Review Special Focus Issue: Forensic and clinical toxicology

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Current role of ICP–MS in clinical toxicology and forensic toxicology: a metallic profile

As metal/metalloid exposure is inevitable owing to its omnipresence, it may exert toxicity in humans. Recent advances in metal/metalloid analysis have been made moving from flame atomic absorption spectrometry and electrothermal atomic absorption spectrometry to the multi-elemental inductively coupled plasma (ICP) techniques as ICP atomic emission spectrometry and ICP–MS. ICP–MS has now emerged as a major technique in inorganic analytical chemistry owing to its flexibility, high sensitivity and good reproducibility. This in depth review explores the ICP–MS metallic profile in human toxicology. It is now routinely used and of great importance, in clinical toxicology and forensic toxicology to explore biological matrices, specifically whole blood, plasma, urine, hair, nail, biopsy samples and tissues.

Background Toxicological analysis has made significant advances based on chromatographic techniques (i.e., GC and LC coupled to MS) during past decades. These methods are excellent for many organic xenobiotics (drugs, drugs of abuse, pesticides and other organic substances), but are not suitable for minerals in general as they require other techniques, such as ion-exchange chromatography (IC) and x-ray diffraction (XRD) for example, and are not suitable for metal or metalloid analysis. The inductively coupled MS (ICP–MS) method used for multi-elemental clinical sample analysis since the 1980s [1] has now been extensively developed for metal and metalloid quantification, such as arsenic and selenium, as the result of the development of new high performance instruments made in the 2000s with compact table-top equipments (Figure 1) [2] . It has now emerged as a major technique in inorganic analytical chemistry owing to its flexibility, high sensitivity and good reproducibility. In a recent review, Heuveln et al. reported current challenges of the applications of ICP–MS in life science applications and its various hyphenations in drug development (i.e., speciation/metabolism and proteomic studies and the analysis of the various

10.4155/BIO.14.190 © 2014 Future Science Ltd

sample matrices applicable to these fields) [3] . In another paper concerning quadrupole and high-resolution ICP–MS methods of analysis, which are now being used to quantify inorganic elements contained in pharmaceutical compounds and biomatrices, Ammerman et al. focused on the technical aspects regarding quantifying pharmaceutically derived inorganic elements in bio­ matrices and the elements occurring endogenously in biomatrices, affecting quantification of blanks, standard curve samples, quality control (QC) samples and the selection of appropriate levels for the limit of quantification (LOQ) [4] . Metal and metalloid are certainly one of the oldest toxicants known to man and were used in 2000 BC. Present in the environment, in the air, in water and in various products including food, they are also employed in many industrial processes and, thus, human exposure is inevitable. They totally differ from other substances, such as drugs or drugs of abuse, as they are not metabolized in the human body. Therefore, a very important characteristic of metals is that they may react in biological systems by losing electrons to be oxidized as cations, or form oxyanions, such as the metalloids arsenic and selenium, and then exert their toxicity;

Bioanalysis (2014) 6(17), 2245–2259

Jean-Pierre Goullé*,1, Elodie Saussereau2, Loïc Mahieu2 & Michel Guerbet1 Faculté de Médecine et de Pharmacie, Université de Rouen, 22 Boulevard Gambetta, 76183 Rouen CEDEX 1, France 2 Laboratoire de Pharmacocinétique et de Toxicologie Cliniques, Groupe Hospitalier du Havre, BP 24, 76083 Le Havre CEDEX, France *Author for correspondence: Tel.: +33 235 148 612 [email protected] 1

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ISSN 1757-6180

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Review  Goullé, Saussereau, Mahieu & Guerbet

Key term ICP–MS: Inductively coupled plasma MS is a technique to quantify one or simultaneously many elements after ionization and mass separation with a mass spectrometer.

