- Email: [email protected]
Atomic Spectroscopy, Biomedical Applications☆
Atomic Spectroscopy, Biomedical Applications☆ A Taylor, Royal Surrey County Hospital, Guildford, UK; University of Surrey, Guildford, UK ã 2017 Elsevier Ltd. All rights reserved.
Introduction Other articles in this Encyclopaedia, describe the fundamental features of the techniques that together comprise analytical atomic spectroscopy. These demonstrate the very wide range of elements that may be determined and biomedical applications require the measurement of almost all these. Terminology varies but it is helpful to call a limited number as those that fulfill structural and functional roles that are essential to maintain health and well-being, and those that are non-essential. Among the essential elements some are present in body tissues at relatively high concentrations, the major elements. All other elements, which individually account for less than 0.01% of the dry weight of the organism are generally termed the trace elements, some of which are essential but most such as lead are present as contaminants from the environment. Undue exposure to both essential and non-essential elements may be injurious to health and may result in morbidity or death. With sufficiently sensitive analytical techniques, almost all elements of the periodic table can be detected in biological specimens. Tables 1 and 2 list those elements known to be essential to biological systems though not all are required by all species. Figure 1 indicates that essential and non-essential elements are relevant to a large number of disciplines which may be regarded as ‘biomedical’. Some of these are described in detail in related articles and are not considered further here. From the preceding discussion it is evident that measurements of minerals and trace elements in biomedical samples are appropriate to investigations related to:
• • • •
the biological importance of essential trace and major elements; the effects of deficiencies of essential elements; undue effects, that is the harmful consequences of a sufficiently large exposure to any element, whether essential or non-essential; the pharmacology of certain metals which are administered as the active principal of a therapeutic agent.
(XRF). For inorganic MS, ionised analyte atoms are separated within electrical or magnetic fields according to their mass-tocharge (m/z) ratio. AAS, AES and AFS involve interactions between UV–visible light and the outer shell electrons of free, gaseous, uncharged atoms. In XRF, high energy particles collide with inner shell electrons of atoms, initiating transitions with eventual emission of X-ray photons. It was the development of flame AAS followed by electrothermal AAS (ETAAS), which is also known as graphite furnace AAS that allowed the investigations mentioned above to really commence. These techniques provided for considerably lower detection limits than had previously been achievable. Although widely used, AAS is essentially a single-element technique and separate measurements must be made if more than one metal is to be determined. Trace element analysis advanced dramatically when inductively coupled plasma–mass spectrometry (ICP-MS) was developed, especially following the introduction of collision and dynamic reaction cells to remove some important interferences. Not only are the detection limits and speed of analysis possible with ICP-MS equal to or better than those seen with AAS, but it is also a powerful multi-element technique.
Table 1 Major elements found in biological specimens (all are essential to man) Minerals
Non-minerals
Calcium Iron Magnesium Potassium Sodium
Carbon Chlorine Hydrogen Nitrogen Oxygen Phosphorus Sulfur
Table 2 Essential trace elements (not all are proven to have essential roles in man)
Atomic Spectroscopy Quantitative analytical atomic spectrometry includes the techniques of inorganic mass spectrometry (MS), atomic absorption spectrometry (AAS), atomic emission spectrometry (AES), atomic fluorescence spectrometry (AFS) and X-ray fluorescence ☆ Change History: June 2013. A Taylor revised Introduction, reorganised the structure of text, deleted detailed technical material and expanded sections on applications. Updated further reading. Andrew Taylor, Biomedical Applications of Atomic Spectroscopy, In Encyclopedia of Spectroscopy and Spectrometry (Second Edition), edited by John C. Lindon, Academic Press, Oxford, 1999, Pages 174-182.
76
Minerals
Non-minerals
Chromium Cobalt Copper Iron Manganese Molybdenum Nickel Selenium Silicon Vanadium Zinc
Fluorine Iodine
Encyclopedia of Spectroscopy and Spectrometry, Third Edition
http://dx.doi.org/10.1016/B978-0-12-409547-2.04972-6
Atomic Spectroscopy, Biomedical Applications
In most laboratories ICP-MS serves as a routine technique that affords high throughput, single- or multi-element analysis with very good sensitivity. The very low detection limits for rare earth elements and the actinides permit occasional studies relating to the biochemistry and unusual sources of exposure to these elements. Similarly, low levels of occupational and environmental exposure to platinum and other noble metals may be investigated. The additional facility afforded by mass spectrometric techniques to measure individual isotopes is usefully exploited in special investigations. Chemical vapour generation techniques have been described. These were originally developed as sample delivery accessories for FAAS but have since also been applied to AES, AFS, and ICP-MS. Most practical applications involve either volatile hydride generation or the formation of mercury vapour, although some recent reports have demonstrated that it is possible to measure copper and zinc in this way. The analyte is separated from the matrix as an atomic or chemical vapour that is transported to a suitable instrument for detection and measurement.
77
Applications Numerically, the most important investigations are those undertaken in clinical laboratories, usually with biological fluids. Measuring the major elements is usually accomplished by non-atomic spectrometric techniques such as colorimetry or with ion specific electrodes. Atomic spectrometry is more usually applied to assess trace element status i.e. deficiency of an essential element or toxicity of any element(s). While AAS is still widely employed, ICP-MS has become the technique of choice especially where there is a large workload. Instrumentation for ICP-AES now provides for useful detection limits and while this technique is not widely used within the clinical sector there is considerable potential for the future. X-ray fluorescence is usually applied to quantitative analysis of solid samples and for imaging to show the distribution of elements in tissues. In a few centres, equipment for in vivo measurement of elements in tissues such as bone and skin using XRF has been developed.
