Advances in atomic spectroscopy, volume 5


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Advances in atomic spectroscopy, volume 5

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  3. 3. ADVANCES IN ATOMIC SPECTROSCOPY Editor: JOSEPH SNEDDON Department of Chemistry McNeese State University Lake Charles, Louisiana VOLUME5 9 1999 JAI PRESSINC. Stamford,Connecticut
  4. 4. Copyright 91999by JAI PRESSINC 1O0 ProspectStreet Stamford, Connecticut 06904-0811 All rights reserved. No part of thispublication may be reproduced, stored on a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, filming, recording, or otherwise without prior permission in writing from the publisher. ISBN: 0-7623-0502-9 ISSN: 1068-5561 Manufactured in the United Statesof America
  5. 5. CONTENTS LIST OF CONTRIBUTORS vii PREFACE Joseph Sneddon ix SPECIATION STUDIES BY ATOMIC SPECTROSCOPY M. de la Guardia, M.L. Cervera, and A. Morales-Rubio NEW TYPESOF TUNABLE LASERS Xiandeng Hou, JackX. Zhou, KarlX. Yang, PeterStchur, and Robert G. Michel 99 DEVELOPMENTS IN DETECTORSIN ATOMIC SPECTROSCOPY Frank M. Pennebaker,RobertH. Williams, John A. Norris, and M. Bonner Denton 145 GLOW DISCHARGEATOMIC SPECTROMETRY $ergio Caroli, OresteSenofonte, and Gianluca Modesti 173 LASER-INDUCED BREAKDOWN SPECTROMETRY Yong-II!Lee and JosephSneddon 235 INDEX 289
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  7. 7. LIST OF CONTRIBUTORS Sergio Caroli M.L. Cervera M. Bonner Denton M. de la Guardia Xiandeng Hou Yong-lllLee Robert G. Michel Gianluca Modesti A. Morales-Rubio Applied Toxicology Department Istituto Superioredi Sanit~ Rome, Italy Departmentof Analytical Chemistry Universityof Valencia Valencia, Spain Departmentof Chemistry Universityof Arizona Tuscon,Arizona Departmentof Analytical Chemistry Universityof Valencia Valencia, Spain Departmentof Chemistry Universityof Connecticut Storrs,Connecticut Departmentof Chemistry Changwon National University Changwon, Korea Departmentof Chemistry Universityof Connecticut Storrs,Connecticut Applied Toxicology Department Istituto Superioredi Sanit~ Rome, Italy Departmentof Analytical Chemistry Universityof Valencia Valencia, Spain vii
  8. 8. viii LISTOF CONTRIBUTORS JohnA. Norris Department of Chemistry University of Arizona Tuscon, Arizona FrankM. Pennebaker Department of Chemistry University of Arizona Tuscon, Arizona Oreste5enofonte JosephSneddon Applied Toxicology Department Istituto Superioredi Sanit~ Rome, Italy Department of Chemistry McNeese StateUniversity Lake Charles, Louisiana PeterStchur Department of Chemistry University of Connecticut Storrs, Connecticut RobertH. Williams Department of Chemistry University of Arizona Tuscon, Arizona Kad X. Yang JackX. Zhou Wandsworth ResearchCenter New York StateDepartment of Health Albany, New York CVI LasersCorporation Putnam, Connecticut
  9. 9. PREFACE Element speciation determines the different forms or species a chemical metal can have within a given compound. It is well known that different forms of a metal have different toxicity effects. Chapter 1 by Miguel de la Guardia and coworkers describes the use of various atomic spectroscopic methods and applications of speciation studies in atomic spectroscopy. The emphasis is on combining atomic spectroscopy with gas and liquid chromatography. While the laser has been around for close to 40 years, new lasers are becoming available which have the potential to directly impact atomic spectroscopy. In Chapter 2, Bob Michel and coworkers describe new developments in tunable lasers for use in atomic spectroscopy. Traditional methods of detection such as photography and the photomultiplier tube are being replaced by new detectors which have potential for multielement detection and more. Chapter 3 describes the developments in detectors in atomic spectroscopy from M. Bonner Denton and coworkers. Chapter 4 is on the very active area of glow discharge atomic spectrometry presented by Sergio Caroli and coworkers. After a brief introduction and historical review, the use of glow discharge lamps for atomic spectrometry and mass spec- trometry is discussed. This includes a discussion on the geometry and the use of radiofrequency power. A discussion on recent applications including metals and alloys, nonconductive solid materials, and liquid and gaseous samples is presented. Finally the future of this source in atomic spectrometry is discussed.
  10. 10. x PREFACE Chapter 5 covers the use of a laser-induced or laser-ablated plasma as a spectro- chemical source for atomic emission spectrometry. The technique is referred to as laser-induced breakdown spectrometry (LIBS). A brief introduction is followed by a description of the instrumentation, in particular the laser and detection device. A brief outline of the plasma physics is followed by a description of the applications of LIBS, particularly where it is advantageous over conventional atomic emission techniques. Joseph Sneddon Editor
  11. 11. SPECIATION STUDIES BY ATOMIC SPECTROSCOPY M. de la Guardia, M.L. Cervera, and A. Morales-Rubio Abstract ...................................... 2 I. A Definition and Several Approaches ....................... 2 II. Importance of Speciation ............................. 4 III. Factors Affecting Speciation ............................ 6 IV. Speciation in Waters: The Methodology ..................... 8 V. Metal Speciation in Biological Fluids: Some Specific Problems ...................................... 15 A. Speciation in Blood .............................. 18 B. Speciation in Urine .............................. 25 C. Speciation in Milk .............................. 28 D. Speciation of Miscellaneous Biological Fluids ............... 30 VI. Speciation in Solid Samples: The Challenge .................... 41 A. Speciation of Soil and Sediment Samples .................. 41 B. Speciation of Solid Biological and Food Materials ...... . ...... 49 C. Speciation of Miscellaneous Solid Samples ................. 56 VII. Speciation Studies of Different Metals ...................... 57 A. Aluminium ........... ....................... 58 B. Antimony .................................... 68 Advances in Atomic Spectroscopy Volume 5, pages 1-98. Copyright 9 1999 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0502-9
  12. 12. 2 M. DE LA GUARDIA, M.L. CERVERA,and A. MORALES-RUBIO C. Arsenic .................................... 70 D. Cadmium .................................. 71 E. Calcium ................................... 72 E Chromium .................................. 72 G. Copper .................................... 74 H. Germanium ................................. 75 I. Iodine .................................... 76 J. Iron ..................................... 76 K. Lead ..................................... 77 L. Mercury ................................... 79 M. Nickel .................................... 81 N. Platinum ................................... 81 O. Selenium ................................... 82 P. Tin ...................................... 83 Q. Zinc ..................................... 85 References .................................... 85 ABSTRACT The term speciation is used to describe the oxidation state or chemical form of a metal in a sample. The importance of speciation, particularly in clinical or biological and environmental samples, is described. Sample preparation and preservation is consid- ered to be the key to accurate determination of the species and is discussed in detail. The use of various atomic spectroscopy techniques, coupled with both gas and liquid chromatography, to allow speciation studies of natural or real samples is presented. The application of these hyphenated techniques to selected metals is further presented. I. A DEFINITION AND SEVERAL APPROACHES The term speciation is commonly employed in geochemistry to differentiate dis- solved metals from particulated solid forms. In the past it has been used to describe the different oxidation states of a metal in the same sample. Furthermore, speciation has been used to interpret the results obtained in the electrochemical analysis of complex samples as a function of their chemical forms. This is achieved through the availability of metals to be reduced on the electrodes. In the analysis of soils, the use of sequential extraction schemes based on different reagents provide an excellent way to discriminate the chemical form of metals as a function of their suitability to be extracted by plants. In clinical analysis and toxicological studies, it was clear that the availability of metals to be absorbed by humans is a function of the specific chemical form of the metal. This is used to explain the biogeochemical cycle of trace metals and dramatic accidents, such us the 1954 methyl mercury intoxication in Minamata (Japan) (Smith and Smith,
  13. 13. Speciation Studies 3 1975). A series of different approaches, based on toxicity, bioavailability, bioaccu- mulation, mobility, or biodegradability of metals are of interest to analytical chemists for speciation studies. Recently, the International Union of Pure and Applied Chemistry (IUPAC) has defined speciation as the process yielding evidence of the atomic or molecular form or configuration in which a metal can occur in a compound or a matrix. From this point of view it is clear that to perform actual speciation studies, it is absolutely necessary to quantitatively determine the amount of a chemical form of a metal in a sample (Krull, 1991). The analytical process offers tremendous possibilities to perform speciation studies, based on the use of selective detection systems, chemometrics, and the exploitation of analytical data. Additionally, it is possible to improve speciation during the sampling and sample pretreatment by introducing specific collector devices or appropriate chemistries, as indicated in Figure 1. Using the IUPAC definition, it is clear that selectivity in front of the determination of a species is the key problem. Conventional atomic spectroscopy, because of the high temperatures used in the measurement cells, offers only limited applicability for speciation. The atomic signals are based on the presence of free atoms in the fundamental or the excited states, or on the previous formation of free gaseous ions. It makes it difficult to obtain different signals as a function of the chemical form of the metal to be determined. However, it is possible to improve the performance of atomic spectros- copy, from the ability to accurately determine the total metal content to the specific determination of species at trace levels by means of hyphenation between the atomic detector and some separation processes. The main objective of this chapter is to provide an analytical perspective about the state-of-the-art of speciation by atomic spectroscopy. Furthermore, a look at the SAMPLING l SAMPLE PRETREATMENT lSEPARATION lDETECTION l CHEMOMETRICS * Specific collection and/or preconcentration of species * Selective leaching * Derivatization * Extraction in specific conditions * Volatilization of compounds * Chromatographic separations * Specific molecular methods * Selective atomic spectrometry methods hyphenated with separation procedures * data deconvolution Figure 1. Possibilitiesoffered by the analytical processto do speciation studies.