consequently, it is necessary to measure their content in biological matrices. Among these elements, some are major toxics. Cases of intoxication have been reported with arsenic, thallium, lead, mercury and chromium, but also cadmium, platinum, nickel, aluminium or gadolinium. Others are essential elements (i.e., cobalt, copper, iron, magnesium, selenium or zinc), but they will also become toxic with increasing exposure, or due to pathologic metabolism, such as in Wilson Disease (WD). Furthermore, the metal toxicity may vary with the oxidation state of the element, for example, hexavalent chromium (CrVI) is a powerful toxic element [5] , whereas trivalent chromium (CrIII), less toxic, is essential and plays a major role in glucose metabolism. The element toxicity may also substantially vary with the chemical state or the chemical species, such as mercury or arsenic. Elemental mercury (Hg°), even if a somewhat high amount is ingested has only a low toxicity [6,7] ; by contrast mercury vapors and organic compounds, methylmercury (MeHg) for example, are highly toxic for humans [8] . The famous metalloid arsenic is “the poison of Kings and the king of poisons”; while organic arsenic compounds in seafood (arsenocholine, arsenobetaine) are safe, inorganic arsenic is particularly reactive [9] , therefore it is essential to differentiate the two species. During recent years the effects of arsenic on health have been a constant concern in many countries, ­particularly in Bangladesh [10] . Among emerging important topics in clinical toxicology, metals and metalloids have a large place owing

to the various sources of exposure. How the environment contributes to human exposure is currently better understood and highlighted by the contamination of the air due to tetraethyl lead that was previously extensively used as a gasoline additive and had an impact on human health [11] . Also MeHg, with its extraordinary bioaccumulation in carnivorous fish [12] , arsenic water pollution in more than 80 countries all over the world [13] , as well as the former wide industrial use of cadmium [14] , have subsequently led to the monitoring of these elements in biological matrices being required. Metals and metalloids are present in a many pharmaceuticals, such as the platinum-based antineoplastic drugs or platinum chemotherapeutic agents to treat cancer [15] , and gadolinium contrast media, which is injected to improve the visibility of internal body structures in MRI, and the potential toxicity of these elements must not be ignored [16] . The aluminium compounds, which are used as adjuvants to enhance the immunologic response of many vaccines [17] and the preservative organomercury compound thiomersal or thimerosal added to some vaccines remain topics of concern [18] . Various metals used for implants, such as cobalt or chromium alloys, and titanium for joint replacements may be detrimental to human health [19] . More recently, nanotechnology, which is one of the most active research areas in the science of modern materials, includes the production of metal nanoparticles [20] . Advances in nanotechnology and metals have impacted on each another in a number of ways, so that metals play a very important role in this new industry but the potential environmental health impact for humans is of major concern [21] . Metal/metalloid reviews have been developed in detail by Taylor and colleagues in their last extensive annual atomic spectrometry update ‘Review of

Plasma Interface

Plasma gas

Quadrupole

h Torch Auxiliary gas

ayy Spray mber chamber

Carrier gas

Sample

Nebulizer

Ion lens

Detector

Figure 1. Principle of ICP–MS. Reproduced with permission, courtesy of Agilent Technologies Inc.

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Current role of ICP–MS in clinical toxicology & forensic toxicology: a metallic profile

advances in the analysis of clinical and biological materials, foods and beverages’ [22] . The volume of atomic spectrometry work surveyed each year between 1985 and 2010 has increased approximately fivefold [23] . For metal/metalloid measurement, significant progress has occurred, particularly over past decades, that has progressively moved from the classic techniques flame atomic absorption spectrometry (FAAS) and electrothermal atomic absorption spectrometry (EAAS) to the ICP techniques ICP atomic emission spectrometry (ICP-AES) and ICP–MS [24] . The transition first from FAAS to ICP-AES was driven by a greater degree of automation and the migration to ICP–MS due to the throughput capabilities of ICP-AES coupled with the sensitivity of EAAS [24] . The ICP–MS technique was originally described in 1981 by Date and Gray [25] . The first evaluations of the technique for simultaneous multi-element trace analysis in clinical chemistry were published at the end of the 1980s [1] , but the technique “has now reached a level of maturity that precludes the emergence of ‘standout’ new developments” [24] . Since that time it has been progressively applied to clinical toxicology and forensic toxicology techniques. As ICP–MS is a powerful analytical tool for the determination of metals and metalloids, this paper extensively explores the current role of ICP–MS for metal and metalloid analysis in biological fluids and matrices regarding human toxicology. The ICP–MS technique is now routinely used to establish the metallic profile in clinical toxicology and forensic toxicology. It is currently the best way to explore and monitor professional or environmental exposure in clinical toxicology, poisoning or death involving metals or metalloids in forensic cases [2,26] . ICP–MS performance for multi-elemental biological applications & single analysis The first basic ICP–MS biological applications were devoted to clinical samples [1] . However, at this time

