Chapter 16 HPLC-ICP-MS screening for forensic applications

M.J. Bogusz (Ed.). Forensic Science Handbook of Analytical Separations, Vol. 6 r 2008 Elsevier B.V. All rights reserved.

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HPLC-ICP-MS screening for forensic applications Kevin M. Kubachka, Douglas D. Richardson and Joseph A. Caruso Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221-0172, USA

16.1 INTRODUCTION There is a wide range of substances that pose a threat to human safety. These toxins exist in the environment both naturally and created by human activities. Some man-made toxins are designed with destructive applications in mind (anthrax, chemical warfare agents), others with good intentions, are toxic to humans (pesticides), while more are byproducts from industry/manufacturing (lead from fossil fuels). Regardless of their route to the environment and consequentially humans, scientific methods for the detection of these compounds are of great importance. In regards to forensics, analytical methods are needed with the following expectations in mind: 1. Establishing environmental levels of contaminants to ensure safety by: a. Assessing risk b. Minimizing exposure c. Developing countermeasures 2. Determining the identification of unknown substances at a crime scene. 3. Early detection of toxins in humans with the hopes of preventing chronic exposure. 4. Establishing the cause of death, as in the case of a poisoning. These methods need to be applicable in a variety of sample matrices including environmental (water, soil, air, etc.), foods and beverages, and biological samples (hair, blood, urine, etc.). Due to the large variety of sample types, methods should be selective and able to resolve analytes from possible interferences. Qualitative analysis is important when identifying unknowns, while quantitation is necessary to establish toxic levels and enforce regulatory limits of various compounds. Also analysis at low levels is needed as many compounds are considered a hazard at even

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part per billion levels. For example, in the US, as mandated by the Environmental Protection Agency (EPA) in 2001, arsenic (As) in drinking water is limited to 10 parts per billion [1]. There are several elements of high concern as they are occupational or residential exposure hazards. Often times, to monitor these elements they are analyzed with respect to their total element concentration. Many EPA restrictions are currently based on this information, as in the case of uranium, in which the limit in water is at 30 mg L
1 [1]. However, there are several elements in which more information is useful based on the fact that an element may have varying toxicities in different ‘‘forms.’’ One specific example is the toxicity of chromium (Cr), as CrVI is considered carcinogenic and mutagenic and CrIII is an essential nutrient [2]. Research devoted to the investigation of various chemical forms or species of the same element has been termed ‘‘elemental speciation.’’ In this chapter, the elements antimony (Sb), arsenic (As), chromium (Cr), lead (Pb), mercury (Hg), selenium (Se), tellurium (Te), tin (Sn) and vanadium (Vn) will be discussed as they are of most interest and relate most heavily to forensic applications. Typical analysis of the previously mentioned elements is performed through the use of instruments with element-specific detection capabilities, where these elements are used as ‘‘elemental tags.’’ One of the more common elemental analysis techniques includes atomic absorption spectroscopy (AAS) with flame ionization (FAAS) or electrothermal ionization (ETAAS). Optical emission spectroscopy used other ionization sources including as microwave-induced plasma (MIP) and inductively coupled plasma (ICP). The inability of these spectroscopic methods to reach low detection limits and the presence of spectral interferences enhanced the need for more sensitive and selective detectors [3]. With the use of ICP with mass spectrometry (ICP-MS) for element-specific detection experiments, sensitivity, and selectivity are significantly improved. Other methods such as molecular mass spectrometry (MS) with softer ionization methods, such as electrospray ionization (ESI), chemical ionization (CI), or atmospheric pressure ionization (API) can also be used, however, a decrease in both sensitivity and selectivity are typically observed compared to ICP-MS element-specific detection [3]. ICP-MS is very sensitive with detection limits for some analytes at low part per trillion (ppt) levels. As previously mentioned, it is an element-specific detection method, with the ability to detect almost all elements in the periodic table virtually simultaneously depending upon the mass analyzer. The use of this harsh ionization technique in conjunction with a mass selective detector provides a tool for isotopespecific experiments. Quantitation is easily achieved using an external calibration curve, standard addition, or isotope dilution methods, with a dynamic range of up to 9 orders of magnitude. Also, when speciation analysis is required, it is easily interfaced with chromatographic techniques such as high performance liquid chromatography (HPLC), gas chromatography (GC), and capillary electrophoresis (CE). This chapter will focus upon forensic applications of HPLC with ICP-MS elemental-specific detection.

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16.2 INSTRUMENTATION 16.2.1 Inductively coupled plasma mass spectrometer What follows is a brief summary of ICP-MS from sample introduction to analyte detection as explained from left to right in accordance with Fig. 16.1. The initial part is the sample introduction, typically involving nebulization of the liquid sample, in which gas forces the solvent out of the end of a narrow inner diameter tube, thus forming a ‘‘mist’’ of sample. The sample then is transported into the ionization source, which in most cases is an argon plasma, by the carrier gas, typically argon. Here, the sample is desolvated, atomized, and ionized to positive charge by the plasma which is at temperatures from 6000 to 10,000 K. The ions then pass through the interface region consisting of both a sampler and skimmer cone (composed of either nickel or platinum depending upon the application). The interface region is the transition point from atmospheric pressure to the vacuum region of the instrument. This vacuum region consists of ion optics for focusing, collision/reaction cell for interference removal (described in the next section), mass analyzer (quadrupole, time of flight, sector field), and detector (electron multiplier). 16.2.1.1 Resolving interferences As previously mentioned, ICP-MS is an element-specific technique with the mass analyzer detecting an analyte based on its mass to charge ratio (m/z). However, there are interferences present including isobaric interferences (54Cr and 54Fe) and poly80 + Se ). The 40Ar+ atomic interferences (40Ar+ 2 and 2 polyatomic interference at 80 m/z is always present at high levels in an argon plasma and interferes with the most abundant isotope of Se at 80 m/z (49.61%). There are several approaches to solving this problem. One involves the use of introducing a collision or reaction gas in conjunction with an energy barrier (He, H2, Xe, NH3), to collide/react with the

Fig. 16.1. Agilent ICP-MS block diagram. Reaction/collision cell is located after the ion lens and before the mass analyzer (in this case, a quadrupole). From Agilent Technologies with permission.