Diagnosis of Deficiency and Toxicity
Samples and Sample Preparation Typical biological fluids include blood and blood serum, blood plasma, urine and saliva. Other specimens, for example dialysis fluids, intestinal contents, parenteral nutrition solutions, seminal plasma, and cerebrospinal fluid may be analysed on rare occasions. The concentrations of many elements in plant, animal or human tissues are usually much higher than in biological fluids and very often the weight of an available specimen is such that a relatively large mass of analyte is recovered into a small volume of solution, thus enhancing the concentration still further. The objectives for preparation of biomedical specimens are to (1) remove interfering components from the matrix and (2) adjust the concentration of analyte to facilitate the actual measurement. These objectives may be realized by a number of approaches (Table 3) which in general are appropriate to all the techniques described in this article.
As a consequence of their roles within enzymes and in structural tissues deficiencies of essential elements give rise to a wide range of symptoms involving all the major organs and tissues of the body. Circumstances in which the body content of these micronutrients may become reduced include inadequate dietary intake, poor intestinal absorption or increased excretion. In addition, there are genetic disorders that result in a failure to properly utilize a specific element e.g. acrodermatitis entreopathica which causes zinc deficiency. The essential elements that are most often measured in biological fluids are zinc, copper and selenium. Situations leading to undue accumulation of an element include occupational exposure, contaminants present in the natural environment (air, drinking water, foods and beverages), domestic items, proprietary medicines and supplements, cosmetics, deliberate poisoning (suicide or homicide), iatrogenic exposure. Legislation to protect the health of workers exposed to chemical agents exists in most countries. The associated
Table 3
Approaches to sample preparation
Procedure
Remarks
Dilution, protein precipitation Dry ashing
Using simple off-line arrangements or flowinjection manifold Using a muffle furnace or a low-temperature asher (i) In open vessels with convection or microwave heating (ii) In sealed vessels to increase the reaction pressure Using quaternary ammonium hydroxides For analyte enhancement and removal of interferences For analyte enhancement and removal of interferences
Acid digestion
Figure 1 The relevance of minerals and trace elements to biomedical sciences.
Base dissolution Chelation and solvent extraction Trapping onto solidphase media
78
Atomic Spectroscopy, Biomedical Applications
regulations define maximum allowable exposure and procedures that should be implemented to prevent harm to individuals. These procedures may include regular biological monitoring, for example the measurement of blood lead concentrations, with defined levels at which further action must be taken including suspension from the work that involves exposure to the agent in question. Lead, mercury, chromium and cadmium are among the metals liable to be hazardous to health and are used most extensively at work and require monitoring. Other metals associated with specific industries or tasks may require their measurement in blood and/or urine. Different sample types such as saliva, condensates of exhaled breath or bronchial lavage are proposed for special projects. Numerous examples of environmental contamination, with consequences upon individuals, families or entire populations have been recorded. Further examples are regularly seen. These include airborne emissions of metals from smelters or refineries, factory discharges into rivers, lakes or seas with subsequent contamination of foods, accidental additions or deliberate adulteration to drinking water or foods. Measurements of concentrations in biological fluids follow from either the response to a recognized event, to assess whether anyone might be at risk or have suffered real harm, or where individuals are symptomatic of metal poisoning and the source of exposure is being sought. Following discovery of metallic mercury beneath floorboards in a community centre in England, urine specimens were collected from everyone in the village. Measurement of the mercury concentrations confirmed that there was no undue exposure. An example of the second scenario concerned the inhabitants of areas in Bangladesh who developed chronic arsenic poisoning which was eventually shown to have been caused by natural contamination of drinking water contained from deep tube wells. Many examples of real or potential cases of poisoning within the domestic environment are known. Thus analysis of biological fluids along with sources of exposure – such as toys, medicines and cosmetics, drinking water, foods and beverages, painted surfaces, gardening products etc. – is a critical feature in investigating these incidents. The source of a problem may be quite obscure. The cause of profound copper deficiency in a number of patients was found to be a consequence of extensive use of a dental fixative that had a high concentration of zinc (which inhibits the intestinal absorption of copper). While ICP-MS and AAS will be the most obvious technique employed for the analyses, XRF is also very useful, especially to examine surfaces and solid samples. Analysis of biological fluids, usually, blood and urine, are also important in population surveys aimed at establishing normal concentrations, and in epidemiological research. The importance of veterinary medicine should not be overlooked. It was the financial implications of problems in livestock that prompted investigations leading to several elements being shown to be essential. Biopsy specimens from liver and some other tissues are analysed for the diagnosis of genetic diseases that cause deficiency or accumulation of metals. Analysis of tissues collected at post mortem can form part of investigations in cases of homicide, suicide or accidental poisonings.
