorption, atomic fluorescence, and atomic emission spectrometers, but improvements in atomic mass spectrometric instrumentation have led to the widespread use of mass spectrometry in hyphenated techniques. In particular, HPLC-ICP-MS has been widely applied and is becoming routine for speciation studies w8x. Alternative separation techniques have been employed as well, including gas chromatography w5x, obviously limited to volatile compounds unless derivatization is employed, capillary electrophoresis (CE) w70–72x, which can be sampled online, and flatbed electrophoresis w35,73x, which can be coupled offline via techniques such as laser ablation and electrothermal vaporization. CE has been gaining in popularity in recent years due to its ultra-high separation efficiency and the small sample volumes it requires w72x. A particularly mild separation technique that has been applied by few researchers for speciation but may have promise for future investigations is field flow fractionation (FFF), which is known to be capable of separating macromolecules, colloids and even cells w74,75x. The wide variety of hyphenated techniques that have been used for speciation analysis and the applications for which they have been employed have been reviewed w2,3,5,7x. In conjunction with HPLC or CE, ICP-MS has been the most widely used instrument in recent years for element-specific detection, and ESI-MS has been the most widely used for compound identification. Both ionization sources can be coupled in a fairly straightforward manner to a number of separation techniques, and both have been used with a wide variety of mass analyzers. 3.2. ICP-MS for elemental detection ICP-MS has become the most widely used detection technique for speciation analysis. A number of speciation studies have employed optical spectrometric techniques, including atomic absorption spectroscopy (AA) and ICP atomic emission spectroscopy (ICP-AES), but AA does not provide simultaneous multielemental detection, and ICP-AES is in general less sensitive than ICPMS. Routinely achievable sub-ngyl detection limits for ICP-MS w3x have permitted the detection of ultra-trace species in biological and environmental matrices. ICPMS can detect trace-element-containing species, even when a particular trace element is distributed amongst a large number of species. In particular, improvements in ICP-MS sensitivity have allowed the detection of more and more arsenic and selenium compounds in biological matrices and food supplements w27,76x. In addition to its low detection limits, the dynamic range of ICP-MS routinely exceeds six orders of magnitude, allowing detection of both major constituents and trace components at the same sample dilution. Furthermore, the multielemental capability of ICP-MS enables the observation of individual isotopes, which permits the use of isotopic-dilution techniques for internal standardization and also to monitor species transformations that may occur during sample pre-treatment or separation w40,77x. In addition to all of these advantages, there are a number of difficulties that are often encountered in using ICP-MS for speciation analysis. A consideration that is particularly important when ICP-MS is coupled with HPLC or CE is the solvent composition. It is well documented that high concentrations of some organic solvents can result in plasma instability as well as a build-up of carbon residue on the sampling cone w78x. These problems can be alleviated to some degree by addition of oxygen to the nebulizer-gas flow, by increasing the RF power to the plasma, by using a platinum sampling cone, and by using either a low-flow directinjection nebulizer (DIN) or a nebulizer with an efficient desolvation unit (such as a membrane desolvator). If analyte concentrations are high enough, post-column dilution with water (or dilute HNO3) may also be an option. The use of solvent gradients can also pose problems for ICP-MS analysis. A change in solvent composition alters the plasma temperature and electron number density, which can result in different ionization efficiencies and ion energies. If a spray chamber is used, changing solvent concentrations can also result in memory effects due to solvent adhesion and revolatilization to and from the spray chamber walls. Thus, if quantifi- A.L. Rosen, G.M. Hieftje / Spectrochimica Acta Part B 59 (2004) 135–146 cation is desired, solvent gradients should be avoided or closely monitored w15,65x. The other common source of difficulties arising from the coupling of ICP-MS with separation techniques is the sample matrix. Biological and environmental samples contain very complex matrices, and many HPLC and CE techniques require the use of a buffer or a solution of high ionic strength. In the ICP, high salt concentrations can result in signal suppression due to increased space-charge effects which defocus the ion beam w15,79x. Thus, tradeoffs may be required with respect to separation efficiency and detection sensitivity, although matrix