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TABLE 16.1 ANALYTES OF INTEREST WITH THEIR MAJOR ISOTOPES. THE RELATIVE ISOTOPIC ABUNDANCE OF EACH ISOTOPE AND MAJOR CORRESPONDING POLYATOMIC INTERFERENCES ARE ALSO LISTED. THERE ARE NO POLYATOMIC INTERFERENCES FOR MERCURY Isotopes

Percent Abundance

Polyatomic Interferences

75

100 83.8 24.1, 57.2, 23.5, 14.5, 19.0, 99.8

40

As Cr 206 Pb, 207Pb, 208Pb 121 Sb, 123Sb 78 Se, 80Se 116 Sn, 118Sn, 120Sn 126 Te, 128Te, 130Te 51 Vn 52

Ar35Cl+ Cl16O1H+ Pt oxides Pd oxides 40 Ar+ 2 Ru and Pd oxides Ru and Pd oxides 35 16 + 34 16 Cl O , S OH+ 35

22.1, 52.4 47.8 49.8 24.2, 32.6 31.7, 33.8

Adapted from reference [9].

interference or analyte. Further specifics on this topic can be found in the literature [4–7]. For example, the use of H2 can reduce the background from 10,000,000 cps to 10 cps, producing a background equivalent concentration of approximately 1 ppt for 80Se [8]. There are other strategies of working around these interferences such as monitoring a less abundant isotope (82Se) or calculating the contribution of possible interferences based isotopic information (40Ar35Cl+ and 75As+ by monitoring 40Ar37Cl+). Another option is the use of a high-resolution sector field detector that can resolve, for example, the 80Se+ at (79.9165 Da) from 40Ar40Ar+ at (79.9248 Da) [5,6]. For elements discussed in this chapter, the common interferences are shown in Table 16.1. 16.2.1.2 Hydride generation As previously mentioned above, the most common sample introduction technique for HPLC-ICP-MS is pneumatic nebulization. A major drawback of this sample introduction method is the transport efficiency of the sample aerosol to the plasma ionization source. Efforts to overcome this limitation have led to the development of alternative sample introduction methods such as laser ablation [10], electrothermal evaporation [11], and hydride generation [12,13]. These techniques allow for improved analyte transport efficiency, separation from matrix interferences, and improved detection limits by a factor of 10 or more compared to conventional pneumatic nebulization. Story and Caruso, in their critical review of hydride generation techniques reported a decrease in detection limits for hydride forming elements of up to three orders of magnitude compared to pneumatic nebulization techniques [14]. Hydride generation (HG), the most popular alternative sample introduction technique, has been utilized for nearly 40 years as a derivatization method for the analysis of trace elements capable of forming hydrides (As, Bi, Ge, Hg, Pb, Sb, Se, Sn, Te, In, and Tl). Generation of volatile metal hydrides can be accomplished in a variety of ways including: electrochemical generation, photoinduced generation,

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thermochemical generation, metal-acid reduction, and sodium borohydride reduction [15–17]. The suspected mechanism for hydride generation is discussed elsewhere [15,16]. Arsenic is the most common element subjected to analysis by HPLC-HG-ICP-MS [12,18–23]. Arsenic speciation experiments utilizing this technique have been performed in a wide range of sample matrices including; urine [12], river water [18], seawater [22], pepper plants [19], biological tissue [20,23], and soil [21]. Typically, hydride generation for arsenic was thought to be limited to the two inorganic forms (AsIII and AsV) as well as three methylated forms (methylarsonate (MA), dimethylarsinate (DMA), and trimethylarsine oxide). This work demonstrated quantitative analysis of four arsenosugars without a decomposition step by HPLC-HG-ICP-MS for the first time [13]. Selenium speciation by HPLC-HG-ICP-MS has previously been applied to multiple inorganic and organic species [24–27]. These species include selenite, selenate, selenocyanate, trimethylselenonium, selenomethionine, and multiple selenosugars. Analysis of mercury species with element-specific detection through the generation of a volatile species is typically referred to as cold vapor generation [15]. Developed in the 1960s this process results in the reduction of mercury species to the elemental state Hg0 [15]. The resulting Hg0 possesses an extremely high vapor pressure allowing simple separation from an aqueous matrix and efficient transport to the detector. Typical reducing agents for this reaction include tin chloride and sodium tetrahydroborate [15]. Analysis of mercury species by HPLC-CV-ICP-MS has previously been applied to seawater, soil, and fish tissue matrices [28–31]. Hydride generation was used to help eliminate NaCl from the matrix prior to ICPMS detection when analyzing antimony, thus leading to better detection limits [32].

16.2.2 Interfacing HPLC with ICP-MS With all the advantages of ICP-MS, one main disadvantage is that only elemental information is gained. Therefore, speciation was developed by which the theory is to separate various compounds, based on its species-specific properties using some type of chromatography, then pass the eluent into the ICP-MS for mass-specific detection. The most common separation technique coupled to ICP-MS is HPLC. The interfacing is very simple and well documented in the literature [33], as just a PEEK tubing capillary from the end of the column to the nebulizer of the ICP-MS is typically all that is needed. The liquid eluent is ideal for ICP-MS nebulizers, which are chosen based on their compatibility with the method specific chromatographic flow rate. Compositions of the mobile phases in HPLC are the most crucial of factors when interfacing with ICP-MS. Problems occur with high concentrations of organic modifiers such as methanol (MeOH) and acetonitrile (ACN). Typically these should be kept r20% v/v as higher levels can result in plasma instability, thus leading to a noisy baseline, and at high enough levels, the plasma will extinguish. Some research has cited that the addition of MeOH can actually increase the signal to noise of the References pp. 559–563

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analyte as it can increase ionization efficiency of the plasma source. Due to this reason and less instability of the plasma, as caused by MeOH compared to ACN, MeOH is typically the organic solvent of choice for liquid chromatographic methods applied to ICP-MS. This problem can be overcome by desolvation or dilution of the mobile phase prior to introduction into the ICP-MS [34] among other methods [35,36]. This premise also applies to solutions with high salt concentrations, as high enough levels will cause the similar problems and introduction of easily ionized elements such as sodium and potassium can lead to decreased ionization for analytes of interest. It is also important to note that exceeding any of these solution limitations could cause instrument components to dirty faster thus decreasing ion transmission and lowering sensitivity. In HPLC-ICP-MS, species identification is accomplished through comparison to standards both by matching retention times and/or spiking a sample with a known standard and monitoring an increase in response. If standards are not commercially available or capable of being synthesized, the eluting unknown compound may be collected from the HPLC and analyzed with molecular mass spectrometry. This may require special sample preparation prior to analysis due to the difference in ionization source and mass analyzer, typically involving the removal of mobile phase constituents. HPLC-ICP-MS has many advantages that make it very powerful for speciation analysis. HPLC is less time consuming and simple in regards to sample preparation in that no derivatization is needed either prior to analysis or post-column. The interfacing of the two techniques is quite user-friendly. ICP-MS is the ultimate in element-specific detection as far as analyzing a wide range of elements with high sensitivity and selectivity. A plethora of HPLC separation schemes can be interfaced with ICP-MS, thus leading to a wide variety of applications. Applications are constantly being improved upon and new samples/matrices are being explored all the time. Most of the applications discussed in this chapter involve analytical scale chromatography (flow rates 0.1 – 2.0 mL min
1). With the growing use of capillary scale (0.002–0.1 mL min
1) and nano scale (2–0.05 mL min
1) chromatography and compatible low flow nebulizers, these applications are being published more often, but the current lack of developed methods precludes them from this chapter.