  14. 14. 4 M. DE LA GUARDIA, M.L. CERVERA,and A. MORALES-RUBIO future of speciation by considering the tremendous possibilities offered by flow injection analysis (FIA)-microwave-assisted sample treatments, and the combina- tion of different approaches will be presented. It has been proposed that the hyphenation between chromatography and the most sensitive of atomic detectors is the main approach to achieve speciation studies. However, other approaches, such as the old practice of sequential extraction schemes, and some specific leaching or chemical treatments, could also be suitable to improve atomic measurements. They must be considered in order to make speciation available for complex samples and eventually control laboratories. In fact, the challenge is to provide suitable methodologies, far from the reduced perspective of pure academic studies. We will try to harmonize the rigorous application of the IUPAC definition with a touch of imagination in order to incorporate as many possible analytical tools from both classical chemical knowl- edge and modern instrumentation. II. IMPORTANCE OF SPECIATION The main characteristics of the behavior of chemical metals, such as their solubility, mobility, availability or toxicity, are strongly dependent on their specific form. It is clear that for life science studies, it is necessary to know the concentration of each species, as well as determination of the total metal content. The interest in speciation studies has been growing from the 1950s and particu- larly during the last two decades. A series of conferences have specifically focused on speciation and related topics. In April in 1983 in Gtittingen (Germany), a symposium on "Forms of Binding of Chemical Elements in Environmental and Biological Materials" was organized by Schwedt (1983). In September 1984 in Berlin (Germany), a new congress organized by the Association of Sponsors of German Science was devoted to "The Importance of Chemical Speciation in Environmental Processes," thus highlighting the tremendous interest on this aspect of environmental pollution. In September 1988 in Karlsruhe (Germany), EH. Frimmel organized a symposium about "Elements and their Chemical State in the Environment." In 1989, a North Atlantic Treaty Organization (NATO) workshop organized in Izmur (Turkey) focused "International Conference on Metal Specia- tion in the Environment." Since 1990, BCR have organized several meetings and workshops on speciation. These include those at Arcachon (France) on "Improve- ments in Speciation Analysis in Environmental Materials," in 1992 at Sitges (Spain) on "Sequential Extraction of Trace Metals in Soils and Sediments" and in February 1994 in Rome (Italy) on "Trends in Speciation Analysis." In March 1993, Schwedt organized a new meeting in Clausthal on "Advances in Elemental Species Analysis: Concepts, Findings and Evaluation" which focused on the methodological prob- lems of speciation. In June 1991 in Loen (Norway) it was organized as a post-sym- posium of the XXVII Colloquium Spectroscopic International (C.S.I), entitled "Speciation of Elements in Environmental and Biological Sciences." In June 1994,
  15. 15. Speciation Studies 5 the 2nd International Symposium on "Speciation of Elements in Toxicology and Biological Sciences" assured the continuity of international conferences on this topic. From the pioneering German interest in this field, the international scientific community has been quick to accept speciation as a major topic of current analytical chemistry. This is not only due to its extreme importance in evaluating the environ- mental impact and mobility of metals and their toxicological behavior, but also from the methodological point of view in order to assure the accuracy, repeatability and ruggedness of methods of speciation. A good parameter for the evaluation of interest in a research topic is the number of published books. The first publication was in 1983 of the book edited by Leppard on "Trace Element Speciation in Surface Water and its Ecological Implications" (Leppard, 1983). Since that time several books have been published: 1984 (Kramer and Duinker), 1986 (Bernhard et al.), 1987 (Lander), 1988 (Buffle; Kramer and Allen), 1989 (Batley; Harrison and Rapsomanikis), 1990 (Patterson and Passino), 1991 (Krull), and 1996 (Caroli). This regular appearance is a strong indication of the continuous attention paid to this topic by major scientific publishers. Figure 2 shows the accumulated number of published papers from 1980 until the present (late 1998). There is an exponential growth in this area, with the total number of papers published during the period of time considered (from Analytical Abstract Data Base) of more than 700. Considering the metals studied, it can be seen from Figure 3 that the major interest has focused on metals with different stable oxidation states such as As, Se, Cr, and Sb, or with highly stable specific organic forms, commonly used in industrial applications, such as Hg, Sn, and Pb. Figure 2. Chronological evolution of the scientific literature about speciation (from Analytical Abstracts January 1980-March 1998).
  16. 16. 6 M. DE LA GUARDIA,M.L. CERVERA,and A. MORALES-RUBIO Sb Se As 2% 8% Pb Cr .......... 11% Miscellaneous ~~N 19% g 12% Sn 14% Figure 3. Distribution of the literature on speciation as a function of the elements determined. Throughout this chapter the reader will find a guide to follow the scientific literature about speciation studies. It includes a final section providing a summary of speciation of individual metals. III. FACTORS AFFECTING SPECIATION All analytical steps preceding speciation are extremely critical to assure the stability of a chemical species present in natural samples. Sampling, sample preservation, storing, and sample treatment must be carefully controlled in order not to disturb the equilibria established among the species. Therefore, practical studies on spe- ciation must involve several protocols to ensure the accuracy and representivity of laboratory data. Additionally, typical problems which can occur during the deter- mination of total concentration of trace metals, such as the contamination of samples from the material employed for sampling by reagents employed for sample preservation or losses during storing and sample pretreatment, must be taken into account. The instability of redox species changes in a sample erwironment, may also be significant. Due to theses concerns, it is necessary that changes involved by sample handling do not modify or change the ratio between species. A good example of this potential problem can be found during the speciation of Fe in deep lakes. The absence of 0 2 and the presence of high quantities of dissolved CO2 involve a sample environment totally different than that found outside; Fe (II) being frequently oxidized to Fe (III) during the sampling step. Redox conditions, macro- constituents, ionic strength, pH, temperature, and pressure are some of the factors affecting speciation and species preservation. Additionally, synergistic and antago- nistic effects between trace compounds present in the sample must also be taken into consideration. When biological fluids are to be analyzed, the conditions of a
  17. 17. Speciation Studies 7 minimum and soft pretreatment procedure must be applied to attain quick and accurate analysis, paying particular attention to the type of compound used for calibration (Dawson, 1986). During the sampling step, a number of potential problems should be considered during the collection of biological fluids. The major problem in such work is due to the contamination and losses of the trace metal. The effect of contaminants may alter the amount of analyte bound to a given fraction. For example, zinc and copper ions added in-vitro to a serum sample result in an increase in the albumin-bound fraction (Cornelis et al., 1993). It must also be considered that some factors, such as person-to-person differences, region-to-region variations, occupational expo- sure, and physiological state of the subject, would influence the chemical speciation of a given metal in body fluids (Negretti de Br~itter et al., 1995). Sampling of urine in studies devoted to metal speciation is commonly performed on urine collected during a 24-h period. One reason for this is that many constituents exhibit diurnal variation, with variable peak excretions as a result of variation in drinking patterns. At the beginning of the collection period (usually when the person awakens), the bladder should be emptied, the specimen discarded, and the time noted. All urine specimens passed during the next 24-h period are collected in a pre-cleaned polyethylene or polypropylene container. At the end of the collection period the bladder is emptied and the specimen is added to those already collected. Transfer of the urine from the body into the container may introduce contamination from clothes and skin (Robberecht and Deelstra, 1984). The timing of body fluids taken from a subject can have a significant influence on the concentration of total metal and species. Thus, the diurinal variation of zinc concentration in human blood plasma, and the history of the tissue before sampling, e.g. the use of cosmetic agents (shampoo or conditioner) in the treatment of hair, affects the result (Dawson, 1986). It has been shown that the concentration of protein-bound serum zinc in human blood plasma varies depending on whether the sample was taken from a patient who was standing or lying in a recumbent position (Behne, 1981). Based on the previous comments, it is absolutely necessary in the determination of speciation of metals in biological fluids to include a detailed description of both, the samples analyzed and the procedures for sampling and storage, in order to make possible a critical evaluation of the published literature. The key to successful speciation work is the preservation of the species. Often this is impossible and the integrity of an organometallic compound is violated (Woittiez et al., 1991). Nevertheless, collected samples are usually frozen at -20 ~ until required. Next, the frozen substance is allowed to thaw to room temperature. At this stage, pretreatment needs the addition of preservatives, stabilizers, and other additives. If these substances are complexing agents or their presence could change the pH, ionic strength, redox potential, and dielectric constant of the medium, it could result in some changes in the distribution of chemical species. For example, methylmercury may be lost from blood on freeze-drying (Horvat and Byrne, 1992).
  18. 18. 8 M. DELAGUARDIA,M.L.CERVERA,and A. MORALES-RUBIO Lyophilization is a common process applied to natural or real samples. It converts the material into a form that can be easily stored for longer periods. It is necessary to ensure that the analytes (metals) are not lost and the material characteristics as well as the stability of the analyte species are maintained. In fact, it has been reported that lyophilization could cause the destruction of lipoproteins (Kroll et al., 1989) and also result in denaturation and aggregation of other proteins. Evidently, this will change the speciation of metals like selenium which is associated with lipoproteins (Gardiner, 1993). A problem, studied in depth, is the effect of storage conditions of sediments for species of Cd, Cu, Fe, Mn, Ni, Pb, and Zn. Typical methods of sample preservation available are (1) wet at room temperature, (2) wet at low temperature, (3) frozen, (4) freeze-drying, (5) oven-drying, (6) microwave- assisted drying, and (7) air-drying at room temperature. It was found that wet preservation methods produced a reducing soil or sediment environment, whereas drying procedures provided an oxidizing environment with important conse- quences for speciation. Drying at 90 ~ promotes the crystallization of amorphous oxides and the formation of new solid mineral phases. It appears that freeze-drying and microwave-assisted drying could be more appropriate for geochemical material preservation. During a study on preparation of reference materials of clays and sediments, it was concluded that microwave-assisted drying provided excellent precision but did not produce identical results than those found after conventional drying. The composition changes and sample instability could be produced during the microwave treatment (Beary, 1988). However, in spite of changes introduced by drying, due to the inevitable oxidation of samples, which can be dramatic in the case of oxidizing sediments, it is clear that drying inhibits further changes in speciation mediated by microbiological action. It was concluded, in a rigorous study, that air-drying preservation of soils and sediments facilitates sample han- dling, homogenization, and preservative subsampling without affecting chemical species (Ure, 1994). In order to improve the methodologies for species separation and determination, efforts must be made in order to assure correct protocols, including all the steps of the analytical process. This will lead to accurate results in speciation studies. Appropriate methodologies for sampling, which avoid species transformation, and a complete guide for sample preservation, homogenization, and subsampling must be developed, and accompanied by a careful check on their effect on species stability. In the following sections problems regarding speciation in complex liquid samples, such biological fluids, and particularly in solid samples, for which a drastic sample pretreatment is often required, will be presented. These areas will be discussed in practical applications of speciation studies. IV. SPECIATION IN WATERS: THE METHODOLOGY All atomic spectrometric methods work for the direct analysis of liquid or dissolved samples. All the main atomic spectrometry detectors have been employed in
  19. 19. Speciation Studies 9 speciation studies. From the techniques indicated in Figure 4, cold vapor atomic absorption spectrometry (CVAAS) is preferred for Hg determination and micro- wave-induced plasma-atomic emission spectrometry (MIP-AES) can be applied to easy volatile compounds or easily derivatizable metals, from which a gaseous phase, free from the solvent, can be obtained. On the other hand, the lack of an appropriate commercially available atomic fluorescence spectrometry (AFS) in- strumentation (this has been changed recently) requires a strong reduction of the metals to determine Hg, As, Se, Sb and Te (Corns et al., 1993). In fact, the evolution of the literature about speciation in atomic spectrometry clearly shows that, in the case of atomic emission measurements, the recent development of hot atomization systems such as plasma, has completely replaced flame emission procedures. The inductively coupled plasma (ICP) is the most commonly and widely employed plasma source. Regarding the use of atomic absorption techniques, flame atomic absorption spectrometry (FAAS) continues to be employed to a larger extent than electrother- mal atomization atomic absorption spectrometry (ETAAS). This is due to the easily coupling between FAAS and dynamic separation systems. This is despite the reduced sensitivity of FAAS as compared with ETAAS. An important aspect to be considered in order to improve the sensitivity of speciation studies by FAAS is the possibility to generate on-line volatile derivatives, like covalent hydrides. This avoids problems related to the reduced nebulization efficiency of classical continu- ous introduction systems. This dramatically increases the sensitivity and will add /EMISSION [FLAME PHOTOMETRY(FP) I PLASMAEMISSION | MICROWAVEINDUCEDPLASMA(MIP) | INDUCTIVELYCOUPLEDPLASMA(ICP) I DIRECTCOUPLEDPLASMA(DCP) /FLUORESCENCE I ATOMIC FLUORESCENCE(AFS) I/ION COUNTING i ICP.MASSSPECTROMETRY~ lioBsORPTiON LD VAPOURATOMICABSORPTION(OVAAS) AME ATOMICABSORPTIOM(FAAS) LECTROTHERMALATOMICABSORPTION(ETAAS) Figure 4. Atomicspectrometrymethodscurrentlyemployedin speciationstudies.