problems in the ICP can be alleviated somewhat by performing sample pretreatment, by altering argon flow rates, by modifying interface configurations and voltages, or by post-column dilution w79,80x. A final potential problem is the appearance of isobaric overlaps from polyatomic ions occurring at the same nominal myz as the ions of interest. This problem is particularly acute when selenium is to be detected, since its most abundant isotope (80Se) has the same nominal mass as 40Ar2 w22x. Very high resolution mass analyzers are capable of overcoming isobaric overlaps to some degree, and a number of methods have been developed to either reduce the appearance of the isobaric overlap, or to move the ion of interest to a different myz via a chemical reaction w81x. 3.3. ESI-MS for compound identification Because the ICP is a destructive ionization source, conventional ICP-MS serves as an ultra-sensitive elemental detector only, and it provides no inherent information about chemical species. Thus, most speciation studies making use of ICP-MS have employed a separation step and have used standards for compound identification via matching of retention times. However, standards are not always available, methods such as CE produce retention times that are notoriously dependent on factors such as ionic strength, and it is not always possible to predict the compounds that one expects to find. Indeed, progressively lower detection limits for ICP-MS are providing more and more peaks for newly detected, unidentified compounds. Standards are not yet available for the wide variety of metallothionein subisoforms or the minor arsenic- and selenium-containing species in biological samples w27,55,76x. Furthermore, the approach based upon retention-time matching of chromatographic peaks detected by ICP-MS assumes that complete chromatographic separation has been achieved and that each peak corresponds to a single pure compound, which may not be the case. If identification of all compounds containing the metal or metalloid of interest is to be achieved, a technique must be used that can provide information about the intact molecular species. One of the most commonly used 141 techniques for this purpose is electrospray ionization mass spectrometry. The ‘soft’ ionization of ESI produces molecular ions without any significant fragmentation. For many heavy compounds, a series of multiply charged peaks are observed, permitting a very accurate determination of molecular weight. In the past, many studies that have focused on compound identification have made use of nuclear magnetic resonance (NMR) spectroscopy. ESI-MS requires significantly smaller sample volumes and less preliminary purification than are needed by NMR and has been preferred by most researchers in recent years w47x. In situations where ESI has proven unsuccessful, other mass spectrometric methods, including matrix assisted laser desorption ionization (MALDI), have been used for molecular weight determination w82x. However, MALDI has been applied less frequently because its coupling to separation techniques such as HPLC and CE is not as straightforward since the sample must be immobilized. When one uses ESI-MS or other mass spectrometric techniques that produce intact molecular ions, structural information can be obtained by using tandem mass spectrometry (MSyMS), in which collision-induced dissociation (CID) is used to reproducibly fragment a selected molecular ion. MSyMS has been used by a large number of researchers for positive identification and characterization of detected species w2,46,47,52,76x. Depending upon instrument configuration, it is often also possible to remove weakly bound metals from metallocomplexes by increasing interface potential differences or by post-column acidification. The metalbinding ligand can then be observed on its own, allowing the determination of its molecular weight and elucidation of metal:ligand stoichiometry w18,19,83x. As with any technique, there are some disadvantages to using ESI-MS. The main disadvantage is its relatively poor detection limits compared to ICP-MS. Routinely achievable detection limits for ESI-MS are generally two to three orders of magnitude poorer than those for ICP-MS w27x. This discrepancy becomes a problem when one wishes to identify ultra-trace species that have been detected by ICP-MS. In fact, most studies aimed at compound identification in real biological samples have resorted to preconcentration of chromatographic fractions preceding analysis by ESI-MS w27,29,52x. The other major complication that has been observed with ESI-MS is the suppression of the analyte signal by complex matrices. The presence of high concentrations of salts, which may be present in HPLC buffers or CE electrolytes, has been shown to suppress ionization of analyte compounds and thus to decrease the observed signal w27x. Pneumatically assisted ESI, also known as Ion-spray䉸, has been used by many to overcome these matrix effects. Ion-spray䉸 is capable of handling