Basic Physiology and Biochemistry Topics such as absorption, distribution to tissues and excretion, and the mechanisms of action/toxicity at a biochemical or molecular level are generally of research interest, rather than focusing on specific clinical problems. Very often this research involves adding an element to a model system or volunteer and monitoring the fate, or effects, of that addition. Aside from straightforward measurement of the concentration of the element(s) of interest, further insight may be obtained with a number of additional analytical strategies. Radioactive tracers such as 14C, have long been used to follow the metabolism of natural compounds or drugs. Similar work has been undertaken with elements but is not possible with some groups e.g. children and pregnant women, or with elements where there is no radioactive isotope. With the development of sensitive mass spectrometric assays and sources of enriched stable isotopes fundamental physiological and biochemical studies are possible. Experiments with Fe, Ni, Se and Zn, for example, to establish the kinetics and other parameters following exposure and absorption of normal, as well as reduced or enhanced, amounts have been performed. Within biological systems, few elements are present as the simple ion or uncharged atom. Even if exposure is to the inorganic form the processes of absorption involve incorporation into a molecular species with binding to a protein or other organic moiety, often with further metabolism. There may also be changes to the oxidation state of the atom. These changes influence the further effects of the element. Metallomics or elemental speciation is the area of research to understand these events and among the techniques employed are those for molecular separation, species identification and for in situ analysis. The imaging of tissues offers insights into metabolic pathways and disease states with most work exploiting laser ablation in combination with ICP-MS (LA-ICP-MS), or XRF. Although there are considerable technical issues associated with quantification due to strong matrix effects, useful data may be obtained. One example with LA-ICP-MS used enriched Zn isotopes as bio-tracers to create Zn isotope ratio images of rat brain tissue slices to investigate Zn kinetics at the microscale. Synchrotron-based XRF microscopy was employed for Se imaging in mouse liver and kidney tissues, leading to the discovery of localised pools of Se present as glutathione peroxidase 3. These examples show how mapping of tissue slices for example reveal sites at which accumulation occurs either naturally, with disease or following therapeutic interventions.
Miscellaneous Applications The excellent sensitivity and resolution of LA-ICP-MS is exploited to determine the profile of an element along a single strand of hair in very small increments. This is often applied to show when an individual may have received lethal exposure to an element such as arsenic. Similarly, in association with the speciation studies referred to above LA-ICP-MS is used to identify the small amounts of metallo-proteins separated by gel electrophoresis or similar techniques.
Atomic Spectroscopy, Biomedical Applications
79
While the natural proportions of different isotopes of an element are usually constant, the isotopes of lead vary in distribution depending upon the initial content of thorium and uranium when the earth’s crust was first laid down. These elements decay to produce lead isotopes and the ratios of e.g. 206Pb:207Pb are characteristic of the site of extraction. These subtle variations, which are measurable by ICP-MS, are used to aid identification of sources of exposure by comparing their isotopic ratios with those in the patient’s blood. Analysis of tissue samples is usually possible only following removal at surgery or post mortem. Small biopsy specimens can be obtained from a limited number of sites but this represents an invasive procedure which is not necessarily acceptable. The technique of in vivo XRF has been developed which provides for analysis of tissues close to the surface of the body, typically bone (knee cap, shin, fingers) and skin although some work with liver has been reported. Most studies are with lead to assess lifetime occupational accumulation, relocation from bone during pregnancy, and childhood environmental exposures. Other elements determined in this way include iron, and silver. Metal-containing pharmaceuticals are widely used to prevent infections and in the treatment of many disorders (cancer, depression, rheumatoid arthritis etc.). For some the dose regime has to be adapted in individual patients so that plasma concentrations are maintained within a defined therapeutic range. For others, efficacy and toxicity are closely related and it is necessary to avoid overdosing. Thus, regular monitoring of lithium for example accompanies the use of this agent when used to treat depression. Aluminum, which may be given to patients with chronic renal failure is similarly determined in these patients. Even before a drug is licensed for use in treatment preliminary studies are required to establish doses necessary to produce an effective response and without unacceptable side-effects. Initial pharmacokinetic investigations and toxicity studies in animals, followed by formal clinical trials involve measurements of the drug and/or metabolites in plasma and other samples. Where a new metallo-drug is investigated these analyses could include determination of the metal concentrations. A further activity for which sector field ICP-MS or multicollector ICP-MS are supremely appropriate involves the characterization and certification of biomedical reference materials where the high degree of accuracy required is achieved by inclusion of isotope dilution analysis into the measurement(s).