16.3 APPLICATIONS 16.3.1 Arsenic Arsenic has been an element of much concern throughout history. One source of infamy arises from the speculation that Napoleon met his demise at the hands of arsenics’ toxicity. Although arsenic poisonings are not frequent, it will always carry high interest in the publics’ eye. Arsenic is also under heavy surveillance as it is a common environmental pollutant. The main source of arsenic contamination for the aquatic environment is from geological sources, either surface weathering or

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underground deposits [37]. Humans play a part in the problem with the use of pesticides and herbicides, pressure-treated lumber, industrial manufacturing, and roxarsone (a growth promoter in chicken feed). The problem is especially prevalent in less developed countries, in which arsenic contamination of village water supplies is all too common. Because of these reasons, scientific methods are needed to analyze body fluids for diagnosing exposure as a result of a criminal poisoning (i.e. determining the cause of death) or from environmental sources. Methods should also be applicable for environmental samples to assess risk of contamination. As mentioned in the introduction, many regulatory limits of arsenic (arsenic in drinking water) are based on total arsenic levels. However, different species of arsenic have varying levels of toxicity. Arsenic is most commonly found in these forms: arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMAV), dimethylarsinic acid (DMAV). AsIII and AsV are viewed as the acutely toxic species. MMAV and DMAV compounds are carcinogenic. However, arsenobetaine (AsB) and arsenocholine (AsC) are viewed as virtually non-toxic [37]. There are several other arseniccontaining compounds that are of interest in various areas of research; they include, but are not limited to the species in Table 16.2. The most common route of exposure for arsenic is oral ingestion. The commonly accepted detoxification pathway of arsenic is methylation. Fig. 16.2 shows a proposed methylation pathway in the body [38]. When a normal person is exposed to trace levels of inorganic arsenic it is partly methylated into MMA and DMA, which are excreted largely in the urine [39]. Arsenic also frequently enters the blood. One study explored the distribution of various arsenic species in human organs following fatal acute intoxication by arsenic trioxide. The majority of the arsenic found in the organs was AsIII [39]. Arsenic is commonly analyzed in food and water samples, with a wide distribution of species

TABLE 16.2 COMMON ARSENIC SPECIES FOUND IN SPECIATION ANALYSIS Name Inorganic compounds Arsenite (arsenous acid) Arsenate (arsenic acid) Organic compounds Monomethylarsonous acid Monomethylarsonic acid Dimethylarsinous acid Dimethylarsinic acid Arsenobetaine Arsenocholine Trimethylarsine oxide Tetramethylarsonium ion Arsenic-containing ribosides

Abbreviation

AsIII AsV

As(OH)3 AsO(OH)3

MMAIII MMAV DMAIII DMAV AsB AsC TMAO Me4As+ Arseno sugars

CH3As(OH)2 CH3AsO(OH)2 (CH3)2AsOH (CH3)2AsO(OH) (CH3)3As+CH2COO
(CH3)3As+CH2COO
(CH3)4As+ (CH3)4As+ Various sugar structures

Reprinted from [37] with permission from Elsevier.

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Fig. 16.2. Reduction and methylation pathways for arsenic. From reference [38] with permission.

present throughout various samples. For example, arsenic is found in high levels in seafood, but the majority of the arsenic is in AsB [40,41]. 16.3.1.1 Sample preparation One main problem in any speciation analysis is the preservation of the natural species at time prior and right up to analysis. This is a particular problem in the case of oxidation of AsV to AsIII. In a study of urine samples, it was concluded that samples could be kept at low temperatures (4
201C) with no additives or acidification for up to 2 months without inter-species conversion [42]. For liquid samples such as drinking water and urine, analyte extraction is viewed as unnecessary. Typically, the samples are diluted with deionized water (DIW), filtered through 0.45 mm filters to remove any particulate1 and then injected into the HPLC-ICP-MS system. This is typically referred to as the ‘‘dilute and shoot’’ method and has been used by Shraim et al. [43] (5X dilution) among others [44]. He et al. [45] analyzed blood samples by partitioning the blood into serum and erythrocyte portions, followed by de-proteinization and filtration through a 0.45 mm membrane. No other pretreatment procedures were needed before injection. These methods can only be used for samples in which the matrix is liquid based and are compatible with the chromatographic method. When dealing with a more complicated matrix, extraction of the analytes is necessary. The key to extraction for arsenic speciation is not to alter the natural

1

It goes without saying that all liquid samples/extracts should be filtered through a 0.45 mm or 0.2 mm filter prior to injection into the HPLC system, even if not mentioned. This also applies to mobile phases and is especially crucial with capillary and nano HPLC applications.