a wider range of solvent compositions than standard ESI, 142 A.L. Rosen, G.M. Hieftje / Spectrochimica Acta Part B 59 (2004) 135–146 although matrix effects can still be a problem w71x. MALDI-MS has also been applied as an alternative to ESI-MS in situations where matrix effects were deemed to be a problem w82x. 3.4. ESI-MS for elemental analysis of small molecules One of the primary advantages of ESI-MS, as mentioned above, is that it is a ‘soft’ ionization technique, producing little fragmentation. Most researchers utilizing ESI-MS have taken advantage of this feature, detecting intact molecular ions and selecting specific molecular ions for fragmentation via CID. However, some researchers have successfully applied ESI-MS for elemental analysis. It has been well demonstrated that the ions observed in an ESI-MS spectrum are highly dependent upon the conditions imposed within the ESI unit and the atmosphere-vacuum interface. ‘Mild’ interface conditions, including moderate desolvation temperatures, low interface voltages and low curtain-gas flow rates, have been found to preserve the species found in solution, while ‘harsh’ interface conditions, including higher desolvation temperatures, interface voltages and gas flow rates, have been found to induce declustering and fragmentation w84x. This fragmentation within the interface region is believed to result from a collisioninduced dissociation process and has been called by a number of names, including ‘high-pressure,’ ‘up-front,’ and ‘source’ CID w6,85x. Several researchers have successfully manipulated interface conditions to study inorganic and relatively small organic molecules w85x. Agnes and Horlick have identified three sets of parameters that result in the observation of three distinct types of mass spectra for inorganic solution ions: the ion cluster mode, the metalion mode, and an intermediate mode w84x. A number of other researchers have applied well-defined interface conditions to achieve partial fragmentation, complete fragmentation, and removal of metal ions from relatively small organometallic compounds, such as ferrocene, tributyltin, and arsenobetaine w83,86,87x. Despite these achievements, however, elemental spectra have not been obtained using ESI-MS for large molecules such as proteins. Interface conditions have been found under which non-covalent interactions can be selectively broken, and under which functional groups can be cleaved, but to the authors’ knowledge complete fragmentation of a protein molecule to its elements using only an electrospray source has not been achieved w88,89x. When compared with ICP-MS, ESI-MS for elemental analysis has advantageous and disadvantageous features. ESI-MS in the elemental mode can provide a complete elemental spectrum free from the spectral interferences often encountered in ICP-MS w6,87,90x, and it is capable of providing information about valence state for solution ions that is unobtainable with conventional ICP-MS w84,91x. Its use can potentially reduce the cost of analysis due to decreased sample consumption, lack of argon consumption, and because elemental and molecular information can be obtained on the same instrument, although not at the same time w89,90x. The major shortcoming of ESI-MS relative to ICP-MS is poor detection limits, which are observed in both molecular and elemental modes w90x. Additionally, it is difficult to eliminate oxides without significant loss of signal, highpressure CID is generally difficult to control and can result in decreased resolution, and optimal interface parameters vary substantially from element to element, making multielement determinations difficult w85,86,90,92x. Finally, and quite importantly for applications in the field of biochemistry, elemental ESI-MS has not been shown to work for large molecules w89x. It is clear that elemental ESI-MS may have a promising future for the analysis of inorganic and small organic compounds, particularly for those species that are difficult to detect by ICP-MS, but at this point it does not appear to play a role in the analysis of biomolecules. 3.5. Mass analyzers for mass spectrometric detection The most common mass analyzer used in conjunction with ICP and ESI ionization sources in hyphenated techniques has been the quadrupole mass filter. Its robustness, modest vacuum requirements, and compact size have made the quadrupole attractive for routine analysis. However, current research is turning increasingly toward other types of mass analyzers, including ion-trap w93x, sector-field w94x, and time-of-flight w95,96x units. These mass analyzers generally have the advantage of improved resolution, and all have the potential to detect or extract all ions simultaneously. The elimination of spectral skew afforded by non-scanning instruments permits accurate representation of transient signals, which is necessary when they are coupled directly to a chromatographic or electrophoretic separation. With respect to speciation, techniques for simultaneous