  20. 20. 10 M. DE LA GUARDIA, M.L. CERVERA,and A. MORALES-RUBIO new variables. These can be suitable to improve the selective determination of different species, as a function of their ability to form hydrides and the kinetics of this process. However, as indicated previously, the low residence time of sample particles in the measurement zone and the high temperature of the atomization cells can cause problems in discriminating the chemical forms of a metal. As a consequence, the single use of atomic spectrometry is not convenient for "in situ" analysis of chemical species with only a few exceptions (Arpadjan and Krivan, 1986; de la Guardia, 1996). This has led to the general strategy for speciation by atomic spectrometry being based on its hyphenation with separation techniques. Figure 5 summarizes the techniques most often employed to preconcentrate selectively or to isolate a specific chemical form of a metal to be determined by atomic spectrometry. From these techniques, a general consensus has been reached that gas chromatography (GC) (Schwedt and Russel,1973; Fernandez, 1977; Van Loon, 1979; Segar, 1984; Ebdon et al., 1986; Chan and Wong, 1989) and liquid chromatography (LC) (Van Loon, 1981; Fuwa et al., 1982; Chau, 1986; Irgolic, 1987; Ebdon et al., 1987a and 1988) are the best alternatives to provide accurate determination of the different species of an metal. Recent studies have focused on the development of appropriate interfaces between chromatography and flame spectrometers (Aue and Hill, 1973; Kawaguchi et al., 1973; Jones and Managhan, 1976; Parris et al., 1977; Ebdon et al., 1988), electrothermal atomizers (Brickman et al., 1977; Ebdon et al., 1982 and 1987b) or plasmas (Beenakker, 1977; Windsor and Denton, 1979; Gast et al., 1979; De Jonghe et al., 1980; Krull and Jordan, 1980; Hansler and Taylor, 1981; Duebelbeis et al., 1986; Bushee, 1988; Bushee et al., 1989; Crews et al., 1989; Heitkemper et al., 1989). Gas chromatographic (GC) separation requires that the species to be determined are volatile and thermally stable under the conditions employed for separation. Figure 5. Separationmethods commonly employed in speciation studiesclassified as a function of the phasesinvolved (reproduced from de la Guardia, 1996).
  21. 21. Speciation Studies 11 Speciation through GC-atomic spectrometry is limited to the analysis of volatile organic compounds such as lead or mercury alkylderivatives, which commonly occur in natural samples. Additionally, volatile derivatives can be prepared before the chromatographic separation, as was the case in the arsenic speciation through methyl thioglycolate formation (Haraguchi and Takatsu, 1987; Ebdon et al., 1988). Compared with GC, procedures based on liquid chromatography (LC) separation followed by atomic spectrometry detection are more simple and suitable to the direct speciation of many natural occurring species. In general, LC-AS speciation studies involve separations on C18 bonded silica columns, or the use of ion- exchange resins. Porous gels based on the use of size exclusion mechanisms (Van Loon and Barefoot, 1992) are suitable to be applied to differentiate between inorganic and organic species, cationic and anionic species, and also species of the same type but with different molecular sizes. However, the simplest applicability of LC for speciation studies in atomic spectrometry is compounded by difficulties found in the development of appropriate interfaces between the LC and the atomic detector. The flow rate of the carrier gas or liquid phase through the chromatographic column must be adjusted to the gas or liquid uptake of the detector. A simple heated stainless steel tube, with minimal dimensions, can be employed in the case of GC. For LC separations, an auxiliary supply of the mobile phase is commonly required to supplement the column effluent. New nebulizers (Gustavsson and Nygren, 1987), jet separator interfaces (Gustavsson, 1987) and thermospray interfaces (Robinson and Choi, 1987)have been proposed for this reason. The time of species separation must be reduced to a minimum in order to save both time and gas consumption, particularly when inductively coupled plasma-atomic emission spectrometry (ICP- AES) or inductively coupled plasma-mass spectrometry (ICP-MS) determination is performed. In the literature there are examples on fast chromatographic separation and atomic spectrometry detection of several species of a metal. Figure 6 depicts a gas chromatogram of seven organotin species separated after ethylation with NaBET4 (Martin-Lecuyer and Donard, 1996). It can be seen in Figure 7 by an appropriate selection of both, the chromatographic column and the mobile phase, it is possible to clearly separate different mercury species, by liquid chromatography. This allows the quantitative analysis of traces of these species by using an extremely sensitive detector such as ICP-MS (Pastor, 1998). The hyphenation between chromatography and atomic spectrometry provides suitable tools for speciation in liquid and dissolved samples. However, additional efforts must be achieved in order to improve the multimetal capabilities of some atomic detectors, like ICP-AES, or ICP-MS in order to develop methodologies suitable for multimetal speciation studies in natural samples. There are some precedents on the use of typical single metal techniques, such as ETAAS, for multi-speciation analysis. These are based on the use of a gold trap for retention of Se and Te species and sequential leaching with H20, 1 M HC1 and 3 M HNO3,
  22. 22. 12 M. DE LA GUARDIA, M.L. CERVERA, and A. MORALES-RUBIO (MV) 20,0 - 19,5 - 19,0 - 18,5! 18,0 17,5~ 17,0 16,5 - 16,0 - 15,5 - 15,0 - 14,5 - 3 i 5 I o 15 I 10 Time (min) Figure 6. Gas chromatogram of seven organotin species separated by GC after ethylation with NABEt4 and detected by flame photometry (reproduced from Martin- 3+ 2+ 3+ + Lecuyer and Donard, 1996). (1) BuSn , (2) Bu2Sn , (3) PhSn , (4) Bu3Sn , (5) Bu4Sn, (6) Ph2Sn2+, (7)Ph3Sn+, and (e) Internal Standard (Pr3Sn+). combined with ion chromatography (Muangnoicharoen et al., 1988) or based on extraction in CHC13and CC14of As(III), Sb(III), Se(IV) and Te(IV) with 4% APDC followed by a separate treatment of another sample aliquot after an appropriate reduction of oxidized forms of these four metals (Chung et al., 1984a, b). However, multi-speciation studies has been achieved using ICP-AES (McCarthy et al., 1983; Gjerde et al., 1993; Sanz-Medel et al., 1994), MIP-AES (Sadiki and Williams, 1996), and ICP-MS (Haraldsson et al., 1993; Jantzen and Prange, 1995; Kumar et al., 1995; De Smaele et al., 1996; Krupp et al., 1996; Guerin et al., 1997). Gas chromatography has been employed in the multimetal speciation of organo- tin and organolead compounds. It is based on their extraction with pentane. This is followed by derivatization with pentylmagnesium bromide and butylmagnesiun
  23. 23. Speciation Studies 60000 50000 - Ions/s 40000 - 30000 - 20000 - HgCI2 2.56 EtHg 4.36 13 1MeHg I~ ~3.15 JI PhHg 10000 0 0 200 400 600 800 Time (s) Figure 7. Liquid chromatogram of four mercury species separated by HPLC and direct ICPMSdetection of 25 ng of each specie (from Pastor, 1998). chloride, and MIP-AES (Sadiki and Williams, 1996). During the direct separation of organotin, organolead and organomercury species in environmental samples (De Smaele et al., 1996) and sediment samples (Jantzen and Prange, 1995), ICP-MS was used in the determination. Methylated and alkylated species of As, Sn, Sb and methylated derivatives of Bi, Hg, Ge and Te can be also separated and determined by GC-ICP-MS (Krupp et al., 1996). Supercritical fluid chromatography (SFC) with ICP-MS detection has been proposed for the separation of trimethylarsine, triphenylarsine, triphenyl arsenic oxide, triphenylantimony, and diphenylmercury (Kumar et al., 1995). Liquid chromatography coupled to ICP-AES (McCarthy et al., 1983; Gjerde et al., 1993; Sanz-Medel et al., 1994) and coupled to ICP-MS (Haraldsson et al., 1993; Guerin et al., 1997) provides an excellent way for multimetal speciation. Direct injection nebulization in ion chromatography-ICP- AES provides a good way for As, Se and Cr multispeciation (Gjerde et al., 1993). Using a nucleosil dimethylamine column and a mobile phase of ammonium
  24. 24. 14 M. DE LA GUARDIA, M.L. CERVERA,and A. MORALES-RUBIO phosphate buffer with gradient elution from pH 4.6 to pH 6.9, it has been possible to separate AsO]-, SeO~-, AsO3-, and SeO2-. Absolute detection limits of 52, 140, 57, and 91 ng, respectively, were obtained in these studies (McCarthy et al., 1983). The use of a C18 bonded silica column, modified with didodecylodimethylam- monium bromide in 50% methanol, has been proposed to achieve the separation of As, Se, Hg, and Sn species using surfactant vesicles mobile phase and ICP-AES determination (Sanz-Medel et al., 1994). The technique of ICP-MS provides a highly selective and sensitive detector for multimetal speciation. It has been proposed as a method for A1, Cd, Co, Cu, Pb, Mn, Mo, Ni, Zn, and Fe speciation in waters using Chelex 100, Fractogel TSK DEAE-650 and C18 to fractionate the free metal ions or easily dissociate complexes, humic complexes on nonpolar organic compounds (Haraldsson et al., 1993). Monomethylarsonic acid, dimethy- larsinic acid, selenite, selenate, tellurate and antimonate can be separated using a PRP • 100 anion exchange columns and determined accurately by ICP-MS (Guerin et al., 1997). Figure 8 shows an example of the chromatograms which can be obtained for simultaneous speciation of arsenic and selenium compounds using a microcolumn of anion exchange for the quantitative separation of monomethylar- sonic, As (III) and As (V) and that of Se (IV) and Se (VI) using ICP-AES as a detector. The excellent sensitivity of AFS and ICP-MS detection and, in the latter case, its exciting possibilities for multimetal and isotopic analysis offer excellent possibilities for a rigorous speciation of liquid samples. An additional effort is required in order to improve both analyte separations and detection, in order to be 800 - Se +4 600 - t-- 400- < 200 - MMA :~ Se+6 I 000- 0 I I I 0 50 100 150 200 Time (s) Figure 8. Simultaneousspeciation of As and Se species by using anion exchange chromatography directly coupledto ICP-AESthrough a direct injection nebulization system (reproducedfrom CETAC,1993).
