05 Trace Elements PDF

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403CHAPTER OUTLINE■ INSTRUMENTATION AND METHODS Sample Collection and Processing Atomic Emission Spectroscopy Atomic Absorption Spectroscopy Inductively Coupled Plasma Mass Spectrometry Interferences Elemental Speciation Alternative Analytic Techniques ■ ARSENIC Health Effects Absorption, Transport,...

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CHAPTER

Trace Elements Alan L. Rockwood, Elzbieta (Ela) Bakowska

C H A P T E R ■ INSTRUMENTATION AND METHODS Sample Collection and Processing Atomic Emission Spectroscopy Atomic Absorption Spectroscopy Inductively Coupled Plasma Mass Spectrometry Interferences Elemental Speciation Alternative Analytic Techniques ■ ARSENIC Health Effects Absorption, Transport, and Excretion Toxicity Laboratory Evaluation of Arsenic Status ■ CADMIUM Health Effects Absorption, Transport, and Excretion Toxicity Laboratory Evaluation of Cadmium Status ■ LEAD Health Effects Absorption, Transport, and Excretion Toxicity Laboratory Evaluation of Lead Status ■ MERCURY Health Effects Absorption, Transport, and Excretion Toxicity Laboratory Evaluation of Mercury Status ■ CHROMIUM Health Effects Absorption, Transport, and Excretion Deficiency Toxicity Laboratory Evaluation of Chromium Status ■ COPPER Health Effects Absorption, Transport, and Excretion Deficiency

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Toxicity Laboratory Evaluation of Copper Status IRON Health Effects Absorption, Transport, and Excretion Deficiency Toxicity Laboratory Evaluation of Iron Status Total Iron Content (Serum Iron) Total Iron-Binding Capacity Percent Saturation Transferrin and Ferritin MANGANESE Health Effects Absorption, Transport, and Excretion Deficiency Toxicity Laboratory Evaluation of Manganese Status MOLYBDENUM Health Effects Absorption, Transport, and Excretion Deficiency Toxicity Laboratory Evaluation of Molybdenum Status SELENIUM Health Effects Absorption, Transport, and Excretion Deficiency Toxicity Laboratory Evaluation of Selenium Status ZINC Health Effects Absorption, Transport, and Excretion Deficiency Toxicity Laboratory Evaluation of Zinc Status BIBLIOGRAPHY REFERENCES

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PART 2 ■ CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES

A

lmost half of the elements listed in the periodic table have been found in the human body.1 The essential and nonessential toxic trace elements included in this chapter all have biochemical importance, whether minor or major. The essential trace elements are usually associated with an enzyme (metalloenzyme) or another protein (metalloprotein) as an essential component or cofactor. Deficiencies typically impair one or more biochemical functions and excess concentrations are associated with at least some degree of toxicity. Although trace elements, such as iron, copper, and zinc, are found in mg/L concentrations, ultratrace elements, such as selenium, chromium, and manganese, are found in less than ␮g/L concentrations. An element is considered essential if a deficiency impairs a biochemical or functional process and replacement of the element corrects this impairment. Decreased intake, impaired absorption, increased excretion, and genetic abnormalities are examples of conditions that could result in deficiency of trace elements. The World Health Organization has established the dietary requirement for nutrients as the smallest amount of the nutrient needed to maintain optimal function and health. Any element that is not considered essential is classified as nonessential. Nonessential trace elements are of medical interest primarily because many of them are toxic. This chapter presents information on the laboratory techniques for trace element determination. The absorption, transport, distribution, and removal biochemical functions will be described and related to the clinical significance of disease states or toxicity.

INSTRUMENTATION AND METHODS For many years, the most commonly used instrumentation for trace metal analysis has been the atomic absorption spectrometer, either with flame (FAAS) or flameless (i.e., graphite furnace, GFAAS) atomization. Atomic emission spectrometry is also useful for some elements, particularly if used in the form of inductively

coupled plasma atomic emission spectroscopy (ICPAES) for atomization and excitation. Recently, inductively coupled plasma mass spectrometry (ICP-MS) is becoming more widely used because of its sensitivity, wide range of elements covered, and relative freedom from interferences. There is no single technique that is best for all purposes. A matrix summarizing the relative advantages and disadvantages of the main techniques is given in Table 17-1.