detection permit identification of species containing more than one type of metal as well as determination of stoichiometry within these compounds. This capability is of particular interest in the study of metallothioneins, which can bind many different metals at a number of coordination sites w19,20x. Furthermore, multielemental detection can allow rapid, simultaneous speciation analysis of multiple elements in a complex sample, and can enable the observation of isotopic patterns for confirmation of metal content in molecular spectra, and for improved structural analysis capability in MSyMS w97x. The ability to resolve and simultaneously detect multiple isotopes also enables the use of isotope-dilution methods w40x for improved quantification. The availability of quadrupole time-of-flight (QTOF) tandem mass spectrometers offers structural A.L. Rosen, G.M. Hieftje / Spectrochimica Acta Part B 59 (2004) 135–146 analysis without the disadvantages of low-resolution scanning techniques. 3.6. Multidimensional separation techniques The need to identify species from increasingly complex mixtures that elute in individual chromatographic fractions has underscored the difficulty in obtaining chromatographically pure fractions. An effective and increasingly popular way of ensuring complete chromatographic (or electrophoretic) separation is to use multiple orthogonal (complementary) separation techniques in series. Such ‘multidimensional’ approaches have been recently reviewed with respect to speciation analysis w97x, and have been applied to the speciation of arsenic w47,98x and selenium w26x. The most common combinations incorporate size-exclusion chromatography (SEC) followed by cation-exchange, anionexchange, or reversed-phase HPLC, or CE. The initial SEC separation facilitates removal of large biological polymers, partial desalting of analytes, and a rough fractionation based upon molecular weight w97x. The separations that follow must be chosen based upon the analytes of interest, the sample matrix, and the detection technique to be used. Two- and three-dimensional approaches are generally sufficient for adequate separation. An additional benefit of multidimensional separation techniques is that they provide a more simplified matrix, which reduces matrix interferences seen in the MS detectors. A critical consideration when one uses separation techniques is preservation of the original chemical species. If the speciation analysis is to accurately represent the species distribution in the sample, changes in chemical composition and oxidation state must be minimized in every step of the procedure, including sample preparation, separation and detection. The separation steps used for a particular application must be chosen carefully to minimize interactions with the species of interest, to maintain appropriate pH, and to minimize loss of analyte. When chemical interactions pose a problem, CE can be used as an alternative to HPLC, because the lack of analyte-stationary phase interactions provide less opportunity for chemical changes w71x. Ion exchange chromatography has been used by some in place of more traditional reversed phase chromatography due to reduced interactions with the matrix w15x. Once again, the best chromatographic or electrophoretic technique for a given experiment should be chosen based upon knowledge of the analytes of interest, the metals or metalloids they contain, the matrix within which they exist, and the detection technique to be used. 4. Concluding statements It is evident from the above overview that although a great deal of speciation work has been done, a tremen- 143 dous amount of additional information about the speciation of trace metals in biological and environmental samples must be obtained. Significant advances have been made in developing instrumentation that has improved sensitivity and versatility, and can provide more complete and conclusive information. ICP-MS and ESI-MS have been applied by a large number of researchers as complementary techniques, providing elemental and molecular information that together provide a complete picture of elemental speciation. Future work in the development of instrumentation for speciation analysis will include not only improvement of existing technologies, but also a streamlining of the process. Instrumental designs have been conceived which allow the simultaneous use of ICP-MS and ESI-MS instruments in parallel w92x, or the incorporation of ICP and ESI sources into a single mass spectrometer w99x. Advances such as these will provide improved speciation capabilities, and continuing collaboration among the fields of analytical, bioinorganic, and environmental chemistry will facilitate the application of new technologies to real-world problems. Acknowledgments Supported in part by the US Department of Energy through grant DOE DE-FG02-98ER14890. Support was also provided by the US Department of Energy, Office of Non-proliferation Research and Engineering, through Pacific Northwest National Laboratory (PNNL). PNNL is operated by Battelle Memorial Institute for the Department of Energy under contract DE-AC06-76RLO-1830. References w1x D.M. Templeton, F. Ariese, R. Cornelis, L.