  25. 25. Speciation Studies 15 able to perform direct multimetal speciation in natural samples. For that reason it will be necessary to develop additional strategies for on-line precolumn and postcolumn derivatization and a careful validation of the academic methodology by the analysis of complex samples with the use of reference and spiked samples. V. METAL SPECIATION IN BIOLOGICAL FLUIDS: SOME SPECIFIC PROBLEMS Speciation of trace metals in biological fluids and tissues has been approached in many different ways during the last decades, but it is extremely difficult because of the complexity of biological systems. Metal speciation in biological fluids implies investigation of the bond between trace metals and available ligands, mostly proteins or compounds with relatively low molecular mass, as a basis of kinetic and metabolic studies (Cornelis and De Kimpe, 1994) and must take into consideration the complexity of clinical matrices. The most common way for speciation of metal ions in biological fluids is the identification and quantification of the biologically active compounds to which the metal is bound and the quantification of the metal in relation to those particular molecules. Initially, many difficulties were encountered while determining the total content of trace metals in a biological fluid or tissue sample, such as the elimination of matrix interferences, the development of effective and fast sample decomposition methods, or the control of sample contamination. These problems are now under- stood and under control. However, a new problem is to define the various biocom- partments to which the trace metals are linked. As a matter of fact, speciation of trace metals in biological fluids consists of defining the various biocompartments to which these metals are linked and to explain their mobility, storage, retention, and toxicity. The toxicity of chemical species of different metals is a function of the target metal and the chemical structure of the compounds considered and depends on the absorption path of the metals. Most of the species of interest in the toxicology of trace metals are small molecules. Many metals are capable of forming organomet- allic compounds and their toxic effects, in some cases, exceed by far those of the inorganic forms of the metals or the compounds formed with large molecules. In general, in the investigation of the toxic effects, the speciation of small molecules is of concern, whereas in the investigation of biological functions, the determination of large molecules has priority (Das et al., 1995). In the case of speciation studies it is very important to maintain the integrity of the metal-ligand binding and to check the mass balance of the protein and the trace metal throughout the isolation steps (Cornelis et al., 1993). Again, in view of the recent problems in environmental and clinical fields all over the world, fast and reliable analytical techniques for chemical speciation in biological fluids are needed urgently (Dunemann, 1992).
  26. 26. 16 M. DE LA GUARDIA, M.L. CERVERA,and A. MORALES-RUBIO Chemical speciation of trace metals in biological matrices has been discussed in some review articles. Metal ion speciation in biological fluids (Das et al., 1996) and in solid matrices including foodstuff and biological materials (Das et al., 1995) have been discussed recently by our group. Gardiner (1988) discussed the role of atomic spectrometry techniques in chemical speciation in biology and medicine. They also described considerations in the preparation of biological and environmental refer- ence materials for use in the study of the chemical speciation of trace metals (Gardiner, 1993). Behne (1992) depicted the trends and problems of speciation of trace metals in biological materials. Van Loon and Barefoot (1992) reviewed a selection of major developments in the field of analytical methodology for metal speciation in various matrices including biological samples. Ebdon et al. (1986) has extensively reviewed the coupling between GC and atomic spectrometry and of LC and atomic spectrometry. An excellent review is available on flow injection and speciation (Luque de Castro, 1986). Although there are some review articles on speciation of a particular metal, like selenium in human urine (Robberecht and Deelstra, 1984),and arsenic in biological fluids (Violante et al., 1989), the literature on speciation of metal ions in biological fluids is rather limited. In Figure 9 the number of published papers as a function of the year of their publication is shown. From this data it is evident that the scientific literature Figure 9. Development of the literaturepublished about metal speciation in biologi- cal fluids.
  27. 27. Speciation Studies 17 concerning this topic has been increasing in recent years. The papers published could be classified into four groups depending on the type of biological fluid: blood, urine, milk, and miscellaneous biological fluids. In Figure 10 the percentage of analytical papers devoted to different body fluids is shown. The use of atomic spectrometric methods in clinical samples is well established. Alcock (1993) has published a review on this aspect. It is clearly evident from Figure 11 that the feasibility of various atomic spectrometric techniques for the determination of chemical species in biological fluid samples are in the order: ETAAS > cold vapor atomic spectrometry (CVAAS) > hydride generation atomic absorption spectrometry (HGAAS) > FAAS > ICP-AES = ICP MS > direct current plasma-atomic emission spectrometry (DCP-AES) = MIP-AES. All these tech- niques are useful for the analysis of liquid samples (except MIP-AES which is more convenient for gaseous samples). In recent years, hyphenated methods like those involving chromatographic methods linked to atomic ones have emerged. All of the hyphenated methods have shown high promise for specific metal species in specific sample matrices, with different degrees of selectivity, specificity and sensitivity/de- tection limit. All these techniques have provided the analyst a choice of methods for trace metal species determination. Actually, there is no simple way to decide which specific hyphenated or direct method of analysis is the best for a particular metal species in a particular sample matrix. Perhaps that remains as a lacuna in the whole scheme of trace metal speciation. Urine Milk 11% Blood 36 % ~%~176"/~o /- ~'*~.,@'~q'r162 ~ ~ LBr~176176 1% 9e ..g d Figure 10. Distributionof published papersaboutspeciation in biological fluids as a function of the typeof sampleconsidered.
  28. 28. 18 M. DE LA GUARDIA,M.L. CERVERA,and A. MORALES-RUBIO ETAAS 22.4% ICPMS 1 8 . 1 ~ ~ . ~ x ~ DCPAES 1.7% ~:I~HGAFS 2.6% ~ i i![,~,PAES3.4*/. FAAS 9.5% ICPAES 16.4% 2.1% HGAAS 13.8% Figure 11. Atomicspectrometricmethodsappliedto speciationof biological fluids. In several cases, atomic spectrometric measurements can be employed for speciation purposes without requiring the use of chromatographic techniques (de la Guardia, 1996). In these cases, the procedures could be based on (1) the different atomization yields obtained for different chemicals, (2) the use of selective extrac- tion, (3) derivatization procedures performed previously to the measurement step, (4) a selective volatilization of the different chemical forms of the elements to be determined, or, (5) other less commonly used separation methodsme.g, ultrafiltra- tion, coprecipitation, electrodeposition, or electrophoresis. The development of automated procedures of analysis based on flow injection analysis (FIA) techniques offers new possibilities for the on-line treatment of samples. It can be expected to provide the availability of simple and low-cost procedures for the metal speciation based on on-line separation and atomic spec- trometric determination (Luque de Castro, 1986; de la Guardia, 1996). Thus, by introducing FIA, a 5.5- to 60-fold increase in the sensitivity is obtained for FAAS and a 15- to 50-fold increase for ETAAS. Examples for other speciation methods proposed for the analysis of various trace metal species are based on high-performance liquid chromatography (HPLC) separation followed by determination by differential pulse anodic stripping voltam- metry (DPASV) (Michalke and Schramel, 1990) or radiotracer labeling (Cornelis, 1992). To date, there is no generalized method for speciation of protein-bound trace metals (Cornelis et al., 1993). To provide an idea of such a method, an outline of procedures applied to trace metal speciation in blood is presented in Figure 12. A. Speciation in Blood Blood is the most important indicator for humans and domestic animals as an illness diagnostic. For clinical purposes, serum is commonly used but in some cases
  29. 29. Speciation Studies 19 ,I, ISearat,ono.t.e prote,ns! ~ o., 0erm.a,,on-,.n,on-. ca,,on-,a,,in,,,-ch,oma,o0r,0hy..,o~ !i iii i ~1 9 ii Major fraction for trace element analysis ,, ~AtomicspectrometryI i 1 $ i M,no;,raot,onI i I Proteinidentification ~Nephelometry1 ~On-linemonitoring-1|UVand~or LVisible spectrometry 1 I Proteinquantification i1 ~.Electrophoresis~ Figure 12. Scheme for trace element speciation in blood (reproduced from Das et al., 1996). whole blood samples may be analyzed (Pais, 1994). Blood contains thousands of different compounds, although with little variability in total ionic strength. A series of procedures have been proposed in the bibliography for trace metal speciation involving aluminum, arsenic, chromium, copper, iron, mercury, lead, platinum, selenium, silicon and zinc. Most studies have been made with aluminum and mercury followed by selenium and chromium and then the other metals. There are only a few studies focused on simultaneous speciation of several metals (Br~itter et al., 1988a). Aluminum Aluminum toxicity is now well recognized and gaining more and more interest. The only aluminum oxidation state in biological samples is (+III). Metal speciation is a crucial feature in directing the biological effects of aluminum. In blood plasma, citrate is the main small carrier and transferrin the main protein carrier for AI(III or +3). In fluids where the concentrations of these two ligands are low, nucleoside mono- and diphosphates become aluminum binders and when they are absent, then catecholamines are the major ligands. Double-stranded deoxynucleic acid (DNA) binds AI(III) weakly, and in general, is unable to compete with other ligands for its complexation. In the cell nucleus, AI(III) is probably bound to nucleotides or to phosphorylated proteins (Martin, 1992). Nevertheless, organically complexed forms of aluminum appear to be much less toxic than inorganic forms. An aluminum