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species. Again, the problem of the conversion of AsV into AsIII is the main concern and sometimes cannot be avoided. Some reports will only state the sum of the inorganic arsenic compounds rather than the individual species due to the uncontrollability of the AsV to AsIII conversion. Solid samples incur more challenges, in which extractions are necessary. Typically samples are lyophilized, made as homogeneous as possible, and then extracted with some type of solvent, typically DIW and/or organic solvents such as acetonitrile, methanol, and/or chloroform. Supercritical fluid extractions (SFE) and microwaveassisted extractions (MAE) are also among those used [46,47]. Once the analytes are in the liquid portion, it can be injected into the HPLC. When analyzing human organs, extractions were performed using a 50:50 MeOH/ DIW (v/v) solution. [39] There are a variety of extraction procedures involving food and environmental samples. A multi-step extraction for arsenic from algae uses methanol, acetone, and diethyl ether as used by Madsen et al. [48] Poultry waste was extracted with DIW, and solid phase extraction was used to clean up the samples by removing the hydrophobic organic compounds that would complicate the separation. [49] For freeze-dried apples, several extraction solvents were evaluated including 50:50 MeOH/DIW (v/v), a-amylase+50:50 MeOH/DIW (v/v), and 40:60 ACN/DIW (v/v). Each was in combination with sonication and all three produced high percent recoveries. [50] In work done by Ackerman et al. [51] an extraction using trifluoroacetic acid (TFA) is compared with an enzymatic extraction using pepsin and pancreatin. These methods were applied to rice samples with the TFA extraction proving slightly more efficient; however, the use of TFA will convert AsV into AsIII. 16.3.1.2 Chromatography As arsenic is of high concern, there are a great number of speciation techniques; only several of the more common methods are discussed here. For a more complete listing refer to the review by B’Hymer et al. [37] Ion exchange is the most commonly used HPLC technique for arsenic speciation. Anion-exchange is often used to separate AsIII, AsV, MMAV, and DMAV, while cation-exchange is frequently used to separate AsB, AsC, trimethylarsine oxide (TMAO) and tetramethylarsonium ion (Me4As+). In ion exchange, the main factors that influence the separation are pH of the buffer, ionic strength and concentration of the buffer solutions, and temperature. As previously mentioned, buffers that are compatible with ICP-MS must be chosen. The most commonly used buffers for ion exchange with ICP-MS are phosphate, [44,50] carbonate, [52,53] phthalic acid, [54] tetramethylammonium hydroxide, [55] formate, [38] and nitrate [56] buffers. Ammonium is the cation of choice as it is the most volatile and leaves less residual material on the detector’s components. A few specific examples of ion exchange methods are present in the following paragraphs. The Dionex IonPak AS7 is commonly used and Kohlmeyer et al. [57] were able to separate 17 arsenic compounds using a nitric acid, 0.05 mM benzene-1, 2-disulfonic acid dipotassium salt, 0.5% MeOH v/v mobile phase with gradient altering the nitric acid concentration. This type of separation is used in several applications with nitric acid levels typically from 0.1 to 50 mM. Other additives to the References pp. 559–563

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544 MMA 12000

Signal intensity/counts

As(V)

8000 AsB As(III)

t=0 DMA

4000 STD

Cl-

0 1.00

2.00

3.00

4.00

5.00 Time/min

6.00

7.00

8.00

9.00

Fig. 16.3. Anion exchange chromatogram of five arsenic species in a 0.1% Cl-matrix resulting from gradient elution with ICP-MS detection. AsB, 2 ppb; DMA, 1 ppb; AsIII, DMA and AsV, 5 ppb. From reference [59], reproduced with permission of The Royal Society of Chemistry.

mobile phases have been used such as acetate buffers including ion pairs like benzene1,2-disulfonic acid. Small levels of MeOH have been added to increase sensitivity (0.5% v/v in DIW). Creed and coworkers reported (NH4)2CO3 mobile-phase-based separations using a Hamilton PRP-X100 column and InterAction Ion-120 column interfaced with ICP-MS (shown in Fig. 16.3) and ESI-MS for identification of previously uncharacterized arsenosugars [58,59]. Madsen et al. [48] developed a cationic exchange methods for algal extract using a Hewlett Packard Zorbax 300 SCX column using 20 mM pyridine, pH 2.2 with ICP-MS and ESI-MS for detection. Reversed-phase ion-pairing chromatography is also commonly used for arsenic speciation and is explained in depth elsewhere [60–65]. Le and Ma [66] used a C18 column with 10 mM propanesulfonate, 4 mM malonic acid, and 0.1% MeOH v/v to separate 7 arsenic standards (shown in Fig. 16.4). 16.3.1.3 Detection limits Detection limits for arsenic species are very low at part per trillion levels with absolution detection limits reported around 50–200 pg of arsenic [37].

16.3.2 Selenium High interest involving selenium centers on its dual personality as it can be both toxic or beneficial. Most recently it was discovered that Se has anti-cancer properties as shown in research by Clark et al. [67]. Selenium has also been investigated in slowing progression from AIDS and HIV along with proper immune functioning [68]. The problem with selenium is that its toxic and the beneficial range is quite

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800 DMA 600

counts

AsB 400

As(III) MA As(V)

200

0 0

200

400 time s

600

800

Fig. 16.4. Anion exchange chromatogram of an aqueous standard solution containing As, DMA, AsIII, MMA (MA), and AsV (1 mg As L
1 each). From reference [55] with kind permission of Springer Science and Business Media.

small, in general it is considered to be between 40 and 100 mg kg
1 of body weight per day [69]. Due to this narrow toxicity range, accurate and precise methods are needed to correctly assess selenium levels. Selenium reaches the environment mainly from coal mining and irrigation water that extracts the selenium from underground shale. It is generally accepted that the inorganic forms, selenate and selenite, are considered toxic while the organic forms are non-toxic or even beneficial. Many supplements are now sold containing the most common beneficial Se compound, selenomethionine (SeMet). Other compounds, such as selenomethylcysteine (SeMeCys), are also thought to have anti-cancer properties. Recently, there have been several selenosugars that have been reported. [70,71] A more complete list of common selenium compounds is shown in Table 16.3. Selenium also has the capability of entering proteins by both specific coding via the UGA codon as selenocysteine, and nonspecifically in the form of SeMet as selenium replaces sulfur in methionine. There are approximately 35 selenoproteins that have been found in mammals [72], while others have been found in yeast [73]. Cases of selenium poisoning are very rare [75]. Such cases involving selenic acid or sodium selenite have been reported [76]. The bulk of the research involving humans and selenium focuses on the bioavailability of various selenium species and metabolism. Environmental samples and food stuffs receive the most attention related to selenium speciation. Important applications of selenium speciation include insuring References pp. 559–563

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TABLE 16.3 SOME INORGANIC AND ORGANIC COMPOUNDS OF INTEREST IN SPECIATION ANALYSIS Chemical Name Hydrogenselenide Selenous acid (selenite) Selenocyanate Trimethylselenonium cation Dimethylselenide Dimethyldiselenide Dimethylseleniumsulfide Dimethylseleniumdioxide Dimethylselenopropionate Methylselenol Methylseleninic acid Methylseleninic acid Selenocysteine Selenomethylcysteine Selenocystine Selenomethionine Selenoethionine g-Glutamyl-Se-methylselenocysteine Selenocystathionine Selenohomocysteine Se-adenoxylselenohomocysteine Selenosugars Selenoproteins