-G. Danielsson, H. Muntau, H.P. Van Leeuwen, R. Lobinski, Guidelines for terms related to chemical speciation and fractionation of elements. Definitions, structural aspects, and methodological approaches, Pure Appl. Chem. 72 (2000) 1453–1470. w2x R. Lobinski, J. Szpunar, Biochemical speciation analysis by hyphenated techniques, Anal. Chim. Acta 400 (1999) 321–332. w3x J. Szpunar, Bio-inorganic speciation analysis by hyphenated techniques, Analyst 125 (2000) 963–988. w4x O.F.X. Donard, J.A. Caruso, Trace metal and metalloid species determination: evolution and trends, Spectrochim. Acta Part B 53 (1998) 157–163. w5x B. Bouyssiere, J. Szpunar, R. Lobinski, Gas chromatography with inductively coupled plasma mass spectrometric detection in speciation analysis, Spectrochim. Acta Part B 57 (2002) 805–828. w6x H. Chassaigne, V. Vacchina, R. Lobinski, Elemental speciation analysis in biochemistry by electrospray mass spectrometry, Trends Anal. Chem. 19 (2000) 300–313. w7x P.C. Uden, Element-specific chromatographic detection by atomic absorption, plasma atomic emission and plasma mass spectrometry, J. Chrom. A 703 (1995) 393–416. w8x K. Sutton, R.M.C. Sutton, J.A. Caruso, Inductively coupled plasma mass spectrometric detection for chromatography and capillary electrophoresis, J. Chrom. A 789 (1997) 85–126. 144 A.L. Rosen, G.M. Hieftje / Spectrochimica Acta Part B 59 (2004) 135–146 w9x C. Sarzanini, E. Mentasti, Determination and speciation of metals by liquid chromatography, J. Chrom. A 789 (1997) 301–321. w10x R. Lobinski, Speciation-targets, analytical solutions and markets, Spectrochim. Acta Part B 53 (1998) 177–185. w11x H.M. Crews, Speciation of trace elements in foods, with special reference to cadmium and selenium: is it necessary?, Spectrochim. Acta Part B 53 (1998) 213–219. w12x A. Sanz-Medel, Trace element analytical speciation in biological systems: importance, challenges and trends, Spectrochim. Acta Part B 53 (1998) 197–211. w13x US Department of Health and Human Services Food and Drug Administration website http:yywww.fda.gov. w14x National Academy of Sciences Institute of Medicine (IOM) website http:yywww.iom.edu. w15x E.H. Larsen, Method optimization and quality assurance in speciation analysis using high performance liquid chromatography with detection by inductively coupled plasma mass spectrometry, Spectrochim. Acta Part B 53 (1998) 253–265. w16x FAOyWHO Food Standards Codex Alimentarius Comission official website http:yywww.codexalimentarius.nety. w17x R.R. Barefoot, Speciation of platinum compounds: a review of recent applications in studies of platinum anticancer drugs, J. Chrom. B 751 (2001) 205–211. w18x J. Szpunar, R. Lobinski, Speciation in the environmental fieldtrends in analytical chemistry, Fresenius’ J. Anal. Chem. 363 (1999) 550–557. w19x A. Prange, D. Schaumloffel, Hyphenated techniques for the characterization and quantification of metallothionein isoforms, Anal. Bioanal. Chem. 373 (2002) 441–453. w20x C.N. Ferrarello, M. Montes Bayon, R. Fernandez de la Campa, A. Sanz-Medel, Multi-elemental speciation studies of trace elements associated with metallothionein-like proteins in mussels by liquid chromatography with inductively coupled plasma time-of-flight mass spectrometric detection, J. Anal. At. Spectrom. 15 (2000) 1558–1563. w21x V. Vacchina, K. Polec, J. Szpunar, Speciation of cadmium in plant tissues by size-exclusion chromatography with ICP-MS detection, J. Anal. At. Spectrom. 14 (1999) 1557–1566. w22x P.C. Uden, Modern trends in the speciation of selenium by hyphenated techniques, Anal. Bioanal. Chem. 373 (2002) 422–431. w23x T. Lindemann, H. Hintelmann, Identification of seleniumcontaining glutathione S-conjugates in a yeast extract by twodimensional liquid chromatography with inductively coupled plasma MS and nanoelectrospray MSyMS detection, Anal. Chem. 74 (2002) 4602–4610. w24x C. Ip, M. Birringer, E. Block, M. Kotrebai, J.F. Tyson, P.C. Uden, D.J. Lisk, Chemical speciation influences comparative activity of selenium-enriched garlic and yeast in mammary cancer prevention, J. Agric. Food Chem. 48 (2000) 2062–2070. w25x L.C. Clark, G.F. Combs Jr., B.W. Turnbull, E.H. Slate, D.K. Chalker, J. Chow, et al., Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin: a randomized controlled trial, J. Am. Med. Assoc. 276 (1996) 1957–1963. w26x S. McSheehy, F. Pannier, J. Szpunar, M. Potin-Gautier, R. Lobinski, Speciation of seleno compounds in yeast aqueous extracts by three-dimensional liquid chromatography with inductively coupled plasma mass spectrometric and electrospray mass spectrometric detection, Analyst 127 (2002) 223–229. w27x C. Casiot, V. Vacchina, H. Chassaigne, J. Szpunar, M. PotinGautier, R. Lobinski, An approach to the identification of selenium species in yeast extracts using pneumatically-assisted w28x w29x w30x w31x w32x w33x w34x w35x w36x w37x w38x w39x w40x w41x w42x electrospray tandem mass spectrometry, Anal. Commun. 