Review

Key term Metallic profile: In various biological matrices is determined by multi-elemental analysis of approximately 30 elements by ICP–MS.

most of the results obtained, particularly for the first row transition elements as well as for arsenic and selenium, were severely degraded by polyatomic interferences [1] . This problem is nowadays solved by different approaches to overcome this (see section on critical evaluation of the ICP–MS techniques). ICP–MS not only allows individual element analysis but also the simultaneous determination of approximately 30 elements of toxicological interest in a single run. Furthermore, this approach permits an optimal gain in sensitivity in all biological matrices: specifically, whole blood, plasma or serum, red cells, urine, hair, nail, biopsy samples or tissues [2,27–29] . The LOQ is very low as it ranges from 2 ng/l for uranium to 700 ng/l for selenium in plasma for a 25 multi-element analysis [27] and from 0.2 ng/g for platinum, thallium and uranium to 500 ng/g for boron in nail for a 32 multi-element analysis [28] . In whole blood, compared with the EAAS, the LOQ is much better for all elements, except for aluminium [27] . Table 1 shows a typical sample preparation used for whole blood, plasma, urine, hair, nail or tissue [2,27–29] . Briefly, for each sample, a 0.3 ml whole blood, plasma or urine specimen is used. Otherwise, 20 mg of nail/hair or 40 mg of tissue, is digested with pure nitric acid during 1 hour at 70°C, and a part of the digest solution is then diluted in a dilution solution before analysis (Table 1) . Furthermore, today ICP–MS can be used to generate semiquantitative data for elements even if they are not present in calibration solutions. In plasma MS we can take profit from the fact that the ionization and transmission characteristics of elements are relatively predictable. Then, there are a number of factors to consider when converting the integrated ion intensity to an element concentration:

Table 1. ICP–MS sample preparation.  

Whole blood

Plasma

Urine

Hair/nail

Tissue

Sample

0.3 ml

0.3 ml

0.3 ml

20 mg

40 mg

Nitric acid (pure)

No

No

No

0.2 ml

0.2 ml

Digestion

Freeze 1 h to hemolyze

No

No

70°C 1 h

70°C 1 h

Digested sample

No

No

No

0.1 ml

0.1 ml

Nitric acid (2 %)

No

0.1 ml

No

0.1 ml

0.1 ml

Standard addition

0.1 ml

No

0.1 ml

No

No

Dilution solution

2.6 ml

2.6 ml

2.6 ml

3.8 ml

3.8 ml

Dilution solution (v/v): nitric acid 1.0 %, butanol 0.5 %, triton 0.1 % for whole blood and 0.01 % for plasma, nail and tissue, rhodium and indium as internal standards (1 μg/l). Standard addition calibration for whole blood and urine, aqueous calibration for other matrices.

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Review  Goullé, Saussereau, Mahieu & Guerbet

Key term HR-ICP–MS: High resolution or sector field/inductively coupled plasma MS is used to determine the mass of an element with a higher precision (0.01 mu or less instead of 0.1 amu) as ICP–MS can be affected by polyatomic mass interferences due to matrix.