H2Se (volatile) SeO3H2 (SeO2
3 ) SeO4H2 (SeO2
4 ) HSeCN (CH3)3Se+ (CH3)2Se (volatile) (CH3)Se-Se(CH3) (volatile) (CH3)Se-S(CH3) (volatile) (CH3)2SeO2 (volatile) (CH3)2Se+CH2CH2COOH CH3SeH CH3Se(O)OH CH3SeOH HOOCCH(NH2)CH2-Se-H HOOCCH(NH2)CH2-Se-CH3 HOOCCH(NH2)CH2-Se-Se-CH2CH(NH2)COOH HOOCCH(NH2)CH2CH2-Se-CH3 HOOCCH(NH2)CH2CH2-Se-CH2CH3 H2NCH2CH2-CO-NHCH(COOH)CH2-Se-CH3 HOOCCH(NH2)CH2CH2-Se-CH2CH(NH3)COOH HOOCCH(NH2)CH2CH2-Se-H HOOCCH(NH2)CH2CH2-Se-CH2C4H5C5N4NH2 Various sugar structures Various proteins and enzymes (i.e., GPX, Selenoprotein P, TR)

that foods contain the beneficial forms of selenium rather than toxic species and confirming the selenium content of commercial selenium supplements. 16.3.2.1 Sample preparation Liquid samples that have been analyzed include urine, blood, and natural waters. Urine samples can simply be diluted [71]. Blood samples can be centrifuged down, and their supernatant collected, then injected [77]. Water samples can just be injected directly. Extraction of selenium must be performed to preserve the original species. Typically for food samples, hot water extracts are performed to mimic food preparation techniques [78]. This is typically deemed adequate for freeing selenium species not associated with larger molecules [79]. There is a wide range of sample preparation techniques used to extract selenoamino acids (SeMet, SeMeCys, etc.) from proteins and other larger molecules. These include the use of various combinations sodium dodecyl sulfate, driselase, proteinase K [80], protease [81], trypsin [82], and lipase [83]. Yang et al. [84] evaluated 14 extraction techniques applied to yeast samples and determined that the use of 4 M methanesulfonic acid was the most efficient at extracting SeMet. Concern should be taken when looking at previous research in

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regards to SeMet content, as when enzymes are used, the SeMet content could be from proteins rather than native SeMet [79]. 16.3.2.2 Chromatography Several speciation methods have been designed. A few of the more commonly used methods are summarized here, for a more complete listing refer reviews by B’Hymer and Caruso [74] and Polatajko et al. [79]. Anionic exchange is not as commonly used, due to the relatively high pKa’s of the majority of selenium compounds of interest, but it is able to resolve selenite and selenate. Cationic exchange is more frequently used. Larsen et al. [85] have achieved the separation of a mixture of 12 selenium species comprising of selenoamino acids, selenonium ions, and inorganic selenium. Separation of anionic species was carried out with an anion-exchange column with isocratic elution with an aqueous salicylate-TRIS mobile phase at pH ¼ 8.5. The cationic species were separated using cation-exchange column with gradient elution with aqueous pyridinium formate, pH ¼ 3 [79]. Perhaps, the most commonly used separation method of selenium species is ion-pairing reversed phase chromatography. Although selenate and selenite typically cannot be resolved [79], a wide variety of selenium compounds can be separated. An assortment of ion-pairs have been used including formic acid, trifluoroacetic acid (TFA), heptafluorobutyric acid (HFBA), hexane sulfonic acid, citric acid, and malonic acid. TFA and HFBA concentrations are typically r1% (0.1%) but up to 5% has been used. Isocratic runs have been used with the addition of an organic modifier. Kotrebai et al. [86] compared different ion pairs, with the eventual separation of 22 selenium compounds using ion-pairing RP chromatography, shown in Fig. 16.5. Montes-Bayon et al. [87] and Uden and coworkers [86,88] use the combination of ICP-MS and ESI-MS which can help to identify selenium species which could not be identified by ICP-MS due to the lack of available standards. For this, mobile phases must be compatible with both methods including at or below 0.1% (v/v) TFA or HFBA (for electrospray) and low organic (for ICP). Only the relatively high abundant selenium species can be identified by ESI-MS due to sensitivity differences or preconcentration is necessary. 16.3.2.3 Detection limits Common detection limits for selenium species are typically low part per billion levels. Encinar et al. [89] reported detection limits below 0.5 ppb for human serum samples using a collision cell. B’Hymer and Caruso reported detection limits of 0.5–2 ppb.

16.3.3 Chromium Chromium is a perfect example of this variation in species toxicity in that the chromic ion (CrIII) is an essential nutrient involved in the regulation of glucose, cholesterol, and fatty acid metabolism, while hexavalent chromium, typically chromate, is mutanogenic and carcinogenic [90]. Alternatively, chromium particles in air References pp. 559–563

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Fig. 16.5. HPLC-ICP-MS chromatograms of selenium standards using 0.1% TFA, PFPA or HFBA as ion-pairing agents. SeIV, SeVI, and selenocystine elute before 7 min. Se-methylselenocysteine (9) and selenomethionine (11). Reprinted from [86] with permission from Elsevier.

aid in the oxidation of sulfur dioxide (SO2) resulting in the production of acidic gases which produce acid rain [90]. Hexavalent chromium is widely utilized in a variety of industries including: plastics, metal working, and paints/inks/dyes [91]. Due to the widespread use and health risk associated with this element, environmental and occupational regulatory agencies have mandated strict guidelines for the storage, use, and disposal of anything containing hexavalent chromium [91]. Moreover, the toxicity differences between CrIII and CrVI requires an analytical method capable of species differentiation and low level detection. 16.3.3.1 Sample preparation Separation of the two chromium species is a challenging task in that the chromic ion (CrIII) exists as a cationic aqua-hydroxo complex, while CrVI exists as anionic chromate. The two redox forms of chromium pose limitations in sample collection, storage, and pretreatment procedures due to possible interconversion between the two oxidation states. Pantsar-Kallio and Manninen [93] stress the importance of sample analysis immediately following collection. 16.3.3.2 Chromatography Efforts to separate both redox species of chromium have led to the investigation of reverse-phase chromatography or ion chromatography coupled with element-specific