some loss of sensitivity. The most usual procedure, however, is to use a collision, or dynamic reaction, cell. The cell is a multipole – a quadrupole, hexapole or octapole – aligned between the ion optics and the quadrupole mass filter. Interactions between polyatomic ions and a gas e.g. nitrogen, helium, or methane fed into the cell, greatly reduce spectral interferences. These interactions lead to the formation of secondary species, which are removed from the ion path on the basis of kinetic energy discrimination or mass discrimination so that only the analyte ion is transmitted to the detector. Other approaches to the elimination of spectral interferences may be appropriate. Addition of an organic solvent to the sample diluent can reduce some of the argon-based interferences. Separation of analyte ions from those involved in the formation of polyatomic species prior to the measurement is also effective. Separation may be achieved by vaporisation of the sample, for example by hydride generation or by electrothermal vaporisation, or by prior use of a chromatographic or other separation step such as liquid–liquid extraction. Non-spectral interferences, associated with sample introduction and fluctuations in the inductively coupled plasma, are effectively eliminated by using an internal standard. This should be an element not present in the original sample, not subject to spectral interferences, and with mass and ionisation energy close to those of the analyte(s). Two types of interference are associated with the analysis of biomedical specimens by FAAS. Typical biological fluids contain protein and other macromolecules which increase the sample viscosity compared with simple aqueous solutions. The viscosity may reduce the aspiration rate through the narrow capillary tubing of the pneumatic nebulizer, and absorbance signals will be attenuated compared with aqueous calibration solutions, giving falsely low results. This matrix effect may be removed by dilution of the sample, precipitation of the protein or by matching the viscosity of the calibration and sample solutions. Calibration by standard additions can be adopted if there is sufficient sample. A second problem occurs in samples with a high content of dissolved solids which are liable to produce falsely high results because of non-atomic absorption or light scattering. If this occurs the analyte can be removed from the matrix by extraction into an organic solvent, or a background correction technique (see below) can be employed. The topic of interferences found with ETAAS is dealt with in more detail in other articles. Of special significance to biomedical specimens, which typically contain large amounts of sodium, chloride and carbon-based biomolecules, are;
Interferences Seen in the Analysis of Biomedical Sciences by Atomic Spectroscopy
•
ICP-MS is subject to spectral interferences. These are isobaric, e.g. 64Niþ on 64Znþ, 156Gd2þ on 78Seþ, or from polyatomic ions formed from interactions between matrix components and the plasma gas such as 40Ar on 40Caþ, 31P16Oþ 2 on 63 80 þ Cuþ, 40Ar35Clþ on 75Asþ and 40Arþ Se . If a non2 on affected isotope is present with sufficient abundance this may be counted to avoid the interference, e.g. 82Seþ in place of 80 þ 66 Se , Znþ in place of 64Znþ. Sector field ICP-MS is not subject to most of these interferences, although there may be
• •
Drying; protein-rich viscous specimens may dry unevenly with ‘explosive sputtering’ at the start of the ash phase, causing poor reproducibility. Ashing; volatile halides, for example AlCl3 may form, causing pre-atomization loss of the analyte. Atomization; (i) stable compounds such as carbides can be produced, giving low atomization rates (e.g., for Mo), (ii) vapour phase reactions, especially molecular condensation at the cooler ends of the furnace (e.g., Pb(g) þ 2NaCl(g) ! PbCl2(g) þ 2Na(g)) resulting in non-atomic absorption of incident radiation and scattering of incident radiation by particulates (carbon, smoke, salts).
80
Atomic Spectroscopy, Biomedical Applications
Background correction is essential for almost all biomedical applications to correct for the non-atomic absorption of the incident radiation. The three systems used in commercial instruments are effective although the Smith-Hieftje variation is included by very few manufacturers. Deuterium background correction was the first system to be introduced and is widely used. The Zeeman-effect technique is ideal for dealing with structured background which is of particular significance in the measurement of cadmium in urine and elements such as arsenic and selenium in iron-rich samples, that is blood. Without Zeeman-effect background correction, these applications are difficult to carry out successfully by ETAAS. In energy dispersive XRF the signal may be complicated by contributions from the sample matrix, making calibration difficult. Techniques to overcome the interferences include the use of reference materials, matrix-matched standardization and/or internal standardisation. with total reflection XRF, or when samples are prepared as very thin films, there is effectively no absorption by the matrix and problems are reduced and simpler to resolve.
See also: Atomic Absorption, Methods and Instrumentation; Atomic Absorption, Theory; Atomic Emission, Methods and Instrumentation; Atomic Fluorescence, Methods and Instrumentation; Atomic Spectroscopy, Pharmaceutical Applications; Biological Applications of Hyperpolarized 13C NMR; Circular Dichroism and ORD, Biomacromolecular Applications; Counterfeit Drugs Studied by NMR; Drug Metabolism Studied Using NMR Spectroscopy; Fragment-Based Drug Design by NMR; HPLC–NMR, Pharmaceutical Applications; IR, Biological Applications; IR, Medical Science Applications; Mass Spectrometry in Drug Metabolism: Principles and Common Practice;
Medical Applications of Mass Spectrometry; MRI Applications, Biological; NMR Spectroscopy in the Evaluation of Drug Safety; Raman Optical Activity, Macromolecule and Biological Molecule Applications; Raman Spectroscopy, Medical Applications: A New Look Inside Human Body With Raman Imaging; Spectroscopic Methods in Drug Quality Control and Development; Spectroscopy for Process Analytical Technology (PAT); Spectroscopy in Biotechnology Research and Development; Structural Chemistry Using NMR Spectroscopy, Pharmaceuticals; UV-Visible Absorption Spectroscopy, Biomacromolecular Applications; Vibrational Spectroscopy Applications in Drugs Analysis.
Further Reading Clough R, Drennan-Harris L, Harrington C, Hill S, and Tyson J (2012) Atomic Spectrometry Update: Elemental Speciation. J. Anal. At. Spectrom. 27: 1185–1224. Evans E, Palmer C, and Smith C (2012) Atomic Spectrometry Update: Advances in Atomic Spectrometry and Related Techniques. J. Anal. At. Spectrom. 27: 909–927. Taylor A, Day M, Marshall J, Patriarca M, and White M (2012) Atomic Spectrometry Update: Clinical and Biological Specimens, Foods and Beverages. J. Anal. At. Spectrom. 27: 537–576. West M, Ellis A, Potts P, Streli C, Vanhoof C, Wegrzynek D, and Wobrauschek P (2011) Atomic Spectrometry Update: X-ray Fluorescence. J. Anal. At. Spectrom. 26: 1919–1963. Butler O, Evans H, Fisher A, Hill S, Harrington C, Taylor A, West M, and Ellis A (2010) Atomic Spectrometry Updates: A 25 Year Perspective. J. Anal. At. Spectrom. 25: 1546–1566. Flanagan R, Taylor A, Watson I, and Whelpton R (2008) Fundamentals of Analytical Toxicology. Chichester: Wiley. Taylor A (2011) Atomic Absorption Spectroscopy, Inductively Coupled Plasma–Mass Spectrometry and Other Techniques for Metals Analysis. In: Widdop B (ed.) Clarke’s Textbook of Poisons. British Pharmaceutical, Society.