  30. 30. 20 M. DE LA GUARDIA, M.L. CERVERA,and A. MORALES-RUBIO species of particular concern are A13§ AI(OH)2§ AI(OH)~, and AI(O H)4 (Gardiner et al., 1984). Aluminum species present in human blood serum have been separated by gel permeation chromatography (GPC) and by ultrafiltration (Blanco Gonzalez et al., 1989). The former procedure was applied for dialysis patients to fractionate aluminum-bound species. Aluminium determination was performed by ETAAS. Several inferences have been drawn by these authors including: (1) albumin and probably transferrin are the major proteins that bind aluminium; (2) added alu- minium is taken up by serum constituents only after incubation and a slow exchange of aluminium between the species occurs in serum; (3) the low molecular mass fraction is made up of mainly inorganic aluminium complexes; and, (4) the pH of the serum determines the level of ultrafiltrable aluminium. In experiments with patients undergoing desferrioxamine (DFO), chelation ther- apy showed that the ultrafiltrable aluminium in their serum increased by up to 74% because of the formation of a relatively low molecular mass chelate with the DFO. HPLC separation of serum proteins was performed with an ion-exchange column ofTSK DEAE-3SW using a sodium acetate gradient (0-0.5 M) at pH 7.4 (Tris-HC1 buffer). Proteins were detected spectrophotometrically at 280 nm and the alu- minium determined by ETAAS in 0.5 mL fractions collected from the HPLC column. Results obtained with this system suggests that transferrin is the only aluminium binding protein in normal serum, but in the presence of DFO in serum, most of the aluminium was found to be bound to this drug. Further work in this area using ultramicrofiltration and HPLC techniques by Sanz-Medel and Fairman (1992) has revealed that more than 90% of the aluminium in serum is bound to the protein transferrin. To have a clear idea about the presence of A1 species in blood the following studies at clinically relevant concentrations must be performed: (1) identification of the aluminium binding serum proteins; (2) quantification of the amount of these protein-bound aluminium as well as non-protein bound fraction; (3) study of the effect of the Fe-transferrin saturation on the transferrin binding of aluminium which is important in view of the large number of iron-depleted patients; and, (4) investigation of the effect of DFO on the binding of A1 and Fe to transferrin (Van Landeghem et al., 1994). Mercury There has been a continuous work on mercury species present in biological fluids. For the determination of mercury one of the most selective and sensitive methods is the use of CVAAS based on the low boiling point of Hg~and the easy reduction of mercurials to the zero-oxidation state (Magos, 1971). From a number of publish- ed papers on mercury speciation in blood, it can be concluded that the reduction of mercurials has been selectively carried out by using different reduction steps with SnC12 and with a mixture of SnC12 + CdC12. Recently, most of the speciation methods for mercury are based on chroma- tographic separations with detection by means of atomic spectrometry. A procedure
  31. 31. SpeciationStudies 21 for determination of organic and inorganic mercury in various biological materials including blood by ETAAS has been reported (Filippelli, 1987). Organic mercury was extracted as the chloride in benzene and reextracted by a thiosulphate solution. The organic mercury thiosulfate extract was next treated with cupric chloride, reextracted in the benzene layer, and analyzed by GLC for speciation. Inorganic mercury was converted into a methyl chloride derivative by methanolic tetra- methyltin prior to extraction. Speciation of mercury in human whole blood by capillary gas chromatography with a MIP-AES system following complexometric extraction and derivatization has been described by Bulska et al. (1992). In this method, methyl- and inorganic mercury were extracted in toluene from whole blood samples as their diethyldithiocarbamate (DDTC) complexes. The product was butylated and the mercury species were then separated and detected. Selenium The accurate speciation of selenium has been a major challenge for analytical chemists and knowledge of its pathways in the environment and living organisms is still limited. The metal can either be considered as essential; 10 to 40 lxg mL-1 in serum and 0.1 I.tgmL-1 in urine or can be toxic when it is in excess. Br~itter et al. (1988a) have developed a procedure for establishing profiles of selenium protein in various body fluids via the on-line coupling of gel permeation chromatography and ICP-AES after performing the acid digestion treatment and the formation of Se(IV) with the subsequent hydride generation in the connecting flow. Recovery studies have been performed to examine and optimize the wet ashing conditions with the aid of various selenium compounds. To demonstrate the usefulness of this technique for the speciation of selenium, its distribution in samples of human origin has been measured. In serum, selenium was found distributed among three different fractions. The location of these peaks seemed to be similar to those of zinc. The highest selenium peak at 90 + 15 kDa is in the same region but shows a broader shape when compared to zinc. This may be due to the presence of selenium containing enzyme glutathione peroxidase (molecular mass 88 kDa) which elutes in the same region. The selenium content bound to different fractions were as follows: 90 kDa fraction 77 + 4%, 200 kDa fraction 10 + 3% and the high molecular mass (>600 kDa) fraction 13 + 5%. Chromium Speciation of chromium in blood has become an important area of research after it was known that patients with terminal renal failure treated with hemodialysis or continuous ambulatory peritoneal dialysis become iatrogenically loaded with chro- mium. The fact was revealed through observation of very high chromium concen- trations (4.25 ng mL-1) in their serum in comparison to normal healthy persons (0.16 ng mL-1) (Wallaeys et al., 1986). Urasa and Nam (1989) developed a method for chromium speciation using both anion- and cation-separator columns. The
  32. 32. 22 M. DE LA GUARDIA, M.L. CERVERA,and A. MORALES-RUBIO procedure used DCP-AES detection and required a preconcentration step to achieve a detection limit of 1 ng mL-1. The method was applied to human serum and other samples. The authors found 0.05 gg mL-1 of Cr(VI) and 0.06 l-tgmL-1 of Cr(III) in a standard reference material of human serum in freeze-dried form obtained from the National Institute of Standards and Technology (SRM 909); although the reducing capacity of saliva and stomach involves that any intake of Cr(VI) can be readily reduced to Cr(III) (De Flora and Wetterhahn, 1989). Chromium speciation in plasma was reported by Cornelis and her group. Chro- mium is known to be mainly bound to two plasma proteinsmtransferrin (molecular mass = 80 kDa) and albumin (molecular mass = 64.5 kDa). A suitable separation procedure was developed for these proteins with in vitro 51Cr(III) labeled plasma from healthy persons as well as from dialysis patients (Cornelis et al., 1992). Speciation studies were also undertaken with the aid of in vitro and in vivo 51Cr-labeled rat and rabbit plasma. It consisted of a combination of fast protein liquid chromatography with cation- and anion-exchange system, ensuring a com- plete resolution of both proteins and a total recovery of chromium. The metabolized Cr was measured by using NaI (T1) detector of the 51Cr label. On the other hand, identification and quantification of proteins were done by isoelectrofocusing and nephelometry, respectively (Cornelis and De Kimpe, 1994). The 51Cr has been found to distributed as follows: 85% is associated with the transferrin, 8% seems to be bound to albumin and 6% appears to be spread over the other components (Cornelis et al., 1992). Since transferrin is usually saturated at 30% only with iron, this protein should indeed be considered as a potential binding site for chromium (Wallaeys et al., 1987). Recent studies demonstrated that 51Cr can shift from albumin to an unidentified low molar mass complex in ambulatory peritoneal dialysis patients (Borguet et al., 1995). Arsenic Arsenic occurs in both inorganic and organic forms which exhibit large differ- ences in their metabolism and toxicity. Elimination kinetics has shown that arsenic is removed very quickly from blood to urine with a half-life in the body of about 30 h (Chana and Smith, 1987). The determination of inorganic arsenic and or- ganoarsenicals in biological fluids was reviewed by Violante et al. (1989). This chapter emphasizes the necessity for distinguishing between As of nutritional origin and that from water or the environment and for guarding against possible intercon- version of the inorganic oxidation states during sample treatment. Speciation studies for arsenic in blood are comparatively less numerous than in urine. Specia- tion of As (III) and As (V) in biological materials including blood was studied by HNO3-H2SO4 digestion followed by hydride generation AAS technique. It was found that the extent of change of the original valency of As was not reproducible (Weigert and Sappl, 1983). Fast protein liquid chromatography cation and anion exchange separation scheme (Cornelis et al., 1993) were applied to serum incubated
  33. 33. SpeciationStudies 23 in-vitro with carrier-free 74ASO43for 24-hr. All 74As was completely eluted from the cation exchanger together with the negatively charged unbound proteins. The ultraviolet (UV) responses of the separated species in combination with the metal- specific responses can be used for correlating the arsenic species with the bulk amount of potential arsenic-binding partners in serum. The protein fractions were identified as asialotransferrin, sialotransferrin and albumin carrying, respectively, 17.7, 25.3, and 56.3 of total 74Asradioactivity, i.e. arsenic is bound to these proteins in these proportions. However, when the albumin fraction was subjected to gel permeation chromatography on a Superose column for differentiating the mole- cules according to molecular mass instead of charge, the elution patterns of the albumin and the 74As did not coincide anymore. Copper and Zinc Recently, several reports have appeared in the literature on the speciation of zinc. Faure et al. (1990) separated the human serum fractions by ultrafiltration with the use polyacrylonitrile membranes. Loosely bound zinc (bound to albumin and some other proteins) was separated with ultrafiltrable zinc after treatment of the serum with ethylene diamine tetraacetic acid (EDTA). The difference between the loosely bound and total zinc gave the content of strongly bound zinc, i.e. to t~2-macroglobu- lin. The zinc content in each fraction was measured by conventional ETAAS. Zinc in serum is bound to macroglobulin (720 kDa) and albumin (66.5 kDa) whereas Cu is bound to ceruloplasmin (160 kDa) and albumin (66.5 kDa) (Gardiner et al., 1981). Sch/3ppenthau and Dunemann (1994) have reported the separation of serum for characterization of metals (including copper and zinc) and nonmetal species by size-exclusion chromatography (SEC). The coupling of HPLC to ICP-AES was performed by connecting the column outlet of the chromatographic system with the nebulizer of the metal-specific detection systems of ICP-AES or ICP-MS. The metal distribution patterns in serum samples indicate a Cu maximum at 68 kDa which again correlates with the first major sulfur maximum at 75 kDa. Thus Cu may be bound to the albumin fraction. The Zn maximum has been recorded at the 49 kDa region. The absence of the high molecular mass proteins in the investigated samples was explained by the method of sample preparation (i.e. centrifugation and filtration through a 0.2 ~m membrane). Iron In a pioneering study, for the determination of iron species in serum samples, an HPLC-ETAAS hyphenated method with an on-line metal scavenger for studying protein binding has been reported (Van Landeghem et al., 1994). Due to the currently introduced procedures for the treatment of anemia in dialysis patients involving a relative iron deficiency in these subjects, the study on the competition between A1 and Fe for binding to transferrin presents a renewed interest. As shown in this study, over 80% of the total serum A1 and over 97% of the total serum Fe is