• The atomic mass of the element to express the results in mass concentration; • The abundance of the isotope used for analysis (which is known); • The relative sensitivity factor to establish the response curve of the instrument. Once it has been established, the sensitivity of any element may be deduced from the curve to calculate the semiquantitative concentrations for more than 30 elements in a few minutes. For uncalibrated elements, most recoveries are in the 70–130 % range [Goullé J-P, Unpublished Data] . The semiquantitative mode may also be used with only one point calibration. With this last mode, particularly good results concerning accuracy and reproducibility were obtained by Chen et al. [30] . Regarding the use of the semiquantitative mode, without any calibration point, we were able to measure whole blood gadolinium in a case of nephrogenic systemic fibrosis, with a fair level of precision [31] . ICP–MS analytical considerations This section deals with metal multi-elemental and individual studies that are based on the standard technique of ICP–MS and derived techniques. As polyatomic interferences may be produced by the combination of two or more atomic ions that have the same atomic mass as the element to be analyzed, instruments have been developed to overcome this problem and enhance specificity (see section on critical evaluation of the ICP–MS techniques). The use of a dynamic reaction cell (DRCICP–MS with quadrupole, hexapole or octopole) or a triple quadrupole ICP–MS/MS that breaks the atomic ions combination, or the sector field ICP–MS that are the high resolution equipment (HR-ICP–MS) and the multicollector machines (MC-ICP–MS), with a high precision in mass determination, will solve this problem [32–34] . With ICP–MS/MS in MS/MS mode, the first MS operates as a mass filter, allowing only the target analyte mass to enter the cell and rejecting all other masses. With all non-target plasma and sample matrix ions excluded from the cell, sensitivity and interference removal efficiency in collision mode is improved compared with ICP-QMS. In the MC-ICP–MS the quadrupole is replaced by a magnetic sector, this equipment with a 3,000 resolution suits well for isoto-

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pic measurements. The HR-ICP–MS, which includes both the magnetic sector and an electric sector, has a higher resolution, which can reach 10,000, but when the resolution increases sensitivity decreases. Preparation of biological sample by ICP–MS Sample preparation is a crucial stage in order to assure accurate results and many parameters are essential to produce high quality degree analysis. The following are the basic rules: • Before analysis a short-term preservation of the biological material at -20°C is recommended [35] ; • The quality of biological collection, particularly for whole blood and plasma, is important. ‘metal-free’ devices are required, such as 6 ml special vials [27] ; • The high purity of the reagents used and argon as a source of plasma is required [27] ; • A short preparation stage is recommended to limit the risk of metal contamination [27] ; • Participation in a QC scheme and use of reference or certified materials are required [27] . There is a broad consensus as regards the dilution of the biological sample with a diluted nitric acidic solution containing a detergent such as Triton X-100 and metal internal standards [27,35,36] . The mode of calibration includes either a water calibration or a standard matrix addition [27,36] . As shown in Table 1, a typical sample size used is 0.3 ml for whole blood, plasma or urine for multi-elemental quantification [27] . However, there is a real challenge to reduce the volume sample and therefore recent studies have employed microflow nebulizers to overcome this difficulty. A new aerosol generation system with ultra-low flow rates of 1 μl min1 for samples, such as urine or serum, based on thermal inkjet technology termed ‘drop-on-demand’; with a microvolume sample introduction system consisting of a 20 μl injection loop with a high-performance concentric nebulizer coupled to a heated cyclonic spray chamber that produces a flow rate of 10 μl min-1, has recently been reported in the literature [37,38] . A microfluidic chip-based nanoflow injection system with 200 nl sampling with a flow rate of 20 μl min-1 has also been reported by Wu et al. for platinum in human plasma determination. The detection limit was 2.5 fg, which was improved by a factor of 3,200 in comparison with a conventional sampling system [39] . Practical solutions to overcome interferences Advances in the ICP–MS method include a collision or DRC for the determination of some elements, such