Introduction Other articles in this Encyclopaedia, describe the fundamental features of the techniques that together comprise analytical atomic spectroscopy. These demonstrate the very wide range of elements that may be determined and biomedical applications require the measurement of almost all these. Terminology varies but it is helpful to call a limited number as those that fulfill structural and functional roles that are essential to maintain health and well-being, and those that are non-essential. Among the essential elements some are present in body tissues at relatively high concentrations, the major elements. All other elements, which individually account for less than 0.01% of the dry weight of the organism are generally termed the trace elements, some of which are essential but most such as lead are present as contaminants from the environment. Undue exposure to both essential and non-essential elements may be injurious to health and may result in morbidity or death. With sufficiently sensitive analytical techniques, almost all elements of the periodic table can be detected in biological specimens. Tables 1 and 2 list those elements known to be essential to biological systems though not all are required by all species. Figure 1 indicates that essential and non-essential elements are relevant to a large number of disciplines which may be regarded as ‘biomedical’. Some of these are described in detail in related articles and are not considered further here. From the preceding discussion it is evident that measurements of minerals and trace elements in biomedical samples are appropriate to investigations related to:
• • • •
the biological importance of essential trace and major elements; the effects of deficiencies of essential elements; undue effects, that is the harmful consequences of a sufficiently large exposure to any element, whether essential or non-essential; the pharmacology of certain metals which are administered as the active principal of a therapeutic agent.
(XRF). For inorganic MS, ionised analyte atoms are separated within electrical or magnetic fields according to their mass-tocharge (m/z) ratio. AAS, AES and AFS involve interactions between UV–visible light and the outer shell electrons of free, gaseous, uncharged atoms. In XRF, high energy particles collide with inner shell electrons of atoms, initiating transitions with eventual emission of X-ray photons. It was the development of flame AAS followed by electrothermal AAS (ETAAS), which is also known as graphite furnace AAS that allowed the investigations mentioned above to really commence. These techniques provided for considerably lower detection limits than had previously been achievable. Although widely used, AAS is essentially a single-element technique and separate measurements must be made if more than one metal is to be determined. Trace element analysis advanced dramatically when inductively coupled plasma–mass spectrometry (ICP-MS) was developed, especially following the introduction of collision and dynamic reaction cells to remove some important interferences. Not only are the detection limits and speed of analysis possible with ICP-MS equal to or better than those seen with AAS, but it is also a powerful multi-element technique.
Table 1 Major elements found in biological specimens (all are essential to man) Minerals
Non-minerals
Calcium Iron Magnesium Potassium Sodium
Carbon Chlorine Hydrogen Nitrogen Oxygen Phosphorus Sulfur
Table 2 Essential trace elements (not all are proven to have essential roles in man)
Atomic Spectroscopy Quantitative analytical atomic spectrometry includes the techniques of inorganic mass spectrometry (MS), atomic absorption spectrometry (AAS), atomic emission spectrometry (AES), atomic fluorescence spectrometry (AFS) and X-ray fluorescence ☆ Change History: June 2013. A Taylor revised Introduction, reorganised the structure of text, deleted detailed technical material and expanded sections on applications. Updated further reading. Andrew Taylor, Biomedical Applications of Atomic Spectroscopy, In Encyclopedia of Spectroscopy and Spectrometry (Second Edition), edited by John C. Lindon, Academic Press, Oxford, 1999, Pages 174-182.
76
Minerals
Non-minerals
Chromium Cobalt Copper Iron Manganese Molybdenum Nickel Selenium Silicon Vanadium Zinc
Fluorine Iodine
Encyclopedia of Spectroscopy and Spectrometry, Third Edition
http://dx.doi.org/10.1016/B978-0-12-409547-2.04972-6
Atomic Spectroscopy, Biomedical Applications
In most laboratories ICP-MS serves as a routine technique that affords high throughput, single- or multi-element analysis with very good sensitivity. The very low detection limits for rare earth elements and the actinides permit occasional studies relating to the biochemistry and unusual sources of exposure to these elements. Similarly, low levels of occupational and environmental exposure to platinum and other noble metals may be investigated. The additional facility afforded by mass spectrometric techniques to measure individual isotopes is usefully exploited in special investigations. Chemical vapour generation techniques have been described. These were originally developed as sample delivery accessories for FAAS but have since also been applied to AES, AFS, and ICP-MS. Most practical applications involve either volatile hydride generation or the formation of mercury vapour, although some recent reports have demonstrated that it is possible to measure copper and zinc in this way. The analyte is separated from the matrix as an atomic or chemical vapour that is transported to a suitable instrument for detection and measurement.
77
Applications Numerically, the most important investigations are those undertaken in clinical laboratories, usually with biological fluids. Measuring the major elements is usually accomplished by non-atomic spectrometric techniques such as colorimetry or with ion specific electrodes. Atomic spectrometry is more usually applied to assess trace element status i.e. deficiency of an essential element or toxicity of any element(s). While AAS is still widely employed, ICP-MS has become the technique of choice especially where there is a large workload. Instrumentation for ICP-AES now provides for useful detection limits and while this technique is not widely used within the clinical sector there is considerable potential for the future. X-ray fluorescence is usually applied to quantitative analysis of solid samples and for imaging to show the distribution of elements in tissues. In a few centres, equipment for in vivo measurement of elements in tissues such as bone and skin using XRF has been developed.