Sample Collection and Processing Specimens for analysis of trace elements must be collected with scrupulous attention to details such as anticoagulant, collection apparatus, and specimen type (serum, plasma, or blood). By the low concentration in biologic specimens and the ubiquitous presence in the environment, extraordinary measures are required to prevent contamination of the specimen. This includes using special sampling and collection devices, specially cleaned glassware, and water and reagents of high purity. The selection of needles, evacuated blood collection tubes, anticoagulants and other additives, water and other reagents, pipettes, and sample cups must be carefully evaluated for use in trace and ultratrace analyses. In addition, the laboratory environment must be carefully controlled. Recommended measures include placing the trace elements laboratory in a separate room incorporating rigorous contamination control features, such as sticky mats at doors, nonshedding ceiling tiles, carefully controlled air flow to minimize particulate contamination, disposable booties worn over shoes, particle monitoring equipment, etc. Many useful measures are borrowed from those employed in semiconductor clean rooms.

Atomic Emission Spectroscopy The simplified principle of the atomic emission spectroscopy (AES) instruments is presented in Figure 17-1.

TABLE 17-1 RELATIVE ADVANTAGES AND DISADVANTAGES OF MAIN TECHNIQUES FOR ELEMENTAL ANALYSIS FLAME AA

GFAA

ICP-AES

ICP-MS

Sensitivity

Moderate

Excellent

Moderate

Excellent

Selectivity

Excellent

Good

Poor

Good

Elemental coverage

Moderate

Good

Good

Excellent

Speed for one analyte

Fast

Slow

Fast

Fast

Multi-element capabilities

No

No

Yes

Yes

Initial cost of instrument

Low

Moderate

Moderate

High

Cost of consumables

Very Low

Very High

Low

Moderate

Ease of operation

Excellent

Poor

Moderate

Moderate

CHAPTER 17 ■ TRACE ELEMENTS

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Light emission Mixing chamber with burner head

Monchromator Flow spoiler Detector

Flame or plasma FIGURE 17-1. Simplified schematic of AES.

Nebulizer

The three most important components of AE spectrophotometer are: 1. A source, in which the sample is atomized in a suffi-

ciently hot source to produce an excited-state species. Those species will emit radiation upon relaxation back to the ground state. 2. A wavelength selecting device (monochromator), for the spectral dispersion of the radiation and separation of the analytic line from other radiation 3. A detector permitting measurement of radiation intensity A liquid sample, containing element(s) of interest, is converted into an aerosol and delivered into the source, where it receives energy sufficient to emit radiation. The intensity of the emitted radiation is correlated to the concentration of an analyte and is basis for quantitation. The most commonly currently used sources in AES are flame and inductively coupled plasma. Flames are capable of producing temperatures up to 3000 K. Typical fuel gases include hydrogen and acetylene, while oxidant gases include air, oxygen, and nitrous oxide. The gases are combined in a specially designed mixing chamber. A sample is also introduced into the mixing chamber using a nebulizer that converts liquid into a fine spray. The mixing chamber and burner assembly are shown in Figure 17-2. The same assembly can be used for atomic absorption instrumentation. Inductively coupled plasma torches (described in more detail in a later section) are capable of much higher temperatures and are therefore applicable to a wider range of elements. In AES, both atomic and ionic excited states can be produced (depending on the element and the source), which leads to production of complicated emission spectrum. The “emission spectrum” of an element is composed of a series of very narrow peaks (sometimes known as “lines”), with each line at a different wavelength and each line matched to a specific transition. Each element has its own characteristic emission spectrum. For example, sodium can be detected by tuning the monochromator to a wavelength of 589 nm. Ideally, each emission line of a given element would be distinct from

Impact bead End cap FIGURE 17-2. Mixing chamber burner for flame AA. (Courtesy of Perkin-Elmer. Waltham, MA.).

all other emission lines of all other elements. However, there are many cases where emission lines from certain elements overlap the emission lines of certain other elements, resulting in interferences. The choice of interference-free wavelength (atomic or ionic line) may be challenging. While there are several possible wavelengths for a given element, wavelengths producing suitable analytic performance, such as limit of quantitation, freedom from interferences, robustness, etc. are selected. The first detectors in AES used photographic film. Contemporary AES instruments feature photomultiplier tubes or array-based detector systems.