  34. 34. 24 M. DE LA GUARDIA, M.L. CERVERA,and A. MORALES-RUBIO bound to transferrin, indicating both elements do not dissociate from transferrin during gradient elution. The postelution Fe recovery was found to be 97.3 + 2.0%. Lead The speciation of lead has attracted great deal of interest particularly with respect to organolead compounds. A high-resolution GC-ETAAS method for the determi- nation of trimethyllead salts in human blood samples has been described by Nygren and Nilsson (1987). The method involves several steps including complexation with sodium diethyldithiocarbamate (pH 9), extraction with pentane, evaporation, and butylation by using a Grignard reagent in tetrahydrofuran. Platinum In recent years, platinum compounds, especially cisplatin, are used for the treatment of cancer. In the analysis of biological fluids of patients treated with platinum salts, it is desirable to identify free platinum species and those bound to macromolecules. Ion exchange (Bannister et al., 1978) and ultrafiltration (Pinta et al., 1978) techniques coupled to traditional atomic spectrometry have been applied to study the different platinum species in blood plasma and serum. Cisplatin, its hydrolysis products, and two methionine-platinum complexes were studied by reversed phase ion-pair chromatography with on-line ICP-AES and applied to the analysis of (bio)transformation products originating from cisplatin in human and rat plasma in vitro and in vivo (De Waal et al., 1987). Free platinum (not bound to proteins) in plasma has been separated by ultrafiltration and the metal concentration was determined by ICP-AES (Dominici et al., 1986). Silicon Silicon is becoming a biological trace metal of increasing scientific interest, particularly in connection with neurological disorders associated with aluminium in dialysis encephalopathy and in Alzheimer's disease. Relatively few analytical data exist on the concentration of Si in physiological fluids in health and disease (P6rez Paraj6n and Sanz-Medel, 1994). To provide evidence on the possible correlation between A1 and Si levels in the serum of renal failure patients, and the possibility of the reduction of aluminium bioavailability by the presence of silicon in biological fluids, the effects of different factors including storage conditions, administration of desferrioxamine, and kidney transplantation on the total A1 and Si contents and on their distribution in the same serum samples were examined and compared by Wr6bel et al. (1994). Ultramicrofiltration was used for the separation of low molecular mass and high molecular mass serum fractions and ETAAS for the determination of Si. Distribution of Si in serum proved to be affected only by the storage conditions. When the sample is stored properly (pH < 7.8), the ultrafil- trable Si content results were consistent and reproducible. It was found that 43 +
  35. 35. SpeciationStudies 25 3% of total serum Si in the low molecular mass fraction was ultrafiltrable. Never- theless, Si binding to serum proteins must be different from that observed for aluminium. Various Metal Speciation Speciation of various metals in human serum by anion exchange and size exclusion chromatography (SEC) with detection by ICP-MS was reported by Shum and Houk (1993). A direct injection nebulizer was used with packed microcolumns for anion exchange chromatography and SEC. Proteins in human serum were separated by SEC without sample pretreatment. The metals present in each molecu- lar mass fraction were determined by ICP-MS with detection limits of 0.5 to 3 pg of metal. Six metal-binding molecular mass fractions determined in human serum were assigned as follows: (1) >650 kDa fraction for Pb, Cd, Zn, Cu; (2) 300 kDa fraction for Pb, Zn, Cu; (3) 130 kDa fraction for Pb, Cd, Zn, Ba, Cu, Na; (4) 85 kDa fraction for Fe; (5) 50 kDa fraction for Zn, and (6) 15 kDa fraction for Pb and Zn. There was only one Fe-binding molecular mass fraction found at 85 kDa and this could be serum transferrin. The proteins responsible for the other molecular mass fractions required identification. In summary, speciation studies in blood must take into consideration the low metal contents in clinical samples, the presence of free ions and protein-bounded metals, and the effect of added compounds, like EDTA or DFO. Additionally, natural occurring organometalic species must be considered. B. Speciation in Urine In the case of intoxication or to provide information on the balance between intake and output, the level of trace metals like arsenic, mercury, or selenium in urine is frequently taken as an indicator, since the kidney is an important feature in body homeostasis. Like blood, urine is a complex matrix often causing analytical problems because of its high salt content and a range of organic constituents. When applied to a chromatographic system, the urine matrix may create column overload problems. This can result in peak splitting or broadening of the analyte signals. Thus samples must be subjected to a desalting process, before subsequent separa- tions, to allow control of the elution parameters. The major work that has been performed on metal speciation in urine samples concerns speciation of arsenic followed by selenium and then mercury. Reports on the speciation of other metals like tin, lead, cadmium, chromium, zinc, iron, and magnesium in this biological fluid have been also reported. Arsenic The presence of As in the human body mainly occurs through food and/or occupational exposure. After absorption in the gastrointestinal tract or in the lungs,
  36. 36. 26 M. DE LA GUARDIA, M.L. CERVERA,and A. MORALES-RUBIO this metal is eliminated through urine. Inorganic arsenic undergoes considerable biotransformation in the body; both monomethylarsonic acid (MMA) and dimethy- larsinic acid (DMA) derivatives are formed. The proportion of inorganic and methylated species in urine may vary, although DMA is generally the major metabolite (Buchet et al., 1981). Although total urinary arsenic determinations are often used to assess occupational exposure to inorganic arsenic, specific measure- ments of DMA, MMA, and inorganic arsenic provide a more reliable indicator or exposure than total urinary arsenic levels (Chana and Smith, 1987). Again, or- ganoarsenic compounds such as arsenobetaine have been found to be present in urine following the ingestion of seafood. The decreasing order of toxicity of arsenic compounds is now well known: arsenite > arsenate > MMA > DMA > As~ > arsenobetaine. Different types of methods have been investigated for the speciation of arsenic compounds in urine. Initially for speciation studies of arsenic compounds by atomic spectrometry, a series of procedures based on the use of a selective liquid-liquid extraction or the use of chromatography were reported. Several reports deal with the separation and detection of arsenic species present in urine. This has been performed by HPLC coupled with hydride generation AAS for the determination of arsenobetaine (AB), arsenocholine (AC), and tetramethylarsonium cations in human urine (Momplaisir et al., 1991), and also for the determination of arsenite, arsenate, DMA, and MMA at IxgL-l As level (Chana and Smith, 1987). There are several reports (Jimenez de Bias et al., 1994a; Le et al., 1994) on the determination and speciation of arsenic in human urine by ion-exchange chromatography-flow- injection analysis with hydride generation AAS. Eight arsenic compounds (four anionic, such as arsenite, arsenate, monomethylarsonate, and dimethylarsinate, and four cationic, such as arsenobetaine, trimethylarsine oxide, arsenocholine, and tetramethylarsonium ion) in urine were separated by anion- and cation-exchange HPLC and detected by ICP-MS at m/z = 75. Hexahydroxyantimonate(III) was used as an internal standard for their qualitative analysis. Arsenite was unstable in both urine samples and standard mixtures when diluted with a basic (pH 10.3) mobile phase used for anion chromatography. Interference due to 4~247 was eliminated by chromatographic separation of the chloride present in the sample from the arsenic analytes (Larsen et al., 1993a). Selenium Nearly all information on selenium species in urine refers to rats and mice. Studies on selenium metabolites in human urine are less numerous. Trimethyl selenonium ion (TMSe§ a detoxification metabolite of selenium that is excreted in the urine, was first isolated and identified in rat urine (Palmer et al., 1969). The determination in human urine by cation exchange chromatography and ETAAS was reported by Tsunoda and Fuwa (1987). Fodor and Barnes (1983) were able to speciate selenate and selenite from urine samples by using different pH values for
  37. 37. SpeciationStudies 27 complexation with a poly(dithiocarbamate) resin. It was found that for the urine of 11 healthy persons, the selenite content (8.6 I.tgL-1) was on average about three times more than the selenate concentration (3.1 lag L-l). Laborda et al. (1993) analyzed chromatographic effluents containing selenium species of urine samples by ETAAS using a sampling procedure based on fraction collection and hot injection into an electrothermal atomizer. The HPLC separation of TMSe§ SeO32, SeO42was performed by anion-exchange chromatography using 0.01M ammonium citrate at pH 3 and 7 as eluent. Mercury For mercury speciation in urine samples similar methods have been suggested. In work on mercury speciation in urine and blood, the reduction of mercurials were selectively performed by using different reduction steps with SnC12 and with a mixture of SnC12+ CdC12.Several workers (Robinson and Wu, 1985; Seckin et al., 1986) have separated the organomercury species of urine primarily by gas chroma- tography followed by AAS detection. Samples of urine were injected directly on to a Chromosorb W AW-DMCS column interfaced with ETAAS. Inorganic Hg was the major form of mercury excretion. The results indicated the presence of uniden- tified nonvolatile Hg species in the samples (Robinson and Wu, 1985). Mercury and its species have been determined in urine samples from humans after breathing air in a dental workplace. The urine was treated with concentrated nitric acid and digested in a polytetrafluorethylen (PTFE) reactor at 140 ~ for 90 min. The resulting solution was mixed with SnC12and the Hg vapor produced was analyzed by CVAAS. For GLC analysis of urine, samples were extracted with toluene and then with benzene. The extract was analyzed with temperature programming from 110 to 220 ~ at 15 ~ min-l. Nitrogen as carrier gas and 63Ni-electron capture detection was used (Seckin et al., 1986). Urine samples have also been analyzed by Shum et al. (1992) after separation with ion-pair liquid chromatography followed by ICP-MS detection with direct injection nebulization. A 24 h urine specimen was analyzed by this method and Hg2§ methylHg§ and ethylHg§ species were found. Other Metals Trialkyltins are the organotins having the greatest biocidal activity in mammali- ans. A purge and trap flame photometric gas chromatographic technique for speciation of trace organotin and organosulphur compounds in human urine stand- ard reference material was reported by Olson et al. (1983). Urine samples were purged with nitrogen, with or without prior treatment with NaBH4, and the volatile compounds were trapped on Tenax GC. These compounds were desorbed by heating the Tenax and were analyzed by GLC. The flame photometric detector was used in either the sulfur mode (394 nm) or the tin mode (600 to 2000 nm) with a hydrogen-rich flame. Neutral volatile organotin and organosulfur compounds did not require a pretreatment with NaBH4. This is necessary for alkylchlorotins.