Diagnosis of Deficiency and Toxicity
Samples and Sample Preparation Typical biological fluids include blood and blood serum, blood plasma, urine and saliva. Other specimens, for example dialysis fluids, intestinal contents, parenteral nutrition solutions, seminal plasma, and cerebrospinal fluid may be analysed on rare occasions. The concentrations of many elements in plant, animal or human tissues are usually much higher than in biological fluids and very often the weight of an available specimen is such that a relatively large mass of analyte is recovered into a small volume of solution, thus enhancing the concentration still further. The objectives for preparation of biomedical specimens are to (1) remove interfering components from the matrix and (2) adjust the concentration of analyte to facilitate the actual measurement. These objectives may be realized by a number of approaches (Table 3) which in general are appropriate to all the techniques described in this article.
As a consequence of their roles within enzymes and in structural tissues deficiencies of essential elements give rise to a wide range of symptoms involving all the major organs and tissues of the body. Circumstances in which the body content of these micronutrients may become reduced include inadequate dietary intake, poor intestinal absorption or increased excretion. In addition, there are genetic disorders that result in a failure to properly utilize a specific element e.g. acrodermatitis entreopathica which causes zinc deficiency. The essential elements that are most often measured in biological fluids are zinc, copper and selenium. Situations leading to undue accumulation of an element include occupational exposure, contaminants present in the natural environment (air, drinking water, foods and beverages), domestic items, proprietary medicines and supplements, cosmetics, deliberate poisoning (suicide or homicide), iatrogenic exposure. Legislation to protect the health of workers exposed to chemical agents exists in most countries. The associated
Table 3
Approaches to sample preparation
Procedure
Remarks
Dilution, protein precipitation Dry ashing
Using simple off-line arrangements or flowinjection manifold Using a muffle furnace or a low-temperature asher (i) In open vessels with convection or microwave heating (ii) In sealed vessels to increase the reaction pressure Using quaternary ammonium hydroxides For analyte enhancement and removal of interferences For analyte enhancement and removal of interferences
Acid digestion
Figure 1 The relevance of minerals and trace elements to biomedical sciences.
Base dissolution Chelation and solvent extraction Trapping onto solidphase media
78
Atomic Spectroscopy, Biomedical Applications
regulations define maximum allowable exposure and procedures that should be implemented to prevent harm to individuals. These procedures may include regular biological monitoring, for example the measurement of blood lead concentrations, with defined levels at which further action must be taken including suspension from the work that involves exposure to the agent in question. Lead, mercury, chromium and cadmium are among the metals liable to be hazardous to health and are used most extensively at work and require monitoring. Other metals associated with specific industries or tasks may require their measurement in blood and/or urine. Different sample types such as saliva, condensates of exhaled breath or bronchial lavage are proposed for special projects. Numerous examples of environmental contamination, with consequences upon individuals, families or entire populations have been recorded. Further examples are regularly seen. These include airborne emissions of metals from smelters or refineries, factory discharges into rivers, lakes or seas with subsequent contamination of foods, accidental additions or deliberate adulteration to drinking water or foods. Measurements of concentrations in biological fluids follow from either the response to a recognized event, to assess whether anyone might be at risk or have suffered real harm, or where individuals are symptomatic of metal poisoning and the source of exposure is being sought. Following discovery of metallic mercury beneath floorboards in a community centre in England, urine specimens were collected from everyone in the village. Measurement of the mercury concentrations confirmed that there was no undue exposure. An example of the second scenario concerned the inhabitants of areas in Bangladesh who developed chronic arsenic poisoning which was eventually shown to have been caused by natural contamination of drinking water contained from deep tube wells. Many examples of real or potential cases of poisoning within the domestic environment are known. Thus analysis of biological fluids along with sources of exposure – such as toys, medicines and cosmetics, drinking water, foods and beverages, painted surfaces, gardening products etc. – is a critical feature in investigating these incidents. The source of a problem may be quite obscure. The cause of profound copper deficiency in a number of patients was found to be a consequence of extensive use of a dental fixative that had a high concentration of zinc (which inhibits the intestinal absorption of copper). While ICP-MS and AAS will be the most obvious technique employed for the analyses, XRF is also very useful, especially to examine surfaces and solid samples. Analysis of biological fluids, usually, blood and urine, are also important in population surveys aimed at establishing normal concentrations, and in epidemiological research. The importance of veterinary medicine should not be overlooked. It was the financial implications of problems in livestock that prompted investigations leading to several elements being shown to be essential. Biopsy specimens from liver and some other tissues are analysed for the diagnosis of genetic diseases that cause deficiency or accumulation of metals. Analysis of tissues collected at post mortem can form part of investigations in cases of homicide, suicide or accidental poisonings.