36 (1999) 77–80. M. Kotrebai, M. Birringer, J. Tyson, E. Block, P.C. Uden, Selenium speciation in enriched and natural samples by HPLCICP-MS and HPLC-ESI-MS with perfluorinated carboxylic acid ion-pairing agents, Analyst 125 (2000) 71–78. A.P. Vonderheide, K. Wrobel, S.S. Kannamkumarath, C. B’Hymer, M. Montes-Bayon, C. Ponce de Leon, J.A. Caruso, Characterization of selenium species in Brazil nuts by HPLCICP-MS and ES-MS, J. Agric. Food Chem. 50 (2002) 5722–5728. S.S. Kannamkumarath, K. Wrobel, K. Wrobel, A. Vonderheide, J.A. Caruso, HPLC-ICP-MS determination of selenium distribution and speciation in different types of nut, Anal. Bioanal. Chem. 373 (2002) 454–460. J. Zheng, M. Ohata, N. Furuta, Reversed-phase liquid chromatography with mixed ion-pair reagents coupled with ICPMS for the direct speciation analysis of selenium compounds in human urine, J. Anal. At. Spectrom. 17 (2002) 730–735. S.C.K. Shum, R.S. Houk, Elemental speciation by anion exchange and size exclusion chromatography with detection by inductively coupled plasma mass spectrometry with direct injection nebulization, Anal. Chem. 65 (1993) 2972–2976. T.H. Cao, R.A. Cooney, M.M. Woznichak, S.W. May, R.F. Browner, Speciation and identification of organoselenium metabolites in human urine using inductively coupled plasma mass spectrometry and tandem mass spectrometry, Anal. Chem. 73 (2001) 2898–2902. D. Behne, C. Hammel, H. Pfeifer, D. Rothlein, H. Gessner, A. Kyriakopoulos, Speciation of selenium in the mammalian organism, Analyst 123 (1998) 871–873. T.W.M. Fan, E. Pruszkowski, S. Shuttleworth, Speciation of selenoproteins in Se-contaminated wildlife by gel electrophoresis and laser ablation-ICP-MS, J. Anal. At. Spectrom. 17 (2002) 1621–1623. M. Montes-Bayon, E. Yanes, C. Ponce de Leon, K. Jayasimhulu, A. Stalcup, J. Shann, J.A. Caruso, Initial studies of selenium speciation in Brassica juncea by LC with ICPMS and ES-MS detection: an approach for phytoremediation studies, Anal. Chem. 74 (2002) 107–113. S.C.K. Shum, H.-m. Pang, R.S. Houk, Speciation of mercury and lead compounds by microbore column liquid chromatography-inductively coupled plasma mass spectrometry with direct injection nebulization, Anal. Chem. 64 (1992) 2444–2450. J. Lintschinger, O. Schramel, A. Kettrup, The analysis of antimony species by using ESI-MS and HPLC-ICP-MS, Fresenius’ J. Anal. Chem. 361 (1998) 96–102. J. Gui-Bin, Z. Qun-Fang, H. Bin, Speciation of organotin compounds, total tin, and major trace metal elements in poisoned human organs by gas chromatography-flame photometric detector and inductively coupled plasma-mass spectrometry, Environ. Sci. Technol. 34 (2000) 2697–2702. J.I. Garcia Alonso, J. Ruiz Encinar, P. Rodriguez Gonzalez, A. Sanz-Medel, Determination of butyltin compounds in environmental samples by isotope dilution GC-ICP-MS, Anal. Bioanal. Chem. 373 (2002) 432–440. J. Zheng, A. Iijima, N. Furuta, Complexation effect of antimony compounds with citric acid and its application to the speciation of antimony(III) and antimony(V) using HPLCICP-MS, J. Anal. At. Spectrom. 16 (2001) 812–818. T. Bantan, R. Milacic, B. Mitrovic, B. Pihlar, Combination of various analytical techniques for speciation of low molecular weight aluminum complexes in plant sap, Fresenius’ J. Anal. Chem. 365 (1999) 545–552. A.L. Rosen, G.M. Hieftje / Spectrochimica Acta Part B 59 (2004) 135–146 w43x M. Van Hulle, C. Zhang, X. Zhang, R. Cornelis, Arsenic speciation in Chinese seaweeds using HPLC-ICP-MS and HPLC-ES-MS, Analyst 127 (2002) 634–640. w44x S.A. Pergantis, E.M. Heithmar, T.A. Hinners, Speciation of arsenic animal feed additives by microbore high-performance liquid chromatography with inductively coupled plasma mass spectrometry, Analyst 122 (1997) 1063–1068. w45x S. McSheehy, P. Pohl, R. Lobinski, J. Szpunar, Investigation of arsenic speciation in oyster test reference material by multidimensional HPLC-ICP-MS and electrospray tandem mass spectrometry (ES-MS-MS), Analyst 126 (2001) 1055–1062. w46x S.A. Pergantis, W. Winnik, D. Betowski, Determination of ten organoarsenic compounds using microbore high-performance liquid chromatography coupled with electrospray mass spectrometry-mass spectrometry, J. Anal. At. Spectrom. 12 (1997) 531–536. w47x S. McSheehy, J. Szpunar, R. Lobinski, V. Haldys, J. Tortajada, J.S. Edmonds, Characterization of arsenic species in kidney of the clam Tridacna derasa by multidimensional liquid chromatography-ICPMS and electrospray time-of-flight tandem mass spectrometry, Anal. Chem. 74 (2002) 2370–2378. w48x A.D. Madsen, W. Goessler, S.N. Pedersen, K.A. Francesconi, Characterization of an algal extract by HPLC-ICP-MS and LCelectrospray MS for use in arsenosugar speciation studies, J. Anal. At. Spectrom. 