Atomic Absorption Spectroscopy Atomic absorption spectroscopy (AAS) is an analytic procedure for the quantitative determination of elements through the absorption of optical radiation by free atoms in the gas phase. The spectra of the atoms are line spectra that are specific for the absorbing elements. Absorption is governed by the Beer-Lambert law: A ⫽ ⫺log 10

( II ) ␧LC 1

g

(Eq. 17-1)

0

where A is the absorbance of the sample, I0 is the incident light intensity, 1I is the transmitted light intensity, ␧ is the molar absorptivity of the target analyte for the wavelength being used, L is the path length, and Cg is the gas-phase concentration of the target analyte. Under some simplifying assumptions, this equation takes the form: A ⫽ KC

(Eq. 17-2)

where K is a constant determined by calibration, and C is the solution phase concentration of the analyte.

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PART 2 ■ CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES

Light absorption Monchromator Detector

Flame or graphite furnace Light source FIGURE 17-3. Simplified schematic of AAS instrumentation.

The simplified principle of the AAS instruments is presented in Figure 17-3. The four most important components of AA spectrophotometer are : 1. Radiation (light) source, which emits the spectrum of

the analyte element 2. Atomizer, in which the atoms of the element of inter-

est in the sample are formed

Selenium and cadmium are often measured by GFAAS. GFAAS allows for measurements of both liquid and solid samples. A common problem in GFAAS is that analyte volatility depends on the molecular form of the analyte and the sample’s matrix. To overcome this limitation, chemical modifiers (palladium nitrate, magnesium nitrate, or mixture of both) are frequently added to samples, calibrators, and controls.

3. Monochromator, for the spectral dispersion of the ra-

Hydride Generation and Cold Vapor AAS

diation and separation of the analytic line from other radiation 4. Detector permitting measurement of radiation intensity

There are two techniques of chemical vapor generation: cold vapor (CVAAS) and hydride generation (HGAAS). CVAAS is used exclusively for the determination of mercury. Mercury is reduced to its elemental form and transferred into a vapor phase, concentrated with the assistance of amalgamation, and measured in the absorption cell. In HGAAS the sample undergoes a chemical reaction; as a result, the analyte is converted into a gaseous hydride. The hydrides are then atomized either in flame or graphite furnace. This technique is only applicable to determination of antimony, arsenic, bismuth, selenium, tellurium, and tin. The specificity of the chemical vapor generation techniques can allow for the speciation of the analytes. If the parameters are chosen properly, it may be possible to distinguish between different forms of the analytes, especially between their organic and inorganic forms.

Typical radiation sources for AAS are hollow cathode lamps (HCLs) and electrodeless discharge lamps (EDLs). The HCL contains a quantity of the target element in the form of a hollow cylinder. During operation, a small quantity of the target element is vaporized, and some of the gasphase atoms of the target element become electronically excited and emit photons with the right wavelength to be absorbed by atoms of the target element in the atomizer. While HCLs are an ideal source for determining most elements by atomic absorption, for volatile elements the use of electrodeless discharge lamps is recommended. The most common sources in AAS are flame (FAAS) and graphite furnace (GFAAS, also called flameless or electrothermal AAS). The mixing chamber burner, which produces laminar flames of high optical transparency, was already described in the section on AES in this chapter. Copper, iron, and zinc are often measured by FAAS. The graphite tubes are most commonly used atomizers in flameless AAS. Tubes are made of high-purity polycrystalline electrographite and coated with pyrolytic graphite and can be heated to a high temperature by an electrical current. A small aliquot (usually 20 ␮L) of liquid sample is placed in the tube at the ambient temperature. The heating program (specifying the temperatures and times) is designed to first dry the sample, then pyrolize, vaporize, and atomize the sample, followed by a cleaning step.