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detection [90–101]. Byrdy et al. [95] were the first to demonstrate HPLC-ICP-MS for chromium speciation. This work consisted of a reverse-phase separation on a Dionex AS7 column with an NG1 guard [95]. The mobile phase was comprised of 35 mM ammonium sulfate with ammonium hydroxide at pH 9.2 [95]. Prior to analysis, a chelation procedure was utilized to stabilize the Cr3+ in the standard solutions with EDTA. The authors cited previous work of successful application of the developed method to dietary supplements, urine standard reference material, and chromium dyes [95,96]. Pantsar-Kallio and Manninen performed anion exchange for the separation of chromium species from multiple interfering ions such as chloride, sulfate, carbonate, cyanide, and some organic species was discussed. They used a Waters IC-Pak A column with a 4–40 mM HNO3 gradient [93]. Seby et al. [94] investigated the influence of interfering ions for chromium speciation following work by Barnowski et al. [97] in which the use of a IonPac CS5A with a IonPac CG5A guard column with both anion and cation exchange capabilities using a nitric acid eluent. Separation of the two chromium species was achieved in less than 7 min [94]. This is shown in Fig. 16.6. Due to the increase in pH accompanied with the addition of hydrogen carbonate resulted in hydrolysis of the CrIII into various hydroxycomplexes (Fig. 16.7). The newly formed and Cr(OH)03 due to the instability of peaks were suspected to be Cr(OH)(H2O)2+ 5 4+ Cr(OH)2(H2O) . 16.3.3.3 Detection limits Byrdy et al. [95] concluded that the single ion monitoring capability of ICP-MS provided ultra-trace detection levels (ng) previously never achieved. Seby et al. [94] achieved detection limits of 0.38 and 0.20 mg L
1 for CrIII and CrVI respectively. Several other HPLC-ICP-MS techniques were reported with varying detection limits [98–101], with the lowest being 0.063 and 0.061 mg mL
1 for CrIII and CrVI, respectively [100].

16.3.4 Lead In the past, lead was used in paints and as an anti-knocking agent in fuels. However, its use is now heavily regulated, due to its many health concerns. Of the utmost importance are its implications to the health of children as they are at the highest risk for lead contamination according to the EPA. The main routes of exposure include deteriorating lead-based paint, lead-contaminated dust, and leadcontaminated residential soil [1]. Lead species of high interest include inorganic Pb (Pb2+), trialkyl lead and tetraalkyl lead compounds. Tetraethyl lead (TTEL) is a central nervous system toxin that produces an acute toxic psychosis [102]. Inorganic lead is much less toxic than its organic forms with varying toxicities within organic lead compounds, for example tetraethyl lead is approximately 10 fold more toxic than tetramethyl lead in rats [103]. When coming from References pp. 559–563

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Fig. 16.6. Description of the effect of hydrogen carbonate ions on the developed chromium speciation method. From reference [94], reproduced with permission of The Royal Society of Chemistry.

Fig. 16.7. Hydrolysis of CrIII in solution over the pH range of 4–10. From reference [94], reproduced with permission of The Royal Society of Chemistry.

auto emissions lead enters the environment as tetraalkyl lead compounds and is easily broken down by the environment into trialkyl lead compounds. Eventually, biological systems will metabolize the lead into the least toxic form, inorganic lead.

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16.3.4.1 Sample preparation Ideally, samples for lead speciation should be analyzed as soon as possible. If not, they should be stored in Teflon or polyethylene containers. When analyzing biological samples concern should be taken because acidification changes the physicochemical distribution of lead species, and therefore must not be used prior to speciation [104]. Ebdon and coworkers [105] found that using properly cleaned containers, unacidified natural water samples stored at 41C in absence of light for up to 3 months exhibited no measurable inter-species conversion. 16.3.4.2 Chromatography In recent years, the speciation of lead has been analyzed more using GC rather than HPLC. The majority of HPLC-ICP-MS methods for lead speciation methods were developed prior to 1999. HPLC methods for the separation of lead compounds have dealt almost exclusively with the separation of the trialkyl forms. These separations exploit the difference in hydrophobicity of the species, typically utilizing an ion-pair in combination with high percent organic with a RP column. Ebdon et al. [105] used an acetate buffer system with sodium 1-pentanesulfonic acid (SPSA) and 60% MeOH (v/v) (shown in Fig. 16.8). Pan et al. [106] used similar conditions without SPSA. In two studies by Al-Rashdan et al. [107,108] Pb (Pb2+), trimethyllead chloride (TML), triethyllead chloride (TEL), triphenyllead chloride (TPhL), and (TTEL) were separated with isocratic or gradient elution involving the use of 30% v/v MeOH. Brown et al. [109] used only a DIW/MeOH mobile phase system to separate PbII, TML and TEL using isotope dilution for quantification. Shum et al. [110] separated the same using a microbore RP column with 5 mM ammonium pentanesulfonate in 20% ACN (v/v) (shown in Fig. 16.9).

Fig. 16.8. Isocratic ion-pairing chromatogram of inorganic lead, TML and TEL. Reprinted from [105] with permission from Elsevier.

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Fig. 16.9. Isocratic ion-pairing chromatogram of separation of Pb species in NIST SRM 2670 freeze-dried urine (normal level). EDTA was added to the sample at 10 mg L-1 before injection. Injection 1: NIST SRM 2670 freeze-dried urine (normal level). Injection 2: NIST SRM 2670 freeze-dried urine spiked with 40 pg (as Pb) of each of the trialkyllead species. From reference [110] with permission.

16.3.4.3 Detection limits Although baseline resolution is somewhat difficult to achieve for each of the above mentioned methods, detection limits were reported at 0.5
2 ppb.

16.3.5 Mercury Mercury is one of the more infamous metals of concern. Mercury mainly enters the environment from industrial applications. Coal-fired power plants are the largest remaining source of human-caused mercury emissions [1]. Criminal mercury poisonings are extremely rare and in one reported case only total Hg in blood was examined rather than mercury speciation [111]. Organomercury compounds are more toxic than inorganic mercury, HgII [112]. Mercury compounds of high interest include methyl mercury (CH3Hg+), ethyl mercury (C2H5Hg+), dimethyl mercury, diethyl mercury and phenyl mercury (C6H5Hg+). 16.3.5.1 Sample preparation The containers most suitable for Hg sample storage are Pyrex or Teflon containers and must be cleaned rigourously (e.g., with aqua regia, chromic acid, nitric acid, and BrCl). A final soaking of Teflon in 1% HCl at 70 1C removes all traces of oxidizing compounds that may interfere with CH3Hg+ [113].