15 (2000) 657–662. w49x G. Samanta, U.K. Chowdhury, B.K. Mandal, D. Chakraborti, N.C. Sekaran, H. Tokunaga, M. Ando, High-performance liquid chromatography inductively coupled plasma mass spectrometry for speciation of arsenic compounds in urine, Microchem J. 65 (2000) 113–127. w50x K. Wrobel, K. Wrobel, B. Parker, S.S. Kannamkumarath, J.A. Caruso, Determination of As(III), As(IV), monomethylarsonic acid, dimethylarsinic acid, and arsenobetaine by HPLC-ICPMS: analysis of reference materials, fish tissues and urine, Talanta 58 (2002) 899–907. w51x K.T. Suzuki, B.K. Mandal, Y. Ogra, Speciation of arsenic in body fluids, Talanta 58 (2002) 111–119. w52x M. Montes-Bayon, D.L. LeDuc, N. Terry, J.A. Caruso, Selenium speciation in wild-type and genetically modified Se accumulating plants with HPLC separation and ICP-MSyESMS detection, J. Anal. At. Spectrom. 17 (2002) 872–879. w53x V. Nischwitz, B. Michalke, A. Kettrup, Identification and quantification of metallothionein isoforms and superoxide dismutase in spiked liver extracts using HPLC-ESI-MS offline coupling and HPLC-ICP-MS online coupling, Anal. Bioanal. Chem. 375 (2003) 145–156. w54x X. Yu, M. Wojciechowski, C. Fenselau, Assessment of metals in reconstituted metallothioneins by electrospray mass spectrometry, Anal. Chem. 65 (1993) 1355–1359. w55x H. Chassaigne, R. Lobinski, Speciation of metal complexes with biomolecules by reversed-phase HPLC with ion-spray and inductively coupled plasma mass spectrometric detection, Fresenius J. Anal. Chem. 361 (1998) 267–273. w56x E. Grill, E.L. Winnacker, M.H. Zenk, Phytochelatins: the principal heavy-metal complexing peptides of higher plants, Science 230 (1985) 674–676. w57x I. Leopold, D. Gunther, J. Schmidt, D. Neumann, Phytochelatins and heavy metal tolerance, Phytochemistry 50 (1999) 1323–1328. w58x U. Kramer, I.J. Pickering, R.C. Prince, I. Raskin, D.E. Salt, Subcellular localization and speciation of nickel in hyperaccumulator and non-accumulator Thlaspi species, Plant Physiol. 122 (2000) 1343–1353. w59x N.S. Pence, P.B. Larsen, S.D. Ebbs, D.L.D. Letham, M.M. Lasat, D.F. Garvin, D. Eide, L.V. Kochian, The molecular w60x w61x w62x w63x w64x w65x w66x w67x w68x w69x w70x w71x w72x w73x w74x 145 physiology of heavy metal transport in the ZnyCd hyperaccumulator Thlaspi caerulescens, Proc. Natl. Acad. Sci. 97 (2000) 4956–4960. U. Kramer, A.N. Chardonnens, The use of transgenic plants in the bioremediation of soils contaminated with trace elements, Appl. Microbiol. Biotechnol. 55 (2001) 661–672. R.L. Chaney, M. Malik, Y.M. Li, S.L. Brown, E.P. Brewer, J.S. Angle, A.J.M. Baker, Phytoremediation of soil metals, Curr. Opin. Biotechnol. 8 (1997) 279–284. S.J. Berners-Price, P.J. Sadler, Coordination chemistry of metallodrugs: insights into biological speciation from NMR spectroscopy, Coord. Chem. Rev. 151 (1996) 1–40. B.P. Esposito, R. Najjar, Interactions of antitumoral platinumgroup metallodrugs with albumin, Coord. Chem. Rev. 232 (2002) 137–149. J. Szpunar, A. Makarov, T. Pieper, B.K. Keppler, R. Lobinski, Investigation of metallodrug-protein interactions by size-exclusion chromatography coupled with inductively coupled plasma mass spectrometry (ICP-MS), Anal. Chim. Acta 387 (1999) 135–144. W.R.L. Cairns, L. Ebdon, S.J. Hill, A high performance liquid chromatography-inductively coupled plasma-mass spectrometry interface employing desolvation for speciation studies of platinum in chemotherapy drugs, Fresenius’ J. Anal. Chem. 355 (1996) 202–208. O. Corcoran, J.K. Nicholson, E.M. Lenz, F. Abou-Shakra, J. Castro-Perez, A.B. Sage, I.D. Wilson, Directly coupled liquid chromatography with inductively coupled plasma mass spectrometry and orthogonal acceleration time-of-flight mass spectrometry for the identification of drug metabolites in urine: application to diclofenac using chlorine and sulfur detection, Rapid Commun. Mass Spectrom. 14 (2000) 2377–2384. R.R. Barefoot, J.C. Van Loon, Determination of platinum and gold in anticancer and antiarthritic drugs and metabolites, Anal. Chim. Acta 334 (1996) 5–14. J.C. Van Loon, Metal speciation by chromatographyyatomic spectrometry, Anal. Chem. 51 (1979) 1139A–1150A. K.T. Suzuki, Direct connection of high-speed liquid chromatograph (equipped with gel permeation column) to atomic absorption spectrophotometer for metalloprotein analysis: metallothionein, Anal. Biochem. 102 (1980) 31–34. B. Michalke, O. Schramel, A. Kettrup, Capillary electrophoresis coupled to inductively coupled plasma mass spectrometry (CEyICP-MS) and to electrospray ionization mass spectrometry (CEyESI-MS): an approach for maximum species information in speciation of selenium, Fresenius’ J. Anal. Chem. 363 (1999) 456–459. J.W. Olesik, J.A. Kinzer, E.J. Grunwald, K.K. Thaxton, S.V. Olesik, The potential and challenges of elemental speciation by capillary electrophoresis-inductively coupled plasma mass spectrometry and electrospray or ion spray mass spectrometry, Spectrochim. Acta Part B 53 (1998) 239–251. M. Moini, Capillary electrophoresis mass spectrometry and its application to the analysis of biological mixtures, Anal. Bioanal. Chem. 373 (2002) 466–480. C.C. Chery, H. Chassaigne, L. Verbeeck, R. Cornelis, F. Vanhaecke, L. Moens, Detection and quantification of selenium in proteins by means of gel electrophoresis and electrothermal vaporization ICP-MS, J. Anal. At. Spectrom. 