  38. 38. 28 M. DE LA GUARDIA, M.L. CERVERA,and A. MORALES-RUBIO Speciation studies of mono-, di- and tributyltin compounds in urine have been performed by extraction, GC separation, and ETAAS determination (Dyne et al., 1991). Preconcentration and determination of ionic alkyl lead compounds in human urine has been described by Neidhart and Tausch (1992). Preconcentration and cleanup steps were followed by HPLC. Ionic species were preconcentrated by solid-phase extraction and detection was performed on-line. The alkyllead species were eluted from the HPLC column, partially dealkylated by iodine solution to form dialkyllead species, and detected as [4-(2-pyridylazo) resorcinol] complexes after the reduction of excess iodine with thiosulfate. Various cationic species of lead as Pb2§ Me3Pb§ and Et3Pb§ were separated as ion pairs by reversed-phase liquid chromatography-ICP-MS with a direct injection nebulization system (Shum et al., 1992). The method was tested for measurement of Pb species in National Institutes of Science & Technology-Standard Reference Material (NIST-SRM) 2670 freeze- dried human urine. Since this reference material contained only Pb2§ and did not contain measurable levels of Me3Pb§ and Et3Pb§ these compounds were spiked in the NIST urine to test the suitability of the method. Reasonable chromatographic peaks were seen for Pb2§ Me3Pb§ and Et3Pb§ species in the spiked urine sample. A thermospray-interfaced HPLC-flame AAS system was developed for studies of cadmium speciation in human body fluids by Chang and Robinson (1993). The cadmium compounds in urine were separated by HPLC on a Zorbax C8 column with water as the mobile phase. The column eluate was fed directly to the thermospray interface and the analyte delivered directly into the base of the flame for effective atomization and detection by AAS. Successful separations were achieved for a large number of nonvolatile cadmium compounds. C. Speciation in Milk Human milk, cow milk, or infant formulas are the main nutrient fluids for newborn infants. Assuming that the composition of breast milk may satisfy the growing demand of healthy babies during their early months of age, it could be a reference to evaluate the nutritional value of alternative milk formulas. The protein and composition as well as the kind of trace metal of breast milk have been known for the last several years. But the absorption and utilization of the trace metals depend not only on the total amount in the milk but also on the availability of the chemical form in which they occur. Hence the speciation of metals in milk is very meaningful. Few metal speciation studies have been performed in this body fluid. Only several reports on speciation of zinc, cadmium, selenium, mercury, lead, and a few other metals in milk may be found in the literature. Babies at their early ages are susceptible to selenium deficiency. This may arise due to intake of milk or infant formulas with low selenium content. Again, the total amount of this metal does not ensure the overall utilization or bioavailability of selenium. Thus the chemical forms of selenium and its distribution in food are
  39. 39. Speciation Studies 29 important factors of selenium bioavailability. The distribution patterns of selenium species in cow and human milk were compared by Van Dael et al. (1988). Milk samples were fractionated into fat, whey, and casein parts by ultracentrifugation. In order to separate the lipid components, milk fat globule membranes were solubilized with sodium dodecyl sulphate. Further centrifugation separated the outer and inner fat globule membranes from triglycerides. After separation all fractions were lyophilized and stored at -20 ~ Samples were digested with a mixture ofHNO 3and HC104and then analyzed for selenium by hydride generation AAS. The study revealed that the whey fraction represented 40 and 72% of the total selenium content of cow and human milk, respectively. The lipid fraction contained approximately 10% of either cow or human milk's total selenium. After solubiliza- tion of milk fat globule membranes, 61 and 80% of selenium was found in the outer fat globule membrane for cow and human milk, respectively. Determination of the selenium content in individual proteins of cow's milk revealed that the highest selenium concentrations were present in 13-1actoglobulinand in K-casein. Br~itteret al. (1988a) developed a procedure to determine selenium protein profiles in skimmed human breast milk via on-line coupling of gel permeation chromatogra- phy and ICP-AES after performing the acid digestion procedure and the formation of Se(IV) with the subsequent generation of selenium hydride in the connecting flow. In total, four selenium peaks (molecular mass at >600, 90, 25, and 10 kDa) have been detected in breast milk. A method for the preparative separation of human breast milk proteins was developed by Michalke (1993), keeping metal-protein complexes intact, especially with respect to zinc and cadmium species. Separations were performed on TSK columns, using HW 55 gel, with double distilled water as the mobile phase. The metals were determined in native human milk, the protein pellet, and supernatant (without fat fraction) as well as in peak related HPLC-fractions of protein pellet and supernatant with differential pulse anodic stripping voltammetry (DPASV) for cadmium and DCP-AES for zinc, respectively. Cadmium content of whole breast milk, the protein pellet, and the peak-fraction corresponding to metallothionein was determined to be 1 Ixg L-1, whereas no cadmium was found in the supernatant. On the other hand, the amount of zinc was found to be about 3.5 mg L-1 in human milk and only a small quantity (160 ~g L-1) could be detected in the protein pellet. Zinc content could be related to several breast milk proteins (e.g. metallothionein) in different amounts. In the case of the supernatant, zinc was related only to citrate. On-line combination of gel filtration chromatography and ICP-AES has been applied for the fractionation and identification of metal-containing species in skimmed human milk including background control and its subtraction. Subtraction yielded a selenium peak of lower intensity and its shift to position around 10 kDa. At this position, iron, manganese, and zinc elute exactly in the same fraction. Another chromatogram of defatted human milk showed the distribution of copper, iron, manganese, and zinc. Fromthis study, the binding of citrate was established
  40. 40. 30 M. DE LA GUARDIA, M.L. CERVERA,and A. MORALES-RUBIO with zinc. There was also some evidence for the binding of iron to citrate (Br~itter et al., 1988a). The technique of ICP-AES coupled to a gel filtration chromatography system was used by Br~itter et al. (1988b) to characterize the species containing copper, zinc, iron, manganese, magnesium, and calcium in human milk and milk formulas based on cow's milk. The distribution profiles clearly indicate that the metal (Cu, Zn, and Fe)-protein binding in human milk and milk formulas are different. Investigations of protein-bound trace metals in human milk before and after the birth of a baby showed marked differences in zinc and iron bound to citrate. An increase of citrate concentration by a factor of 25 + 3 takes place after birth at the first day of milk secretion. About 80% Cs, 22% Sr, and 1.5% Eu were recovered in pectin when fresh pasteurized skimmed cow's milk spiked with the respective radionuclides (85Sr, 137Cs,and 152Eu)was shaken with a 4% aqueous solution of apple pectin at an initial milk/pectin volume ratio of 7:3. The recovery fraction was proportional to the abundance of radionuclides in milk. Extraction of the spiked milk was performed with Aerosol OT in isooctane (Mac~igek and Gerhart, 1994). Binding of added strontium by milk proteins under native conditions was also investigated using pectin of various degrees of esterification. Upon partition of Sr, Cs, and Eu in aqueous two-phase milk-pectin system performed by membraneless dialysis, it was compared with the distribution between cation exchanger and milk, milk formula, or pectin solutions. The low molecular mass fraction of added Sr in milk assessed from Dowex 50 x 8 sorption data was found to be 31% and that of Cs and Eu were 58 and 40%, respectively (Mac~igek et al., 1994). D. Speciation of Miscellaneous Biological Fluids Some papers concerning different biological fluids in which speciation studies have been carried out other than blood, urine, and milk have been also published. Some examples are on blood cell lysate, sweat, saliva, cerebrospinal, seminal, tear, and bronchoalveolar fluids which have been developed in recent years are presented below. Several reports varying in manipulative complexity have been proposed for determining As, Fe, Zn, and Se species of blood cell lysate. The binding of arsenic in the red blood cells of rat were investigated by Cornelis and De Kimpe (1994) because it was thought that most of this metal binds to hemoglobin (Hb). The radiotracer 74As was used throughout the experiment. The red blood cells lysate was submitted to size exclusion chromatography (SEC) using Superose HR 12 column and 0.15 M NaCI + 25 mM Tris (pH 8) eluent and cation-exchange chromatography using Mono S HR 5/5 column and 10 mM malonic acid (pH 5.8) and 10 mM malonic acid + 0.3 M LiC1 (pH 5.8) eluents. The study of binding of As in the red blood cells lysate with SEC showed that 87.7% of the metal is associated with a protein with a relative molecular mass of around 60 kDa and a
  41. 41. Speciation Studies 31 strong absorption band at 420 nm. This compound is Hb. Cation exchange of the SEC fraction showed that the signal peak observed in the SEC chromatogram consisted of several Hb species, each carrying part of the 74As. On-line combination of gel filtration chromatography and ICP-AES for studying metal speciation in blood serum and human milk has already been discussed in previous sections. The same technique was also used for red blood cell lysate (Br~itter et al., 1988a). From the gel filtration chromatography of human erythro- cyte-lysate on a TSK column, it may be found that selenium is eluted in two peaks which correspond to a molecular mass of 90 kDa and 33 kDa. The high molecular mass compound could be classified with the selenoenzyme glutathione peroxidase. Iron and zinc were also monitored with selenium. Fe indicates the position of Hb in erythrocytes and Zn being in the position of the enzyme carbonic anhydrase. Owing to the narrowness of these two markers, a clear identification of the selenium binding complex at 33 kDa is not possible. Accumulation of iron in the myocardium in circumstances of transferrin satura- tion is associated with heart failure in iron-loaded patients. To characterize the underlying causes of this phenomenon, Parks et al. (1993) measured the flux as well as the speciation of iron in normal and iron-loaded cultures of rat myocardio- cytes. Iron loading of cultured myocytes induced shifts in iron speciation. Thus the ratio of iron bound in hemosiderin-like compounds to ferritin-bound iron increased twofold from a range of 0.84 to 1.44 in control cells to 1.96 to 3.3 in iron-loaded cells. Only few analytical methods are known for determining chemical species of mercury, cadmium and sodium in sweat (Robinson and Wu, 1985; Chang and Robinson, 1993). Calcium and magnesium species have been studied in saliva (Lagerlof and Matsuo, 1991). Speciation of iron, potassium, sodium, and calcium has been reported in cerebrospinal fluid (Gutteridge, 1992). Ferrous ion has been detected in cerebrospinal fluid by using bleomycin and DNA damage. Normal cerebrospinal fluid samples were centrifuged and the supernatant liquid was stored at-20 ~ For the determination of Fe(II) species the following reagents were added in order, into metal-free plastic tubes: (1) 0.4 mL of DNA solution (1 mg mL-1) stored over 0.05 volumes of Chelex-100 resin, (2) 0.1 mL of bleomycin sulphate (1.5 mg mL-1) stored over a solution of conalbumin (5%, w/v) where 120 mM sodium azide was added to inhibit the ferroxidase activity of ceruloplasmin, (3) 0.1 mL of cerebro- spinal fluid, and (4) 0.4 mL of sodium phosphate buffer (pH 7) treated with conalbumin. The mixture was incubated at 37 ~ for 30 min to allow Fe2§ - ent degradation of DNA and then treated with 0.5 mL of thiobarbituric acid (1%, w/v in 50 mM NaOH) and 0.5 mL of HC1 (25%, v/v). The mixture was heated at 100 ~ for 5 min, cooled and extracted with 1.5 mL of butan-1-ol. The mixture was centrifuged and the thiobarbituric acid-reactive material in the clear upper organic layer was determined spectrofluorometrically.