Basic Physiology and Biochemistry Topics such as absorption, distribution to tissues and excretion, and the mechanisms of action/toxicity at a biochemical or molecular level are generally of research interest, rather than focusing on specific clinical problems. Very often this research involves adding an element to a model system or volunteer and monitoring the fate, or effects, of that addition. Aside from straightforward measurement of the concentration of the element(s) of interest, further insight may be obtained with a number of additional analytical strategies. Radioactive tracers such as 14C, have long been used to follow the metabolism of natural compounds or drugs. Similar work has been undertaken with elements but is not possible with some groups e.g. children and pregnant women, or with elements where there is no radioactive isotope. With the development of sensitive mass spectrometric assays and sources of enriched stable isotopes fundamental physiological and biochemical studies are possible. Experiments with Fe, Ni, Se and Zn, for example, to establish the kinetics and other parameters following exposure and absorption of normal, as well as reduced or enhanced, amounts have been performed. Within biological systems, few elements are present as the simple ion or uncharged atom. Even if exposure is to the inorganic form the processes of absorption involve incorporation into a molecular species with binding to a protein or other organic moiety, often with further metabolism. There may also be changes to the oxidation state of the atom. These changes influence the further effects of the element. Metallomics or elemental speciation is the area of research to understand these events and among the techniques employed are those for molecular separation, species identification and for in situ analysis. The imaging of tissues offers insights into metabolic pathways and disease states with most work exploiting laser ablation in combination with ICP-MS (LA-ICP-MS), or XRF. Although there are considerable technical issues associated with quantification due to strong matrix effects, useful data may be obtained. One example with LA-ICP-MS used enriched Zn isotopes as bio-tracers to create Zn isotope ratio images of rat brain tissue slices to investigate Zn kinetics at the microscale. Synchrotron-based XRF microscopy was employed for Se imaging in mouse liver and kidney tissues, leading to the discovery of localised pools of Se present as glutathione peroxidase 3. These examples show how mapping of tissue slices for example reveal sites at which accumulation occurs either naturally, with disease or following therapeutic interventions.
Miscellaneous Applications The excellent sensitivity and resolution of LA-ICP-MS is exploited to determine the profile of an element along a single strand of hair in very small increments. This is often applied to show when an individual may have received lethal exposure to an element such as arsenic. Similarly, in association with the speciation studies referred to above LA-ICP-MS is used to identify the small amounts of metallo-proteins separated by gel electrophoresis or similar techniques.
Atomic Spectroscopy, Biomedical Applications
79
While the natural proportions of different isotopes of an element are usually constant, the isotopes of lead vary in distribution depending upon the initial content of thorium and uranium when the earth’s crust was first laid down. These elements decay to produce lead isotopes and the ratios of e.g. 206Pb:207Pb are characteristic of the site of extraction. These subtle variations, which are measurable by ICP-MS, are used to aid identification of sources of exposure by comparing their isotopic ratios with those in the patient’s blood. Analysis of tissue samples is usually possible only following removal at surgery or post mortem. Small biopsy specimens can be obtained from a limited number of sites but this represents an invasive procedure which is not necessarily acceptable. The technique of in vivo XRF has been developed which provides for analysis of tissues close to the surface of the body, typically bone (knee cap, shin, fingers) and skin although some work with liver has been reported. Most studies are with lead to assess lifetime occupational accumulation, relocation from bone during pregnancy, and childhood environmental exposures. Other elements determined in this way include iron, and silver. Metal-containing pharmaceuticals are widely used to prevent infections and in the treatment of many disorders (cancer, depression, rheumatoid arthritis etc.). For some the dose regime has to be adapted in individual patients so that plasma concentrations are maintained within a defined therapeutic range. For others, efficacy and toxicity are closely related and it is necessary to avoid overdosing. Thus, regular monitoring of lithium for example accompanies the use of this agent when used to treat depression. Aluminum, which may be given to patients with chronic renal failure is similarly determined in these patients. Even before a drug is licensed for use in treatment preliminary studies are required to establish doses necessary to produce an effective response and without unacceptable side-effects. Initial pharmacokinetic investigations and toxicity studies in animals, followed by formal clinical trials involve measurements of the drug and/or metabolites in plasma and other samples. Where a new metallo-drug is investigated these analyses could include determination of the metal concentrations. A further activity for which sector field ICP-MS or multicollector ICP-MS are supremely appropriate involves the characterization and certification of biomedical reference materials where the high degree of accuracy required is achieved by inclusion of isotope dilution analysis into the measurement(s).