17 (2002) 576–580. M. Hassellov, B. Lyven, C. Haraldsson, W. Sirinawin, Determination of continuous size and trace element distribution of colloidal material in natural water by on-line coupling of flow field-flow fractionation with ICPMS, Anal. Chem. 71 (1999) 3497–3502. 146 A.L. Rosen, G.M. Hieftje / Spectrochimica Acta Part B 59 (2004) 135–146 w75x J.F. Ranville, D.J. Chittleborough, F. Shanks, R.J.S. Morrison, T. Harris, F. Doss, R. Beckett, Development of sedimentation field-flow fractionation-inductively coupled plasma mass-spectrometry for the characterization of environmental colloids, Anal. Chim. Acta 381 (1999) 315–329. w76x P.A. Gallagher, X. Wei, J.A. Shoemaker, C.A. Brockhoff, J.T. Creed, Detection of arsenosugars from kelp extracts via ICelectrospray ionization-MS-MS and IC membrane hydride generation ICP-MS, J. Anal. At. Spectrom. 14 (1999) 1829–1834. w77x J. Bettmer, Elemental speciation, Anal. Bioanal. Chem. 372 (2002) 33–34. w78x H.E. Taylor, R.A. Huff, A. Montaser, Novel applications of ICPMS, in: A. Montaser (Ed.), Inductively Coupled Plasma Mass Spectrometry, Wiley-VCH, New York, NY, 1998, pp. 711–712. w79x G. Horlick, A. Montaser, Analytical characteristics of ICPMS, in: A. Montaser (Ed.), Inductively Coupled Plasma Mass Spectrometry, Wiley-VCH, New York, NY, 1998, pp. 543–547. w80x H. Niu, R.S. Houk, Fundamental aspects of ion extraction in inductively coupled plasma mass spectrometry, Spectrochim. Acta Part A 51 (1996) 779–815. w81x S.H. Tan, G. Horlick, Background spectral features in inductively coupled plasmaymass spectrometry, Appl. Spectrosc. 40 (1986) 445–460. w82x J. Ruiz Encinar, R. Ruzik, W. Buchmann, J. Tortajada, R. Lobinski, J. Szpunar, Detection of selenocompounds in a tryptic digest of yeast selenoprotein by MALDI time-of-flight MS prior to their structural analysis by electrospray ionization triple quadrupole MS, Analyst 128 (2003) 220–224. w83x W. Hang, V. Majidi, Evaluation of ion transport processes in a heated capillary tube interface for electrospray ionization timeof-flight mass spectrometry, J. Anal. At. Spectrom. 16 (2001) 938–944. w84x G.R. Agnes, G. Horlick, Determination of solution ions by electrospray mass spectrometry, Appl. Spectrosc. 48 (1994) 655–661. w85x I.I. Stewart, Electrospray mass spectrometry: a tool for elemental speciation, Spectrochim. Acta Part B 54 (1999) 1649–1695. w86x P.P. Mahoney, J.P. Guzowski Jr, S.J. Ray, G.M. Hieftje, Electrospray ionization time-of-flight mass spectrometer for elemental analysis, Appl. Spectrosc. 51 (1997) 1464–1470. w87x J.J. Corr, Measurement of molecular species of arsenic and tin using elemental and molecular dual mode analysis by ionspray mass spectrometry, J. Anal. At. Spectrom. 12 (1997) 537–546. w88x Y.-T. Li, Y.-L. Hsieh, J.D. Henion, Studies on heme binding in myoglobin, hemoglobin, and cytochrome c by ion spray mass spectrometry, J. Am. Soc. Mass Spectrom. 4 (1993) 631–637. w89x J.C.Y. Le Blanc, Use of ionspray mass spectrometry in the speciation and elemental characterization of metallothioneins, J. Anal. At. Spectrom. 12 (1997) 525–530. w90x F. Byrdy Brown, L.K. Olson, J.A. Caruso, Comparison of electrospray and inductively coupled plasma sources for elemental analysis with mass spectrometric detection, J. Anal. At. Spectrom. 11 (1996) 633–641. w91x G.R. Agnes, I.I. Stewart, G. Horlick, Elemental speciation measurements with electrospray mass spectrometry: an assessment, Appl. Spectrosc. 48 (1994) 1347–1359. w92x R.S. Houk, Electrospray and ICP-mass spectrometry: enemies or allies?, Spectrochim. Acta Part B 53 (1998) 267–271. w93x M. Miguens-Rodriguez, R. Pickford, J.E. Thomas-Oates, S.A. Pergantis, Arsenosugar identification in seaweed extracts using high-performance liquid chromatographyyelectrospray ion trap mass spectrometry, Rapid Commun. Mass Spectrom. 16 (2002) 323–331. w94x S. Hann, A. Zenker, M. Galanski, T.L. Bereuter, G. Stingeder, B.K. Keppler, HPIC-UV-ICP-SFMS study of the interaction of cisplatin with guanosine monophosphate, Fresenius’ J. Anal. Chem. 370 (2001) 581–586. w95x P.P. Mahoney, S.J. Ray, G.M. Hieftje, Time-of-flight mass spectrometry for elemental analysis, Appl. Spectrosc. 51 (1997) 16A–28A. w96x I.V. Chernushevich, W. Ens, K.G. Standing, Orthogonal-injection TOFMS for analyzing biomolecules, Anal. Chem. 71 (1999) 452A–461A. w97x J. Szpunar, R. Lobinski, Multidimensional approaches in biochemical speciation analysis, Anal. Bioanal. Chem. 373 (2002) 404–411. w98x S. McSheehy, P. Pohl, R. Lobinski, J. Szpunar, Complementarity of multidimensional HPLC-ICP-MS and electrospray MSMS for speciation analysis of arsenic in algae, Anal. Chim. Acta 440 (2001) 3–16. w99x Rosen A.L., On the Feasibility of a Dual-Source Time-ofFlight Mass Spectrometer for Elemental Speciation, MS Thesis, Indiana University, Bloomington, IN, 2003.