  42. 42. 32 M. DE LA GUARDIA, M.L. CERVERA,and A. MORALES-RUBIO In seminal fluid, iron and zinc species have been studied by various authors (Caldini et al., 1986; Gavella, 1988). Atomic absorption techniques for direct determination of multimetal species in whole tear film were described by Giordano et al. (1983). Dissolved sodium, lithium, potassium, calcium, and magnesium were determined by flame AAS. No matrix effect was observed. ETAAS was used for determination of chromium, manganese, iron, lead, zinc, copper, nickel, cobalt, arsenic, and aluminium. These metals were present in the range between less than 0.01 to 1 l.tgmL-1 and probably, following this methodology the total content is determined. The reader is invited review Table 1 for the analytical details of the selected metal ion species in biological fluids. It suggests that chromatography is the method of choice for the preferential separation of various components in biological fluids. After separation, metals are usually analyzed by atomic spectrometry techniques suitable for trace analysis such as AAS (flame and electrothermal) inductively coupled plasma spectrometry (ICP-AES and ICP-MS). However, in some work, radiochemical methods like neutron activation analysis (NAA) and electrochemical techniques like DPASV have been used. It is clearly evident from Table 1 that serum and urine are the more commonly studied matrices. There has been more interest in speciation studies of arsenic, mercury, and selenium in different biological fluids. Aluminium in blood has also been studied widely. Only few reports are available on metals like cadmium, chromium, iron, lead, platinum, zinc, copper, and others in these matrices. An examination of Table 1also reveals that the detailed information about the analytical performance of the developed methodologies is not very often reported. Speciation studies of biological fluids are very significant in clinical chemistry to understand the physiological behavior of trace elements in the living system. We have tried to highlight some of the achievements realized until now for the separation and quantification of the metal species which might be present below sub-lxg L-1 level in biological fluids. From the published literature it can be concluded that it is not possible to detect a general methodology for trace metal speciation in biological fluids. However, some important aspects of the most appropriate methodology could be taken into consideration, such as the importance of mechanical separation. This is a useful preliminary step to identify the different size of the chemical species. This provides information about the molecular mass of the proteins or the other molecules to which the metal ions are bound. This step, commonly performed by ultrafiltration or centrifugation, can be considered as a specific methodology for speciation studies in biological materials as a difference of similar studies carried out in other matrices, such as sediments, soils, or environmental samples. After the physical separation of different molecular mass fractions, the combi- nation between chromatographic method and sensitive atomic spectrometric detec- tion, as indicated for water analysis, seems to be the best alternative for the identification and quantitative analysis of different species. However, there is a lack
  43. 43. Table 1. Analytical Details of the Atomic Spectrometry Procedures Developed for Speciation in Biological Fluids Element Species Matrix Methodology DetectionLimit RSD Recovery Ref. AI Transferrin Serum HPLC-ETAAS 0.12 l~g L-1 8% 101.1 + 15.3% Van Landeghem et al. (1994) -- Gardiner et al. (1984) 60-125% Keirsse et al. (1987) Blanco Gonzalez et al. (1989) Wrobel et al. (1994) -- Le et al. (1996) OJ AI Albumin, transferrin Serum GPC-ETAAS AI Proteins with different MM Serum SEC-ETAAS AI Albumin, transferrin Serum Ultrafiltration, HPLC-ETAAS As AsC~3-,AsC)34-, MMA, DMA, Urine HPLC-HGAFS -- -- AB, AC, TMA +, As-sugars AS AsO~3-,AsO34-, MMA, DMA, Urine HPLC-HGAAS 8-15 l~g L-1 2.5-5.3% -- AB, AC AS AsC~3-,AsO34-, MMA, DMA Blood IEC-HGAAS 0.44-0.92 Ilg L-1 3.6-6.8% 90-110% As AsO~3-,AsO34-, MMA, DMA Urine Extraction-ETAAS -- -- 94.9-107.7% HPLC-ICPMS Serum IEC-HGAASAs MMA, DMA, AB, AC 1-1.5 Mg L-1 4.2-4.8% >90% As AsO~3-,AsO34-, MMA, DMA Urine HPLC-MIPAES 1 ng mL-1 (AsO:]-) 2.8% (AsC~-) -- 5 ng mL-1 (AsCP4-) 2.5% (AsO34-) 6 ng mL-1 (MMA) 3.1% (MMA) 1.2 ng mL-1 (DMA) 2.1% (DMA) AS AsO~3-, DMA Urine IC-ICPMS -- -- -- As Toxic-As (AsC~3-+ AsOa4- + Urine Microwave 4-6 I~g L-1 4-7% -- MMA + DMA) digestion-HGAAS Non-toxic-As (AB + AC) Lopez Gonzalvez et al. (1996) Zhang et al. (1996a) Bavazzano et al. (1996) Zhang et al. (1996b) Costa Fernandez et al. (1995) Feldmann (1996) Lopez Gonzalvez et al. (1995) (continued)
  44. 44. Table 1. Continued Element Species Matrix Methodology Detection Limit RSD Recovery Ref. As AsO]3-, AsO~-, MMA, DMA Urine Micellar LC- 90-300 pg 3.4-5% ~ Ding et al. (1995) ICPMS As Inorganic-As, MMA, DMA Urine IEC-HGAAS 0.9 ~g L-1 (Inorg-As) 3.5% (Inorg-As) ~ Jimenez de Bias et al. (1994b) 4~ As AsO]-, AsCP4-,MMA, DMA, Urine IC-ICPMS AB, TMAO As AsO]-, AsCP4-, MMA, DMA Urine HPLC-HGAAS ,~o~-,,~- As As As As As As Inorganic and organic-As AsO:]3-, AsO43-, MMA, DMA, AB, AC, TMAO, TMA+ Inorganic and organic-As Inorganic, MMA, hydroxydimethylarsine oxide AB, AC, TMA + Inorganic, MMA and DMA Blood Digestion-HGAAS Blood, Derivatization-GC urine Urine HPLC-ICPMS Urine Extraction-ETAAS Urine IEC-HGAAS Urine HPLC-HGAAS Urine IEC-HGAAS 1.3 l~g L-1 (MMA) 3.2% (MMA) 0.5 I~g L-1 (DMA) 4.6% {DMA) 0.22-0.44 l~g L-~ 3.2-4.9% Inoue et al. (1994) 2 14gL-1 5.1% (AsC~3-}; -- Chana and Smith 3.8% (MMA); (1987) 5.3% (DMA); 7.2% (AsO34-) m -- 90-105% Weigert and Sappl (1983) 0.1-5 ng L-1 m -- Dix et al. (1987) 3-6 ng m1-1 (cations) 23% (AB}; 96-108% Larsenet al. (1993) 7-10 ng m1-1 (anions) 5.4-8.9% (others) 10 l~g L-1 7.9% (inorg-As); 93% (inorg-As}; Fitchett et al. 3.6% (DMA} 88.4% (DMA) (1975) 0.5 l~g L-1 3-6% 95-102% Buratti et al. (1984) I -- 85-97% Momplaisir et al. (1991} 2 l~g L-1 3.2-4.6% 85-93% Jimenezde Bias et al. (1994a)
  45. 45. M'I As AsO~3-, AsO43-,MMA, DMA, AB Urine As As As AsO~3-,AsO34-, MMA, DMA Urine AsO~3-,AsO34-, MMA, DMA Urine AsO~3-,AsO34-, MMA, DMA Urine Cd Proteins with different MM Urine, sweat Cr Cr(lll), Cr(VI) Blood, urine, serum Cr Cr(lli), Cr(VI) Urine Cr Cr(lll), Cr(VI) Urine Cr Cr(Vl) Blood, urine Cr Cr(lll), Cr(VI) Serum Fe Transferrin Serum Hg Hg, MeHg Urine Hg Hg, MeHg Urine HPLC-ICPMS HPLC-ICPAES HPLC-ICPMS HPLC-ICPMS HPLC-FAAS FAAS HPLC-ICPMS IC-ICPAES IC-ICPMS Extraction-AAS IEC-DCPAES HPLC-ETAAS CV-ICPAES HPLC-MIPAES 10 I~g L-1 (AsO~3-, DMA, AB); L-1 (MMA); 15 l~g L-1 (AsC~4-)20 llg 0.5 l~g L-1 36-96 pg 63 pg (AsC~-); 37 pg (AsC~4-); 80 pg (MMA, DMA) 1 I~gL-1 24 l~g L-1 (CrItt) 75 llg L-1 (Crvl) 3 pg 12-15 I~g L-1 35-47 l~g L-1 3-15 l~g L-1 0.17 l~gL-1 4 ng mL-1 0.15 ng mL-1 35 ng mL-1 10% 0.9% (Crlll), 1.3% (Crvl) 0.3-1.6% 1.4% 5% 6.7% 6.8% 97.3 + 2% Le et al. (1994) Low et al. (1986 and 1987) Heitkemper et al. (1989) Sheppard et al. (1992) Chang and Robinson (1993) Gaspar et al. (1996) Zoorob et al. (1995) Jensen and Bloedorn (1995) Devoto (1968) Urasa and Nam (1989) VanLandeghemet al. (1994) Menendez Garcia et al. (1996) Costa Fernandez et al. (1995) (continued)
  46. 46. Table T. Continued Element Species Matrix Methodology DetectionLimit RSD Recovery Ref. Hg MeHg, EtHg, PhHg Urine HPLC-CVAFS 5-7 ng m >95% Yoshino et al. (1995) Aizpun et al. (1994) Palmisano et al. (1993) Coyle and Hartley (1981) Lind et al. (1993) Filippelli, 1987 Bulska et al. (1992) M,; O~ Hg Hg, MeHg Urine HPLC-CVAAS Hg Hg, MeHg, MeEtHg, diEtHg Blood GC-CVAFS 0.3-0.6 ng g-1 Hg Hg Hg Inorganic and organic-Hg Inorganic and organic-Hg Inorganic and MeHg, PhHg Hg Inorganic and MeHg Hg Hg Inorganic and organic-Hg Inorganic and organicoHg Hg . Inorganic and MeHg, EtHg, PhHg Blood, Reduction-CVAAS urine Blood Reduction-CVAAS Blood, GC-ETAAS milk Whole GC-MIPAES blood Urine Reduction-CVAAS Urine, GC-ETAAS sweat Urine GC-CVAAS Hg Inorganic and organic-Hg Urine Ion-pair LC-ICPMS Pb TrimethyI-Pb Blood GC-ETAAS Pb Ionic alkyI-Pb Urine HPLC <4 gg L-1 3 ~g L-1 (MeHg); 125 ~g L-' (PhHg) 1 pg 3-5 ng L-1 0.3 ng 1.7 ~g L-1 (total); 12 ~g L-' (MeHg); 2.4 gg L-' (EtHg); 21 Mg L-1 (PhHg) 7 pg 3 l~g L-1 6.9% 90-114% 3.5% 89-103% 5% m 1-8% 2.8-5.2% 5-10% 11% 86-106% 97-102% 95.2 + 2.7% (MeHg); 99.5 + 4.3% (inorg-Hg) 93% Oda and Ingle (1981) Robinson and Wu (1985) Seckin et al. (1986) Shum et al. (1992) Nygren and Nilsson (1987) Neidhart and Tausch (1992)
  47. 47. ",4 Se Se Se Se Se Se Se Se Se Se Sn Sn Cisplatin, hydrolysis products Free, complexed Se-Cys, Se-Met, TMSe§ Inorganic-Se, total selenoamino acids SeO~-,SeO~-, selenoamino acids SeO]3- Inorganic-Se TMSe§ TMSe+, total Se TMSe§ selenonocholine TMSe§ SeO]3-, SeO24- Proteins with different MM Proteins with different MM Organotins Mono-, di-, and tri-alkyltins Plasma Plasma Serum, urine Urine Urine Blood Blood Urine Urine Spiked urine Urine Milk Serum Urine Urine HPLC-ICPAES Ultrafiltration- ETAAS HPLC-ICPMS HPLC-HGAAS -ICPAES -ICPMS HPLC-HGAAS HPLC-ETAAS GC GC-MIPAES IEC-ETAAS IEC-ICPMS HPLC-AAS HPLC-ETAAS Ultrafiltration- HGAAS HPLC-ETAAS GLC GC-ETAAS 35 Mg L-1 L-1 (Se-Cys), 0.2 ~g L-1 0.6 l~g (Se-Met), 0.2 l~g L-1 (TMSe§ 6.8 l~g L-1 (inorg-Se) 30 l~g L-1 0.16 l~g L-1 1 ~gL -1 5 l~g L-~ 2 llg L-1 1.2 ILtgL-1 40 ng Se 44 ng (TMSe+); 31 ng (selenonocholine) 1.83 ng (TMSe+); 1.15 ng (SeO24-) 1-3 pg L-1 5% m m 96-104% 91 +12% m B De Waal et al. (1987) Dominici et al. (1986) Muffoz Olivas et al. (1996) Gonzalez LaFuente et al. (1996a) Marchante Gayon et al. (1996) Kurahaschi et al. (1980) Harrison and Rapsomanikis (1989) Tsunoda and Fuwa (1987) Sun et al. (1987) Blais et al. (1991 ) Laborda et al. (1993) lyengar (1987) Wrobel et al. (1994) Olson et al. (1983) Dyne et al. (1991 )
  48. 48. Table 1. Continued Element Species Matrix Methodology DetectionLimit RSD Recovery Ref. Zn Albumin, 0s Serum Ultrafiltration . . . . Faure et al. (1990) ETAAS Cu, Zn Dissolved species Blood HPLC-ETAAS 1 ~g L-1 (for both) -- -- Gardiner et al. (1981) SchOppenthau and Dunemann (1994) Wu and Robinson (1986) Buchberger and Rieger (I 989) Qo Cu, Zn Ceruloplasmin and albumin Serum HPLC-ICPMS, -- (for Cu); macroglobulin and HPLC-ICPAES albumin (for Zn) Mg, Zn Mg2§ predominant, zinc Urine HPLC-FAAS J m species could not be identified Ca, Mg, Dissolved species Tear Centrifugation-IC Ca (0.60 l~g mL-1); 7% (Ca); Na, K Mg (0.25 I~g mL-1); 3-4% (Mg,Na,K) Na (0.07 ~g mL-1); K (0.12 ~g mL-1) Cd, Cu, Proteins with different MM Serum HPLC-ICPMS Cu (0.7 pg); Fe (3 pg); m m Shum and Houk Fe, Pb, Cd and Pb (0.5 pg); (1993) Zn Zn (1 pg) Cu, Fe, Mn, Se, Zn Ca, Cu, Fe, Mg, Mn, Zn Proteins with different MM Proteins with different MM Serum, HPLC-ICPAES milk and red blood cell lysate Milk GPC-ICPAES Br~tter et al. (I 988a) Arts and Hafkenscheid (1984)
  49. 49. AI, As, Ca, Co, Cr, Cu, Fe, Li, Mg, Mn, Na, Ni, K, Pb, Zn Dissolved species Tear FAAS, ETAAS E 7-I 2% 93-I 05% Giordano et al. (1983) t,D Notes: Abbreviations: RSD - relative standard deviation; HPLC - high performance liquid chromatography; ETAAS - electrothermal atomic absorption spectrometry; GPC - gel permeation chromatography; $EC - size exclusion chromatography; HGAFS - hydride generation atom ic fluorescence spectrometry; HGAAS - hydride generation atomic absorption spectrometry; IEC - ion exchange chromatography; ICP MS - inductively coupled plasma mass spectrometry; MIP AES - microwave induced plasma atomic emission spectroscopy; IC - ion chromatography; LC - liquid chromatography; GC - gas chromatography; ICP AES - inductively coupled plasma atomic emission spectroscopy; FAAS - flame atomic absorption spectrometry; AAS - atomic absorption spectrometry; DCP AES - direct current plasma atomic emission spectroscopy; CVICP AES - cold vapor inductively coupled plasma atomic emission spectroscopy; CVAFS - cold vapor atomic fluorescence spectrometry; CVAA5 - cold vapor atomic absorption spectrometry; GLC - gas liquid chromatography; ~ - Molecular Mass; MMA - monomethyl arsonic acid; DMA - dimethyl arsinic acid; AB - arsenobetaine; AC - arsencx:holine; TMA + - tetramethylarsonium ion, T,~O - trimethylarsine oxide; MeHg - methylmercury; EIHg - Ethylmercury; PhHg - phenylmercury; MeEIHg - methylethylmercury; diEtHg - diethylmercury; Se-Cys - selenocystine; Se-Met - selenomethionine; TMSe+ - trimethyl selenonium ion.