some loss of sensitivity. The most usual procedure, however, is to use a collision, or dynamic reaction, cell. The cell is a multipole – a quadrupole, hexapole or octapole – aligned between the ion optics and the quadrupole mass filter. Interactions between polyatomic ions and a gas e.g. nitrogen, helium, or methane fed into the cell, greatly reduce spectral interferences. These interactions lead to the formation of secondary species, which are removed from the ion path on the basis of kinetic energy discrimination or mass discrimination so that only the analyte ion is transmitted to the detector. Other approaches to the elimination of spectral interferences may be appropriate. Addition of an organic solvent to the sample diluent can reduce some of the argon-based interferences. Separation of analyte ions from those involved in the formation of polyatomic species prior to the measurement is also effective. Separation may be achieved by vaporisation of the sample, for example by hydride generation or by electrothermal vaporisation, or by prior use of a chromatographic or other separation step such as liquid–liquid extraction. Non-spectral interferences, associated with sample introduction and fluctuations in the inductively coupled plasma, are effectively eliminated by using an internal standard. This should be an element not present in the original sample, not subject to spectral interferences, and with mass and ionisation energy close to those of the analyte(s). Two types of interference are associated with the analysis of biomedical specimens by FAAS. Typical biological fluids contain protein and other macromolecules which increase the sample viscosity compared with simple aqueous solutions. The viscosity may reduce the aspiration rate through the narrow capillary tubing of the pneumatic nebulizer, and absorbance signals will be attenuated compared with aqueous calibration solutions, giving falsely low results. This matrix effect may be removed by dilution of the sample, precipitation of the protein or by matching the viscosity of the calibration and sample solutions. Calibration by standard additions can be adopted if there is sufficient sample. A second problem occurs in samples with a high content of dissolved solids which are liable to produce falsely high results because of non-atomic absorption or light scattering. If this occurs the analyte can be removed from the matrix by extraction into an organic solvent, or a background correction technique (see below) can be employed. The topic of interferences found with ETAAS is dealt with in more detail in other articles. Of special significance to biomedical specimens, which typically contain large amounts of sodium, chloride and carbon-based biomolecules, are;
Interferences Seen in the Analysis of Biomedical Sciences by Atomic Spectroscopy
•
ICP-MS is subject to spectral interferences. These are isobaric, e.g. 64Niþ on 64Znþ, 156Gd2þ on 78Seþ, or from polyatomic ions formed from interactions between matrix components and the plasma gas such as 40Ar on 40Caþ, 31P16Oþ 2 on 63 80 þ Cuþ, 40Ar35Clþ on 75Asþ and 40Arþ Se . If a non2 on affected isotope is present with sufficient abundance this may be counted to avoid the interference, e.g. 82Seþ in place of 80 þ 66 Se , Znþ in place of 64Znþ. Sector field ICP-MS is not subject to most of these interferences, although there may be
• •
Drying; protein-rich viscous specimens may dry unevenly with ‘explosive sputtering’ at the start of the ash phase, causing poor reproducibility. Ashing; volatile halides, for example AlCl3 may form, causing pre-atomization loss of the analyte. Atomization; (i) stable compounds such as carbides can be produced, giving low atomization rates (e.g., for Mo), (ii) vapour phase reactions, especially molecular condensation at the cooler ends of the furnace (e.g., Pb(g) þ 2NaCl(g) ! PbCl2(g) þ 2Na(g)) resulting in non-atomic absorption of incident radiation and scattering of incident radiation by particulates (carbon, smoke, salts).
80
Atomic Spectroscopy, Biomedical Applications
Background correction is essential for almost all biomedical applications to correct for the non-atomic absorption of the incident radiation. The three systems used in commercial instruments are effective although the Smith-Hieftje variation is included by very few manufacturers. Deuterium background correction was the first system to be introduced and is widely used. The Zeeman-effect technique is ideal for dealing with structured background which is of particular significance in the measurement of cadmium in urine and elements such as arsenic and selenium in iron-rich samples, that is blood. Without Zeeman-effect background correction, these applications are difficult to carry out successfully by ETAAS. In energy dispersive XRF the signal may be complicated by contributions from the sample matrix, making calibration difficult. Techniques to overcome the interferences include the use of reference materials, matrix-matched standardization and/or internal standardisation. with total reflection XRF, or when samples are prepared as very thin films, there is effectively no absorption by the matrix and problems are reduced and simpler to resolve.
See also: Atomic Absorption, Methods and Instrumentation; Atomic Absorption, Theory; Atomic Emission, Methods and Instrumentation; Atomic Fluorescence, Methods and Instrumentation; Atomic Spectroscopy, Pharmaceutical Applications; Biological Applications of Hyperpolarized 13C NMR; Circular Dichroism and ORD, Biomacromolecular Applications; Counterfeit Drugs Studied by NMR; Drug Metabolism Studied Using NMR Spectroscopy; Fragment-Based Drug Design by NMR; HPLC–NMR, Pharmaceutical Applications; IR, Biological Applications; IR, Medical Science Applications; Mass Spectrometry in Drug Metabolism: Principles and Common Practice;
Medical Applications of Mass Spectrometry; MRI Applications, Biological; NMR Spectroscopy in the Evaluation of Drug Safety; Raman Optical Activity, Macromolecule and Biological Molecule Applications; Raman Spectroscopy, Medical Applications: A New Look Inside Human Body With Raman Imaging; Spectroscopic Methods in Drug Quality Control and Development; Spectroscopy for Process Analytical Technology (PAT); Spectroscopy in Biotechnology Research and Development; Structural Chemistry Using NMR Spectroscopy, Pharmaceuticals; UV-Visible Absorption Spectroscopy, Biomacromolecular Applications; Vibrational Spectroscopy Applications in Drugs Analysis.
Further Reading Clough R, Drennan-Harris L, Harrington C, Hill S, and Tyson J (2012) Atomic Spectrometry Update: Elemental Speciation. J. Anal. At. Spectrom. 27: 1185–1224. Evans E, Palmer C, and Smith C (2012) Atomic Spectrometry Update: Advances in Atomic Spectrometry and Related Techniques. J. Anal. At. Spectrom. 27: 909–927. Taylor A, Day M, Marshall J, Patriarca M, and White M (2012) Atomic Spectrometry Update: Clinical and Biological Specimens, Foods and Beverages. J. Anal. At. Spectrom. 27: 537–576. West M, Ellis A, Potts P, Streli C, Vanhoof C, Wegrzynek D, and Wobrauschek P (2011) Atomic Spectrometry Update: X-ray Fluorescence. J. Anal. At. Spectrom. 26: 1919–1963. Butler O, Evans H, Fisher A, Hill S, Harrington C, Taylor A, West M, and Ellis A (2010) Atomic Spectrometry Updates: A 25 Year Perspective. J. Anal. At. Spectrom. 25: 1546–1566. Flanagan R, Taylor A, Watson I, and Whelpton R (2008) Fundamentals of Analytical Toxicology. Chichester: Wiley. Taylor A (2011) Atomic Absorption Spectroscopy, Inductively Coupled Plasma–Mass Spectrometry and Other Techniques for Metals Analysis. In: Widdop B (ed.) Clarke’s Textbook of Poisons. British Pharmaceutical, Society.