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Liquid samples are quite simplistic in analysis by HPLC-ICP-MS as samples can be directly injected or diluted, then injected. Extracting mercury species from non-liquid samples is the major source of difficulty in mercury speciation. One problem is extracting both organic and inorganic species in the sample extraction. Also it has been shown that conversion of HgII into CH3Hg+ is possible in many extraction techniques, thus leading to overestimates of CH3Hg+ levels [113]. Most extraction procedures focus on removing CH3Hg+ and HgII from the matrix. Mercury species were extracted from hair samples using 2 mL of HNO3 (concentrated) and 1 mL of H2O2 (30% v/v) overnight at room temperature. The solution was diluted to 10 mL with DIW and injected directly onto the column [114]. Most extraction techniques derive from one presented by Westoo in which the sample is heated in acid (HCl) at high temperatures (microwave-assisted extraction), followed by non-polar (toluene) extraction. Some authors have recommended back extraction using some type of aqueous phase to further clean up the sample [112,113,115]. Two of the more successful procedures in terms of extraction efficiency of CH3Hg+ are by Horvat et al. [116] with 9574% extracted from soil via a distillation method; and by Tseng et al. [117] with 95–105% extracted from fish tissue by alkaline digestion using tetramethyl ammonium hydroxide (TMAH) with focused microwave power. 16.3.5.2 Chromatography As with lead, the majority of Hg speciation is currently performed using GC. However, there are still several HPLC methods, most of which involve the use of some type of ion pair with RP separation (IP-RP) and an organic modifier to help elute hydrophobic analytes from the column. Morton et al. [114] separated HgII from CH3Hg+ using a C18 RP column with 0.06 M ammonium acetate, 5% v/v methanol, 0.1% v/v 2-mercaptoethanol as a mobile phase. Shum et al. [110] separated HgII, CH3Hg+, and methyl ethyl mercury using a microbore RP column with 5 mM ammonium pentanesulfonate in 20% ACN (v/v). Separation of 5 mercury compounds was presented by Falter and Ilgen using a C18 column with 65% ACN (v/v) with ammonium acetate to adjust pH to 5.5 [118] (shown in Fig. 16.10). Tu et al. [31] used a Dionex PCX-500 guard column with 65% MeOH v/v with 0.45 M HCl. 16.3.5.3 Detection limits Detection limits of species are quite low at 41 ppb per compound. Tu et al. [31] reported detection limits of 35 pg Hg mL
1 and 73 pg Hg mL
1 for HgII and CH3Hg+, respectively. Preconcentration was used by Falter and Ilgen [118] to get absolute detection limits of 10–20 pg of Hg for various mercury species. Shum et al. [110] reported absolute detection limits for HgII, CH3Hg+ and MEM of 6–18 pg of Hg or 3–9 ppb in regards to Hg. 16.3.6 Tin Tin is also a heavily regulated element. The toxicity of tin is different from species to species as the organic form is far more toxic than the inorganic forms, which are References pp. 559–563

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Fig. 16.10. Separation of 5 mercury standards. The concentrations of each standard are: mersalyl acid at 50 pg Hg mL
1; CH3Hg+, C2H5Hg+, and C6H5Hg+ are at 200 pg Hg mL
1; and HgII at 220 pg Hg mL
1. From reference [118] with kind permission of Springer Science and Business Media.

regarded as virtually non-toxic. The toxicity of the organic forms increase as the alkyl chains increase. Maximum toxicity is achieved at tributyltin (TBT) [119]. The plastics industry is the source of most organotin usage [120]. They are also found in antifouling paints and pesticides [121]. These compounds tend to accumulate in the soil, thus making them a constant hazard [122]. Species of interest include the butyltins (mono (MBT), di (DMT), and (TBT)), phenyltins (mono (MPhT), di (DPhT), and tri (TPhT)), and triorganotins (methyl (TMT), ethyl (TET), and propyl (TPT)). 16.3.6.1 Sample preparation Sample preparation remains a challenge for extracting organotins from solid samples. Solvent extractions have some success at approximately 70–100%. These usually involve combinations of sonication, stirring, hydrochloric acid, and/or acetic acid with sequential non-polar extraction [122,123]. A method used by Arnold et al. [124] involving pressurized liquid extraction (PLE) offers high recovery with little degradation of original organotin species. Microwave-assisted extraction was shown to cause extensive degradation of phenyltin compounds [125]. Optimum solvents for PLE were shown to be methanol with 0.5 M acetic acid and 0.2% (w/v) tropolone [122] and extraction efficiencies were above 90%, except for DPhT. 16.3.6.2 Chromatography For HPLC-ICP-MS, a few genres of separations have been applied to the separation of organotin compounds. Again, as with lead and mercury, the majority of current speciation analysis is carried out by GC. The main species of interest are TBT and the phenyltin compounds. The use of ion exchange is popular but currently, none have separated all the triorganotins, butyl tin species, and phenyl tin species. This is most likely due to the incompatibility of the mobile phases to elute everything off the column with ICP-MS [121]. Reversed phase separations have also been used. Chiron et al. [122] used a TSK gel ODS TM column and a combination of tropolone and triethylamine at an optimum ratio of 0.075% (w/v):0.1% (v/v) in a

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Fig. 16.11. Chromatogram obtained after extraction of a spiked sediment by PLE. Spiking level: BTs— 1 mg g
1; PhTs—0.5 mg g
1. (IS) internal standard; (A) Monophenyltin; (B) monobutyltin; (C) triphenyltin; (D) diphenyltin; (E) tributyltin; (F) dibutyltin. Reprinted from [122] with permission from Elsevier.

methanol–DIW–acetic acid (72.5:21.5:6, v/v/v) mobile phase (shown in Fig. 16.11). Sodium pentane sulfonate has been employed as an ion pair in IP-RP [126,127]. The majority of these methods for speciating tin on RP columns use high organic levels, which require special considerations, previously mentioned, when interfacing with ICP-MS. A method by White et al. [36] used chromatography that was compatible with both API-MS and ICP-MS thus achieving molecular confirmation in conjunction with low detection limits. They used a C18 column with ACN, acetic acid, DIW, and triethylamine (at 65:10:25:0.5 v/v) to resolve DPhT, TPhT, DBT, and TBT. 16.3.6.3 Detection limits Detection limits for species of tin in terms of absolute detection limits vary from 1.5 to 55 pg of Sn [121].