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Current role of ICP–MS in clinical toxicology & forensic toxicology: a metallic profile

as chromium (52 Da) due to a mass interference as the torch uses the argon (40 Da) element that combines with the carbon (12 Da) present in usual biological matrices (blood, urine). To overcome this problem, the DRC device uses a gas that breaks this polyatomic interference [40,41] . The DRC-ICP–MS method has also been applied to mult-ielement analysis [42] . A more effective solution, in order to overcome the matrix-induced polyatomic mass interference problems, is to use more expensive HR-ICP–MS equipment that has been extensively applied by many authors over the past few years. This has led to good results that have been evaluated in two papers by Bocca et al. [43,44] . Despite the increase in the sensitivity of instruments, unfortunately ICP–MS techniques are not always sensitive enough to quantitate certain elements present at the ultra-trace level in biological fluids. Furthermore, these matrices have a complex composition and a high salt content that subsequently requires preconcentration techniques prior to elemental analysis. In a very recent review, Lum et al. discussed four aspects of the recent development in metal preconcentration in clinical samples before analysis, for example, the use of ionic liquids in extraction, sorption by nanomaterials, preconcentration using ­surfactants and automation [45] . Critical evaluation of the ICP–MS methods One of the most important limitations of the ICP–MS methods is the presence of several types of interference: • Non-spectral interferences due to matrix effects; • Spectral interferences that may be: –– Isobaric ions (two elements that share the same isotopic mass); –– Double-charged ions; –– Polyatomic ions. The polyatomic interference may be produced by the combination of two (or more) atomic ions. For example, the gas argon (Ar: 40 Da) may combine with the chloride ion (Cl: 35 Da) of the biological matrix to produce ArCl (75 Da), which overlaps with arsenic (75 Da) determination. There are several different ways to compensate for these polyatomic interferences in ICP– MS. Three commonly used methods to overcome these interferences are: • The use of correction equations that are built into the machine software to facilitate automatic correction, which is successfully used for the determination of arsenic in whole blood for example [27] ;

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• When a correction equation cannot be applied, the gas of a DRC may broke the combination of the atomic ions Ar and carbon (C: 12 Da): ArC (52 Da) overlaps with chromium (52 Da) determination  [40] ; • The use of another isotope if the abundance is sufficient as for 53chromium (9.5% of the element), which is used to analyze the metal in hair and nail. This is due to its concentration, which is 1,000 times higher than in blood [28] . Another solution that is not common because of the high cost of the equipment is the use of high resolution apparatus (HR-ICP–MS or MC-ICP–MS), which is able to achieve an improved mass accuracy. With regards to the critical evaluation of ICP–MS, it is important to consider the ionization potential, the higher it is, and the more difficult it is to produce ions. Once ions are generated the transmission efficiency depends upon its mass-to-charge ratio. Therefore, low mass isotopes tend to be transmitted with the lowest efficiency due to scattering effects. For example, the LOQ of aluminium in whole blood is lower by EAAS compared with ICP–MS [Goullé J-P, Unpublished Data] . Mid-mass isotopes tend to be transmitted with better efficiency and high-mass with greatest efficiency. For example, the ICP–MS whole blood lead LOQ is 0.1 μg/l and 10 μg/l with EAAS [27] . ICP–MS applications to whole blood, plasma & urine in clinical toxicology The ICP–MS was extensively applied to multi-element determinations in whole blood, plasma serum and urine as these fluids are the clinical samples most commonly used for metal and metalloid analysis. Blood and urine analysis are particularly useful to document acute and chronic exposure to elements as this may produce both blood and urine elevated levels. Regarding ICP–MS multi-element and individual element determination, a large quantity of data and their application were published in recent years, mainly in the area of clinical toxicology. The main topics were: Normal multi-elemental values in biological fluids

Normal multi-elemental values in biological fluids have been reported from several specific geographical locations all over the world [27,46–56] . Various exposure or pathologies have also been explored [46,49,54] . Plasma and whole blood ICP–MS typical analytical performance and reference values in 106 adult volunteers, recently published by our group, are reported in Tables 2 & 3 [27] .

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Review  Goullé, Saussereau, Mahieu & Guerbet

Table 2. Whole blood ICP–MS typical analytical performance and reference values in 106 adult volunteers. Element Isotope

LOD μg/l

LOQ μg/l

Median (n=106) μg/l

Range 5th−95th percentile μg/l

7

Lithium

0.02

0.05

Current role of ICP-MS in clinical toxicology and forensic toxicology: a metallic profile.

As metal/metalloid exposure is inevitable owing to its omnipresence, it may exert toxicity in humans. Recent advances in metal/metalloid analysis have...
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