UBC Theses and Dissertations
Development of analytical methods for arsenic speciation Pergantis, Spiridon
Metal speciation has become increasingly important in making risk assessments of metal toxicities. No longer is the knowledge of the total elemental content adequate for such a purpose, it is now essential to know the chemical form and concentration of all species of the element, including oxidation state of the metal and the type and number of substituents. Mass spectrometry is a very powerful tool for the identification of arsenic species in environmental samples. In particular Desorption chemical ionization - mass spectrometry (DCI-MS) produces satisfactory spectra with 100 ng arsenic placed on the DCI filament, making the technique eminently suitable for the investigation of environmental samples. The spectra of the arsenicals exhibit molecular ions as well as characteristic fragment ions. To extend the usefulness of low resolution DCI-MS, an analytical method was developed that permitted the use of mass deficient reference standards for calibration purposes in accurate mass measurements of positive ions under ammonia DCI conditions. This was accomplished by employing a mixture of ammonia and methane as reagent gases. In the high resolution accurate mass measurement experiment, this gas mixture allows for simultaneous detection of the mass spectrum of perfluorokerosene (calibration substance) adequate for calibration purposes, and the spectrum of the analyte which contains molecular weight information. For positive ion accurate mass measurements of higher masses (up to m/z=2300), Fomblin 18/8 oil was used successfully as a reference standard under ammonia, methane, and iso-butane desorption chemical ionization conditions. Both low and high resolution DCI-MS were used to identify arsenic compounds present in Mytilus californianus. This was accomplished by first extracting the arsenicals from the mussel flesh with methanol, and then isolating and purifying the compounds by means of conventional chromatography. Subsequently the mass spectrometric techniques described above were used to analyze the purified materials and provide spectra suitable for the structural characterization of two principal arsenic species present in the mussels; arsenobetaine (AsB) and the tetramethylarsonium ion. Matrix assisted laser desorption ionization - time of flight - mass spectrometry (MALDI-TOF-MS) is a very sensitive mass spectrometric technique which can detect as little as 0.3 ng of arsenic or 4 pmole of the arsenical under investigation. This feature suggests that the method could be used for the identification of minor arsenic components in environmental samples. Additional advantages of the technique are its capability of providing molecular ions as well as fragment ions for a variety of arsenicals, and allowing for a certain degree of control in compound fragmentation, by adjusting the N₂ laser power. Quantitative analysis by MALDI-TOF-MS is still considered a highly unreliable procedure since many variables associated with sample preparation and analytical procedure can seriously affect the results. Hydride generation - gas chromatography - mass spectrometry (HG-GC-MS), developed in this work, was used to provide conclusive evidence of -CD₃ incorporation from L-methionine-methyl-d₃ into arsenic compounds produced from arsenate by alga cell cultures. These findings strongly support the notion that the oxidation-reduction pathway involving carbonium ions, as originally suggested by Challenger for the alkylation of arsenic by microorganisms, applies to marine unicellular alga and probably other marine organisms. Micro - liquid chromatography (LC) columns (0.32 mm inner diameter) fabricated and packed in house can be conveniently coupled on-line to a variety of mass spectrometric systems, mainly because of the extremely low flow rates they require. It was shown that a 99 % reduction in the volume of solvent waste is achieved by switching from conventional to micro-LC, and a 87.5 % reduction by switching from microbore to micro-LC. Compared to previous work, lower detection limits for arsenic compounds were also obtained when micro-LC columns were employed in conjunction with ultraviolet (UV) and electrothermal atomic absorption (ETAA) detectors. The micro-LC columns are able to efficiently separate arsenicals that are used as animal feed additives as well as their potential metabolites. The separation efficiency achieved on this type of column was shown to be similar to those achieved on microbore (1 mm inner diameter) and conventional (4.5 mm inner diameter) high performance liquid chromatography (HPLC) columns. Simplex optimization was used to efficiently delineate the optimum experimental conditions to be used for the electrothermal atomic absorption spectrometric analysis of arsenic in a standard reference material of marine origin. Four experimental variables, were considered: ashing temperature, atomization temperature, modifier concentration, and atomization ramping time. This combination of methods and materials provides a powerful means of rapidly optimizing the experimental conditions used for the analysis of arsenic in a wide variety of samples of environmental origin. Excellent recoveries of arsenic were obtained when using the optimum electrothermal atomic absorption spectrometry conditions to analyze standard solutions of arsenobetaine, arsenocholine and tetramethylarsonium iodide. This procedure also allowed for the accurate determination of arsenic in Californian mussels collected from a variety of locations along the B.C. coast, which was found to range from 9 to 16 μg.g⁻¹ Finally, in Chapter 6 it was demonstrated that methylarsonic acid (MAsA) is a likely precursor to AsB in the marine environment. Mytilus calfornianus exposed for 9 days to a seawater system containing [³H]-methylarsonic acid, was found to contain [³H]-. methylarsonic acid along with [³H]-arsenobetaine and two unknown ³H-labeled compounds in the tissue parts of this mussel. A linear increase with time in the specific activity present in the flesh of Mytilus calfomianus was also observed indicating uptake of the labelled compound or its metabolites. The highest specific activity was found in the visceral mass and the gills of the mussel.
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