16.3.7 Vanadium The beneficial and harmful effects of vanadium are not well understood. At trace levels, vanadium is an essential element for normal cell growth, but at higher concentrations, it can be toxic [128]. Vanadium is mainly used in steel manufacturing and is also released into the environment from the burning of fossil fuels. The possibility of vanadium’s insulin type behavior has also been explored [129]. The daily estimated recommended daily amount for humans is 10–60 mg [130]. One big concern is the presence of vanadium in natural waters. In natural waters, it normally References pp. 559–563

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exists as VV or VIV, with VV being more toxic than VIV [131]. These compounds usually are present at part per billion levels, therefore selective and sensitive methods of analysis are necessary to evaluate vanadium contamination. 16.3.7.1 Sample preparation VanadiumIV, as the vanadyl cation VO+ 2 , may be present in reducing environment. It is most stable in acidic solution below pH 2, but is oxidized to the pentavalent state by atmospheric oxygen at higher pH values [128]. This conversion between oxidation states becomes one of the main problems facing vanadium speciation analysis. Since the majority of the applications in the literature involve water, the main sample preparation techniques are to acidify the sample, keep at low temperatures, and in an oxygen-free environment. Other reports believe it is necessary to analyze the samples immediately after collection without acidification [128]. Other techniques involve the complexation of the vanadium species with ethylenediaminetetraacetic acid (EDTA) [132] or (1,2-cyclohexylene-dinitrilo)tetraacetic acid (CDTA) [133] to stabilize each species. 16.3.7.2 Chromatography There are only a limited number of HPLC-ICP-MS methods in the literature. Wann and Jiang [132] used EDTA to complex and stabilize VIV and VV to form [VOY]2
and [VO2Y]3
(Y represents deprotonated EDTA). Separation was then carried out using RP C8 column with 3 mM EDTA, 0.5 mM tetrabutyl ammonium phosphate (TBAP), pH 6.5 and 12% MeOH (v/v) as a buffer. The separation is shown in Fig. 16.12. Other HPLC methods have been presented and could be adapted to use ICP-MS for Vn detection. Work by de Beer and Coetzee [133] separated VIV and VV using a Dionex Ionpac AG5 guard column. The buffer was 5.6 mM NaHCO3,

Fig. 16.12. HPLC-ICP-MS chromatogram for: ClO+, VIV, and VV. Vanadium species at 5 ng mL
1 and Cl
at 0.2 M. Reprinted from [132] with permission from Elsevier.

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4.6 mM Na2CO3, and 20 mM CDTA. In this method, UV detection was used, but using ammonium salts instead of sodium salts would make this method more compatible with ICP-MS. Gaspar and Posta [134] used 0.1 M KH-phthalate in DIW with a RP C18 column using FAAS for detection. 16.3.7.3 Detection limits Gaspar and Posta [134] reported detection limits for VV and VIV as 0.18 and 0.15 mg/mL, respectively, with FAAS detection. With the substitution of ICP-MS, detection limits could certainly be lowered. Of the methods surveyed here, Wann and Jiang [132] had the best detection limits for online HPLC-ICP-MS determination for VIV and VV at 0.025 and 0.041 ppb, respectively.

16.3.8 Antimony Natural sources of antimony (Sb) in the environment come via soil runoff and rock weathering. Mining, fossil fuel combustion, and other industrial processes also contribute to antimony entering the environment [135]. The toxicity of antimony is heavily dependent on the species with the inorganic SbIII oxyanion being the most toxic [135], SbIII is approximately 10 fold more toxic than SbV, with inorganic species typically being more toxic than organic forms [136]. SbIII, SbV, and trimethylantimony dichloride are the most commonly analyzed species. 16.3.8.1 Sample preparation For urine samples, dilutions ranging from 1:3 to 1:50 with DIW and filtration were the only necessary preparation steps prior to injection [32]. Extraction of Sb from solid samples is problematic due to the low extraction efficiency of usually only a few percent [32]. Amerieh et al. [137] extracted Sb from soil using 100 mM citric acid, pH 2.1 at room temperature for 45 min in which 40% total Sb was extracted. Extraction of sewage samples were carried out by Lintschinger et al. [138] using combinations of DIW, MeOH and KOH with adequate extraction efficiencies. When extracting Sb from fly coal ash, 1 M citric acid was used with efficiencies of 22–36% [139]. Krachler and Emons [32] reported that the high NaCl content of urine complicates anionic separation, but they were eventually able to overcome it using a PRP-X100 column (20 mM EDTA, pH 4.7) to separate SbV and SbIII, and an ION-120 column (2 mM NH4HCO3 and 1 mM tartaric acid, pH 8.5) to separate SbV and TMSbCl2. Additionally, hydride generation was used to help eliminate NaCl from the matrix prior to ICP-MS detection thus leading to better detection limits [32]. Some methods involve the complexation of SbIII and SbV with citric acid [137] prior to separation. The PRP-X100 [138,139] was the most commonly used column, with others such as Synchropak Q300 [136] and Dionex AS4A-SC4 [138]. Mobile phases typically consisted of combinations of EDTA [136,137], phthalic acid [138], and tetraethylammonium hydroxide [138], among others [139]. References pp. 559–563

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Fig. 16.13. The separation of SbV, SbIII and TMSb all at 20 ng mL
1. From reference [140], reproduced with permission of The Royal Society of Chemistry.

Nash and coworkers were able to separate all three compounds of interest in less than 10 min. A gradient elution was used at 601C using Alltec HAAX column with 100 mM ammonium tartrate mobile phase with pH gradient from pH 2.3 to 1.5 [140] (shown in Fig. 16.13). 16.3.8.2 Detection limits Detection limits for SbV, SbIII, TMSb ranged between 0.005 and 0.3 ppb with the lowest detection limits being reported by Lintschinger [138]. 16.3.9 Tellurium Tellurium is similar to selenium in terms of its toxicity, as TeIV is 10 times more toxic than TeVI. [141] Tellurium is commonly used in electronics, metallurgy, and pharmaceuticals [141]. Little research has been presented using HPLC-ICP-MS to separate both species simultaneously. Two such methods have been used to detect only TeIV using HPLC-ICP-MS. [142,143] One method by Vin˜as et al. [144] involves complexing the tellurium species with 50 mM citric acid. Speciation is then carried out using a PRP-X100 column with 8 mM EDTA and 2 mM potassium hydrogenphthalate as a mobile phase. The separation is less than 8 min, with atomic fluorescence spectrometry used as the detection method; it is feasible that ICP-MS could easily be substituted. Detection limits were 0.69 mg Te L
1 and 0.76 mg Te L
1 for TeVI and TeIV, respectively.

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16.4 CONCLUDING REMARKS In summation, HPLC-ICP-MS can be applied to large number of sample types while analyzing for numerous elements. There are several important factors to consider when choosing the speciation method. Sample preparation is crucial, as preserving the native form of the species must be balanced with extraction efficiency. A wide range of chromatographic methods can be coupled with ICP-MS, with ion exchange and reverse phase ion pairing being the most popular. Mobile phase constituents must be properly selected to ensure compatibility with ICP-MS. ICP-MS offers low detection limits, which are critical when doing trace analysis, and with the use of hydride generation, further reduction of detection limits for many elements can be achieved. Due to the versatility of HPLC-ICP-MS new applications are constantly being developed and can easily be tailored to fit individual cases. 16.5 REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

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