Inductively Coupled Plasma Mass Spectrometry Inductively coupled plasma mass spectrometry (ICP-MS) is a state-of-the-art analytic technique for elemental analysis. The term plasma in ICP refers to an ionized gas (almost always argon), in which a certain proportion of electrons are free. Like other mass spectrometers, the ICP-MS measures the mass-to-charge ratio (molecular mass divided by ionic charge [m/z]) of selected analyte ions) and includes the following components: (1) an ion source, (2)

CHAPTER 17 ■ TRACE ELEMENTS

Skimmer cone

Ion lenses

407

Quadrupole MS

RF coil

ICP torch

Plasma Sampler cone EM detector

Turbo pumps

Mechanical pump FIGURE 17-4. Simplified schematic of ICP-MS instrumentation.

a mass-to-charge ratio (m/z) analyzer, and (3) an ion detector. A simplified schematic of an ICP-MS is given in Figure 17-4. The argon plasma induced by commercial ICP instruments (both ICP-AES and ICP-MS) generates high temperature, such as approximately 6,000 K to approximately 10,000 K, and serves several purposes. First, it dries the droplets produced by the nebulizer, and then it vaporizes the dried particles. This step is followed by atomization of any molecular species. Finally, atoms are thermally ionized, at which point they are ready for introduction into the mass spectrometer Nearly all ICP torches consist of three concentric quartz tubes surrounded by a coil carrying radiofrequency power. The middle tube of the torch carries the argon (Ar) that forms the plasma. Quantitative analysis for clinical samples is best performed with the use of an internal standard. All patient samples, calibrators, and controls are diluted with an internal standard, usually a solution of an uncommon element (such as yttrium) that is different than the target element. Rather than using the raw signal level of the target elements as the basis for quantitation, the signal for each of target element is divided by the signal of the internal standard to give signal ratios (i.e., normalized intensities).

Quadrupole Mass Spectrometers The typical mass spectrometer used for ICP-MS is a quadrupole mass spectrometer. The analyzer consists of four parallel conducting rods arranged in a square array. By superposition of radiofrequency (RF) and constant (DC) voltages applied to the rods, the instrument can be tuned so that only ions of a specific m/z ratio can pass through the device to reach the detector. This type of instrument tends to be relatively simple to use and maintain, but the resolution (the ability to discriminate between closely spaced m/z values) is limited, being able to

well resolve peaks separated by one m/z unit but not able to resolve peaks separated by a small fraction of an m/z unit.

High-Resolution Mass Spectrometers Other ICP-MS instruments incorporate high-resolution mass spectrometers. These are usually “double focusing sector field” instruments. Such instruments separate ions of different m/z values via deflection in a magnetic field, with ions of greater m/z being deflected to a lesser degree than those of lower m/z. The magnetic field is adjusted so as to allow only ions of a selected m/z to reach the detection system at any given point in time. A second device known as an electrostatic analyzer corrects for certain nonideal effects, allowing the instrument to achieve high resolution. Commercially available high-resolution ICPMS instruments are capable of a resolution of 10,000 (10% valley). This is enough to resolve, for example 75 As ⫹ from 75ArCl⫹, both nominally 75 m/z units, but which differ by 10 ⫻ 10⫺3 units when viewed at high resolution. However, magnetic sector instruments are not capable to resolve elemental isobaric interferences such as 115Sn/ 115In or 40Ca/40Ar, which would require resolution much higher than 10,000.

Interferences In general, the interferences in elemental analysis are classified as spectroscopic or nonspectroscopic.

Spectroscopic Spectral interferences generally result from a spectral overlap with the spectrum of the target analyte. For example, in AA certain molecular species may have broad absorption spectra that may overlap the line spectra of the elements of interest, leading to false elevations of the target element concentrations. A much less common occurrence would be for the absorption spectrum of one element to overlap with that of another.

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Various strategies are used to deal with spectral interferences in AAS. A continuum source background corrector may be included in the instrument design at the cost of some instrument complication. Another alternative is Zeeman background correction, which relies on shifting the atomic spectral lines by the application of a magnetic field. In ICP-MS, spectral interferences include polyatomic species whose m/z may overlap m/z of the target analyte. For example, 56(ArO⫹ ) has the same nominal m/z as 56 Fe ⫹. The argon oxide ion, which can be a significant component of plasma generated by an ICP torch, can potentially interfere with iron analysis by ICP-MS. Another well known polyatomic interference is argon chloride ion 75(ArCl)⫹ on determination of 75As⫹ . An extensive table of polyatomic interferences in ICP-MS has been published.2 A second source of spectral interferences in ICP-MS arises from nearby elements in the periodic table. For example, tin (Sn) and cadmium (Cd) both have isotopes at 114 Da (amu), so they could potentially interfere with each other if the instrument ...